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Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical
spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast,
MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulat...
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... optimal PCR annealing temperature of all primers was 62°C. Primer sequences are listed in Table 2. PCRs were performed with the Bio-Rad MJ mini personal thermal cycler using 60 ng of cDNA, 400 nM each primer, 200 M deoxynucleoside triphosphates (dNTPs), 1 polymerase buffer (Qiagen), and 1 U Taq polymerase (Qiagen) in a 25-l volume. ...
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Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that d...
The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal med...
Streptomyces topoisomerase I (TopA) exhibits exceptionally high processivity. The enzyme, as other actinobacterial topoisomerases I, differs from its bacterial homologs in its C-terminal domain (CTD). Here, bioinformatics analyses established that the presence of lysine repeats is a characteristic feature of actinobacterial TopA CTDs. Streptomyces...
The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene dele...
Bacterial cell division is a highly complex process that requires tight coordination between septum formation and chromosome replication and segregation. In bacteria that divide by binary fission a single septum is formed at mid-cell, a process that is coordinated by the conserved cell division scaffold protein FtsZ. In contrast, during sporulation...
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... For microscopic examination, a sterile coverslip was inserted at a 45° angle into the dual-culture PDA medium plates at 26 ± 2 °C for 3 days to allow the culture to overgrow its surface. The antagonistic mode of action at the Trichoderma-pathogen interaction region was observed using an Olympus CX33 microscope (Olympus, Tokyo, Japan) (Heichlinger et al. 2011). ...
Trichoderma harzianum is a well-known biological control agent (BCA) that shows great potential in controlling many pathogenic fungi. To screen for genes associated with mycoparasitism, we sequenced and analyzed the transcriptome of T. harzianum T4 grown in dual culture with Colletotrichum musae. We analyzed differentially expressed genes (DEGs) of Trichoderma harzianum T4 in three different culture periods: before contact (BC), during contact (C) and after contact (AC). A total of 1453 genes were significantly differentially expressed compared to when T. harzianum T4 was cultured alone. During the three periods of double culture of T. harzianum T4 with C. musae, 74, 516, and 548 genes were up-regulated, respectively, and 11, 315, and 216 genes were down-regulated, respectively. The DEGs were screened using GO and KEGG enrichment analyses combined with differential expression multiples. Six gene categories related to mycoparasitism were screened: (a) pathogen recognition and signal transduction, (b) hydrolases, (c) ribosomal proteins and secreted proteins, (d) multidrug-resistant proteins and transporters, (e) heat shock proteins and detoxification, and (f) oxidative stress and antibiotics-related genes. The expression levels of 24 up-regulated genes during T. harzianum T4’s antagonistic interaction with C. musae were detected via real-time fluorescence quantitative PCR (RT-qPCR). This study provided information on the transcriptional expression of T. harzianum T4 against C. musae. These results may help us to further understand the mechanism of mycoparasitism, which can provide a potential molecular target for improving the biological control capacity of T. harzianum T4.
... Impaired growth under hyperosmotic conditions has been previously reported in Streptomyces mutants lacking cell envelope-related genes, such as mreB, which encodes an actin-like protein, MreB, and is regulated by SigE (see Fig. S6 at https://doi.org/10.6084/m9.figshare.22223797) (15,23,24). MreB is widely conserved among bacteria, including Escherichia coli, Bacillus subtilis, and Streptomyces species (23)(24)(25). ...
... (15,23,24). MreB is widely conserved among bacteria, including Escherichia coli, Bacillus subtilis, and Streptomyces species (23)(24)(25). In many unicellular bacteria, MreB plays a central role in lateral cell wall synthesis by interacting with cell wall synthesis proteins and supporting the proper localization of the cell wall synthesis machinery (26,27). ...
... Although Streptomyces MreB is not a determinant of cell shape (see Fig. S11 at https://doi.org/10.6084/m9.figshare.22223797) (23,24), it is consistently expressed and forms a cell wall-synthesizing complex with other Mre proteins, including MreC, MreD, PBP2, FtsI, and RodZ, that closely resembles the lateral wall-synthesizing complex produced by rod-shaped bacteria (24,30). Importantly, mycelia of the DmreB mutant of S. coelicolor are highly sensitive to osmotic stress, similar to the DsigE mutant with impaired cell envelope integrity (23,24). ...
Recent bioinformatic analyses have revealed that CIS-related gene clusters are highly conserved in Gram-positive actinomycetes, especially members of the genus Streptomyces known for their ability to produce therapeutic antibiotics. While typical CISs are released from the cells and can act as protein translocation systems that inject effector proteins into the target cells, our results indicate the unique intracellular localization of SLPs, CIS-related nanostructures produced by S. lividans . In addition, the direct and indirect interactions of SLPs with cytoplasmic proteins and SLP localization within specific regions of mycelia suggest that the biological significance of SLPs is related to intracellular processes.
