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Pseudomonas aeruginosa: genes and enzymes of alginate synthesis
Abstract
Alginate is an important virulence factor of Pseudomonas aeruginosa, a bacterium that colonizes the pulmonary tracts of cystic fibrosis patients. Alginate is also widely used in the food, pharmaceutical and chemical industries, and consequently there is considerable interest in the molecular biology and biochemistry of alginate synthesis. As well as its therapeutic potential, research on mucoid P. aeruginosa may provide a lead to an alternative source of alginate for industrial use.
... Transcription of these genes is under the strict control of one algD upstream promoter and two internal promoters. Therefore, differential transcription of upstream genes to internal promoters has been proposed as a mechanism that may control the stoichiometry of protein subunits in a complex of several proteins, resulting in different combinations of alginates under different Fig. 2 May and Chakrabarty 1994;Rehm and Valla 1997). ...
... Transcription of these genes is under the strict control of one algD upstream promoter and two internal promoters. Therefore, differential transcription of upstream genes to internal promoters has been proposed as a mechanism that may control the stoichiometry of protein subunits in a complex of several proteins, resulting in different combinations of alginates under different Fig. 2 (Hay et al. 2013;May and Chakrabarty 1994;Rehm and Valla 1997). ...
Despite the extensive application of enzymes in various industries, their instability and non-reusability cause to their limitation. The immobilization of enzymes can be used as a remedy for these limitations. However, supports and immobilization methods affect the activity and stability of enzymes. Here, the effects of alginate support on immobilized enzymes are investigated. Alginate is an unbranched heterogeneous copolymer comprised of 1,4′-linked β-D-Mannuronic acid (M) and α-L-Guluronic acid (G) residues. Enzyme immobilization on alginate support can improve their stability and increase their reusability significantly. Enzymes entrapment through the alginate and its derivatives is an effective method for immobilization. Availability, biocompatibility, resistance against microbial contamination, non-toxicity, and low price, make it an ideal candidate. Green alginate synthesized nanoparticles, and their combination with other materials (organic/inorganic) have introduced a variety of new applications in drug delivery. Moreover, directed immobilization methods, directed mutagenesis-based methods, and the methods based on recombinant fusion protein technology have paved the way for new strategies for the attachment of enzymes on some supports based on alginate for various pharmaceutical and industrial applications.
... Several different roles for the enzyme have been proposed. These include the regulation of the length of secreted alginate polymer by cleaving β-1,4-linkages between ManA residues prior to export (43,44); aiding in biofilm detachment (45); and degradation of alginate that is not exported from the cell to prevent its accumulation within the periplasmic space (46,47). Studies have also suggested that the enzyme is part of a multiprotein complex with AlgG, AlgX, and AlgK and that it assists in transporting the J o u r n a l P r e -p r o o f AlgL clears the periplasm of alginate 3 polymer across the outer membrane (47). ...
Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein’s role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.
... In five SAR92 species, we identified homologues of the known alginate synthesis operons (Fig. 4), previously characterised in non-marine Pseudomonas and Azotobacter (for reviews see refs. [49][50][51]). These loci typically include the genes algAEJX, other cell wall and nucleotide sugar synthesis genes, and characteristically, genes encoding glycosyl transferases of the GT2 family identified as mannuronan synthases. ...
Background
The planktonic bacterial community associated with spring phytoplankton blooms in the North Sea is responsible for a large amount of carbon turnover in an environment characterised by high primary productivity. Individual clades belonging to the Gammaproteobacteria have shown similar population dynamics to Bacteroidetes species , and are thus assumed to fill competing ecological niches. Previous studies have generated large numbers of metagenome assembled genomes and metaproteomes from these environments, which can be readily mined to identify populations performing potentially important ecosystem functions. In this study we attempt to catalogue these spring bloom-associated Gammaproteobacteria , which have thus far attracted less attention than sympatric Alphaproteobacteria and Bacteroidetes .
Methods
We annotated 120 non-redundant species-representative gammaproteobacterial metagenome assembled genomes from spring bloom sampling campaigns covering the four years 2010–2012 and 2016 using a combination of Prokka and PfamScan, with further confirmation via BLAST against NCBI-NR. We also matched these gene annotations to 20 previously published metaproteomes covering those sampling periods plus the spring of 2009.
Results
Metagenome assembled genomes with clear capacity for polysaccharide degradation via dedicated clusters of carbohydrate active enzymes were among the most abundant during blooms. Many genomes lacked gene clusters with clearly identifiable predicted polysaccharide substrates, although abundantly expressed loci for the uptake of large molecules were identified in metaproteomes. While the larger biopolymers, which are the most abundant sources of reduced carbon following algal blooms, are likely the main energy source, some gammaproteobacterial clades were clearly specialised for smaller organic compounds. Their substrates range from amino acids, monosaccharides, and DMSP, to the less expected, such as terpenoids, and aromatics and biphenyls, as well as many ‘unknowns’. In particular we uncover a much greater breadth of apparent methylotrophic capability than heretofore identified, present in several order level clades without cultivated representatives.
Conclusions
Large numbers of metagenome assembled genomes are today publicly available, containing a wealth of readily accessible information. Here we identified a variety of predicted metabolisms of interest, which include diverse potential heterotrophic niches of spring bloom-associated Gammaproteobacteria . Features such as those identified here could well be fertile ground for future experimental studies.
... Not surprisingly, algU and gyrA are defined as pathoadaptive genes, i.e. genes in which functional mutations optimize pathogen fitness 14 . Even though AlgU, GyrA and RpoN are known targets of P. aeruginosa evolution in the CF environment, their role in increasing the within-patient fitness through reduction of growth rate was previously unknown 52,56,57 . Our results suggest that by tailoring global regulators functionality to the CF environment, bacteria increase their fitness in two ways: 'locally' modifying the functionality of specific system such as alginate production, nitrogen metabolism and ciprofloxacin resistance, and 'globally' by decreasing the growth rate which has Fig. 6 Trajectory of adaptation of Pseudomonas aeruginosa to the cystic fibrosis (CF) environment. ...
Long-term infection of the airways of cystic fibrosis patients with Pseudomonas aeruginosa is often accompanied by a reduction in bacterial growth rate. This reduction has been hypothesised to increase within-patient fitness and overall persistence of the pathogen. Here, we apply adaptive laboratory evolution to revert the slow growth phenotype of P. aeruginosa clinical strains back to a high growth rate. We identify several evolutionary trajectories and mechanisms leading to fast growth caused by transcriptional and mutational changes, which depend on the stage of adaptation of the strain. Return to high growth rate increases antibiotic susceptibility, which is only partially dependent on reversion of mutations or changes in the transcriptional profile of genes known to be linked to antibiotic resistance. We propose that similar mechanisms and evolutionary trajectories, in reverse direction, may be involved in pathogen adaptation and the establishment of chronic infections in the antibiotic-treated airways of cystic fibrosis patients.
... Many of the enzymes in the alginate-biosynthetic pathway have been characterized (Table 3), and since several recent reviews (283,284) cover their biochemical and physicochemical properties in significant detail, that information will not be repeated here. The majority of enzymes involved in the synthesis and modifications of the alginate polymer are encoded by the large cluster of genes at 34 min of the P. aeruginosa chromosomal map (77) (Fig. 4). ...
Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.
... The arrangement of the glycolytic pathway through the entner-Doudoroff and pentose phosphate pathway enables assimilation of hexose sugars ensuring a high level of reducing equivalents 114,115 . similarly, it supports the production of the exopolysaccharide alginate, which is one of the virulence factors that P. aeruginosa produces during infection 116 . the tricarboxylic acid (tCa) cycle, funnelled through the assimilation of amino and organic acids, provides building blocks required for cellular growth 117 . ...
Intense genome sequencing of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) airways has shown inefficient eradication of the infecting bacteria, as well as previously undocumented patient-to-patient transmission of adapted clones. However, genome sequencing has limited potential as a predictor of chronic infection and of the adaptive state during infection, and thus there is increasing interest in linking phenotypic traits to the genome sequences. Phenotypic information ranges from genome-wide transcriptomic analysis of patient samples to determination of more specific traits associated with metabolic changes, stress responses, antibiotic resistance and tolerance, biofilm formation and slow growth. Environmental conditions in the CF lung shape both genetic and phenotypic changes of P. aeruginosa during infection. In this Review, we discuss the adaptive and evolutionary trajectories that lead to early diversification and late convergence, which enable P. aeruginosa to succeed in this niche, and we point out how knowledge of these biological features may be used to guide diagnosis and therapy.
... Cellfree supernatant was collected after centrifugation at 10000 rpm for 10 min. Alginate quantification was performed by measuring the uronic acid content from a standard curve of alginic acid of brown algae (Sigma Aldrich, United States), ranging from 10 to 1000 µg.mL −1 (May and Chakrabarty, 1994). Absorbance at A 530 was indicative of a positive uronic acid test. ...
