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A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium
Abstract
PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.
... One cluster of 10 genes, including dmxB, pmxA, and pkn1 as well as the benchmarking mrpC and fruA genes, were induced more than 4-fold (log 2 fold change [FC], $2; adjusted P # 0.05) at one or more time points during development ( Fig. 1B; Table S1B). Pkn1 is a Ser/Thr protein kinase with a C-terminal PilZ domain and is specifically important for development (36,60); it is not known whether the PilZ domain binds c-di-GMP. These observations are in agreement with previous findings that dmxB and pkn1 transcription is upregulated during development (40,60). ...
... Pkn1 is a Ser/Thr protein kinase with a C-terminal PilZ domain and is specifically important for development (36,60); it is not known whether the PilZ domain binds c-di-GMP. These observations are in agreement with previous findings that dmxB and pkn1 transcription is upregulated during development (40,60). A second cluster of 18 genes, including tmoK, pixB, gacA, and pilB, were induced more than 2fold (log 2 FC, $1; adjusted P # 0.05) at one or more time point(s) during development. ...
... pkn1, MXAN_2902, MXAN_6957, and MXAN_7024 are PilZ domain proteins; however, none contain the conserved motifs for c-di-GMP binding (27,36). Except for Pkn1, a lack of any of these four proteins does not cause defects during growth or development (36,60). MXAN_7500 is a MshEN domain protein with the sequence motifs for c-di-GMP binding (17); however, it is not known whether this protein binds c-di-GMP or whether it is important during growth and development. ...
Myxococcus xanthus has a nutrient-regulated biphasic life cycle forming predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. The second messenger 3'-5', 3'-5 cyclic di-GMP (c-di-GMP) is essential during both stages of the life cycle; however, different enzymes involved in c-di-GMP synthesis and degradation as well as several c-di-GMP receptors are important during distinct life cycle stages. To address this stage specificity, we determined transcript levels using transcriptome sequencing (RNA-seq) and transcription start sites using Cappable sequencing (Cappable-seq) during growth and development genome wide. All 70 genes encoding c-di-GMP-associated proteins were expressed, with 28 upregulated and 10 downregulated during development. Specifically, the three genes encoding enzymatically active proteins with a stage-specific function were expressed stage specifically. By combining operon mapping with published chromatin immunoprecipitation sequencing (ChIP-seq) data for MrpC (M. Robinson, B. Son, D. Kroos, L. Kroos, BMC Genomics 15:1123, 2014, http://dx.doi.org/10.1186/1471-2164-15-1123), the cAMP receptor protein (CRP)-like master regulator of development, we identified nine developmentally regulated genes as regulated by MrpC. In particular, MrpC directly represses the expression of dmxB, which encodes the diguanylate cyclase DmxB that is essential for development and responsible for the c-di-GMP increase during development. Moreover, MrpC directly activates the transcription of pmxA, which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3',3'-cGAMP in vitro and is essential for development. Thereby, MrpC regulates and curbs the cellular pools of c-di-GMP and 3',3'-cGAMP during development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity. MrpC is important for this regulation, thereby being a key regulator of developmental cyclic di-nucleotide metabolism in M. xanthus. IMPORTANCE The second messenger c-di-GMP is important during both stages of the nutrient-regulated biphasic life cycle of Myxococcus xanthus with the formation of predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. However, different enzymes involved in c-di-GMP synthesis and degradation are important during distinct life cycle stages. Here, we show that the three genes encoding enzymatically active proteins with a stage-specific function are expressed stage specifically. Moreover, we find that the master transcriptional regulator of development MrpC directly regulates the expression of dmxB, which encodes the diguanylate cyclase DmxB that is essential for development, and of pmxA, which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3',3'-cGAMP in vitro and is essential for development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity and that MrpC plays an important role in this regulation.
... The other has been to seek developmental factors that could play a role similar to that which has been well characterized for sporulation in B. subtilis (138). Both of these approaches have been successful: a large family of serine/threonine kinases has been described (164), and a number of sigma factors, including two that seem to be developmentally regulated, have been isolated (2,3,85). ...
... A number of the PCR products from the M. xanthus chromosome were likely candidates, and these were used for subsequent cloning and sequencing. Munoz-Dorado et al. (164) characterized one such gene, pkn-1, and showed that it indeed has substantial similarity to the catalytic domains of the eukaryotic kinases. When pkn-1 was placed in E. coli, the pkn-1 product was autophosphorylated at its serine and threonine residues. ...
... A subdomain sequence of pkn-1 was used as a further probe, which revealed an additional 23 bands on a Southern blot of an XhoI digest of M. xanthus chromosomal DNA (266). There are now a total of 26 putative kinase genes in M. xanthus, most of which have been cloned and 8 of which have been partially or completely sequenced (164). The details of this family of kinases and their interactions with a possible sigma cascade represent an exciting area of study in the regulation of M. xanthus development. ...
... (or eSTKs/eSTPs) due to their homology to eukaryotic signaling systems. Compared to eukaryotic systems that began to be characterized over 60 years ago, prokaryotic systems were only first identified in the early 1990s (Bakal & Davies, 2000;Munoz-Dorado et al., 1991). These bacterial kinases are likely evolutionarily ancient, sharing a common ancestor with those found in eukarya and archaea (Leonard et al., 1998;Stancik et al., 2018). ...
... Ever since the first bacterial Hanks-type Ser/Thr signaling pathway was identified in M. xanthus(Munoz-Dorado et al., 1991), whole genome sequencing has demonstrated that these evolutionarily ancient pathways are widespread in bacteria. Subsequently, it was discovered that these signaling systems can perform regulation on diverse cellular processes by directly phosphorylation of proteins.Experimentally, this was largely accomplished using a combination of phosphoproteomics and targeted in vitro validation of individual phosphosites. ...
Bacteria possess diverse classes of signaling systems that they use to sense and respond to their environments and execute properly timed developmental transitions. One widespread and evolutionarily ancient class of signaling systems are the Hanks‐type Ser/Thr kinases, also sometimes termed “eukaryotic‐like” due to their homology with eukaryotic kinases. In diverse bacterial species, these signaling systems function as critical regulators of general cellular processes such as metabolism, growth and division, developmental transitions such as sporulation, biofilm formation, and virulence, as well as antibiotic tolerance. This multifaceted regulation is due to the ability of a single Hanks‐type Ser/Thr kinase to post‐translationally modify the activity of multiple proteins, resulting in the coordinated regulation of diverse cellular pathways. However, in part due to their deep integration with cellular physiology, to date, we have a relatively limited understanding of the timing, regulatory hierarchy, the complete list of targets of a given kinase, as well as the potential regulatory overlap between the often multiple kinases present in a single organism. In this review, we discuss experimental methods and curated datasets aimed at elucidating the targets of these signaling pathways and approaches for using these datasets to develop computational models for quantitative predictions of target motifs. We emphasize novel approaches and opportunities for collecting data suitable for the creation of new predictive computational models applicable to diverse species.
... Previously, bioinformatics analyses identified 24 PilZ domain proteins in M. xanthus (9), among which only three have been analyzed experimentally. Pkn1 is a Ser/ Thr kinase that is important for development (65) (Fig. 1). Based on sequence analysis, the PilZ domain of Pkn1 is predicted not to bind c-di-GMP; however, c-di-GMP binding by Pkn1 has not been studied. ...
... In agreement with previous observations (65), the Dpkn1 mutant formed fruiting bodies but was reduced in sporulation; the DpixB mutant did not form fruiting bodies under the two conditions tested and was also reduced in sporulation. We did not observe developmental defects in the DMXAN_2902 mutant under the two conditions tested (Fig. 2). ...
In bacteria, the nucleotide-based second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus , c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ-domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system, and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro , and regulates motility independently of the Frz system likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA and PixB act in independent pathways and have distinct functions in regulation of motility.
Importance
c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts acts in Frz-independent mannerto regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus .
... Eukaryotic-like signaling and gene regulation have been known in M. xanthus for decades (Kroos, 2005;Munoz-Dorado et al., 1991). ...
Upon starvation, rod‐shaped Myxococcus xanthus bacteria form mounds and then differentiate into round, stress‐resistant spores. Little is known about the regulation of late‐acting operons important for spore formation. C‐signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA‐I, exoL‐P, and nfsA‐H. Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA‐binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR‐Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR‐Cas component, which we propose produces spores suited to withstand starvation and environmental insults.
... Eukaryotic-like signaling and gene regulation have been known inM. xanthus for decades (72,73). More recently, combinatorial control, a common theme in eukaryotic GRNs (67)(68)(69), emerged from studies of M. xanthus gene regulation (7,35). ...
Upon starvation rod-shaped Myxococcus xanthus bacteria form mounds and then differentiate into round stress-resistant spores. Little is known about the regulation of late-acting operons important for spore formation. C-signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to increase transcription of many genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA-I , exoL-P , and nfsA-H . Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA initially, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro , but strikingly each promoter region was unique in terms of whether or not MrpC and the DNA-binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR-Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR-Cas component, which we propose thwarts viral intrusion while making spores suited to withstand starvation and environmental insults.
... STPKLRR is a pathogenic protein kinase and shares the same STYKc kinase domain with Akt2. The STYKc domain in bacteria was first discovered in Myxococcus xanthus [51], and evidence indicates a close association between its phosphorylation and bacterial pathogenesis [52]. InlA, the internalin of L. monocytogenes, promotes adhesion and internalization through an actin-mediated process [53]. ...
We previously demonstrated that the flagellin of intracellular Vibrio splendidus AJ01 could be specifically identified by tropomodulin (Tmod) and further mediate p53-dependent coelomocyte apoptosis in the sea cucumber Apostichopus japonicus. In higher animals, Tmod serves as a regulator in stabilizing the actin cytoskeleton. However, the mechanism on how AJ01 breaks the AjTmod-stabilized cytoskeleton for internalization remains unclear. Here, we identified a novel AJ01 Type III secretion system (T3SS) effector of leucine-rich repeat-containing serine/threonine-protein kinase (STPKLRR) with five LRR domains and a serine/threonine kinase (STYKc) domain, which could specifically interact with tropomodulin domain of AjTmod. Furthermore, we found that STPKLRR directly phosphorylated AjTmod at serine 52 (S52) to reduce the binding stability between AjTmod and actin. After AjTmod dissociated from actin, the F-actin/G-actin ratio decreased to induce cytoskeletal rearrangement, which in turn promoted the internalization of AJ01. The STPKLRR knocked out strain could not phosphorylated AjTmod and displayed lower internalization capacity and pathogenic effect compared to AJ01. Overall, we demonstrated for the first time that the T3SS effector STPKLRR with kinase activity was a novel virulence factor in Vibrio and mediated self-internalization by targeting host AjTmod phosphorylation dependent cytoskeleton rearrangement, which provided a candidate target to control AJ01 infection in practice.
... However, understanding the roles of Ser/Thr (S/T) protein kinases (STPKs) in the maintenance of genome integrity and regulation of cell division in bacteria has also gained a considerable importance. For instance, a eukaryotic-type STPK was first identified in the Gram-negative bacterium Myxococcus xanthus (5). STPKs have now been identified in both bioticand abiotic-stress-tolerant bacteria, including pathogens like Pseudomonas aeruginosa (6), Enterococcus faecalis (7), Staphylococcus aureus (8), Mycobacterium tuberculosis (9), Streptococcus pneumoniae (10), Streptococcus agalactiae (11), Streptococcus pyogenes (12), and the superbug-like Deinococcus radiodurans (13,14). ...