... Those genes ought to play crucial roles in the development of Streptomyces. For instance, AdpA, an essential early regulator of the development (Horinouchi, 2007), and MreB, involved in the spore cell wall assembly (Heichlinger et al., 2011) could be identified in all Streptomyces genomes. Nevertheless, nearly a third of the development-related genes showed varying degrees of loss. ...
As the canonical model organism to dissect bacterial morphological development, Streptomyces species has attracted much attention from the microbiological society. However, the evolution of development-related genes in Streptomyces remains elusive. Here, we evaluated the distribution of development-related genes, thus indicating that the majority of these genes were ubiquitous in Streptomyces genomes. Furthermore, the phylogenetic topologies of related strict orthologous genes were compared to the species tree of Streptomyces from both concatenation and single-gene tree analyses. Meanwhile, the reconciled gene tree and normalization based on the number of parsimony-informative sites were also employed to reduce the impact of phylogenetic conflicts, which was induced by uncertainty in single-gene tree inference based merely on the sequence and the bias in the amount of phylogenetic information caused by variable numbers of parsimony-informative sites. We found that the development-related genes had higher congruence to the species tree than other strict orthologous genes. Considering that the development-related genes could also be tracked back to the common ancestor of Streptomyces, these results suggest that morphological development follows the same pattern as species divergence.
... Additionally, ORF76 encodes putative transcriptional regulators, which regulate the gene expression of phage genes more as compared to host genes [30]. Noticeably, ORF217 encodes the MreB-like protein, which is reported as a key component in peptidoglycan synthesis [31]. An ORF89 encodes the chaperone domain of Endosialidase, which are tail spike proteins of bacteriophages for the degradation of host capsules [32]. ...
Xanthomonas oryzae pv. oryzae (X. oryzae) is a bacterial pathovar of rice diseases all over the world. Owing to emerging antibacterial resistance, phage therapies have gained significant attention to treat various bacterial infections. Nevertheless, comprehensive research is needed for their use as a safe biocontrol agent. In this study, isolation and characterization of a novel phage Xoo-sp15, that infects X. oryzae was ascertained through experimental and bioinformatics analyses to determine its virulent potency and reliability. High throughput sequencing demonstrated that Xoo-sp15 has a dsDNA genome with a total size of 157,091 bp and 39.9% GC content lower than its host (63.6%). Morphological and phylogenetic analyses characterized it as a new member of the Bastille-like group within the family Herelleviridae. In silico analysis revealed that it contains 229 open reading frames and 16 tRNAs. Additionally, this novel phage does not contain any resistant determinants and can infect nine X. oryzae strains. Therefore, Xoo-sp15 has the potential to serve as a novel candidate for phage therapy.
... 3). Mbl is a paralogue of MreB mainly involved in elongation during vegetative division, yet a role in sporulation has been reported in the actinomycete Streptomyces coelicolor (Heichlinger et al. 2011). Therefore, this might reflect a role of Mbl in sporulation also in the Firmicutes, as suggested in B. subtilis (Ojkic et al. 2016). ...
The cell cycle is a fundamental process that has been extensively studied in bacteria. However, many of its components and their interactions with machineries involved in other cellular processes are poorly understood. Furthermore, most knowledge relies on the study of a few models, but the real diversity of the cell division apparatus and its evolution are largely unknown. Here, we present a massive in-silico analysis of cell division and associated processes in around 1,000 genomes of the Firmicutes, a major bacterial phylum encompassing models (i.e. Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus), as well as many important pathogens. We analyzed over 160 proteins by using an original approach combining phylogenetic reconciliation, phylogenetic profiles, and gene cluster survey. Our results reveal the presence of substantial differences among clades and pinpoints a number of evolutionary hotspots. In particular, the emergence of Bacilli coincides with an expansion of the gene repertoires involved in cell wall synthesis and remodeling. We also highlight major genomic rearrangements at the emergence of Streptococcaceae. We establish a functional network in Firmicutes that allows identifying new functional links inside one same process such as between FtsW (peptidoglycan polymerase) and a previously undescribed PBP or between different processes, such as replication and cell wall synthesis. Finally, we identify new candidates involved in sporulation and cell wall synthesis. Our results provide a previously undescribed view on the diversity of the bacterial cell cycle, testable hypotheses for further experimental studies, and a methodological framework for the analysis of any other biological system.
... Another gene with upregulated expression in the dispersed strain was mbl2 (sco6166), which encodes one of the three MreB homologs present in S. coelicolor. Previous studies have shown that mbl2 was expressed only in vegetative mycelium and repressed during differentiation (Heichlinger et al., 2011), in contrast to the other two paralogs, MreB and Mbl1, which were found to regulate cell wall deposition during sporogenic cell division, with a similar role to the one played by homologous proteins in unicellular bacteria. ...