Plant growth-promoting rhizobacteria (PGPR) are associated with plant roots and use organic compounds that are secreted from root exudates as food and energy source. Root exudates can chemoattract and help bacteria to colonize the surface of plant roots by inducing chemotactic responses of rhizospheric bacteria. In this study, we show that root colonization of Brachypodium distachyon by Bacillus velezensis strain B26 depends on several factors. These include root exudates, organic acids, and their biosynthetic genes, chemotaxis, biofilm formation and the induction of biofilm encoding genes. Analysis of root exudates by GC-MS identified five intermediates of the TCA cycle; malic, fumaric, citric, succinic, oxaloacetic acids, and were subsequently evaluated. The strongest chemotactic responses were induced by malic, succinic, citric, and fumaric acids. In comparison, the biofilm formation was induced by all organic acids with maximal induction by citric acid. Relative to the control, the individual organic acids, succinic and citric acids activated the epsD gene related to EPS biofilm, and also the genes encoding membrane protein (yqXM) and hydrophobin component (bslA) of the biofilm of strain B26. Whereas epsA and epsB genes were highly induced genes by succinic acid. Similarly, concentrated exudates released from inoculated roots after 48 h post-inoculation also induced all biofilm-associated genes. The addition of strain B26 to wild type and to icdh mutant line led to a slight induction but not biologically significant relative to their respective controls. Thus, B26 has no effect on the expression of the ICDH gene, both in the wild type and the mutant backgrounds. Our results indicate that root exudates and individual organic acids play an important role in selective recruitment and colonization of PGPR and inducing biofilm. The current study increases the understanding of molecular mechanisms behind biofilm induction by organic acids.
... Mucoid phenotype is characterized by overproduction of alginate, a viscous polysaccharide composed of mannuronic and guluronic acid, which is also an important virulence factor (Pedersen et al., 1990;May and Chakrabarty, 1994). The master regulator of alginate biosynthesis is the alternate sigma factor AlgT (alternatively, AlgU or σ 22 ), a homologue of the stress response regulator RpoE from Escherichia coli. ...
Pseudomonas aeruginosa isolates from cystic fibrosis patients are often mucoid (due to the overexpression of exopolysaccharide alginate) yet lost motility. It remains unclear about how P. aeruginosa coordinately regulates alginate production and the type IV pili‐driven twitching motility. Here we showed that sigma 22 factor (AlgT/U), an activator of alginate biosynthesis, repressed twitching motility by inhibiting the expression of pilin (PilA) through the intermediate transcriptional regulator AmrZ, which directly bound to the promoter region of pilA in both mucoid strain FRD1 and non‐mucoid strain PAO1. Four conserved AmrZ‐binding sites were found in pilA promoters among 10 P. aeruginosa strains although their entire pilA promoters had low identity. AmrZ has been reported to be essential for twitching in PAO1. We found that AmrZ was also required for twitching in mucoid FRD1, yet a high level of AmrZ inhibited twitching motility. This result was consistent with the phenomenon that twitching is frequently repressed in mucoid strains, in which the expression of AmrZ was highly activated by AlgT. Additionally, AlgT also inhibited the transcription of pilMNOP operon, which is involved in efficient pilus assembly. Our data elucidated a mechanism for how AlgT and AmrZ coordinately controlled twitching motility in P. aeruginosa. This article is protected by copyright. All rights reserved.
... Brown seaweeds and certain bacteria such as pseudomonads and Azotobacter are known to be potential producers of the polysaccharide. Pseudomonas aeruginosa produces extracellular biofilms containing alginate as virulence factors during lung infections in cystic fibrosis patients (May & Chakrabarty, 1994). Alginate is included in the outer layer of cysts transformed from vegetative cells of Azotobacter vinelandii (Campos et al., 1996). ...
Alginate-assimilating Sphingomonas sp. strain A1 is the gram-negative bacterium first identified to form a single polar flagellum containing lateral-typed flagellin (p6) in the filament. In addition to the p6 flagellin, two polar-typed flagellins (p5 and p5') are also included in the flagellum. Here we show the significant role of p6 as well as p5/p5' in flagellum formation and cell motility toward alginate. A p6 gene disruptant significantly reduced flagellum formation, and it showed no cell motility, whereas each mutant with a disruption in the p5 or p5' gene exhibited cell motility through the formation of a polar flagellum containing p6. The ratio of p6 to p5 decreased in proportion to cell growth, suggesting that strain A1 changes flagellin ratios in the filament depending on the external environment. Each of purified recombinant p5 and p6 proteins formed the filament by in vitro self-assembly, and an anti-p5 antibody reacted with the p5 filament but not with the p6 filament. Immunoelectron microscopy using an anti-p5 antibody indicated that strain A1 formed two types of the filament in a single polar flagellum: p6 alone in the entire filament and p5 elongation filament subsequent to the p6 proximal end. Immunoprecipitation with an anti-p5 antibody directly demonstrated that p5 and p6 coexist in a single filament. Strain A1 cells were also found to exhibit a chemotactic motility in response to alginate. This is the first report on function/location of the lateral-typed flagellin in a single polar flagellum and the bacterial chemotaxis toward alginate.
... In the periplasm, O-acetylation mediat- ed by the three gene products (algF, algI, algJ) as well as epimerization of mannuronate to guluronate catalyzed by a single gene product (AlgG) yield the final polymer. The function of the periplasmic alginate lyase (AlgL) is still unknown; it has been supposed to control chain length of alginate molecules or provide oligosaccharide primers for the polymerization reac- tion (MAY AND CHAKRABARTY 1994). The pore protein AlgE seems to be involved in the transport of nascent alginate molecules across the outer membrane (REHM ET AL. 1994). ...
Different forms of microbial aggregates such as biofilms and flocs in natural and engineered environments have in common that the constituent cells of these structures are embedded in a hydrated matrix of extracellular polymeric substances (EPS). These EPS are supposed to consist predominantly of polysaccharides and proteins, but other macromolecules such as nucleic acids, (phospho)lipids, and humic substances have also been found in varying amounts as components of the EPS matrix in microbial aggregates such as in wastewater biofilms and activated sludges (Urbain et al. 1993; Jahn and Nielsen 1996; Gehrke and Sand, this volume).
... In this process, the enzyme-associated polysaccharide chain is transferred to the UDP-sugar that newly enters the active site of the polymerase [134,135]. A similar mechanism has been proposed for the synthesis of the alginate capsule of P. aeruginosa [136,137], and for that of the CPSs in type 3 and 37 S. pneumoniae strains [81,138]. ...
Capsules are the outmost structures of bacterial and fungal cells. The capsules protect microbial cells from immune recognition and killing during infection of mammalian hosts. Except for the polyγ-glutamate (PGA) capsule of Bacillus anthracis, other known capsules are all composed of polysaccharides. Certain bacteria (e.g. B. anthracis and Streptococcus pyogenes) produce only one capsule structure, whereas many other bacteria express capsules with great biochemical, structural and immunological diversity within the same species. This diversity is driven by immune selection from the mammalian hosts. The genes for capsule synthesis are typically clustered in a single locus of bacterial chromosome. The number of genes associated with capsule synthesis ranges from one in serotype 37 Streptococcus pneumoniae to >20 in serotype 38 S. pneumoniae. Different bacterial species can share similar genes or mechanisms for capsule synthesis. The expression of the capsule genes is often regulated by environmental conditions. The capsular polysaccharides are the antigens of the current polysaccharide-based vaccines for S. pneumoniae, Neisseria meningitidis, Haemophilus influenzae and Salmonella enterica serovar Typhi. Certain capsule polymers also have important industrial applications.
... The pathogenesis of P. aeruginosa infection is attributed to the production of both cellassociated and extracellular vimlence factors. The cell-associated factors include the flagellum (Mahenthiralingam et al., 1994), the adhesion factors (e.g., pill and other possible adhesins [Simpson et al., 1992]), and alginate (Gilligan, 1991;May and Chakrabarty, 1994). The extracellular vimlence factors include exotoxin A, exoenzyme S, elastases (LasA and LasB), alkaline protease, and phospholipase C (Berka et al., 1981;Gilligan, 1991;Homma, 1980;Nicas and Iglewski, 1985b;Woods and Iglewski, 1983). ...
... Furthermore, alginate is also synthesized as an exopolysaccharide by certain bacteria (4). The alginate biofilm produced by Pseudomonas aeruginosa is an important virulence factor during lung infections in cystic fibrosis patients (5). Alginate is a natural linear polysaccharide composed of (1,4)-linked -D-mannuronate and its C5 epimer, ␣-L-guluronate. ...
Bacterial alginate lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles
and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate lyases
are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a β-jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in
the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular
dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for
substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center,
especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr353 may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization
is proposed. Moreover, although bacterial alginate lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they
share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost
insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.
... 23 A maior parte do nosso conhecimento sobre a genética da biossíntese de alginato se origina de estudos de Pseudomonas aeruginosa, principalmente por causa da relevância médica desta bactéria, como um importante microrganismo patogênico oportunista, para humanos, em pacientes sofrendo de fibrose cística. [24][25][26] Nesta, os alginatos têm papel importante como um fator de virulência. A razão para isto parece ser a formação de um biofilme de alginato, o qual facilita a colonização do pulmão. ...