DivIVA is a member of the Min family of proteins that spatially regulates septum formation at the midcell position and cell pole determination in Bacillus subtilis. Deinococcus radiodurans, a Gram-positive coccus-shaped bacterium, is characterized by its extreme resistance to DNA-damaging agents, including radiation. D. radiodurans cells exposed to gamma radiation undergo cell division arrest by as-yet-uncharacterized mechanisms. divIVA is shown to be an essential cell division gene in this bacterium, and DivIVA of D. radiodurans (drDivIVA) interacts with genome segregation proteins through its N-terminal region. Earlier, RqkA, a gamma radiation-responsive Ser/Thr quinoprotein kinase, was characterized for its role in radioresistance in D. radiodurans. Here, we showed that RqkA phosphorylates drDivIVA at the threonine 19 (T19) residue. The phospho-mimetic mutant with a mutation of T19 to E19 in DivIVA (DivIVAT19E) is found to be functionally different from the phospho-ablative mutant (DivIVAT19A) or the wild-type drDivIVA. A DivIVAT19E-red fluorescent protein (RFP) fusion expressed in the wild-type background showed the arrest in the typical dynamics of drDivIVA and the loss of its interaction with the genome segregation protein ParA2. The allelic replacement of divIVA with divIVAT19E-rfp was not tolerated unless drDivIVA was expressed episomally, while there was no phenotypic change when the wild-type allele was replaced with either divIVAT19A-rfp or divIVA-rfp. These results suggested that the phosphorylation of T19 in drDivIVA by RqkA affected its in vivo functions, which may contribute to the cell cycle arrest in this bacterium. IMPORTANCE Deinococcus radiodurans, a radioresistant bacterium, lacks LexA/RecA-mediated DNA damage response and cell cycle regulation as known in other bacteria. However, it adjusts its transcriptome and proteome upon DNA damage. In eukaryotes, the DNA damage response and cell cycle are regulated by Ser/Thr protein kinases. In D. radiodurans, we characterized a gamma radiation-responsive Ser/Thr quinoprotein kinase (RqkA) that phosphorylated DNA repair and cell division proteins in this bacterium. In previous work, the effect of S/T phosphorylation by RqkA on activity improvement of the DNA repair proteins has been demonstrated. This study reports that Ser phosphorylation by RqkA attenuates the function of a cell polarity and plane of cell division-determining protein, DivIVA, and its cellular dynamics in response to DNA damage, which might help to understand the mechanism of cell cycle regulation in this bacterium.
... (iii) secondary messenger-dependent systems, represented by cyclic adenosine monophosphate (cAMP) (4), cyclic diguanylate (c-di-GMP) (5), cyclic diadenylate (c-di-AMP) (6), and more (7,8); (iv) serine/threonine/tyrosine protein kinases (STYKs) and protein phosphatase-mediated pathways (9); (v) extracytoplasmic function s factors (ECFs) as alternative s factors in redirection of transcription (10); and (vi) quorum sensing (QS) in cell-to-cell communication (11). ...
Microbes rely on signal transduction systems to sense and respond to environmental changes for survival and reproduction. It is generally known that niche adaptation plays an important role in shaping the signaling repertoire. However, the evolution of bacterial signaling capacity lacks systematic studies with a temporal direction. In particular, it is unclear how complexity evolved from simplicity or vice versa for signaling networks. Here, we examine the evolutionary processes of major signal transduction systems in Campylobacterota (formerly Epsilonproteobacteria), a phylum with sufficient evolutionary depth and ecological diversity. We discovered that chemosensory system increases complexity by horizontal gene transfer (HGT) of entire chemosensory classes, and different chemosensory classes rarely mix their components. Two-component system gains complexity by atypical histidine kinases fused with receiver domain to achieve multistep or branched signal transduction process. The presence and complexity of c-di-GMP-mediated system is related to the size of signaling network, and c-di-GMP pathways are easy to rewire, since enzymes and effectors can be linked without direct protein-protein interaction. Overall, signaling capacity and complexity rise and drop together in Campylobacterota, determined by sensory demand, genetic resources, and coevolution within the genomic context. These findings reflect plausible evolutionary principles for other cellular networks and genome evolution of the Bacteria domain. IMPORTANCE Bacteria are capable of sensing and responding to environmental changes by several signal transduction systems with different mechanisms. Much attention is paid to model organisms with complex signaling networks to understand their composition and function, but how a complicated network evolved from a simple one or vice versa lacks systematic studies. Here, we tracked the evolutionary process of each signaling system in a bacterial phylum with robust "eco-evo" framework and summarized the general principles of signaling network evolution. Our findings bridge the gaps in bacterial signaling capacity from highly sophisticated to extremely streamlined, shedding light on rational design of genetic circuitry. This study may serve as a paradigm to examine the complex construction of other cellular networks and genome evolution.
... In the early 1990s, the first protein kinase PknL was discovered in Myxococcus xanthus. This enzyme shares a structural similarity with eukaryotic serine/threonine kinases (STKs) and is required for the normal development of M. xanthus (Muñoz-Dorado et al., 1991). Later, the first bacterial phosphatase was discovered by G A Nimmo et al. who reported that isocitrate dehydrogenase (IDH) is regulated by phosphorylation in Escherichia coli . ...
The human oral cavity harbors approximately 1,000 microbial species, and dysbiosis of the microflora and imbalanced microbiota-host interactions drive many oral diseases, such as dental caries and periodontal disease. Oral microbiota homeostasis is critical for systemic health. Over the last two decades, bacterial protein phosphorylation systems have been extensively studied, providing mounting evidence of the pivotal role of tyrosine and serine/threonine phosphorylation in oral bacterial dysbiosis and bacteria-host interactions. Ongoing investigations aim to discover novel kinases and phosphatases and to understand the mechanism by which these phosphorylation events regulate the pathogenicity of oral bacteria. Here, we summarize the structures of bacterial tyrosine and serine/threonine kinases and phosphatases and discuss the roles of tyrosine and serine/threonine phosphorylation systems in Porphyromonas gingivalis and Streptococcus mutans, emphasizing their involvement in bacterial metabolism and virulence, community development, and bacteria-host interactions.
... It then took a while to demonstrate the presence of other types of protein kinases in bacterial genomes. Indeed, it was only in the 1990s that a particular type of tyrosine protein kinase, which shares some similarities with ATPases, and a serine/threonine protein kinase, whose catalytic domain is homologous to that of eukaryotic protein kinases, were discovered [6,7]. Since then, both have been shown to be widespread in bacterial genomes, and a constantly increasing number of studies have reported their crucial roles in regulating bacterial physiology [3,4,8,9]. ...
It has been nearly three decades since the discovery of the first bacterial serine/threonine protein kinase (STPK). Since then, a blend of technological advances has led to the characterization of a multitude of STPKs and phosphorylation substrates in several bacterial species that finely regulate intricate signaling cascades. Years of intense research from several laboratories have demonstrated unexpected roles for serine/threonine phosphorylation, regulating not only bacterial growth and cell division but also antibiotic persistence, virulence and infection, metabolism, chromosomal biology, and cellular differentiation. This review aims to provide an account of the most recent and significant developments in this up and growing field in microbiology.
... The first bacterial STPK and the eligible ST/Y protein kinase superfamily candidate was characterized from M. xanthus, named Pkn1 [75]. Soon after, another STPK (YpkA) encoded by a virulence-specific plasmid of Y. pseudotuberculosis was identified [26]. ...
The roles of Serine/Threonine protein kinases (STPKs) in bacterial physiology, including bacterial responses to nutritional stresses and under pathogenesis have been well documented. STPKs roles in bacterial cell cycle regulation and DNA damage response have not been much emphasized, possibly because the LexA/RecA type SOS response became the synonym to DNA damage response and cell cycle regulation in bacteria. This review summarizes current knowledge of STPKs genetics, domain organization, and their roles in DNA damage response and cell division regulation in bacteria.
... In addition to the TCS system, signal transduction mechanisms are also facilitated by serine, threonine, and tyrosine phosphorylation mediated protein kinases in prokaryotes. This protein family in myxobacteria has been reported to have strong sequence similarity with eukaryotic-like kinases (ELKs) [32]. M. rosea contains 353 ELKs, which is higher than S. cellulosum So ce56 (317) [33], as well as other myxobacteria (Fig. 4B). ...
Background
Myxobacteria harbor numerous biosynthetic gene clusters that can produce a diverse range of secondary metabolites. Minicystis rosea DSM 24000 T is a soil-dwelling myxobacterium belonging to the suborderSorangiineae and family Polyangiaceae and is known to produce various secondary metabolites as well as polyunsaturated fatty acids (PUFAs). Here, we use whole-genome sequencing to explore the diversity of biosynthetic gene clusters in M. rosea .
Results
Using PacBio sequencing technology, we assembled the 16.04 Mbp complete genome of M. rosea DSM 24000 T , the largest bacterial genome sequenced to date. About 44% of its coding potential represents paralogous genes predominantly associated with signal transduction, transcriptional regulation, and protein folding. These genes are involved in various essential functions such as cellular organization, diverse niche adaptation, and bacterial cooperation, and enable social behavior like gliding motility, sporulation, and predation, typical of myxobacteria. A profusion of eukaryotic-like kinases (353) and an elevated ratio of phosphatases (8.2/1) in M. rosea as compared to other myxobacteria suggest gene duplication as one of the primary modes of genome expansion. About 7.7% of the genes are involved in the biosynthesis of a diverse array of secondary metabolites such as polyketides, terpenes, and bacteriocins. Phylogeny of the genes involved in PUFA biosynthesis ( pfa ) together with the conserved synteny of the complete pfa gene cluster suggests acquisition via horizontal gene transfer from Actinobacteria .
Conclusion
Overall, this study describes the complete genome sequence of M. rosea , comparative genomic analysis to explore the putative reasons for its large genome size, and explores the secondary metabolite potential, including the biosynthesis of polyunsaturated fatty acids.
... The third ORF encodes a protein with sequence identity to rat myosin light-chain kinase (180), a member of a eukaryotic class of serine/ threonine kinases. A similar serine/threonine kinase has been described in another prokaryote, Myxococcus xanthus (155). Although this DNA sequence was present on plasmids from independently isolated, complemented strains, recomplementation of the mutant with the isolated plasmid was not achieved. ...
Photosynthetic organisms can acclimate to their environment by changing many cellular processes, including the biosynthesis of the photosynthetic apparatus. In this article we discuss the phycobilisome, the light-harvesting apparatus of cyanobacteria and red algae. Unlike most light-harvesting antenna complexes, the phycobilisome is not an integral membrane complex but is attached to the surface of the photosynthetic membranes. It is composed of both the pigmented phycobiliproteins and the nonpigmented linker polypeptides; the former are important for absorbing light energy, while the latter are important for stability and assembly of the complex. The composition of the phycobilisome is very sensitive to a number of different environmental factors. Some of the filamentous cyanobacteria can alter the composition of the phycobilisome in response to the prevalent wavelengths of light in the environment. This process, called complementary chromatic adaptation, allows these organisms to efficiently utilize available light energy to drive photosynthetic electron transport and CO2 fixation. Under conditions of macronutrient limitation, many cyanobacteria degrade their phycobilisomes in a rapid and orderly fashion. Since the phycobilisome is an abundant component of the cell, its degradation may provide a substantial amount of nitrogen to nitrogen-limited cells. Furthermore, degradation of the phycobilisome during nutrient-limited growth may prevent photodamage that would occur if the cells were to absorb light under conditions of metabolic arrest. The interplay of various environmental parameters in determining the number of phycobilisomes and their structural characteristics and the ways in which these parameters control phycobilisome biosynthesis are fertile areas for investigation.
... We also found pkn1, which encodes a putative eukaryotic-like protein kinase, within this cluster. Deletions of pkn1 result in premature differentiation and poor spore production [48] In cluster-7, we identified 133 genes that are comparatively upregulated during growth in the nutrient-poor medium as compared to the rich medium. These genes are also highly expressed during late development starting from 12 to 72 h. ...
To accurately identify the genes and pathways involved in the initiation of the Myxococcus xanthus multicellular developmental program, we have previously reported a method of growing vegetative populations as biofilms within a controllable environment. Using a modified approach to remove up to ~90% rRNAs, we report a comprehensive transcriptional analysis of the M. xanthus developmental cycle while comparing it with the vegetative biofilms grown in rich and poor nutrients. This study identified 1522 differentially regulated genes distributed within eight clusters during development. It also provided a comprehensive overview of genes expressed during a nutrient-stress response, specific development time points, and during development initiation and regulation. We identified several differentially expressed genes involved in key central metabolic pathways suggesting their role in regulating myxobacterial development. Overall, this study will prove an important resource for myxobacterial researchers to delineate the regulatory and functional pathways responsible for development from those of the general nutrient stress response.