... Another morphogenic protein identified as differentially expressed was the MreB paralog Mbl2. Of the three paralogs encoded in the S. coelicolor genome, Mbl2 seems to be expressed exclusively in the vegetative mycelium and its deletion does not affect sporulation in solid medium (Heichlinger et al., 2011). Given the role of MreB coordinating synthesis of the cell wall, it is an obvious candidate to contribute to the alterations observed in strain 2L12. ...
Streptomyces are mycelial bacteria adapted to grow in soil. They have become important producers of biomolecules with medical applications, but their growth in industrial fermenters is challenged by their peculiar morphology in liquid culture: the hyphae tend to clump and grow as large pellets, which are oxygen- and nutrient-limited, grow slowly and present diminished protein production. Here, by implementing an experimental evolution strategy, a S. coelicolor strain, 2L12, with dispersed morphology and reduced pellet size in liquid culture and no defects in either differentiation or secondary metabolism was selected. Genome sequencing revealed a single amino acid substitution in a sensor kinase, Sco5282, of unknown function to be responsible for the morphological changes. Moreover, genetic and biochemical scrutiny identified Sco5283 as the cognate response regulator and demonstrated that the acquired mutation activates this two-component system. Finally, transcriptomic analysis of the mutant strain revealed changes in expression of genes involved in central processes such as glycolysis, gluconeogenesis, stress-signaling pathways, proteins secretion and cell envelope metabolism. Thus a novel two-component system is proposed to play a key role in the control of Streptomyces extracellular metabolism.
... A hierarchic cascade of whi genes, encoding several transcriptional regulators control initiation of multiple cell division events and conversion of aerial hyphae into spore chains (Kelemen and Buttner, 1998;Bush et al., 2015). Replication of the chromosome is upregulated, generating multi-genomic aerial hyphae (Ruban-Ośmiałowska et al., synthesis of the sporulation septa and the spore envelope involves in addition further cytoskeletal proteins, the actin homologues MreB and Mbl (Heichlinger et al., 2011). With the help of ParAB, FtsK, the FtsKlike SffA and SmeA, which localizes SffA to the sporulation septa, the multiple chromosome copies segregate into the spore compartments (Wang et al., 2007;Ausmees et al., 2007;Jakimowicz et al., 2005). ...
... Spores of single mutants were sensitive to detrimental environmental conditions, like heat, desiccation, high osmolarity, or cell wall damage by lysozyme or vancomycin, indicating that the integrity of the spore envelope was impaired. Moreover, fusion proteins of MreB and the MreB-like protein Mbl of S. coelicolor A3(2) were localized to sites of spore wall synthesis during the conversion of aerial hyphae into spore chains (Heichlinger et al., 2011). These data suggested that the Mre proteins, which direct PG incorporation at the lateral wall during elongation growth of rod-shaped bacteria have a different role in Streptomyces than in other bacteria and are involved in the synthesis of the thickened spore envelope. ...
... Most of the previously isolated differentiation mutants had quite severe defects causing white (whi) and bald (bld) phenotypes (Kelemen and Buttner, 1998;Bush et al., 2015). The mutants affected in SSSC (2019) 151327 genes had been overseen in these previous screens, since their minor defect, aberrant spores with an impaired spore envelope, has no dramatic consequences for the colony morphology (Kleinschnitz et al., 2011, Ladwig et al., 2015. ...
A crucial stage of the Streptomyces life cycle is the sporulation septation, a process were dozens of cross walls are synchronously formed in the aerial hyphae in a highly coordinated manner. This process includes the remodeling of the spore envelopes to make Streptomyces spores resistant to detrimental environmental conditions. Sporulation septation and the synthesis of the thickened spore envelope in S. coelicolor A3(2) involves the Streptomyces spore wall synthesizing complex SSSC. The SSSC is a multi-protein complex including proteins directing peptidoglycan synthesis (MreBCD, PBP2, Sfr, RodZ) and cell wall glycopolymer synthesis (PdtA). It also includes two eukaryotic like serin/threonine protein kinases (eSTPK), PkaI and PkaH, which were shown to phosphorylate MreC. Since unbalancing phosphorylation activity by either deleting eSTPK genes or by expressing a second copy of an eSTPK gene affected proper sporulation, a model was developed, in which the activity of the SSSC is controlled by protein phosphorylation.