... Brown seaweeds and certain bacteria are well-known producers of this polysaccharide. Pseudomonas aeruginosa produces extracellular alginate-containing biofilms that are involved in the expression of virulence factors during lung infections in cystic fibrosis patients (2,3). In contrast, alginate produced by brown seaweeds is used as a gelling agent and thickener in food. ...
Sphingomonas sp. strain A1, a Gram-negative bacterium, directly incorporates alginate polysaccharide into the cytoplasm through a periplasmic alginate-binding protein-dependent ATP-binding cassette transporter. The polysaccharide is degraded to monosaccharides via formation of oligosaccharides by endo- and exotype alginate lyases. The strain A1 proteins for alginate uptake and degradation are encoded in both strands of a genetic cluster in the bacterial genome and inducibly expressed in the presence of alginate. Here we show function of an alginate-dependent transcription factor AlgO and its mode of action on the genetic cluster and alginate oligosaccharides. A putative gene within the genetic cluster seems to encode a transcription factor-like protein (AlgO). Mutant strain A1 (ΔAlgO) cells with a disrupted algO constitutively produced alginate-related proteins. DNA microarray indicated that wild-type cells inducibly transcribed the genetic cluster only in the presence of alginate, while ΔAlgO cells constitutively expressed the genetic cluster. A gel mobility shift assay showed that AlgO binds to the specific intergenic region (algO:S) between algO and algS. Binding of AlgO to algO:S diminished with increasing alginate oligosaccharides. These results demonstrated a novel alginate-dependent gene expression mechanism. In the absence of alginate, AlgO binds to algO:S and represses the expression of both strands of the genetic cluster, while in the presence of alginate, AlgO dissociates from algO:S via binding to alginate oligosaccharides produced through the lyase reaction, and subsequently initiates transcription of the genetic cluster. This is the first report on the mechanism by which alginate regulates the expression of the gene cluster.
Biofilms are complex multicellular communities formed by bacteria, and their extracellular polymeric substances are observed as surface-attached or non-surface-attached aggregates. Many types of bacterial species found in living hosts or environments can form biofilms. These include pathogenic bacteria such as Pseudomonas, which can act as persistent infectious hosts and are responsible for a wide range of chronic diseases as well as the emergence of antibiotic resistance, thereby making them difficult to eliminate. Pseudomonas aeruginosa has emerged as a model organism for studying biofilm formation. In addition, other Pseudomonas utilize biofilm formation in plant colonization and environmental persistence. Biofilms are effective in aiding bacterial colonization, enhancing bacterial resistance to antimicrobial substances and host immune responses, and facilitating cell‒cell signalling exchanges between community bacteria. The lack of antibiotics targeting biofilms in the drug discovery process indicates the need to design new biofilm inhibitors as antimicrobial drugs using various strategies and targeting different stages of biofilm formation. Growing strategies that have been developed to combat biofilm formation include targeting bacterial enzymes, as well as those involved in the quorum sensing and adhesion pathways. In this review, with Pseudomonas as the primary subject of study, we review and discuss the mechanisms of bacterial biofilm formation and current therapeutic approaches, emphasizing the clinical issues associated with biofilm infections and focusing on current and emerging antibiotic biofilm strategies.
The biofilm matrix is a fortress; sheltering bacteria in a protective and nourishing barrier that allows for growth and adaptation to various surroundings. A variety of different components are found within the matrix including water, lipids, proteins, extracellular DNA, RNA, membrane vesicles, phages, and exopolysaccharides. As part of its biofilm matrix, Pseudomonas aeruginosa is genetically capable of producing three chemically distinct exopolysaccharides – alginate, Pel, and Psl – each of which has a distinct role in biofilm formation and immune evasion during infection. The polymers are produced by highly conserved mechanisms of secretion, involving many proteins that span both the inner and outer bacterial membranes. Experimentally determined structures, predictive modelling of proteins whose structures are yet to be solved, and structural homology comparisons give us insight into the molecular mechanisms of these secretion systems, from polymer synthesis to modification and export. Here, we review recent advances that enhance our understanding of P. aeruginosa multi-protein exopolysaccharide biosynthetic complexes, and how the glycoside hydrolases/lyases within these systems have been commandeered for antimicrobial applications.
Given the excellent characteristics of alginate, it is an industrially important polysaccharide. Mannuronan C5-epimerase (MC5E) is an alginate-modifying enzyme that catalyzes the conversion of β-D-mannuronate (M) to its C5 epimer α-L-guluronate (G) in alginate. Both the biological activities and physical properties of alginate are determined by M/G ratios and distribution patterns. Therefore, MC5E is regarded as a biotechnological tool for modifying and processing alginate. Various MC5Es derived from brown algae, Pseudomonas and Azotobacter have been isolated and characterized. With the rapid development of structural biology, the crystal structures and catalytic mechanisms of several MC5Es have been elucidated. It is necessary to comprehensively understand the research status of this alginate-modifying enzyme. In this review, the properties and potential applications of MC5Es isolated from different kinds of organisms are summarized and reviewed. Moreover, future research directions of MC5Es as well as strategies to enhance their properties are elucidated, highlighted, and prospected.
8 عالی عبدی احیا ، 2 قدم پریناز ، 9 غروی سارا ، 9 1 شناسی، زیست گروه پایه، علوم ی دانشکده مالیر، دانشگاه استادیار، 2 میکروبیولوژی، گروه زیستی، علوم ی دانشکده الزهرا، دانشگاه دانشیار، 3 بیوتکنولوژی. گروه زیستی، علوم ی دانشکده الزهرا، دانشگاه دانشیار، چکیده هدف و سابقه : زایی بیماری عوامل مهمترین از یکی آلژینات، آئروجینوسا سودوموناس اثر سنجش هدف با مطالعه این است. های سلول رشد بر سودوموناسی های عفونت درمان در رایج های بیوتیک آنتی و باکتریایی نوترکیب لیاز آلژینات افزایی هم پالنکتونی آئروجینوسا سودوموناس شد. انجام روش و مواد ها : حاضر مطالعه در آئروجینوسا سودوموناس TAG48 تخلللیل منظور به شد. شناسایی و سازی جدا بالینی نمونه از (لیاز آلژینات ژن پالنکتونی، های سلول بر آن اثر و لیاز آلژینات آنزیم algL با و شد بیان و توالی تعیین سازی، همسانه جداسازی،) و توبرامایسیلن سیپروفلوکساسین، های بیوتیک آنتی و شده خال آنزیم اثر گردید. تخلی تمایلی کروماتوگرافی ستون از استفاده پالنکتونی های سلول بر سفکسیم آئروجینوسا سودوموناس غلللظلت حلداقلل ملهلاری، غلظت حداقل های آزمون و شد بررسی شد. ارزیابی شده یاد های سلول بر آنزیم و ها بیوتیک آنتی توام اثر بررسی همچنین و کشندگی یافته ها : که دادند نشان آنزیم و ها بیوتیک آنتی بررسی MIC آللژیلنلات آنزیم و سفکسیم توبرامایسین، سیپروفلوکساسین، به مربوط باکتری در لیاز TAG48 ترتیب به 4 ، 11 ، 121 و 7339 میزان باشد. می لیتر میلی بر میکروگرم MBC با برابر نیز MIC گردید. محاسبه پالنکتونی های سلول رشد بر سفکسیم و توبرامایسین با لیاز آلژینات که داد نشان نتایج همچنین آئروجینوسلا سودوموناس TAG48 نشد. دیده سیپروفلوکساسین با افزایی هم اثر این اما دارند، مهاری افزایی اثرهم گیری نتیجه : زایی بیماری در آلژینات اهمیت به توجه با آئروجینوسا، سودوموناس موثر زایی بیماری کاهش در تواند می آن تخریب جدید های ژن شناسایی باشد. algL رویکرد تواند می میکروبی جوامع در بلا هلایلی للیلاز آلژینات تحقیق و مطالعه برای جدیدی باشد. بالینی میکروبی های نمونه در باکتریایی های آلژینات علیه ویژه های فعالیت کلیدی واژگان : پالنکتونی، های سلول بیوتیکی، مهارآنتی لیاز، آلژینات آئروجینوسا سودوموناس. مقاله: دریافت ماه بهمن 79 چاپ: برای پذیرش ماه اسفند 79)* شناسی زیست گروه پایه، علوم دانشکده مالیر، دانشگاه مالیر، مکاتبه: برای آدرس تلفن: 11133337141 الکترونیک: پست hadistavafi@yahoo.com (خالقانه مالکیت مجوز تحت و آزاد دسترسی با مقاله این است. محفوظ نویسندگان حقوق http://creativecommons.org/licenses/bync/4.0/ در) میکروب دنیای فصلنامه است. مجاز اصلی اثر به ارجاع و استناد با فقط غیرتجاری استفاده هرگونه است. شده منتشر ها دمه م للری للتل للاکل بل للاس للونل للودومل سل للا للوسل للنل للیل للروجل آئل (Pseudomonas aeruginosa پلاتلوژنلی و منفی گرم باسیلی) بله باال سازشی قدرت دلیل به باکتری این است. طلب فرصت ماننلد ها محیط از انواعی در زیست توانایی فرد به منحصر طور (دارد را گیاهان و حیوانات بدن آبی، و خاکی های زیستگاه 1 .)