... Since the discovery of protein kinase N1 (PKN1) [8], a bacterial serine/threonine kinase with a high structural homology to eukaryotic protein kinases, sequencing efforts have uncovered the ubiquitous eukaryotic-like serine/threonine kinases (eSTKs) that phosphorylate multiple protein substrates and affect many areas of bacteria cell biology [9]. A subset of eSTKs are single-pass transmembrane proteins that have extracellular penicillin-binding-protein and serine/threonine kinase-associated (PASTA) domains, which bind muropeptides [10]. ...
Pyrazolo[3,4-d]pyrimidines represent an important class of heterocyclic compounds well-known for their anticancer activity exerted by the inhibition of eukaryotic protein kinases. Recently, pyrazolo[3,4-d]pyrimidines have become increasingly attractive for their potential antimicrobial properties. Here, we explored the activity of a library of in-house pyrazolo[3,4-d] pyrimidines, targeting human protein kinases, against Staphylococcus aureus and Escherichia coli and their interaction with ampicillin and kanamycin, representing important classes of clinically used antibiotics. Our results represent a first step towards the potential application of dual active pyrazolo[3,4-d]pyrimidine kinase inhibitors in the prevention and treatment of bacterial infections in cancer patients.
... Since the first characterization of Pkn1 as a serine/threonine protein kinase in Myxococcus xanthus (Muñoz-Dorado et al. 1991), Hanks-type serine/threonine kinases have been shown to control many physiological processes across all domains of life (Pereira et al. 2011;Stancik et al. 2018). Hanks-type kinase-encoding genes are enriched in the genomes of actinobacteria (Perez et al. 2008). ...
Newly synthesized proteins are subject to several post-translational modifications (PTMs). These PTMs can create diverse proteins from a single gene and are important for the function of certain proteins in a given situation. Recent advances in mass spectrometry (MS)-based proteomics have enabled the global detection of PTMs of bacterial proteins. In Corynebacterium and other actinobacteria, common PTMs (e.g., phosphorylation and acetylation) as well as uncommon PTMs (e.g., pupylation, mycoloylation, and mycothiolation) have been detected. In this chapter, the features of six representative PTMs found in Corynebacterium glutamicum, phosphorylation, acylation, pupylation, mycoloylation, mycothiolation, and glycosylation, and their roles in protein regulation are described.
... Die erste bakterielle STPK (Pkn1), deren katalytische Domäne eine hohen Sequenzhomologie zu eukaryotischen STPK aufwies, wurde 1991 von Munoz-Dorado et al. in Myxococcus xanthus identifiziert. Die STPK Pkn1 wird unmittelbar vor der Sporenbildung exprimiert und unterliegt einer Ser-und Thr-Autophosphorylierung [27]. Infolge von Genomanalysen wurde deutlich, dass STPKs und STPPs eine wichtige Rolle in der Zellphysiologie zahlreicher Bakterien spielen. ...
Staphylococcus aureus ist ein Kommensale, der die menschliche Haut und Schleimhaut der Nase und des Rachens besiedelt. Der Keim verursacht aufgrund zahlreicher Virulenzfaktoren leichte aber auch schwere Infektionen wie Pneumonie, Endokarditis oder Sepsis. Die Behandlung von S. aureus-Infektionen gestaltet sich heutzutage schwierig, da der Keim Resistenzen gegen verschiedenste Antibiotika ausgebildet hat. Zur Bekämpfung dieser Resistenzen werden neue Antibiotika benötigt, die u.a. mit der Zellphysiologie und der Zellwandwandsynthese der Bakterien interferieren. Die Zellphysiologie und Zellwandsynthese wird abhängig von der Wachstumsphase und Umwelt-einflüssen in den Bakterien streng reguliert. Neben den Zweikomponentensystemen sind Serin/Threonin-Proteinkinasen und -Phosphatasen wesentliche Sensoren und Regulatoren der Bakterien. Durch Phosphorylierung und Dephosphorylierung bewirken diese beiden Systeme eine Hemmung oder Aktivierung der entsprechenden Zielproteine. Dadurch kann sich die Bakterienzelle an innere und äußere Reize anpassen. In dieser Arbeit wurde die konservierte Serin/Threonin-Proteinkinase Stk und die Serin/Threonin-Phosphatase Stp von S. aureus untersucht. Die beiden Proteine Stk und Stp haben einen großen Einfluss auf die Signalweiterleitung, den zentralen Metabolismus, die Stressantwort, die Antibiotikaresistenz und die Virulenz von S. aureus. Im ersten Teil dieser Arbeit wird dargelegt, dass Stk und Stp in der bakteriellen Membran lokalisiert sind, dort miteinander interagieren und antagonistisch Zielproteine phosphorylieren bzw. dephospho-rylieren. Die Deletion der Phosphatase Stp bewirkt, dass zahlreiche Proteine in der Zelle permanent phosphoryliert und daher vermutlich nur noch eingeschränkt funktionstüchtig sind. Die ausbleibende Dephosphorylierung der Proteine in der stp-Mutante hat einen dramatischen Effekt auf die Zellwand-synthese und die Virulenz von S. aureus. So hat die stp-Mutante eine verdickte Zellwand und ist weniger virulent als die stk-Mutante und der Wildtypstamm. Im Rahmen dieser Arbeit wird erstmals eine Erklärung präsentiert, die die strukturellen Besonderheiten von Stk und deren Auswirkung auf die Zellwandsynthese zusammenführt: In der stp-Mutante akkumulieren Zellwandvorläufer in der Zelle, da vermutlich die entsprechenden Zellwandsyntheseproteine durch Stk-vermittelte Phosphorylierung gehemmt werden. Die Proteine FemXAB nehmen eine zentrale Rolle in der Zellwandsynthese ein, indem sie die Pentaglycin-Interpeptidbrücke des Zellwandvorläufers Pentaglycin-Lipid II syntheti-sieren. Stk wird durch die Bindung seiner extrazellulären Domänen an Pentaglycin-Lipid II aktiviert. In der vorliegenden Arbeit konnte FemX als in vitro Substrat von Stk und Stp identifiziert werden. Die permanente Phosphorylierung von FemX in der stp-Mutante führt zur verminderten Synthese der Pentaglycin-Brücken am Lipid II und infolgedessen zum Einbau von unvollständigen Muropeptiden in den neuen Peptidoglycanstrang. Diese strukturelle Veränderung führt zur Verdickung der Zellwand und folglich zur verminderten Empfindlichkeit gegenüber der Glycyl-Glycinpeptidase Lysostaphin. Neben FemX interagiert Stk mit weiteren Zellwandsyntheseproteinen wie FemAB und einigen Zellteilungsproteinen. Diese Ergebnisse verdeutlichen, dass Stk das Vorkommen seines extrazellulären Liganden Lipid II detektiert und dementsprechend die Zellwandsynthese über FemX reguliert. Im zweiten Teil der Arbeit wurde anhand verschiedener Omics-Techniken die stk-, stp- und stk/stp-Mutante im Vergleich zum S. aureus NewmanHG Wildtyp charakterisiert. Dabei zeigten sich teilweise große Unterschiede zwischen der stp-Mutante und den anderen Stämmen. Mit diesen Unter-suchungen konnten Ergebnisse aus anderen Studien bestätigt und mit weiteren Daten untermauert werden. So lässt sich die verminderte Virulenz der stp-Mutante mit der reduzierten Expression und Sekretion von Toxinen wie Hämolysinen und Leukozidinen erklären. Dies führt zu einer verminderten Hämolyse von Erythrozyten und einer verminderten Immunantwort gegen diese Toxine im Infektions-versuch. Stk und Stp phosphorylieren bzw. dephosphorylieren Transkriptionsfaktoren und Antwort-regulatoren von Zweikomponentensystemen, was zu der veränderten Expression und Sekretion der Virulenzfaktoren führt. Die Analyse der Mutanten offenbart, dass Stk ein negativer und Stp ein positiver Regulator der Virulenz in S. aureus ist. Außerdem regulieren Stk und Stp zentrale Aspekte des Metabolismus in S. aureus. So ist die Konzentration an Nukleotidtriphosphaten in der stp-Mutante reduziert, was auf eine verminderte Expression der Gene der Pyrimidinsynthese zurückzuführen ist. Anhand dieser Ergebnisse wird deutlich, dass Stk und Stp wesentliche Aspekte der Zellphysiologie wie die Zellwandsynthese, den zentralen Metabolismus und die Virulenz von S. aureus regulieren.
... In 1991, a bacterial serine/threonine kinase with high structural homology to the eukaryotic protein kinase was first identified in Myxococcus xanthus [58]. Broad genomic studies revealed that eukaryotic-like serine/threonine kinases (eSTKs) were found to be nearly ubiquitous across bacterial species. ...
Antibiotics are one of the greatest medical advances of the 20th century, however, they are quickly becoming useless due to antibiotic resistance that has been augmented by poor antibiotic stewardship and a void in novel antibiotic discovery. Few novel classes of antibiotics have been discovered since 1960, and the pipeline of antibiotics under development is limited. We therefore are heading for a post-antibiotic era in which common infections become untreatable and once again deadly. There is thus an emergent need for both novel classes of antibiotics and novel approaches to treatment, including the repurposing of existing drugs or preclinical compounds and expanded implementation of combination therapies. In this review, we highlight to utilize alternative drug targets/therapies such as combinational therapy, anti-regulator, anti-signal transduction, anti-virulence, anti-toxin, engineered bacteriophages, and microbiome, to defeat antibiotic-resistant bacteria.
... Genome sequencing and physiological studies have shown, however, that this is not the case since genes potentially encoding these kinases have been found to exist in a large number of prokaryotic genomes and to be involved in various cellular processes in several bacterial species (Pereira et al., 2011;Stancik et al., 2018). Recent studies have shown, for example, that the life cycle of Myxococcus xanthus is partly controlled by a network of interacting Hanks-type kinases (Munoz-Dorado et al., 1991;Nariya and Inouye, 2006). In Bacillus subtilis, spore development and germination are controlled by the YabT and PrkC kinases, respectively (Shah et al., 2008;Bidnenko et al., 2013). ...
Hanks-type kinases encoding genes are present in most cyanobacterial genomes. Despite their widespread pattern of conservation, little is known so far about their role because their substrates and the conditions triggering their activation are poorly known. Here we report that under diazotrophic conditions, normal heterocyst differentiation and growth of the filamentous cyanobacterium Nostoc PCC 7120 require the presence of the Pkn22 kinase, which is induced under combined nitrogen starvation conditions. By analyzing the phenotype of pkn22 mutant overexpressing genes belonging to the regulatory cascade initiating the development program, an epistatic relationship was found to exist between this kinase and the master regulator of differentiation, HetR. The results obtained using a bacterial two hybrid approach indicated that Pkn22 and HetR interact, and the use of a genetic screen inducing the loss of this interaction showed that residues of HetR which are essential for this interaction to occur are also crucial to HetR activity both in vitro and in vivo. Mass spectrometry showed that HetR co-produced with the Pkn22 kinase in Escherichia coli is phosphorylated on Serine 130 residue. Phosphoablative substitution of this residue impaired the ability of the strain to undergo cell differentiation, while its phosphomimetic substitution increased the number of heterocysts formed. The Serine 130 residue is part of a highly conserved sequence in filamentous cyanobacterial strains differentiating heterocysts. Heterologous complementation assays showed that the presence of this domain is necessary for heterocyst induction. We propose that the phosphorylation of HetR might have been acquired to control heterocyst differentiation.
... This helix is preserved in the PilZ fusion proteins BcsA and Alg44 and PilZ dimers, as well as in the tetrameric PilZ domain (Fig. 2). Based on sequence comparisons, it has also been predicted in PilZ-containing chemotaxis receptors Tlp1 and Aer from Azospirillum brasilense (29,30), Ser/Thr-type protein kinase Pkn1 (MXAN_1467) from Myxococcus xanthus (61), and many other PilZ fusion proteins. This helix is characterized by a large number of charged amino acid residues, particularly Arg and Lys, which makes it a prime candidate for protein-protein interactions. ...
c-di-GMP is a ubiquitous bacterial second messenger that regulates motility, biofilm formation, and virulence of many bacterial pathogens. The PilZ domain is a widespread c-di-GMP receptor that binds c-di-GMP through its RXXXR and [D/N] h SXXG motifs; some PilZ domains lack these motifs and are unable to bind c-di-GMP. We used structural and sequence analysis to assess the diversity of PilZ-related domains and define their common features. We show that the hydrophobic residue h in the second position of the second motif is highly conserved; it may serve as a readout for c-di-GMP binding. We describe three principal classes of PilZ-related domains, canonical, tetramer-forming, and divergent PilZ domains, and propose the evolutionary pathways that led to the emergence of these PilZ types.