... Each branching event involves reorientation of cell polarity and establishment of a new polar growth center in the lateral hyphal wall (Hempel et al., 2008;Richards et al., 2012). Although MreB proteins are present in Streptomyces, they are not required for hyphal growth and are primarily involved in formation of the cell wall in spores (Mazza et al., 2006;Heichlinger et al., 2011). Also FtsZ and the bacterial cell division machinery are dispensable for viability in Streptomyces, and ftsZ null mutants can still grow by hyphal tip extension and branching (McCormick et al., 1994;Santos-Beneit et al., 2017). ...
Streptomyces spp. grow as branching hyphae, building the cell wall in restricted zones at hyphal tips. The organization of this mode of polar growth involves three coiled‐coil proteins: DivIVA and Scy, which form apical protein complexes referred to as polarisomes; and the intermediate filament‐like protein FilP, which influences cell shape and interacts with both Scy and DivIVA. Here, we use live cell imaging of Streptomyces venezuelae to clarify the subcellular localization and dynamics of FilP and its effect on hyphal morphology. By monitoring a FilP‐mCherry fusion protein, we show that FilP accumulates in gradient‐like zones behind the hyphal tips. The apical gradient pattern of FilP localization is dependent on hyphal tip extension and immediately dissipates upon growth arrest. Fluorescence recovery after photobleaching experiments show that FilP gradients are dynamic and subject to subunit exchange during vegetative growth. Further, the localization of FilP at hyphal tips is not directly dependent on scy, even though the strongly perturbed morphology of most scy mutant hyphae is associated with mislocalization of FilP. Finally, we find that filP has an effect on the size and position of the foci of key polar growth determinant DivIVA. This effect likely contributes to the phenotype of filP mutants.
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... The spore's wall thickening is dependent on the so called Streptomyces-sporewall-synthesizing complex (SSSC), a system somewhat similar to the elongasome of rod-shaped bacteria . Members involved in this complex are the cytoskeletal actin-like proteins, MreB and Mbl that cooperate in spore wall synthesis (Mazza et al., 2006;Heichlinger et al., 2011). Maturation is further accompanied by DNA condensation and spore pigmentation, such as the production of gray spore pigment in S. coelicolor (Kelemen et al., 1998). ...
The complex development undergone by Streptomyces encompasses transitions from vegetative mycelial forms to reproductive aerial hyphae that differentiate into chains of single-celled spores. Whereas their mycelial life – connected with spore formation and antibiotic production – is deeply investigated, spore germination as the counterpoint in their life cycle has received much less attention. Still, germination represents a system of transformation from metabolic zero point to a new living lap. There are several aspects of germination that may attract our attention: (1) Dormant spores are strikingly well-prepared for the future metabolic restart; they possess stable transcriptome, hydrolytic enzymes, chaperones, and other required macromolecules stabilized in a trehalose milieu; (2) Germination itself is a specific sequence of events leading to a complete morphological remodeling that include spore swelling, cell wall reconstruction, and eventually germ tube emergences; (3) Still not fully unveiled are the strategies that enable the process, including a single cell’s signal transduction and gene expression control, as well as intercellular communication and the probability of germination across the whole population. This review summarizes our current knowledge about the germination process in Streptomyces, while focusing on the aforementioned points.
... Apical growth in actinobacteria is independent of MreB. In fact, the genomes of actinobacteria that adopt bacillary-type growth lack mreB homologs, and filamentous Streptomyces use an MreB protein only during sporulation [16][17][18]. Apical growth is guided by the protein DivIVA [13][14][15]; DivIVA assembles to form an internal cytoskeletal element at the cell poles that appears to function to recruit proteins for apical cell-wall synthesis. ...
The dissacharide trehalose is an important intracellular osmoprotectant and the OtsA/B pathway is the principal pathway for trehalose biosynthesis in a wide range of bacterial species. Scaffolding proteins and other cytoskeletal elements play an essential role in morphogenetic processes in bacteria. Here we describe how OtsA, in addition to its role in trehalose biosynthesis, functions as an osmotic stress sensor to regulate cell morphology in Arthrobacter strain A3. In response to osmotic stress, this and other Arthrobacter species undergo a transition from bacillary to myceloid growth. An otsA null mutant exhibits constitutive myceloid growth. Osmotic stress leads to a depletion of trehalose-6-phosphate, the product of the OtsA enzyme, and experimental depletion of this metabolite also leads to constitutive myceloid growth independent of OtsA function. In vitro analyses indicate that OtsA can self-assemble into protein networks, promoted by trehalose-6-phosphate, a property that is not shared by the equivalent enzyme from E. coli, despite the latter’s enzymatic activity when expressed in Arthrobacter. This, and the localization of the protein in non-stressed cells at the mid-cell and poles, indicates that OtsA from Arthrobacter likely functions as a cytoskeletal element regulating cell morphology. Recruiting a biosynthetic enzyme for this morphogenetic function represents an intriguing adaptation in bacteria that can survive in extreme environments.