Previously we used microarray analysis to demonstrate that expression of several heat shock protein genes were up-regulated in biofilm-forming Prevotella intermedia (P. intermedia) strain 17 as compared to the biofilm non-forming variant strain 17-2. We employed a real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy to confirm the up-regulation of these genes in strain 17 recorded by a microarray. Total RNA was isolated from 6-, 12-, 18-, 24- and 30-hour cultures of strains 17, 17-2, and ATCC 25611 (a reference strain for P. intermedia). Real-time RT-PCR was performed according to the one-step RT-PCR protocol of iScript^ One-Step RT-PCR Kit with SYBR^[○!R] Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RT-PCR for 16 S rRNA was performed as an internal control. The target mRNA levels in strains 17, 17-2 and ATCC 25611 were defined and compared using Gene Expression Macro (Bio-Rad). The increased expression levels of heat shock protein genes, such as dnaK, grpE, dnaJ, L, groES, and clpB, were validated by real-time RT-PCR. Four out of six of the tested genes showed at least a 10-fold increase in average expression level in strain 17 as compared to that of strain 17-2 after a 12-hour culture period. Considering the up-regulation of the stress response genes in biofilm-forming strain 17, the biofilm formation in this organism might be associated with its stress response.
We have reported that clinically isolated Prevotella nigrescens strain 22 (strain 22) produced large amounts of exopolysaccharide (EPS) and that this EPS might play an important role as a virulent factor. However, EPS production decreaces with repeated subcultures. In this study, we attempted to establish a method of animal passage that maintains the EPS production of strain 22 at high levels. We also examined the protein expression patterns of strain 22 before and after animal passage using 2-D electrophoresis. EPS production of strain 22 increased after the first animal passage, and continued to increase until the third passage. Mesh-like structures around the cells became significantly denser after animal passage. N-terminal sequence analysis of proteins revealed that three conspicuously up-regulated spots on a 2-D gel after animal passage were the homologs of bacterial heat shock proteins : chaperonin protein DnaK (HSP 70), 60kDa chaperonin (GroEL), and 10kDa chaperonin (GroES). We concluded that this method of animal passage could be used to maintain EPS production of P. nigrescens at a high level, and that the bacterial heat shock protein homologs up-regulated by the animal passage might be involved in a regulatory pathway of EPS production in P. nigrescens under stressful conditions.
Most bacteria in natural environments form communities, which are commonly referred to as biofilms. Previously, we reported that biofilm-forming oral bacteria have an impact on pathogenesis and play a key role in development of persistent infections. We investigated how genes relate to biofilm-formation. We determined the capacity of biofilm-formation of Streptococcus intermedius (S. intermedius) clinical isolate (strain H39) by a crystal violet (CV) assay using a 96-well microtiter plate. Because CV analysis showed that the addition of glucose to the culture medium increased the biofilm-forming capacity of strain H39, we attempted to determine the gene involved in glucose metabolism and biofilm-formation in this strain. We isolated the homolog to the pgcA gene, which codes α-phosphoglucomutase in Bacillus subtilis from strain H39. Moreover, we isolated a mutant strain, which is a recombinant of this gene. Scanning electron microscopy indicated that this strain has a mesh-like structure around its cells, which is typical of biofilm-forming bacteria. Inactivation of the pgcA gene was not enough to stop biofilm-formation of strain H39. These results suggest that the genetic basis of biofilm-formation in S. intermedius is multifaceted and that the composition of the polymeric biofilm matrix is complex. Further studies are needed to elucidate the genetic basis for biofilm-formation.
Recent advances in studies of the genetic and molecular basis of microbial life on solid surfaces have brought the realization that bacteria can sense environmental changes, modulate their own gene expression and establish a structure with community properties known as a biofilm. Cell-to-cell communication in biofilm-associated bacteria is involved in successful pathogenic or symbiotic interaction of a variety of bacteria with animal hosts. Acyl-homoserin lacton quorum-sensing in gram-negative bacteria is known to be important not only for sensing population density, but also for controlling a number of virulence factors necessary for continued success in the host. Pseudomonas aeruginosa, one of the top three causes of opportunistic human infections, has been thoroughly studied as a model of biofilm infections regulated with a quorum-sensing system. In this review, we discuss in detail the virulence factors of P. aeruginosa, especially exopolysaccharide (alginate) synthesis along with the multisteps of biofilm formation. Shika Igaku (J Osaka Odontol Soc) 2002 Dec; 65(3/4 combined): 217-226.
Streptococcus constellatus, a member of the anginosus group streptococci, is commonly associated with oral abscesses, bacteremia with subsequent septic shock, and brain abscesses. We isolated S. constellatus strain H39 from an apical abscess lesion. Scanning electron micrographs revealed that strain H39 had dense meshwork-like structures around the cells, which are a characteristic of biofilm-forming bacteria. In this study, we reported a draft genome sequencing of strain H39 which can be used for further genetic study on biofilm formation mechanisms. The genome sequencing was performed by pyrosequencing. The putative gaps between contigs were closed by sequencing of polymerase chain reaction amplicons over the gaps. A high-quality draft genome sequence indicated that strain H39 had 3 ribosomal RNA genes, 45 transfer RNA genes and 1,926 putative protein-coding genes. We also found that strain H39 possessed genes encoding exopolysaccharide biosynthesis proteins and ABC-type polysaccharide transport proteins. These genes might contribute to the biofilm formation of S. constellatus strain H39.
Previously we demonstrated up-regulated transcriptional levels of several stress response genes in biofilm-forming Prevotella intermedia (P. intermedia) using microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). To further investigate the biofilm formation-gene expression relay system in P. intermedia, we obtained a biofilm-non-forming mutant by exposing P. intermedia strain OD 1-16, a biofilm-forming clinical isolate to ethidium bromide (EtBr), and performed comparative genome analyses in OD 1-16 and a mutant by means of random amplified polymorphic DNA assay (RAPD). In this study, we obtained one mutant that lacked the ability to form biofilm from EtBr-treated OD 1-16 cultures and designated it as strain 171. RAPD revealed that strain 171 had a mutation in a gene encoding trpG type glutamine amidotransferase. The results indicated that this gene might be involved in the biofilm formation of this organism.
Apical periodontitis is a chronic inflammatory disorder of periradicular tissues caused by microorganisms surviving in periapical tissues. We isolated Rothia mucilaginosa from a periapical periodontitis lesion where the presence of bacteria was noted in root canals after numerous treatments. Strain DY-18 was randomly selected from this case for further study. Scanning electron micrographs revealed that strain DY-18 possesses unique phenotypic characteristics, exhibiting dense meshwork-like structures on their cells. The results of staining with Calcofluor, a polysaccharide binding dye, suggested that strain DY-18 produces exopolysaccharide (EPS). Our previous studies suggested that EPS is a major component of bacterial biofilms in the oral cavity. High performance liquid chromatography showed that the EPS purified from culture supernatant contained neutral sugars including galactose, mannose, rhamnose and glucose, and amino sugars including glucosamine and galactosamine, but not uronic acid or extracellular DNA. Additionally, there are some genes relating to galactose and mannose metabolism on the genome of strain DY-18. Our results suggest that strain DY-18 has the ability to form a biofilm containing EPS.
Although sucrose-dependent plaque formation by oral streptococci in the oral cavity has been thoroughly studied, little is known about how the biofilm-forming capacity of sucrose-independent oral bacteria impacts their pathogenesis. We found a viscous material-producing, facultatively Gram-positive anaerobic rod in a stock culture collection that was isolated from an oral abscess. The aim of this study was to identify this strain (designated as K20), to determine its ability to form biofilms, and to clarify its potential for pathogenesis.
Strain K20 was identified as Actinomyces viscosus based upon 16S rRNA sequencing. Scanning electron microscopy revealed that strain K20 had dense mesh-like structures on the cell surface, which is typical of biofilm-forming bacteria. Moreover, it was attached to abiotic material and stained with Calcofluor, a polysaccharide-binding dye, suggesting that it produces exopolysaccharide and has the potential to form a biofilm. Additionally, strain K20 induced persistent abscesses in mice at a concentration of 10⁷⁻⁸ cells, as did exopolysaccharide-producing Prevotella intermedia/nigrescens. In conclusion, the ability of this Actinomyces viscosus strain to form a biofilm may contribute to its pathogenic potential to induce abscesses.