... This role seems to be largely independent of MrpC, because we do not observe drastic changes in MrpC or FruA levels (targets of MrpC activity) in the Δpkn14 mutant (Fig. 4C). Pkn14 (along with Pkn8) belongs to a large kinase/ scaffold protein network (Nariya and Inouye, 2005c), which includes at least two other kinases, Pkn9 and Pkn1, that appear to induce and repress aggregation respectively (Munoz-Dorado et al., 1991;Hanlon et al., 1997). We suggest unphosphorylated Pkn14 affects aggregation indirectly through these proteins, whereas when Pkn14 is stimulated to autophosphorylate, it instead represses development by direct phosphorylation of MrpC. ...
The Crp/Fnr family of transcriptional regulators play central roles in transcriptional control of diverse physiological responses, and are activated by a surprising diversity of mechanisms. MrpC is a Crp/Fnr homolog that controls the Myxococcus xanthus developmental program. A long-standing model proposed that MrpC activity is controlled by the Pkn8/Pkn14 serine/threonine kinase cascade, which phosphorylates MrpC on threonine residue(s) located in its extreme amino terminus. In this study, we demonstrate that a stretch of consecutive threonine and serine residues, T21 T22 S23 S24, is necessary for MrpC activity by promoting efficient DNA binding. Mass spectrometry analysis indicated the TTSS motif is not directly phosphorylated by Pkn14 in vitro but is necessary for efficient Pkn14-dependent phosphorylation on several residues in the remainder of the protein. In an important correction to a long-standing model, we show Pkn8 and Pkn14 kinase activities do not play obvious roles in controlling MrpC activity in wild-type M. xanthus under laboratory conditions. Instead, we propose Pkn14 modulates MrpC DNA binding in response to unknown environmental conditions. Interestingly, substitutions in the TTSS motif caused developmental defects that varied between biological replicates, revealing that MrpC plays a role in promoting a robust developmental phenotype.
... Since a eukaryotic-like Ser/Thr protein kinase (STPK) was first characterized in Myxococcus xanthus (29), many STPKs have been identified and characterized in bacteria, and increasing attention has been paid to the importance of STPKs in prokaryotic signaling pathways related to stress responses, development, virulence, the regulation of central metabolism, as well as cell division and morphology (30)(31)(32)(33). In contrast to HKs that have a strict substrate specificity, STPKs can normally phosphorylate multiple substrates, thereby resulting in pleiotropic responses from a single signal in the signal transduction pathway (31). ...
The mycobacterial SenX3-RegX3 two-component system consists of the SenX3 sensor histidine kinase and its cognate RegX3 response regulator. This system is a phosphorelay-based regulatory system that is involved in sensing of environmental inorganic phosphate (Pi) levels and induction of genes required for Pi acquisition under Pi-limiting conditions. Here, we demonstrate that overexpression of the kinase domain of Mycobacterium tuberculosis PknB (PknB-KDMtb) inhibits the transcriptional activity of RegX3 of both M. tuberculosis and Mycobacterium smegmatis (RegX3Mtb and RegX3Ms, respectively). Mass spectrometry results, along with those of in vitro phosphorylation and complementation analyses, revealed that PknB kinase activity inhibits the transcriptional activity of RegX3Mtb through phosphorylation events at Thr-100, Thr-191, and Thr-217. Electrophoretic mobility shift assays disclosed that phosphorylation of Thr-191 and Thr-217 abolishes the DNA-binding ability of RegX3Mtb, and that Thr-100 phosphorylation likely prevents RegX3Mtb from being activated through conformational changes induced by SenX3-mediated phosphorylation. We propose that the convergence of the PknB and SenX3-RegX3 signaling pathways might enable mycobacteria to integrate environmental Pi signals with the cellular replication state to adjust gene expression in response to Pi availability.
... Eukaryotic-like serine/threonine kinases (eSTK) were first described in bacteria approximately 27 years ago. 20 The penicillin-binding protein and serine/threonine associated (PASTA) kinases are a subset of eSTKs found in numerous gram-positive pathogens and mycobacteria that feature a single transmembrane region linking the cytoplasmic kinase domain to the extracellular penicillin-binding protein domain. The extracellular penicillin binding domain is hypothesized to bind cell-wall precursor muropeptides as a monitor of cell-wall homeostasis. ...
Staphylococcus epidermidis and Staphylococcus aureus are important human pathogens responsible for two-thirds of all post-surgical infections of indwelling medical devices. Staphylococci form robust biofilms that provide a reservoir for chronic infection and antibiotic resistant isolates are increasingly common in both healthcare and community settings. Novel treatments that can simultaneously inhibit biofilm formation and antibiotic resistance pathways are urgently needed to combat the increasing rates of antibiotic resistant infections. Herein we report that loratadine, an FDA-approved antihistamine, potently inhibits biofilm formation in both S. aureus and S. epidermidis. Furthermore, loratadine potentiates β-lactam antibiotics in methicillin-resistant strains of S. aureus and potentiates both β-lactam antibiotics and vancomycin in vancomycin-resistant strains of S. aureus. Additionally, we elucidate loratadine’s mechanism of action as a novel inhibitor of regulatory PASTA kinases in staphylococci and describe how their inhibition affects the expression of genes involved in both biofilm formation and antibiotic resistance in S. epidermidis and S. aureus.
... Sci. 2018,19, 2872 ...
Reversible phosphorylation is a key mechanism that regulates many cellular processes in prokaryotes and eukaryotes. In prokaryotes, signal transduction includes two-component signaling systems, which involve a membrane sensor histidine kinase and a cognate DNA-binding response regulator. Several recent studies indicate that alternative regulatory pathways controlled by Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) also play an essential role in regulation of many different processes in bacteria, such as growth and cell division, cell wall biosynthesis, sporulation, biofilm formation, stress response, metabolic and developmental processes, as well as interactions (either pathogenic or symbiotic) with higher host organisms. Since these enzymes are not DNA-binding proteins, they exert the regulatory role via post-translational modifications of their protein targets. In this review, we summarize the current knowledge of STKs and STPs, and discuss how these enzymes mediate gene expression in prokaryotes. Many studies indicate that regulatory systems based on Hanks-type STKs and STPs play an essential role in the regulation of various cellular processes, by reversibly phosphorylating many protein targets, among them several regulatory proteins of other signaling cascades. These data show high complexity of bacterial regulatory network, in which the crosstalk between STK/STP signaling enzymes, components of TCSs, and the translational machinery occurs. In this regulation, the STK/STP systems have been proved to play important roles.
... Pkn1 in Myxococcus xanthus was identified as the first STPK in prokaryotes in 1991 (30). Later, STPKs have been discovered in the gamut of bacterial species like Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Corynebacterium glutamicum, Bacillus subtilis, Mycobacterium Sp., Anabaena, and Streptomyces Sp. ...
The emergence of increasingly drug‐resistant Mycobacterium tuberculosis strains has become a crucial public health concern. In order to effectively treat tuberculosis, it is imperative to find newer drug targets, which are important for the in vivo bacterial survival and persistence. Phosphorylation based signaling cascades modulated by eukaryotic‐like serine/threonine protein kinases and phosphatase in M. tuberculosis, transduce extracellular stimuli to a cellular response ensuing pathogen's growth, persistence and pathogenesis. Of the 11 STPKs that M. tuberculosis genome encodes, three kinases, namely PknA, PknB and PknG and the sole serine/threonine phosphatase PstP are crucial for the intracellular survival of the bacteria. PknA and PknB regulates cell growth, cell wall synthesis and morphological changes during bacterial cell division; while PknG modulates metabolic changes in response to stress and aids in bacterial survival during latency like conditions. PstP functions to dephosphorylate STPKs and their substrates and hence is important at nearly all stages of infection. Here, we review the current knowledge on PstP, PknA, PknB and PknG based on the genetic, biochemical, and functional studies in M. tuberculosis physiopathology. We further explore the potential of these molecules as targets for therapeutic intervention and discuss the advancement made in the development of inhibitors against these targets.
... In bacteria, a group of Ser/Thr kinases (STK) that is homologous to those of eukaryotic STK has been defined as eukaryotelike STK (eSTK) (Pereira et al., 2011). The bacterial eSTK is first identified from the gram-negative soil microorganism Myxococcus xanthus (Munoz-Dorado et al., 1991) and it has been proven to be widespread in different bacteria, including Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Mycobacterium tuberculosis, Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes (Wang et al., 1998;Av-Gay and Everett, 2000;Rajagopal et al., 2003;Echenique et al., 2004;Mougous et al., 2007;Burnside et al., 2010;Molle and Kremer, 2010;Ohlsen and Donat, 2010;Agarwal et al., 2011;Bugrysheva et al., 2011;Cameron et al., 2012;Hall et al., 2013). ...
Streptococcus suis serotype 2 is an important swine pathogen and an emerging zoonotic agent that causes severe infections. Recent studies have reported a eukaryotic-like Ser/Thr protein kinase (STK) gene and characterized its role in the growth and virulence of different S. suis 2 strains. In the present study, phosphoproteomic analysis was adopted to identify substrates of the STK protein. Seven proteins that were annotated to participate in different cell processes were identified as potential substrates, which suggests the pleiotropic effects of stk on S. suis 2 by targeting multiple pathways. Among them, a protein characterized as cell division initiation protein (DivIVA) was further investigated. In vitro analysis demonstrated that the recombinant STK protein directly phosphorylates threonine at amino acid position 199 (Thr-199) of DivIVA. This effect could be completely abolished by the T199A mutation. To determine the specific role of DivIVA in growth and division, a divIVA mutant was constructed. The ΔdivIVA strain exhibited impaired growth and division, including lower viability, enlarged cell mass, asymmetrical division caused by aberrant septum, and extremely weak pathogenicity in a mouse infection model. Collectively, our results reveal that STK regulates the cell growth and virulence of S. suis 2 by targeting substrates that are involved in different biological pathways. The inactivation of DivIVA leads to severe defects in cell division and strongly attenuates pathogenicity, thereby indicating its potential as a molecular drug target against S. suis.
... It has been traced back to 1940s that Cori, et al. identified the first protein Ser⁄Thr phosphatase that inactivated glycogen phosphorylase, which at that time called the 'PR enzyme' as prosthetic group removing enzyme [72]. However, protein Ser/Thr phosphatase in bacteria has not received attention until very recently when eukaryotic-like serine/threonine protein kinases (eSTKs) were identified in Gram-negative bacterium Myxococcus xanthus for their involvement in the normal development of this organism [73]. Advances in genome sequencing has indicated the presence of eukaryotic-like Ser/Thr protein kinases/phosphatases (eSTKs/eSTPs) in several prokaryotic organisms, which include gram-negative human pathogens such as Pseudomonas aeruginosa [74], and many grampositive pathogens such as S. aureus [75][76][77], Mycobacterium tuberculosis [78,79], Enterococcus faecalis [80], Streptococcus pneumonia [81], Streptococcus agalactiae [82] and Streptococcus pyogenes [83]. ...
Background:
The increasing threats of antibiotic resistance urge the need for developing new approaches to combat bacterial infections including those caused by Staphylococcus aureus (S. aureus). Unlike conventional antibiotics that aim to kill bacteria or inhibit their growth, targeting bacterial virulence may be a promising alternative approach, which imposes less selective pressure for antibiotic resistance in future generations.
Objective:
Our goal is to provide a systematic review about developing high-throughput screening (HTS) strategies for the identification of inhibitors targeting virulence of S. aureus. We also describe an overview of virulence regulatory pathways for potential antivirulence targets.
Methods:
We focus on five potential targets or target families, including agr quorum sensing system, SarA/MgrA protein family, sortase A, Clp protease and eukaryotic-like Ser/Thr phosphatase (Stp1). For each target, we introduce its role in virulence regulation, summarize the HTS approaches that are used to identify novel anti-virulence inhibitors, and discuss the advantages and disadvantages of these strategies.