Biofilm formation is an important virulence factor contributing to the chronicity and persistency of oral infections. Biofilms are formed by microbial cells embedded in exopolysac charides (EPS), which are a component of biofilm. We previously isolated EPSproducing Rothia mucilaginosa (strain DY18) from a persistent apical periodontitis lesion. The aim of the present study was to identify genes relating to biofilm formation. High viscosity of spent culture medium obtained from static culture indicated that strain DY18 produces large amounts of EPS and forms biofilms. In contrast, the low viscosity shown in shake culture was correlated with the planktonic mode of growth of this strain. Gene expression of strain DY18 in the biofilm mode (static culture condition) was compared to that in the planktonic mode (shake culture condition) using microarray analysis. The results suggest that the genes encoding DNA polymerase Ⅲsubunit beta (gene tag :RMDY18_00020), signal transduction histidine kinase (RMDY18_00350), and molecular chaper one (RMDY18_16800) were significantly upregulated in the biofilm mode. Bioinformatic analysis showed that RMDY18_16800 has domains relating to stress response, which has been shown to be a regulator of biofilm formation in other bacteria. The results suggest that these genes might contribute to EPS production and biofilm formation of strain DY18.
Nosocomial infection means infection acquired in medical facilities. As nosocomial infection is included among the opportunistic infection, immunocompromised host are infected easily. In the pathogen of nosocomial infection the resistant bacteria against various antibiotics and disinfectants cause nosocomial infection mostly, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and multidrug resistant Pseudomonas aeruginosa (MDRP). Resistance mechanism in MRSA and VRE are variation or alternation of antimicrobial targets. MRSA acquire resistance mechanism by production of penicillin-binding protein 2´ coded mec gene, and VRE acquire by aynthesis of _D-Alanyl^4-_DLactate^5 or _D-Alanyl^4-_D-Serine^5 coded van gene. MDRP show multidrug resistance by various resistance mechanism, that is production of inactivated enzyme, variation or alternation of antimicrobial targets, low outer membrane permeability, accelation of efflux systems and biofilm formation. The pathogen in nosocomial infection have a great variety of resistance mechanisms. In this symposium, I discuss nosocomial infection about MRSA, VRE and MDRP infection broke out even in dentistry and those resistance mechanisms.
A.zo.to.bac' ter . Fr. n. azote nitrogen; M.L. masc. n. bacter the equivalent of Gr. neut. n. bactrum a rod or staff; M.L. masc. n. Azotobacter a nitrogen rod.
Proteobacteria / Gammaproteobacteria / Pseudomonadales / Pseudomonadaceae / Azotobacter
Cells range from straight rods with rounded ends to more ellipsoidal or coccoid , depending on the culture medium and age. Cells are up to 2 μm or more in diameter and 4 μm in length . A. paspali cells are usually longer, 5–10 µm in length, and can be filamentous, up to 60 µm long. Cells are usually single but may occur in pairs, irregular clumps (especially with A. paspali ), or, more rarely, in chains of varying length. Encystment occurs during late stationary phase at low frequency or at high frequency after culturing on butanol. Motile with peritrichous flagella or nonmotile. Aerobic, having a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. Nitrogen is fixed under microaerobic conditions (2% oxygen), under full aerobiosis, or after adaptation in hyperbaric oxygen. N 2 fixation uses Mo‐, V‐, or Fe‐containing nitrogenase enzymes, depending on the environmental metal supply. Water‐soluble and water‐insoluble pigments are produced by some strains of all species . Growth is heterotrophic; sugars, alcohols, and salts of organic acids are used as carbon sources. Ammonium salts, nitrate, and urea are used as sources of fixed nitrogen. Very few amino acids are used, probably due to a general deficiency in amino acid transport. The minimum pH for growth in the presence of fixed nitrogen sources ranges from 4.8 to 6.0 with maximum pH 8.5. The optimum pH for diazotrophic growth is 7.0–7.5. Most isolates are from soil, but a few are from water . One species ( A. paspali ) has been isolated only from roots of the tropical grass Paspalum notatum .
The mol % G + C of the DNA is : 63.2–67.5.
Type species : Azotobacter chroococcum Beijerinck 1901, 567.
In the study of polysaccharides from Pseudomonas, the two most commonly examined species are Pseudomonas aeruginosa and Pseudomonas syringae. The fluorescent pseudomonads that are phytopathogenic are collectively classified as P. syringae and contain more than 40 distinct pathovars (Young et al., 1978). These pathovars damage many agriculturally important plants (Gvozdyak et al., 1989) and generally possess a narrow host range (Fahy and Loyd, 1983). Pseudomonas aeruginosa is commonly found in aquatic environments, and its high adaptive potential is reflected in its importance as an opportunistic animal pathogen. Infections of the respiratory tract, urinary tract, burn wounds, and blood are often lethal (Cross et al., 1983). In patients with cystic fibrosis (CF), P. aeruginosa causes chronic pulmonary infections which are resistant to antibiotic therapy, making this pathogen the major cause of morbidity and mortality in these patients (Thomassen et al., 1987). Cell envelope components may be critical for the pathogenicity of these pseudomonads. Because the literature on these structures is more complete, the structures of the envelope polysaccharides from P. aeruginosa are presented in detail in this review. The structures of polysaccharides from other species, especially the lipopolysaccharide (LPS) of P. syringae, are also presented for comparison.
Short-chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2-keto-3-deoxy-D-gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG-producing SDR enzymes (NADPH-dependent A1-R and NADH-dependent A1-R') involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes, KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD), prefer NADH as a coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5-diketo-3-deoxy-D-gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X-ray crystallography. Both PcaKduD and SpyDhuD consist of a three-layered structure, α/β/α, with a coenzyme-binding site in the Rossmann fold; similar to enzymes A1-R and A1-R', both arrange the two short and long loops close to the coenzyme-binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1-R' but not in A1-R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2' coenzyme region were determined to be structurally crucial for dual-coenzyme specificity in PcaKduD by structural comparison of the NADH- and NADPH-specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual-coenzyme specificity. This article is protected by copyright. All rights reserved.
Alginate was first isolated from marine algae in the 19th century, and was first identified from a bacterial source, namely mucoid Pseudomonas aeruginosa, in the 1960s75. Alginate is also synthesized by Azotobacter vinelandii as part of the encystment process54. Alginate is a simple unbranched polysaccharide that is composed of two kinds of uronic acid residues: β-D-mannuronic acid (M), and its C5 epimer, α-L-guluronic acid (G) (Figure 1A). Excellent reviews on alginate research have been offered by the late Peter Gacesa in 199046 and 199845. The pathogenesis of mucoid, alginate-producing P. aeruginosa in cystic fibrosis (CF) patients was also reviewed in 1996 by Govan and Deretic56. The following chapter presents highlights from these previous reviews and new studies that have recently been described.
Alginates are linear polyuronic acid hydrocolloids. They are produced by some brown seaweeds and certain species of bacteria. The polymer from seaweed is used extensively as thickening, stabilizing, and emulsifying agents in both the chemical and food industries. Alginic acid (algin, alginate) is a heteropolysaccharide composed of linear sequences of D-mannuronic acid and its C5 epimer, L-guluronic acid. The monomeric units are linked 1,4. Alginic acid polymers form interchain associations in the presence of di and trivalent cations (particularly calcium), producing hydrated gels. This ability to gel in the presence of cations has led to a wide range of uses for this industrial polymer.
Pseudomonas aeruginosa is an opportunistic pathogen responsible for numerous nosocomial infections. A wide variety of extracellular enzymes and toxins contribute to the virulence of this bacterium. Most of these virulence factors are not synthesized constitutively and are very often produced in response to multiple environmental stimuli. This adaptation is frequently mediated by two-component regulatory systems. Furthermore, the production of many virulence factors and secondary metabolites is regulated in correlation with cell density through so-called quorum sensing.
A broad variety of bacteria including the Rhizobiaceae are able to secrete polysaccharides. Sugar polymers that form an adherent cohesive layer on the cell surface are designated capsular polysacharides (CPS), whereas the term exopolysaccharide (EPS) is used for polysaccharides with little or no cell association. Due to the variation of monosaccharide sequences, condensation linkages and non-carbohydrate decorations, an infinite array of structures can be provided by this class of macromolecules. Different rheological properties depend on the structure and the molecular weight of EPS. These properties and the location of EPS, forming the outer layer of the cell surface, contribute to the cell protection against environmental influences, attachment to surfaces, nutrient gathering and to antigenicity (Costerton et al., 1987, Sutherland 1988, Whitfield 1988, Beveridge and Graham 1991). The structural diversity of oligosaccharides derived from EPS enables them to function additionally as informational molecules in cell-cell-communications. Finally, many symbiotic bacteria of the Rhizobiaceae use oligosaccharides as signal molecules in the interaction with their host plant.
The genus Pseudomonas comprises a huge diversity of species which are adapted to very different environments. This capability to thrive in various habitats coincides with an enormous metabolic capacity of this genus which is reflected by the ability to use recalcitrant compounds as carbon source as well as to produce a wide range of secondary metabolites and biopolymers. These properties imply the production of a diversity of enzymes which have been also conceived as biocatalysts for various applications. In this review, an overview will be provided describing the current use as well as the potential use of pseudomonads and their enzymes in various biotechnological production processes. Besides the application of Pseudomonas for the production of biocatalysts and recombinant proteins, the biosynthesis pathways of commercially relevant biopolymers/biomolecules, such as alginates, elastomeric bioplastics, and rhamnolipids, will be described. These biosynthesis pathways have been successfully subjected to metabolic engineering for the production of tailor-made biomolecules (biomaterials). Finally, environmental applications of various Pseudomonas species in biodegradation of recalcitrant pollutants as well as biocontrol agents in plant growth promotion will be discussed
(R)-mandelate dehydrogenase (RMDH) has the potential to produce chiral mandelic acid. The present work reports isolation and identification of a Pseudomonas putida NUST506 contained RMDH. The strain was rod shaped with polar flagella, approximately 0.6-0.9 μm wide and 1.7-2.3 μm long. The RMDH was purified from the strain. The molecular weight of the enzyme was calculated to be 61 kDa. The optimal conditions for RMDH were pH 8.5 and 30 °C. At the optimum condition, the Km and kcat of the RMDH were 2.0 × 10−2 mM and 0.9 s−1 for (R)-mandelic acid, 1.8 × 10−2 mM and 0.9 s−1 for NAD+, as well as 1.5 × 10−2 mM and 0.3 s−1 for NADP+, respectively. The enzyme activity was increased by K+ and dithiothreitol (DTT), but obviously inhibited by Zn2+, Hg2+, sodium dodecyl sulfate (SDS) and ethylenediamine tetra-acetic acid (EDTA).
A gram-negative Sphingomonas sp. strain A1 inducibly forms a mouth-like pit on the cell surface in the presence of alginate and directly incorporates polymers into the cytoplasm via the pit and ABC transporter. Among the bacterial proteins involved in import of alginate, a cell-surface EfeO-like Algp7 shows an ability to bind alginate, suggesting its contribution to accumulate alginate in the pit. Here, we show identification of its positively charged cluster involved in alginate binding using X-ray crystallography, docking simulation, and site-directed mutagenesis. The tertiary structure of Algp7 was determined at a high resolution (1.99Å) by molecular replacement, although no alginates were included in the structure. Thus, an in silico model of Algp7/oligoalginate was constructed by docking simulation using atomic coordinates of Algp7 and alginate oligosaccharides, where some charged residues were found to be potential candidates for alginate binding. Site-directed mutagenesis was conducted and five purified mutants K68A, K69A, E194A, N221A, and K68A/K69A were subjected to a binding assay. UV absorption difference spectroscopy along with differential scanning fluorimetry analysis indicated that K68A/K69A exhibited a significant reduction in binding affinity with alginate than wild-type Algp7. Based on these data, Lys68/Lys69 residues of Algp7 probably play an important role in binding alginate.
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Alginate-producing Pseudomonas aeruginosa are usually associated with the cystic fibrosis lung environment and contribute to the high mortality rates observed among these patients. The present paper describes the purification and enzymatic properties of guanosine diphospho-D-mannose dehydrogenase (EC 1.1.1.132), a key enzyme in alginate biosynthesis by mucoid P. aeruginosa. The enzyme was overproduced using a plasmid vector containing algD (the gene encoding this enzyme) under control of the tac promoter. It was purified from cell-free lysates by lowering the pH to 5.0, heating the extract to 57.5 °C for 10 min, and discarding the protein pellet. The enzyme was selectively precipitated from the supernatant fraction with 45% acetone, resuspended in a 100 mM triethanolamine acetate buffer, pH 7.6, and ultimately purified by Bio-Sil TSK-400 gel filtration chromatography. The subunit molecular weight (Mr 48,000) as well as the N-terminal amino acid sequence corresponded to those predicted from the DNA sequence of algD. The native protein migrated as a hexamer of 290,000 molecular weight upon Bio-Gel A-1.5m gel filtration chromatography. Kinetic analysis demonstrated an apparent Km of 14.9 microM for the substrate GDP-D-mannose and 185 microM for the cofactor NAD+. GDP-D-mannuronic acid was identified as the enzyme reaction product. Several compounds (including GMP, ATP, GDP-D-glucose, and maltose) were found to inhibit enzymatic activity. GMP, the most potent of these inhibitors, exhibited competitive inhibition with an apparent Ki of 22.7 εM. Enzyme activity was also sensitive to the sulfhydryl group modifying agents iodoacetamide and p-hydroxymercuribenzoate. The addition of excess dithiothreitol restored enzyme activity, suggesting a possible involvement of cysteine residues in enzymatic activity.
Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.
The exopolysaccharide alginate is a major virulence factor of Pseudomonas aeruginosa strains that infect the lungs of cystic fibrosis patients. The synthesis of alginate is almost uniquely associated with the pathogenicity of P. aeruginosa within the environment of the cystic fibrosis lung. The gene algC is one of the essential alginate biosynthetic genes and codes for the enzyme phosphomannomutase. In this report, we present data on the transcriptional regulation of algC expression. The activity of the algC promoter is modulated by the response regulator, AlgR1, a member of the two-component signal transduction protein family, which also regulates other alginate-specific promoters. In both mucoid (alginate-positive) and nonmucoid (alginate-negative) P. aeruginosa strains, transcriptional activation of algC increased with the osmolarity of the culture medium. This osmolarity-induced activation was found to be dependent on AlgR1. AlgR1 was found to interact directly with the algC promoter. Deletion mapping, in conjunction with mobility shift assays, showed that AlgR1 specifically bound with two regions of algC upstream DNA. A fragment spanning nucleotide positions -378 to -73 showed strong specific binding, while a fragment located between positions -73 and +187 interacted relatively weakly with AlgR1. Phosphorylation of the AlgR1 protein resulted in the stimulation of its in vitro ability to bind to the algC promoter region (a fragment spanning nucleotides -378 to -73). Transcription from the algC promoter, which has significant homology with the RNA polymerase sigma-54 (RpoN) recognition sequence, decreased in an rpoN mutant of P. aeruginosa.
We report here the purification and characterization of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase, a bifunctional enzyme (PMI-GMP) which catalyzes both the phosphomannose isomerase (PMI) and guanosine 5'-diphospho-D-mannose pyrophosphorylase (GMP) reactions of the Pseudomonas aeruginosa alginate biosynthetic pathway. The PMI and GMP activities co-eluted in the same protein peak through successive fractionation on hydrophobic interaction, ion exchange, and gel filtration chromatography. The purified enzyme migrated as a 56,000 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native protein migrated as a monomer of 54,000 molecular weight upon gel filtration chromatography. The apparent Km for D-mannose 6-phosphate was 3.03 mM, and the Vmax was 830 nmol/min/mg of enzyme. For the GMP forward reaction, apparent Km values of 20.5 microM and 29.5 microM for D-mannose 1-phosphate and GTP, respectively, were obtained from double reciprocal plots. The GMP forward reaction Vmax (5,680 nmol/min/mg of enzyme) was comparable to the reverse reaction Vmax (5,170 nmol/min/mg of enzyme), and the apparent Km for GDP-D-mannose was determined to be 14.2 microM. Both reactions required Mg2+ activation, but the PMI reaction rate was 4-fold higher with Co2+ as the activator. PMI (but not GMP) activity was sensitive to dithiothreitol, indicating the involvement of disulfide bonds to form a protein structure capable of PMI activity. DNA sequencing of a cloned mutant algA gene from P. aeruginosa revealed that a point mutation at nucleotide 961 greatly decreased the levels of both PMI and GMP in a crude extract.
The nucleotide sequence of the Pseudomonas aeruginosa algC gene encoding phosphomannomutase (PMM; EC 5.4.2.8) was determined. The codon usage in algC in the wobble base position was 90.4% G+C, typical of Pseudomonas genes. The predicted amino acid sequence of phosphomannomutase (PMM) showed homology over a stretch of 112 amino acids in the carboxyl terminus with rabbit muscle phosphoglucomutase (PGM), an enzyme that catalyzes a reaction analogous to that catalyzed by PMM. In addition, a specific amino acid sequence within PMM showed homology with the catalytic site of PGM. DNA sequence analysis of a defective algC gene (algC') cloned from a mutant of P. aeruginosa that lacked PMM activity revealed one point mutation (a C to T transition) in the carboxyl terminus of PMM which resulted in an amino acid change from arginine 420 to cysteine 420. The mutation identified in the algC' gene was not within the regions of homology with PGM. The algC promoter showed significant homology with the promoters of two other P. aeruginosa genes involved in alginate synthesis, algD and algR1. Both the algD and algR1 promoters are activated by the product of the algR1 gene in P. aeruginosa. The upstream region of the algC gene contained a sequence identical to the algD upstream sequence that is known to be the binding site for the AlgR1 protein. Expression of algC was reduced 5.7-fold in an algR1 mutant of P. aeruginosa compared to its isogenic parent strain (lacking the algR1 mutation), suggesting that the algR1 gene product activates the transcription of the algC gene.