Conclusion:
The discovery of anti-virulence inhibitors via HTS underlines the promising potential of anti-virulence therapy for S. aureus. The development of HTS strategies can facilitate the identification of novel anti-virulence inhibitors for combating S. aureus infection, and may also advance our understanding on virulence regulation in S. aureus.
... In this reaction cascade, adenylate kinase converts ADP to ATP and AMP, and then AMPK is activated by AMP, which results in the induction of catabolic and inhibition of anabolic pathways. M. xanthus contains ~100 eukaryotic-like Ser/Thr protein kinases [8,12], and there is a possibility that some of them may function as AMPK. Given that M. xanthus also expresses Ppk1 with adenylate kinase activity, which is involved in the regulation of fruiting body formation [22], these data suggest that an unknown signaling cascade similar to that found in eukaryotes may operate in M. xanthus. ...
Polyphosphate kinase 1 (Ppk1) catalyzes reverse transfer of the terminal phosphate from ATP to form polyphosphate (polyP) and from polyP to form ATP, and is responsible for the synthesis of most of cellular polyPs. When Ppk1 from Myxococcus xanthus was incubated with 0.2 mM polyP60−70 and 1 mM ATP or ADP, the rate of ATP synthesis was approximately 1.5-fold higher than that of polyP synthesis. If in the same reaction the proportion of ADP in the ATP/ADP mixture exceeded one-third, the equilibrium shifted to ATP synthesis, suggesting that M. xanthus Ppk1 preferentially catalyzed ATP formation. At the same time, GTP and GDP were not recognized as substrates by Ppk1. In the absence of polyP, Ppk1 generated ATP and AMP from ADP, and ADP from ATP and AMP, suggesting that the enzyme catalyzed the transfer of a phosphate group between ADP molecules yielding ATP and AMP, thus exhibiting adenylate kinase activity.
... Another locus that drives divergence between the two P. acuta morphs was serine-threonine protein salt-induced kinase (SIK) 3, a gene involved in the morphogenesis of anatomical structures. While the function of the gene has not been identified for corals, serine-threonine kinases have been shown to regulate morphogenesis and development in a variety of organisms from bacteria and yeasts to Drosophila [43][44][45][46] . In Drosophila, the main pathway which specifies growth and size control is regulated by SIK3 kinases 47 . ...
Processes of cnidarian evolution, including hybridization and phenotypic plasticity, have complicated the clear diagnosis of species boundaries within the phylum. Pocillopora acuta, a species of scleractinian coral that was recently split from the widespread Pocillopora damicornis species complex, occurs in at least two distinct morphs on the Great Barrier Reef. Contrasting morphology combined with evidence of differential bleaching thresholds among sympatrically distributed colonies suggest that the taxonomy of this recently described species is not fully resolved and may represent its own species complex. To examine the basis of sympatric differentiation between the two morphs, we combined analyses of micro- and macro-skeletal morphology with genome wide sequencing of the coral host, as well as ITS2 genotyping of the associated Symbiodinium communities. We found consistent differences between morphs on both the macro- and micro-skeletal scale. In addition, we identified 18 candidate functional genes that relate to skeletal formation and morphology that may explain how the two morphs regulate growth to achieve their distinct growth forms. With inconclusive results in endosymbiotic algal community diversity between the two morphs, we propose that colony morphology may be linked to bleaching susceptibility. We conclude that cryptic speciation may be in the early stages within the species P. acuta.
... Prokaryotic protein phosphorylation was originally thought to occur predominantly on histidine and aspartate residues phosphorylated by two-component systems in a fashion distinct from eukaryotic kinases (8,9). However, since the discovery of pkn1, a bacterial serine/threonine kinase with high structural homology to eukaryotic protein kinases (10), genomic studies have shown eukaryotic-like serine/threonine kinases (eSTKs) 3 to be near ubiquitous in bacteria (11). Specifically, many important Gram-positive pathogens have transmembrane eSTKs known as Penicillin-binding-protein And Ser/Thr kinase-Associated (PASTA) kinases (12). ...
Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.
... Protein phosphorylation was first described as a major regulatory mechanism in eukaryotes [108,109]. Later, the regulation by phosphorylation with serine/threonine protein kinases (STPKs) in particular has been expanded to prokaryotes [110][111][112][113]. While investigating the S. coelicolor homolog of PknB, a STPK, Yeats et al. identified a definitive domain in its C-terminus. ...
Many bacterial diseases are caused by organisms growing together as communities or biofilms. These microorganisms have the capacity to coordinately regulate specific sets of genes by sensing and communicating amongst themselves utilizing a variety of signals. This book examines the mechanisms of quorum sensing and cell-to-cell communication in bacteria and the roles that these processes play in regulating virulence, bacterial interactions with host tissues, and microbial development. Recent studies suggest that microbial cell-to-cell communication plays an important role in the pathogenesis of a variety of disease processes. Furthermore, some bacterial signal molecules may possess immunomodulatory activity. Thus, understanding the mechanisms and outcomes of bacterial cell-to-cell communication has important implications for appreciating host-pathogen interactions and ultimately may provide new targets for antimicrobial therapies that block or interfere with these communication networks.
Microbes rely on signal transduction systems to sense and respond to environmental changes for survival and reproduction. It is generally known that niche adaptation plays an important role in shaping the signaling repertoire. However, the evolution of bacterial signaling capacity lacks systematic studies with a temporal direction. Particularly, it is unclear how complexity evolved from simplicity or vice versa for signaling networks. Here we examine the evolutionary processes of major signal transduction systems in Campylobacterota (formerly Epsilonproteobacteria), a phylum with sufficient evolutionary depth and ecological diversity. Evolution of signaling systems within Campylobacterota shows two opposite trends. During niche expansion, signaling complexity increases with gene expansions through horizontal gene transfer (HGT), gene duplication, fusion and fission, which create opportunities for genetic innovation and pathway integration. In contrast, as the lineages adapt to a specialized niche, complexity decreases with massive gene losses that lead to the decline or disappearance of pathways mediated by multiple transmitters. Overall, signaling capacity and complexity arise and drop together in Campylobacterota, determined by sensory demand, genetic resources and co-evolution within the genomic context. These findings reflects plausible evolutionary principles for other cellular networks and genome evolution of the Bacteria domain.
Myxococcus xanthus has a nutrient-regulated biphasic lifecycle forming predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. The second messenger c-di-GMP is essential during both stages of the lifecycle; however, different enzymes involved in c-di-GMP synthesis and degradation as well as several c-di-GMP receptors are important during distinct lifecycle stages. To address this stage specificity, we determined transcript levels using RNA-seq and transcription start sites using Cappable-seq during growth and development at a genome-wide scale. All 70 genes encoding c-di-GMP associated proteins were expressed, with 28 up-regulated and 10 down-regulated during development. In particular, the three genes encoding enzymatically active proteins with a stage-specific function were expressed stage-specifically. By combining operon mapping with published ChIP-seq data for MrpC (Robinson et al., 2014), the CRP-like master regulator of development, we identified nine developmentally regulated genes as regulated by MrpC. In particular, MrpC directly represses expression of dmxB , which encodes the diguanylate cyclase DmxB that is essential for development and responsible for the c-di-GMP increase during development. Moreover, MrpC directly activates transcription of pmxA , which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3’, 3’ cGAMP in vitro and is essential for development. Thereby, MrpC regulates and curbs the cellular pools of c-di-GMP and 3’, 3’ cGAMP during development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity. MrpC is important for this regulation, thereby being a key regulator of developmental cyclic di-nucleotide metabolism in M. xanthus .
Importance
The second messenger c-di-GMP is important during both stages of the nutrient-regulated biphasic lifecycle of Myxococcus xanthus with the formation of predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. However, different enzymes involved in c-di-GMP synthesis and degradation are important during distinct lifecycle stages. Here, we show that the three genes encoding enzymatically active proteins with a stage-specific function are expressed stage-specifically. Moreover, we find that the master transcriptional regulator of development MrpC directly regulates expression of dmxB , which encodes the diguanylate cyclase DmxB that is essential for development, and of pmxA , which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3’, 3’ cGAMP in vitro and is essential for development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity, and that MrpC plays an important role in this regulation.
Protein phosphorylation is a post-translational modification that affects protein activity through the addition of a phosphate moiety by protein kinases or phosphotransferases. It occurs in all life forms. In addition to Hanks kinases found also in eukaryotes, bacteria encode membrane histidine kinases that, with their cognate response regulator, constitute two-component systems and phosphotransferases that phosphorylate proteins involved in sugar utilization on histidine and cysteine residues. In addition, they encode BY-kinases and arginine kinases that phosphorylate protein specifically on tyrosine and arginine residues respectively. They also possess unusual bacterial protein kinases illustrated here by examples from Bacillus subtilis.
The thought of “Protein phosphorylation” always draws attention to Ser/Thr kinases or phosphatases as they were discovered prior to the mechanism of Tyr phosphorylation. Tyr phosphorylation (and dephosphorylation) mediated by interplay of Tyr kinase (and phosphatases) regulate several aspects of cellular interactions which are often complex in nature. Serving a major role in signal transduction pathways in animals, Tyr phosphorylation is less documented, yet there is evidence for its involvement in plant physiology. However, the presence of bona‐fide Tyr specific plant phosphatases in the plants evidence the utilization of Tyr phosphorylation signaling in plants. If Tyr phosphatases do exist in the plant system then Tyr kinases should also be indispensably present. So far, the existence of canonical Tyr kinases in plants is a controversial topic as evidence is required to prove their functionality in planta. Although, Dual‐specificity protein Tyr Kinases (DsPTK) do exist and are known to play a role in various aspect of plant development and physiology, still they cannot be addressed as “bonafide‐Tyr kinases” for the plant systems. Finding a canonical Tyr kinase in plants can be assumed as a “treasure hunt” where several clues are indeed required to decipher more about the treasure of Tyr kinases in plants.
Myxococcus xanthus has a complex lifecycle that is regulated by nutrient availability. In the presence of nutrients, M. xanthus cells grow, divide, and move to assemble into colonies that feed cooperatively either saprophytically or on prey. In response to starvation, a developmental program is initiated that culminates in formation of multicellular spore-filled fruiting bodies. The nucleotide-based second messenger cyclic di-GMP accumulates in M. xanthus and has critical functions in both stages of the lifecycle. Here, we describe the roles of cyclic di-GMP, its metabolizing proteins, and receptor proteins. During growth, the correct level of cyclic di-GMP is important for type IV pili-dependent motility. During development, the cyclic di-GMP level increases and a threshold concentration of cyclic di-GMP is essential for completion of the developmental program. By individually inactivating the genes involved in cyclic di-GMP synthesis or degradation, two diguanylate cyclases, DmxA and DmxB, were identified to function at specific stages of the lifecycle with DmxA involved in type IV pili-dependent motility and DmxB in development. Similarly, the phosphodiesterase PmxA is specifically important for development but functions independently of DmxB. Bioinformatics analyses suggest the existence of various cyclic di-GMP receptor proteins, a few of which have been confirmed experimentally while the remainder are still uncharacterized. We are only just beginning to understand regulation by cyclic di-GMP in M. xanthus and it will be exciting to identify all the processes regulated by cyclic di-GMP and the underlying mechanisms.
The bacterium Myxococcus xanthus exhibits a complex multicellular life cycle. In the presence of nutrients, cells prey cooperatively. Upon starvation, they enter a developmental cycle wherein cells aggregate to produce macroscopic fruiting bodies filled with resistant myxospores. We used RNA-Seq technology to examine the transcriptome of the 96 hr developmental program. These data revealed that 1415 genes were sequentially expressed in 10 discrete modules, with expression peaking during aggregation, in the transition from aggregation to sporulation, or during sporulation. Analysis of genes expressed at each specific time point provided insights as to how starving cells obtain energy and precursors necessary for assembly of fruiting bodies and into developmental production of secondary metabolites. This study offers the first global view of developmental transcriptional profiles and provides important tools and resources for future studies.