Pulmonary infection by mucoid, alginate-producing Pseudomonas aeruginosa is the leading cause of mortality among patients suffering from cystic fibrosis. Alginate-producing P. aeruginosa is uniquely associated with the environment of the cystic fibrosis-affected lung, where alginate is believed to increase resistance to both the host immune system and antibiotic therapy. Recent evidence indicates that P. aeruginosa is most resistant to antibiotics when the infecting cells are present as a biofilm, as they appear to be in the lungs of cystic fibrosis patients. Inhibition of the protective alginate barrier with nontoxic compounds targeted against alginate biosynthetic and regulatory proteins may prove useful in eradicating P. aeruginosa from this environment. Our research has dealt with elucidating the biosynthetic pathway and regulatory mechanism(s) responsible for alginate synthesis by P. aeruginosa. This review summarizes reports on the role of alginate in cystic fibrosis-associated pulmonary infections caused by P. aeruginosa and provides details about the biosynthesis and regulation of this exopolysaccharide.
The complete nucleotide sequence of a 3.2-kilobase-pair chromosomal region containing the algP and algQ genes was determined. The algQ gene encodes an acidic 18-kilodalton polypeptide required for transcriptional activation of the algD gene. The algD gene product catalyzes a critical step in alginate biosynthesis, and its overproduction is necessary for the emergence of mucoid Pseudomonas aeruginosa during chronic infections in cystic fibrosis. A novel genetic element, algP, was identified immediately downstream of algQ. This gene appears to act synergistically with algQ. Unlike a biosynthetic gene, algD, and another regulatory gene, algR, which undergo transcriptional activation in mucoid cells, both algP and algQ are equally transcribed in mucoid and nonmucoid isogenic strains of P. aeruginosa. The promoter regions of algP and algQ were mapped by using S1 nuclease protection analysis. The algQ promoter was also analyzed and showed activity in an in vitro transcriptional runoff assay with major RNA polymerase species from P. aeruginosa and Escherichia coli. The putative algQ and algP promoter sequences, unlike algD and algR, resemble sigma 70-utilized promoters from E. coli and appeared constitutively transcribed at a low level in P. aeruginosa. The algP gene has an unusual DNA sequence, with multiple direct repeats organized in six highly conserved, tandemly arranged, 75-base-pair (bp) units. At a lower level, this sequence had 45 degenerate repeats of 12 bp overlapping with the 75-bp repeats and extending beyond the region of 75-bp repeats. The algP repeats appeared important for the function of the algQ-algP regulatory region in maintaining mucoidy.
The Pseudomonas aeruginosa capsule, composed of polysaccharide alginate, is an important Pseudomonas virulence factor encountered primarily in cystic fibrosis. The regulatory algR gene positively controls transcription of a key alginate biosynthetic gene, algD. The algR gene was subcloned and sequenced by creating a set of nested deletions in M13 bacteriophage. DNA sequence analysis of algR revealed the homology of its gene product with a recently recognized class of environmentally responsive bacterial regulatory genes, including ompR, phoB, sfrA, ntrC, spoOA, dctD, and virG; these transcriptional activators control cellular reactions to osmotic pressure, phosphate limitations, or specific chemical compounds present in the medium or released from wounded host tissue. These findings indicate that novel conditions in lungs affected by cystic fibrosis may be participating in the control of mucoidy.
Chronic lung infection with mucoid, alginate-producing strains of Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF) patients. Transcriptional activation of the P. aeruginosa algD gene, which encodes GDPmannose dehydrogenase, is essential for alginate synthesis. Activation of algD is dependent on the product of the algR gene. Sequence homology between the P. aeruginosa algR gene and the Escherichia coli ompR gene, which regulates the cellular response to changes in osmolarity of the growth medium, together with the abnormally high levels of Na+ and Cl- in respiratory tract fluid in CF patients suggested that high osmolarity in the lung of the CF patient might be a signal contributing to the induction of alginate synthesis (mucoidy) in infecting P. aeruginosa. In both mucoid and nonmucoid P. aeruginosa strains (containing a functional algR gene), transcriptional activation of algD increased as the osmolarity of the culture medium increased. The increased activation of algD at high osmolarity was not in itself sufficient to induce alginate synthesis in nonmucoid strains, however, suggesting that other environmental factors are involved in full activation of the alginate genes. The targets of AlgR and OmpR, the algD promoter and the ompC and ompF promoters, respectively, were found to have appreciable sequence homology in the -60 to -110 regions. In E. coli, OmpR was capable of activating the algD promoter nearly as well as AlgR, but in both cases, activation occurred only under conditions of high osmolarity.
Strains of Pseudomonas aeruginosa causing chronic pulmonary infections in patients with cystic fibrosis are known to convert to a mucoid form in vivo characterized by the production of the exopolysaccharide alginate. The alginate production trait is not stable, and mucoid strains frequently convert back to the nonmucoid form in vitro. The DNA involved in these spontaneous alginate conversions, referred to as algS, was shown here to map near hisI and pru markers on the chromosome of strain FRD, an isolate from a cystic fibrosis patient. Although cloning algS+ by trans-complementation was not possible, a clone (pJF5) was isolated that caused algS mutants to convert to the Alg+ phenotype at detectable frequencies (approximately 0.1%) in vitro. Gene replacement with transposon-marked pJF5 followed by mapping studies showed that pJF5 contained DNA transducibly close to algS in the chromosome. Another clone was identified called pJF15 which did contain algS+ from mucoid P. aeruginosa. The plasmid-borne algS+ locus could not complement spontaneous algS mutations in trans, but its cis-acting activity was readily observed after gene replacement with the algS mutant chromosome by using an adjacent transposon as the selectable marker. pJF15 also contained a trans-active gene called algT+ in addition to the cis-active gene algS+. The algT gene was localized on pJF15 by using deletion mapping and transposon mutagenesis. By using gene replacement, algT::Tn501 mutants of P. aeruginosa were constructed which were shown to be complemented in trans by pJF15. Both algS and algT were located on a DNA fragment approximately 3 kilobases in size. The algS gene may be a genetic switch which regulates the process of alginate conversion.
The specific activities of phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and GDP-mannose dehydrogenase (GMD) were compared in a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa and in two spontaneous nonmucoid revertants. In both revertants some or all of the alginate biosynthetic enzymes we examined appeared to be repressed, indicating that the loss of the mucoid phenotype may be a result of decreased formation of sugar-nucleotide precursors. The introduction and overexpression of the cloned P. aeruginosa phosphomannose isomerase (pmi) gene in both mucoid and nonmucoid strains led not only to the appearance of PMI levels in cell extracts several times higher than those present in the wild-type mucoid strain, but also in higher PMM and GMP specific activities. In extracts of both strains, however, the specific activity of GMD did not change as a result of pmi overexpression. In contrast, the introduction of the cloned Escherichia coli manA (pmi) gene in P. aeruginosa caused an increase in only PMI and PMM activities, having no effect on the level of GMP. This suggests that an increase in PMI activity alone does not induce high GMP activity in P. aeruginosa. The heterologous overexpression of the P. aeruginosa pmi gene in the E. coli manA mutant CD1 led to the appearance in cell extracts of not only PMI activity but also GMP activity, both of which are normally undetectable in extracts of CD1. We discuss the implications of these results and propose a mechanism by which overexpression of the P. aeruginosa pmi gene can cause an elevation in both PMM and GMP activities.
Pulmonary infection by mucoid, alginate producing, Pseudomonas aeruginosa is a major complication in patients suffering from
cystic fibrosis (CF). To analyze the mechanisms leading to the emergence of mucoid P. aeruginosa in CF lungs, control of the
algD gene coding for GDPmannose dehydrogenase was studied. Transcriptional activation of algD was shown to be necessary for
alginate production. Sequencing of algD and its promoter revealed multiple direct repeats upstream of the transcription start
and throughout the promoter region. Using the algD-xylE transcriptional fusion the algD promoter was demonstrated to be under
positive control by the algR gene. This gene has previously been shown to undergo antibiotic promoted chromosomal amplification
resulting in the emergence of the mucoid phenotype. These findings provide a basis for better understanding the control of
mucoidy in P. aeruginosa.
Pseudomonas aeruginosa region II alginate genes are involved in the biosynthesis of the uronic acid containing exopolysaccharide, alginic acid. We have subcloned and overexpressed various DNA fragments contained within region II in an attempt to further characterize and more precisely localize the genes involved in alginate production. Overexpression of the genes controlling alginate biosynthesis within region II was accomplished by placing various cloned restriction fragments under the transcriptional control of the hybrid trp-lac (tac) promoter, and plasmid encoded proteins were examined in a maxicell expression system. We correlated various region II plasmid constructions with the ability to complement specific alginate negative (alg) mutants and code for polypeptides. Several proteins suspected of being involved in alginate production were encoded by sequences within region II. The results of this study further reveal that the transcriptional orientation of the alg loci within region II appears to be in the direction from argF to pmi. The specific activities of phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), two enzymes involved in the formation of the alginate precursor GDP-mannuronic acid, were measured in region II alg mutants and in cells overexpressing cloned segments from region II. Based on these enzyme measurements, we conclude that the remaining region II alg genes do not encode either PMM or GMP. These results support the suggestion that the remaining alg genes in region II are likely to be involved in post GDP-mannuronic acid processing events such as mannuronic acid transport, polymerization, secretion, epimerization and acetylation.