Upon environmental or nutritional changes, bacteria must adjust their cell cycle with their growth rate. Most particularly, DNA replication initiation events must be controlled and coordinated with cell physiology to ensure faithful chromosome inheritance. In Bacillus subtilis, a model of Gram-positive bacteria, YabA plays a major role in down regulating initiation replication through interaction with the initiator protein DnaA and the clamp polymerase DnaN. However, YabA is a structural hub protein able to interact with other protein partners, indicating it might be multifunctional. Through its unique overall tri-dimentional structure composed of N-terminal four helix-bundle tetramer connected to four monomeric C-terminal domains by a highly flexible linker, YabA is capable to physically interact with more than one protein at a time, thus providing a suitable platform to integrate intracellular signals to replication initiation. Phosphorylation is the most prevalent post translational modification that modulates protein activities in response to cellular signals. Using in vitro phosphorylation and mass spectrometry we demonstrated that YabA is phosphorylated by the Hanks-type serine/threonine kinase YabT at a threonine residue localized within the flexible inter-domain region. YabT is a kinase activated by DNA and up-regulated during glucose starvation, sporulation and stationary phase. We constructed YabA phosphomimetic (yab-AT71D) and non-phosohorylatable (yabA-T71A) mutants to (i) confirm the requirement of T71 for YabT-mediated phosphorylation in vitro and (ii) perform in vivo and in vitro functional studies.We show in vivo that the phosphorylation of YabA is not involved in initiation control, but rather modulates bacillus developmental processes. We found that YabA phosphorylation inversely regulates sporulation and biofilm formation highlighting the multifunctional role of YabA as well as its role in integrating physiological signals to connect chromosomal replication initiation control with cell development. Our results support a role of YabT-mediated phosphorylation of YabA in Bacillus subtilis life-style decision making through the modulation of Spo0A-P intracellular levels. We established that YabA phosphorylation correlates with high cellular levels of Spo0A-P, leading to sporulation stimulation and preventing biofilm formation. Additionally, thin layer chromatography (TLC) analysis and In-Gel assays showed that YabA possess an atypical "ATP / GTPase" activity. This unusual activity seems to be modulated by phosphorylation of the YabA T71 residue. Our functional analysis pointed to a potential role of YabA in the c-di-GMP signaling transduction pathway, known to regulate biofilm formation in many bacteria. This suggesting a complex regulatory role of YabA during development, involving signaling crosstalk. LC-MS analyzes showed that when overexpressed in Escherichia coli, YabA is phosphorylated on the residue Y90 in a YabT independent manner. Y90 belongs to the interaction C-terminal domain, which contacts DnaA and DnaN. We found that Y90 was involved YabA-mediated replication initiation control. We provided evidence that phosphorylation state of YabA at Y90 can potentially modulates a protein-interaction switch with its protein partners DnaA and DnaN in a yeast-two-hybrid-based assay. Although we did not identified a kinase responsible for the phosphorylation of YabA at Y90 in B. subtilis, this finding hint at the possibility of a YabA-mediated control of initiation modulated by phosphorylation in this bacteria. Thus, all of these in vitro and in vivo observations suggest the existence of different modes of regulation of YabA activity by phosphorylation, involving threonine and tyrosine residues. This study established that YabA, apart from its role during replication initiation, plays a key regulatory role in B. subtilis development.
Del.ta.pro.te.o.bac.te′ri.a. Gr. n. delta name of fourth letter of Greek alphabet; Gr. n. Proteus ocean god able to change shape; Gr. n. bakterion a small rod; M.L. fem. pl. n. Deltaproteobacteria class of bacteria having 16S rRNA gene sequences related to those of the members of the order Myxococcales.
Protein kinases C (PKCs) comprise closely related Ser/Thr kinases, ubiquitously present in animal tissues; they respond to second messengers, e.g., Ca2+ and/or diacylglycerol, to express their activities. Two PKCs have been sequenced fromGeodia cydonium, a member of the lowest multicellular animals, the sponges (Porifera). One spongeG. cydonium PKC, GCPKC1, belongs to the “novel” (Ca2+-independent) PKC (nPKC) subfamily while the second one, GCPKC2, has the hallmarks of the “conventional” (Ca2+-dependent) PKC (cPKC) subfamily. The alignment of the Ser/Thr catalytic kinase domains, of the predicted as sequences for these cDNAs with respective segments from previously reported sequences, revealed highest homology to PKCs from animals but also distant relationships to Ser/Thr kinases from protozoa, plants, and bacteria. However, a comparison of the complete structures of the sponge PKCs, which are-already-identical to those of nPKCs and cPKCs from higher metazoa, with the structures of protozoan, plant, and bacterial Ser/Thr kinases indicates that the metazoan PKCs have to be distinguished from the nonmetazoan enzymes. These data indicate that metazoan PKCs have a universal common ancestor which they share with the nonmetazoan Ser/Thr kinases with respect to the kinase domain, but they differ from them in overall structural composition.
Conditions which gave a high yield of oligonucleotide-directed site specific mutagenesis were obtained with use of the lpp gene of Escherichia coli cloned in the versatile expression plasmid vector, pIN-II. This plasmid (5.6-kb) was digested by PstI followed by exonuclease (Klenow fragment of DNA polymerase I) to remove a portion of the gene for -lactamase. Another sample was digested by Xbal which cuts at a unique site in the pIN-II vector, and EcoRI to remove the portion of the lpp gene to be mutagenized. Both fragments were mixed with a synthetic oligonucleotide (the mutagen) and the mixture was denatured at 100°C for 3 min, followed by gradual cooling to reanneal. The mixture of renatured DNA was treated with the Klenow fragment of DNA polymerase I plus deoxynucleotide triphosphates in the presence of T4 ligase, and ampicillin-resistant transformants were isolated. In the case of a single base substitution, the yield of the desired mutant was as high as 13%. The present method can be applied to any gene cloned in a plasmid vector and is more convenient and simpler than the method using an M13 phage vector, since the mutated gene can be expressed directly from the plasmid vector used for the mutagenesis.
Myxobacteria are soil bacteria whose unusually social behavior distinguishes them from other groups of procaryotes. Perhaps the most remarkable aspect of their social behavior occurs during development, when tens of thousands of cells aggregate and form a colorful fruiting body. Inside the fruiting body the vegetative cells convert into dormant, resistant myxospores. However, myxobacterial social behavior is not restricted to the developmental cycle, and three other social behaviors have been described. Vegetative cells have a multigene social motility system in which cell-cell contact is essential for gliding in multicellular swarms. Cell growth on protein is cooperative in that the growth rate increases with the cell density. Rippling is a periodic behavior in which the cells align themselves in ridges and move in waves. These social behaviors indicate that myxobacterial colonies are not merely collections of individual cells but are societies in which cell behavior is synchronized by cell-cell interactions. The molecular basis of these social behaviors is becoming clear through the use of a combination of behavioral, biochemical, and genetic experimental approaches.
We have identified protein kinase genes of Dictyostelium by using highly conserved amino acid sequence motifs to design the synthesis and amplification of DNA fragments by polymerase chain reactions (PCRs). Cloning and sequencing the PCR products have revealed five different members of the protein kinase multigene family. These five putative kinases showed varying degrees of amino acid sequence similarity (40-70%) to protein kinases in data bases and contained invariant amino acid residues characteristic of protein kinases. DNA from PCR was labeled and used to isolate several lambda gt11 cDNA clones, including one full-length one (Dd kinase-2). The nucleotide sequence of Dd kinase-2 contained a region identical to one of the cloned kinase fragments amplified by PCR, and based on the deduced amino acid sequence Dd kinase-2 encodes a protein of 479 amino acids. A 350-amino acid kinase domain at the C-terminal end shows high homology to the catalytic domains of protein kinase A, protein kinase C, S-6 kinase of Xenopus, and the suppressor of cdc25 of yeast. The N-terminal domain is highly basic and also contains alternating threonine/proline residues. The cDNA hybridized to a single copy gene but to two differentially regulated mRNAs--a 2.0-kilobase mRNA that is expressed in vegetative cells and a 2.2-kilobase mRNA that is expressed during development. The larger mRNA is induced by cAMP by using a cell-surface receptor-mediated signal transduction pathway.
The pivotal role that protein-tyrosine kinases (PTKs) play in the growth regulation of eukaryotic cells is manifest in the frequent appearance of members of the PTK family as growth factor receptors or as the transforming agents of acutely transforming retroviruses. A feature common to all members of the PTK family is a highly conserved catalytic domain which is characteristic of the group as a whole and whose activity appears to be tightly regulated within the cell by other domains of the PTK. Degenerate oligonucleotide probes corresponding to two invariant amino acid sequence motifs within the catalytic domains of all PTK family members were synthesized and employed in the polymerase chain reaction (PCR) to amplify cDNA sequences between them. An M13 PCR library was produced in this way from cDNA prepared against mRNA from the murine hemopoietic cell line FDC-P1. The PCR library was then screened by DNA sequencing for PTK-related sequences. Two sequences were identified that, on the basis of sequence comparison with known PTKs, may encode representatives of a new class of PTK.
A precursor RNA molecule (pre-msdRNA) of approximately 375 bases is considered to form a stable secondary structure which serves as a primer as well as a template to synthesize the branched RNA-linked multicopy single-stranded DNA (msDNA) of Myxococcus xanthus. When 3-base mismatches were introduced into the stem structure immediately upstream of the branched rG residue to which msDNA is linked by a 2',5'-phosphodiester linkage, the production of msDNA was almost completely blocked. However, if additional 3-base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored, being consistent with the proposed model of msDNA synthesis. We also found that the branched rG residue of pre-msdRNA could not be replaced with either rC or rA, while the 5' end (dC) of msDNA which is linked to the branched rG could be substituted with a dG residue. Together with several other mutations, the structural requirements of pre-msdRNA are discussed with respect to the mechanism of msDNA biosynthesis.
The frz genes of Myxococcus xanthus are necessary for proper aggregation of cells to form fruiting bodies. Mutations in the frz genes affect the frequency with which individual cells reverse their direction of movement. We have subcloned and determined the nucleotide sequence of three of the frz genes. From the sequence we predict three open reading frames corresponding to frzA, frzB, and frzCD. The putative FrzA protein (17,094 Da) exhibits 28.1% amino acid identity with the CheW protein of Salmonella typhimurium. The putative FrzCD protein (43,571 Da) contains a region of about 250 amino acids which is similar to the C-terminal portions of the methyl-accepting chemotaxis receptor proteins of the enteric bacteria. FrzCD also contains a region with potentially significant similarity to the DNA-binding region of the Bacillus subtilis sigma 43. The putative FrzB protein (12,066 Da) shares no significant identity with known chemotaxis proteins. The sequence similarities between the putative Frz proteins and the chemotaxis proteins of the enteric bacteria strongly support the hypothesis that the frz genes define a system of signal transduction analogous to the enterobacterial chemotaxis systems.
cDNA clones for a fifth polypeptide of rat brain calmodulin-dependent protein kinase II were isolated and sequenced. The cDNA sequence encoded a polypeptide, designated δ, consisting of 533 amino acid residues with a molecular weight of 60,080. Comparison of amino acid sequences of this and α, β, β‵, and γ polypeptides of calmodulin-dependent protein kinase II reveals marked homology among them.
The mRNAs for δ were expressed in rat brain tissues with different regional specificities. The distribution of α, β/β‵, γ, and δ mRNAs in cerebrum, skeletal muscle, diaphragm, heart, small intestine, uterus, aorta, liver, kidney, lung, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.9-kilobase (kb) RNA species hybridizable with a probe for γ was found in all the tissues examined, and 4.0–4.2-kb RNA species hybridizable with a probe for δ were found in all the tissues examined except for liver, while a 4.8-kb RNA species hybridizable with a probe for α and a 4.2-kb RNA species hybridizable with a probe for β were present in brain but not in the other tissues. With the α probe, however, a 4.1- and 2.6-kb RNA species were both detected in skeletal muscle and diaphragm. With the β probe, a 4.3-kb RNA in skeletal muscle and diaphragm, 2.9-kb RNA in small intestine, and 4.0-kb RNA in testis were detected. With the δ probe, a 3.5-kb RNA in heart and 1.8-kb RNA in testis were detected. Thus, γ and δ mRNAs were expressed in various tissues, while α and β/β′ mRNAs were primarily, if not exclusively, expressed in brain.
Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry.
Two Drosophila genes encoding products related to cGMP-dependent protein kinase have been isolated by cross-hybridization to a Drosophila cAMP-dependent protein kinase catalytic subunit gene. Both genes encode products with putative cGMP binding and kinase domains on the same polypeptide chain, as found for the prototypical bovine lung cGMP-dependent protein kinase. The deduced product of one gene (DG1; cytological position, 21D) is 14% larger than the bovine enzyme and differs substantially in sequence at the amino terminus, the region responsible in the bovine enzyme for dimerization. The second gene (DG2; cytological position, 24A) is transcribed into three major RNA species of different size. The largest (DG2; T1) and smallest (DG2;T3) RNAs encode overlapping polypeptides of similar sequence to the whole length of bovine lung cGMP-dependent protein kinase. The translation product of the third major RNA (DG2;T2) lacks sequences similar to those that constitute the dimerization and kinase inhibitory domains of the bovine enzyme. The percentage amino acid identity between DG1 or DG2 and bovine lung cGMP-dependent protein kinase is 55 and 64%, respectively. A common progenitor of the two cGMP-dependent protein kinase genes, DG1 and DG2, is strongly suggested by the conserved positions of introns in these genes.
The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. We have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, we have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, we have used this method to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, we have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.
Mutations in the SRA1 or SRA3 gene eliminate the requirement for either RAS gene (RAS1 or RAS2) in Saccharomyces cerevisiae.
We cloned SRA1 and SRA3 and determined their DNA sequences. SRA1 encodes the regulatory subunit of the cyclic AMP (cAMP)-dependent
protein kinase and therefore is identical to REG1 and BCY1. This gene is not essential, but its deletion confers many traits:
reduction of glycogen accumulation, temperature sensitivity, reduced growth rate on maltose and sucrose, inability to grow
on galactose and nonfermentable carbon sources, and nitrogen starvation intolerance. SRA3 is homologous to protein kinases
that phosphorylate serine and threonine and likely encodes the catalytic subunit of the cAMP-dependent protein kinase. The
wild-type SRA3 gene either triplicated in the chromosome or on episomal, low-copy plasmids behaves like spontaneous dominant
SRA3 mutations by suppressing ras2-530 (RAS2::LEU2 disruption), cdc25, and cdc35 mutations. These findings indicate that the
yeast RAS genes are dispensable if there is constitutive cAMP-dependent protein kinase activity.
Lysine 622 in the ATP-binding domain of P100gag-mil, the translation product of the v-mil oncogene of MH2, has been replaced with methionine using oligonucleotide site-directed mutagenesis. This substitution results in the inactivation of the serine/threonine-specific autophosphorylation of P100gag-mil in vitro, indicating that this activity is an intrinsic property of the viral protein. This substitution also suppresses two of the biological properties of MH2 which have previously been shown to be dependant upon the expression of v-mil, namely, the production of chicken myelomonocytic growth factor (cMGF) by v-myc-transformed chicken macrophages and the sustained proliferation of chicken neuroretina cells. These data strongly suggest that the biological properties of v-mil are mediated by the phosphorylation at serine/threonine residues of key cellular substrates. In contrast to the in vitro situation, both the mutant and wild-type proteins appear to be phosphorylated at the same sites and to the same extent in either transformed fibroblasts or macrophages. This, together with the fact that the sites phosphorylated in vivo and in vitro are essentially different indicate that most of the phosphate associated with P100gag-mil in transformed cells does not result from an obligate autophosphorylation event but from the phosphorylation by as yet uncharacterized cellular kinase(s).
Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
In 1892 Thaxter described the myxobacteria as “possessing a power of slow locomotion ... a distinctly visible movement characterizes the active rods and consists in a sliding locomotion in conjunction with lateral bending.” Almost one century later there are still conflicting ideas about the mechanism of gliding. At a genomic level it is evident that Myxococcus gliding is a complex phenomenon (Chapter 8). Elucidation of the enigma of gliding motility has been much slower than that for swimming motility of flagellated bacteria, since myxobacteria (and other gliders) have no obvious organelles of motility protruding from their cell envelopes. Indeed, gliding has recently been described as a “backwater” in the motility field (Allen 1981).
This chapter reviews the construction of several high-level expression vectors using the Escherichia coli lipoprotein promoter and part of the protein. These vectors are designed to produce a large amount of an inserted gene product and to localize it to a specific compartment of the E. coli cell—cytoplasm, cytoplasmic membrane, periplasm, or outer membrane. These vectors have three restriction enzyme sites—EcoRI, HindIII, and BamHI—in each of the three reading frames at various positions along the lipoprotein gene. The pIN-I vectors constitutively produce a large amount of the inserted gene product. The pIN-II and pIN-III vectors produce the foreign gene product only in the presence of an inducer. Once a gene has been inserted properly into one of these vectors it can easily be moved to the other vectors, like a cassette, with the use of several possible unique restriction-enzyme sites on either side of the gene. The chapter also describes two promoter cloning vectors that produce a protein—tetracycline resistance or β-galactosidase—when a promoter is inserted.
We examined the structure of protein kinase C in an attempt to understand the molecular events connecting protein kinase C activation with the cellular response. Rabbit complementary DNA clones coding for three distinct types of protein kinase C, named alpha, beta and gamma, have been identified and sequenced. The deduced amino acid sequence for alpha, beta and gamma (673, 671 and 672 amino acids, respectively) are closely related. Kinases alpha and beta share an identical N-terminal sequence of 621 amino acid residues and their messenger RNAs arise from a single gene. The C-terminal halves of alpha, beta and gamma are protein kinase domains and are highly homologous to other protein kinases. The mRNAs for alpha, beta and gamma are expressed in various tissues with strikingly different tissue specificities. The one for gamma is found ubiquitously among various tissues, while those for alpha and beta predominate in the brain.
Fruiting body formation in the bacterium Myxococcus xanthus consists of a temporal sequence of cellular aggregation and sporulation. During the period of cellular aggregation, a major new development-specific protein that has lectin-like activity is synthesized. This protein, called myxobacterial hemagglutinin (MBHA), was able to agglutinate sheep or guinea pig erythrocytes but not horse, ox, chicken, or human erythrocytes. MBHA was undetectable in extracts of vegetative cells, cells starved in liquid buffer, or in glycerol-induced cells. However, cells starved on a fruiting medium produced large amounts of MBHA (about 5% of protein synthesis), starting at about 6-8 hr of development. The protein accumulated in the soluble fraction of cells, reaching a peak of 1-2% of total protein at about the time when aggregation was completed. At later times the amount of MBHA present in the soluble fraction declined although synthesis continued. The hemagglutinating activity of MBHA could not be inhibited with simple sugars or aminosugars but could be inhibited with fetuin, a fetal calf serum glycoprotein. The O-glycosidically linked trisaccharide glycopeptide of fetuin was shown to be inhibitory by itself. The penultimate galactose of this glycopeptide was directly implicated in the inhibitory activity, because the inhibition by asialofetuin was reduced to 1/60th by periodate oxidation and to 1/15th after beta-galactosidase treatment. MBHA is an abundant biochemical marker of development in M. xanthus. The fact that it is a lectin suggests that it may play a role in cell-cell recognition or agglutination.
Myxococcus xanthus, a social procaryotic microorganism, forms fruiting bodies and myxospores. We have isolated a collection of mutants of M. xanthus that are defective in fruiting morphogenesis and have studied synergistic interaction in pairwise mixtures of these mutants. Certain pairs of these fruiting-defective mutants can fruit when mixed together. Similarly, certain mutants that cannot sporulate under standard fruiting conditions can form myxospores in the presence of wildtype or other nonsporulating mutants. The pattern of synergism between pairs of conditional nonsporulating mutants defines at least three and probably four groups of mutants, such that members of a group cannot synergize with each other but can synergize with members of other groups.
The pattern of protein synthesis during development of Myxococcus xanthus was investigated. This gram-negative bacterium has a complex life cycle which involves a temporal sequence of cellular aggregation, mound formation, and myxosporulation. At various stages of development, cells were pulse-labeled with a 14C-labeled amino acid mixture. Synthesis of soluble and membrane proteins was then analyzed by SDS-polyacrylamide gel electrophoresis. Of the 30 major soluble proteins, at least 25% showed significant changes in their rates of production during development. Several significant changes were also found in the membrane proteins as analyzed by two-dimensional polyacrylamide gel electrophoresis. The major proteins synthesized during development were classified into four different types: accumulation proteins, peak proteins, late proteins, and constant proteins. The synthesis of protein S, an accumulation protein, increases dramatically during development to a maximum of 15% of total soluble protein synthesis. When methionine was added to the culture medium, cells did not form fruiting bodies. Under these conditions, almost all of the protein changes observed in the early and middle periods of development still occurred. However, the production of late proteins (e.g., protein U) was not observed, suggesting that methionine blocks a late stage of development. During glycerol induction, many of the changes in protein synthesis which normally occur during development were not observed (e.g., protein S did not accumulate). These results indicate that gene expression in M. xanthus is complex and subject to tight regulation.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Myxococcus xanthus is a Gram-negative bacterium that has a complex life cycle including a temporal sequence of cellular aggregation, mound formation, and myxosporulation. During development, protein S (molecuar weight 23,000) is induced and accumulates in very large amounts. Protein S was found in the soluble fraction of early developmental extracts and in the insoluble fraction in later extracts. This insoluble form of protein S can be solubilized by the addition of 1 M NaCl at 0 degrees C to extracts from aggregated cells (mound stage) or by the addition of 1 M NaCl at 30 degrees C to mature spores. Salt extraction (1 M NaCl) of protein S from mature spores was partially inhibited by the addition of Mg(2+) and almost completely inhibited by the addition of Ca(2+). The viability of spores was not changed by a salt extraction that removed their protein S. Examination of thin sections of mature spores and extracted spores by electron microscopy suggested that the protein S-deficient spores lacked a spore surface coat about 300 A thick. Purified protein S will spontaneously self-assemble onto protein S-deficient spores after removal of the NaCl by dialysis or by addition of 10 mM Ca(2+) to undialyzed samples. Glycerol-induced spores did not contain protein S and did not serve as primers for assembly of protein S. Quantitation of the self-assembly process showed almost stoichiometric binding of protein S to the protein S-deficient spores until saturation at 3.3 x 10(6) molecules per spore, a value 1.35 times higher than the normal level of proteins S found in mature spores. Protein S in the "reconstituted" spores was as protease resistant and sonication resistant as the protein S of native spores. Electron microscopy of the reconstituted spores revealed the assembly of new material on the spore surface. Adjacent spores were sometimes observed to be fused to each other through a common protein S layer. These results suggest that protein S serves a function in spore-spore interaction in the fruiting body.
A new bacteriophage, MX4, was isolated from nature and tested for transduction in strains of Myxococcus xanthus. The phage shows a lytic cycle of growth in strain DZ1 with a latent period of 180 minutes at 35 °C and an average burst size of 75 phage per infected cell. The phage was found to adsorb to but not form plaques on wild-type strains of M. xanthus; it could form plaques on a subclass of host mutants which were isolated as spectinomycin-resistant clones. Auxotrophs were isolated in one of these strains, DZF6, using a new growth medium suitable for nutritional selections. The auxotrophs were transduced to prototrophy at a frequency of about 2 × 10−6 to 4 × 10−5 per plaque-forming unit. Transductions were also performed with several antibiotic resistance markers using a temperature-sensitive phage mutant and maintaining all incubations at 35 °C, the restrictive temperature. In this manner, rifampicin and streptolydigin resistances were co-transduced at frequencies of 88 to 100%. A mutant of phage MX4 was isolated which shows high transduction frequencies. At high multiplicities of infection, this mutant should permit generalized transduction of many unselected markers to recipient strains. For test markers, this frequency was as high as 1 in 200 to 1 in 1200 viable recipient cells. Further, a host range mutation (hrm-1) has been introduced into the phage mutant. Thus genetic analysis of developmental genes in wild-type strains of M. xanthus is now possible.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter. Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the pET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase. Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow and produce innocuous target proteins indefinitely. Different configurations of the expression system can maintain several different steady-state levels of target gene expression. The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products.
The gene for a developmentally expressed sigma-factor, sigB, has been isolated from Myxococcus xanthus by use of the sigA gene (formerly rpoD) of the vegetative sigma-factor as a probe. The sequence of sigB has been determined, and an open reading frame of 193 amino acid residues (Mr = 21,551) was identified. The amino-terminal region of SigB contains 69 residues, of which 35 are identical (50% identity) to the region of SigA required for core RNA polymerase binding and initiation of RNA polymerization. SigB also possesses many features commonly found in other prokaryotic sigma-factors. Analysis of an M. xanthus strain carrying a sigB-lacZ fusion gene revealed that sigB is expressed from a middle to late stage of differentiation corresponding to the period from the onset of sporulation to late development. A sigB deletion mutant displayed normal mound formation and sporulation; however, production of the ops gene product in myxospores of the delta sigB strain was shown to be blocked. Myxospores from the sigB deletion strain also exhibited severe defects in stability and viability during late development. Our data indicate that sigB encodes a sigma-factor essential for the maturation of myxospores at a late stage of M. xanthus differentiation. Our results also suggest that differentiation of M. xanthus is regulated by development-specific sigma-factors.
The protein kinase family of enzymes mediates the responses of eukaryotic cells to both inter- and intracellular signals. These enzymes are either serine/threonine-specific or tyrosine-specific. Many of the latter are transmembrane receptors and are important in transduction of extracellular signals across the plasma membrane, whereas few examples of receptor serine kinases have been reported. We have now identified a complementary DNA clone from Zea mays (L.) encoding a putative serine/threonine-specific protein kinase structurally related to the receptor tyrosine kinases. This structural similarity is evidence for a previously undescribed class of transmembrane receptor in higher plants likely to be involved in signal reception and transduction. Furthermore, the catalytic domain of this protein kinase is linked through a transmembrane domain to an extracellular domain similar to that of glycoproteins encoded in the self-incompatibility locus of Brassica which are involved in the self-recognition system between pollen and stigma.
Tyrosine phosphorylation is important in the transmission of growth and differentiation signals; known tyrosine kinases include several oncoproteins and growth factor receptors. Interestingly, some differentiated cell types, such as erythrocytes and platelets contain high amounts of phosphotyrosine. We analyzed tyrosine kinases expressed in the K-562 chronic myelogenous leukemia cell line, which has a bipotential erythroid and megakaryoblastoid differentiation capacity. Analysis of 359 polymerase chain reaction-amplified cDNA clones led to the identification of 14 different tyrosine kinase-related sequences (JTK1-14). Two of the clones (JTK2 and JTK4) represent unusual members of the fibroblast growth factor receptor gene family, and the clones JTK5, JTK11, and JTK14 may also belong to the family of receptor tyrosine kinases but lack a close relationship to any known tyrosine kinase. Each of these different genes has its own characteristic expression pattern in K-562 cells and several other human tumor cell lines. In addition, the JTK11 and JTK14 mRNAs are induced during the megakaryoblastoid differentiation of K-562 cells. These tyrosine kinases may have a role in the differentiation of megakaryoblasts or in the physiology of platelets.
All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295
in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates
both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed
mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an
arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses.
None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation
of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that
it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent
reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly
among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the
cellular src gene.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
Myxobacteria have been shown to produce a peculiar RNA-DNA complex called msDNA, in which a single-stranded DNA is branched out from a RNA molecule (msdRNA) by a 2',5' phosphodiester linkage. It has been predicted that reverse transcriptase is required for msDNA biosynthesis. We identified a gene for reverse transcriptase in M. xanthus in the region that has been demonstrated to code for a cis- or transacting element for msDNA synthesis. This gene is located immediately downstream of the msdRNA coding region, and codes for a polypeptide of 485 amino acid residues. The polypeptide shows sequence similarity with retroviral reverse transcriptases. This fact, together with the mode of msDNA synthesis, suggests a possible relationship between retroviruses and the msDNA system. The analysis of the gene and the distribution of the msDNA system in independent isolates of M. xanthus indicate that the element is as old as other essential genes in M. xanthus and that it was not recently acquired into the genome.
We have examined the developmental regulation and function of two G alpha protein subunits, G alpha 1 and G alpha 2, from Dictyostelium. G alpha 1 is expressed in vegetative cells through aggregate stages while G alpha 2 is inducible by cAMP pulses and preferentially expressed in aggregation. Our results suggest that G alpha 2 encodes the G alpha protein subunit associated with the cAMP receptor and mediates all known receptor-activated intracellular signal transduction processes, including chemotaxis and gene regulation. G alpha 1 appears to function in both the cell cycle and development. Overexpression of G alpha 1 results in large, multinucleated cells that develop abnormally. The central role that these G alpha proteins play in signal transduction processes and in controlling Dictyostelium development is discussed.
Immobilon, a membrane of polyvinylidene difluoride to which gel-fractionated proteins can be transferred electrophoretically, was found to be an excellent matrix for the analysis of the phosphoamino acid content of phosphoproteins. Hydrolysis of 32P-labeled proteins bound to Immobilon with 5.7 N HCl resulted in the release of 90% of the 32P in the form of Pi, phosphoamino acids, and phosphopeptides. Two-dimensional electrophoretic analysis of the released phosphoamino acids yielded undistorted patterns. Because direct hydrolysis of proteins transferred to Immobilon eliminated the need for both preparative extraction of proteins from a gel and recovery by precipitation, analysis was rapid and yields of phosphoamino acids were extremely consistent. The yield of phosphoamino acids from proteins bound to Immobilon, unlike that from proteins eluted from gels, was independent of the size of the protein. The detection of 32P-labeled, phosphotyrosine-containing proteins in sodium dodecyl sulfate-polyacrylamide gels has been shown to be substantially improved by incubation of the gel in 1.0 N KOH for 2 h at 55 degrees C. Base hydrolysis of proteins bound to Immobilon proved to be faster and more sensitive than hydrolysis of proteins in gels. Less than 10% of bound protein was lost from Immobilon during the 2-h incubation at 55 degrees C in 1.0 N KOH. The autoradiographic image after alkaline hydrolysis of proteins on Immobilon was sharper than that obtained after hydrolysis of proteins in the gel. In addition, unlike base-treated gels, the dimensions of the Immobilon filter were unaffected by treatment with base.(ABSTRACT TRUNCATED AT 250 WORDS)
The cellular slime mold Dictyostelium discoideum undergoes a transition from single-celled amoebae to a multicellular organism
as a natural part of its life cycle. A method of cell-cell signaling that controls chemotaxis, morphogenesis, and gene expression
has developed in this organism, and a detailed understanding of this signaling system provides clues to mechanisms of intercellular
communication in the development of metazoans.
In this study we report the isolation and characterization of three overlapping cDNA clones for the type I beta isozyme of cGMP-dependent protein kinase (cGK) from human placenta libraries. The composite sequence was 3740 nucleotides long and contained 58 nucleotides from the 5'-noncoding region, an open reading frame of 2061 bases including the stop codon, and a 3'-noncoding region of 1621 nucleotides. The predicted full-length human type I beta cGK protein contained 686 amino acids including the initiator methionine, and had an estimated molecular mass of 77,803 Da. On comparison to the published amino acid sequence of bovine lung I alpha, human placenta I beta cGK differed by only two amino acids in the carboxyl-terminal region (amino acids 105-686). In contrast, the amino-terminal region of the two proteins was markedly different (only 36% similarity), and human I beta cGK was 16 amino acids longer. In a specific region in the amino-terminus (amino acids 63-75), 12 out of 13 amino acids of the human I beta cGK were identical to the partial amino acid sequence recently published for a new I beta isoform of cGK from bovine aorta. Northern blot analysis demonstrated a human I beta cGK mRNA, 7 kb in size, in human uterus and weakly in placenta. An mRNA of 7 kb was also observed in rat cerebellum, cerebrum, lung, kidney, and adrenal, whereas an mRNA doublet of 7.5 and 6.5 kb were observed in rat heart. Comparison of Northern and Western blot analyses demonstrated that the mRNA and protein for cerebellar cGK increased during the development of rats from 5 to 30 days old, whereas the 6.5 kb mRNA in rat heart declined.
The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) possesses tyrosine-specific protein kinase activity and autophosphorylates at Tyr-1073. Within the kinase domain of P130gag-fps is a putative ATP-binding site containing a lysine (Lys-950) homologous to lysine residues in cAMP-dependent protein kinase and p60v-src which bind the ATP analogue p-fluorosulfonylbenzoyl-5' adenosine. FSV mutants in which the codon for Lys-950 has been changed to codons for arginine or glycine encode metabolically stable but enzymatically defective proteins which are unable to effect neoplastic transformation. Kinase-defective P130gag-fps containing arginine at residue 950 was normally phosphorylated at serine residues in vivo suggesting that this amino acid substitution has a minimal effect on protein folding and processing. The inability of arginine to substitute for lysine at residue 950 suggests that the side chain of Lys-950 is essential for P130gag-fps catalytic activity, probably by virtue of a specific interaction with ATP at the phosphotransfer active site. Tyr-1073 of the Arg-950 P130gag-fps mutant protein was not significantly autophosphorylated either in vitro or in vivo, but could be phosphorylated in trans by enzymatically active P140gag-fps. These data indicate that Tyr-1073 can be modified by intermolecular autophosphorylation.
Tn5 lac is a transposon that fuses the transcription of lacZ to exogenous promoters. We generated 2374 Tn5 lac insertion-containing strains of Myxococcus xanthus, a soil bacterium that undergoes multicellular development which culminates in the formation of spores. Thirty-six strains were identified that specifically increase beta-galactosidase expression at some particular time during development and these expression times range from minutes after starvation initiates development to 24 hr, when sporulation begins. Different maximum levels of beta-galactosidase expression were also observed and the maximum for many strains that begin beta-galactosidase expression late in development was observed only if spores were disrupted. Seven of the 36 strains display mild to severe defects in aggregation and/or sporulation, as did an additional five strains whose beta-galactosidase expression was not developmentally regulated. Restriction maps of the DNA adjacent to the Tn5 lac insertions that are developmentally regulated and/or cause developmental defects show that most of the 41 insertions are in different regions of the Myxococcus genome. The developmentally regulated Tn5 lac insertions described here provide a set of at least 29 new developmental markers for Myxococcus.
We have isolated three genes (TPK1, TPK2, and TPK3) from the yeast S. cerevisiae that encode the catalytic subunits of the cAMP-dependent protein kinase. Gene disruption experiments demonstrated that no two of the three genes are essential by themselves but at least one TPK gene is required for a cell to grow normally. Comparison of the predicted amino acid sequences of the TPK genes indicates conserved and variable domains. The carboxy-terminal 320 amino acid residues have more than 75% homology to each other and more than 50% homology to the bovine catalytic subunit. The amino-terminal regions show no homology to each other and are heterogeneous in length. The TPK1 gene carried on a multicopy plasmid can suppress both a temperature-sensitive ras2 gene and adenylate cyclase gene.
During the early stages of its developmental program, Dictyostelium discoideum expresses cell surface cyclic adenosine monophosphate (cyclic AMP) receptors. It has been suggested that these receptors coordinate the aggregation of individual cells into a multicellular organism and regulate the expression of a large number of developmentally regulated genes. The complementary DNA (cDNA) for the cyclic AMP receptor has now been cloned from lambda gt-11 libraries by screening with specific antiserum. The 2-kilobase messenger RNA (mRNA) that encodes the receptor is undetectable in growing cells, rises to a maximum at 3 to 4 hours of development, and then declines. In vitro transcribed complementary RNA, when hybridized to cellular mRNA, specifically arrests in vitro translation of the receptor polypeptide. When the cDNA is expressed in Dictyostelium cells, the undifferentiated cells specifically bind cyclic AMP. Cell lines transformed with a vector that expresses complementary mRNA (antisense) do not express the cyclic AMP receptor protein. These cells fail to enter the aggregation stage of development during starvation, whereas control and wild-type cells aggregate and complete the developmental program within 24 hours. The phenotype of the antisense transformants suggests that the cyclic AMP receptor is essential for development. The deduced amino acid sequence of the receptor reveals a high percentage of hydrophobic residues grouped in seven domains, similar to the rhodopsins and other receptors believed to interact with G proteins. It shares amino acid sequence identity and is immunologically cross-reactive with bovine rhodopsin. A model is proposed in which the cyclic AMP receptor crosses the bilayer seven times with a serine-rich cytoplasmic carboxyl terminus, the proposed site of ligand-induced receptor phosphorylation.