When discs of the marine brown alga, Fucus gardneri, were infiltrated with uniformly labeled d-mannose-¹⁴C or uniformly labeled d-glucose-¹⁴C, radioactivity was detected in the respiratory CO2, “fucoidin,” “alginic acid,” and in the residual fractions. Radioactivity was also found in the ethanol and in the acid extracts of the leaves. The extracts contained sugar phosphates, sugar nucleotides, and glyconic acids.
Enzyme preparations were obtained from the alga which contained the following enzymic activities: hexokinase, phosphomannomutase, d-mannose 1-phosphate guanylyl-transferase, guanosine diphosphate-d-mannose dehydrogenase, and mannuronic acid transferase. It was shown that, starting with d-mannose, these enzyme systems are involved in the pathway leading to the formation of guanosine diphosphate-d-mannuronic acid and subsequent incorporation of the d-mannuronic acid into a polymer of the uronic acid.
The enzyme activities of the F. gardneri could be observed only when the cell-free preparations were made in the presence of polyvinylpyrrolidone.
Twenty-six Pseudomonas aeruginosa strains from patients with cystic fibrosis were typed by the Fisher immunotyping scheme. Only 6 strains were agglutinated by a single typing serum, whereas 15 strains were agglutinated with more than one serum and 5 were not agglutinated by any serum. Neither the polyagglutinable nor the nonagglutinable strains were typable by hemagglutination inhibition or immunodiffusion, suggesting that these polyagglutinable strains did not express multiple serotype antigens, but were instead being agglutinated by antibody to nonserotype determinants. Four typable isolates were resistant to pooled normal human serum, whereas the 12 polyagglutinable and nonagglutinable isolates studied were very sensitive to normal human serum. The outer membranes of 16 strains were isolated and characterized. The data suggested, in general, strong conservation of outer membrane protein patterns. Lipopolysaccharides (LPS) were purified by a new technique which allowed isolation of both rough and smooth LPS in high yields. Three of four typable, serum-resistant strains examined had amounts of smooth, O-antigen-containing LPS equivalent to our laboratory wild type, P. aeruginosa PAO1 strain H103. In contrast, 10 of 12 polyagglutinable or nonagglutinable, serum-sensitive strains had very little or no smooth, O-antigen-containing LPS, and the other two contained less smooth LPS than our wild-type strain H103. In agreement with this data, five independent, rough, LPS O-antigen-deficient mutants of strain H103 were nontypable and serum sensitive. We suggest that the LPS defects described here represent a significant new property of many P. aeruginosa strains associated with cystic fibrosis.
We investigated the phi PLS27 receptor in Pseudomonas aeruginosa strain PAO lipopolysaccharide (LPS) by analyzing a resistant mutant. This mutant, which was designated AK1282, had the most defective LPS yet reported for a P. aeruginosa rough mutant; this LPS contained only lipid A, 2-keto-3-deoxyoctonate, heptose, and alanine as major components. In addition, this LPS lacked galactosamine, which is present in the inner core of the LPS of other rough mutants. The loss of galactosamine but only a small decrease in the alanine content indicated that the core of strain PAO LPS differed from the core structure which has been suggested for the LPS of other well-characterized P. aeruginosa strains. Our analysis also indicated that galactosamine residues may be crucial for phi PLS27 receptor activity of the LPS. Electrodialysis of LPS and conversion to salt forms (sodium or triethylamine) influenced the phage-inactivating capacity of the LPS, as did the medium in which the inactivation occurred; experiments performed in 1/10-strength broth resulted in much lower PhI50 (concentration of LPS causing a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) values than experiments performed in regular-strength broth. Sonication of the LPS also increased the phage-inactivating capacities of the LPS preparations.
Growth and exoproduct production were examined with sputum from patients with respiratory diseases serving as the growth substrate for mucoid strains of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients. Mucoid strains are uniquely common to chronic respiratory infections of CF patients. The mucoid colonial morphology of P. aeruginosa is due to the biosynthesis of the exopolysaccharide alginate. Alginate-producing (Alg+) strains utilized CF sputum for growth and high yields of alginate; however, sputum from patients with other respiratory diseases produced comparable results. Analysis of CF sputum medium indicated that amino acids and small peptides were major substrates for P. aeruginosa in respiratory secretions. Cultures of Alg+ strains in CF sputum medium were inhibited in growth and reduced in alginate yields by a low concentration (1 mM) of D-mannose, suggesting therapeutic applications. The rates of growth of two Alg+ strains in CF sputum medium were found to be slightly lower compared with their respective spontaneous Alg- mutants, indicating that the mucoid phenotype does not enhance the ability of P. aeruginosa to utilize respiratory secretions. At all stages of growth in CF sputum medium, two Alg+ strains produced lower yields of protease than did their respective Alg- mutants. When seven Alg+ strains of CF origin were compared with their respective Alg- mutants, the Alg+ phenotype correlated with reduced yields of extracellular proteases. These data are consistent with the hypothesis that mucoid strains of P. aeruginosa are more suited to chronic rather than to acute respiratory infections in that reduced yields of proteases temper the level of damage to the lungs and result in a reduced infiltration of phagocytic cells.
Phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase (PMI-GMP), which is encoded by the algA gene, catalyzes two noncontiguous steps in the alginate biosynthetic pathway of Pseudomonas aeruginosa; the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate and the synthesis of GDP-D-mannose and PPi from GTP and D-mannose 1-phosphate. Amino acids that are required for the GMP enzyme activity were identified through site-directed mutagenesis of the algA gene. Mutation of Lys-175 to arginine, glutamine, or glutamate produced an enzyme whose Km for D-mannose 1-phosphate was 470-3,200-fold greater than that measured for the wild type enzyme. In addition, these mutant enzymes had a lower Vmax for the GMP activity as compared with the wild type PMI-GMP. These results indicate that Lys-175 is primarily involved in the binding of the substrate D-mannose 1-phosphate, although it is likely that other residues are required for the specificity of binding. Mutation of Arg-19 to glutamine, histidine, or leucine resulted in a 2-fold lower Vmax for the GMP enzyme activity and a 4-7-fold increase in the Km for GTP as compared with the wild type enzyme. Thus, it appears that Arg-19 functions in the binding of GTP. In addition, chymotryptic digestion of PMI-GMP showed that the carboxyl terminus is critical for PMI activity but not for GMP activity. Taken together, these results support the hypothesis that the bifunctional PMI-GMP protein is composed of two independent enzymatic domains.
The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D. E. Woods, P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri, J. Infect. Dis. 163:143-149, 1991). This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene. To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoid strain), and PAO-rev (a nonmucoid revertant strain). The nucleotide sequence of a 4.8-kb fragment from PAO-muc was determined. A+T-rich regions of approximately 2 kb (IS-PA-4) and 0.4 kb (IS-PA-5) were identified in this fragment. DNA probes constructed internal to these regions hybridized to PAO-muc but not to PAO or PAO-rev, suggesting that PAO-muc contains an insertion element. Sequence analysis of the nonmucoid clones indicated that a 2,561-bp fragment corresponding to IS-PA-4 and a 992-bp fragment corresponding to IS-PA-5 were not present in PAO or PAO-rev. Both nonmucoid clones, however, contained in the same location as IS-PA-4, a 1,313-bp region which was not present in PAO-muc. DNA probes complementary to this sequence, designated IS-PA-6, did not hybridize with PAO-muc, indicating that this sequence had been replaced upon conversion to the mucoid phenotype. Between IS-PA-4 and IS-PA-5 there was a 500-bp sequence which was 94% identical to the 500-bp sequence downstream of IS-PA-6. These insertion elements had some DNA sequence similarity to plasmid and transposon sequences, suggesting that they may be of plasmid origin. IS-PA-4 and IS-PA-5 were shown also to be present in two mucoid isolates from cystic fibrosis patients. The insertions occurred in the same location upstream of the toxA gene, suggesting that this type of genetic recombination may also be associated with mucoid conversion in some P. aeruginosa clinical isolates.
Mucoid strains of Pseudomonas aeruginosa produce a viscous exopolysaccharide called alginate and also express alginate lyase activity which can degrade this polymer. By transposon mutagenesis and gene replacement techniques, the algL gene encoding a P. aeruginosa alginate lyase enzyme was found to reside between algG and algA within the alginate biosynthetic gene cluster at 35 min on the P. aeruginosa chromosome. DNA sequencing data for algL predicted a protein product of ca. 41 kDa, including a 27-amino-acid signal sequence, which would be consistent with its possible localization in the periplasmic space. Expression of the algL gene in Escherichia coli cells resulted in the expression of alginate lyase activity and the appearance of a new protein of ca. 39 kDa detected on sodium dodecyl sulfate-polyacrylamide gels. In mucoid P. aeruginosa strains, expression of algL was regulated by AlgB, which also controls expression of other genes within the alginate gene cluster. Since alginate lyase activity is associated with the ability to produce and secrete alginate polymers, alginate lyase may play a role in alginate production.
Significant activation of promoters of alginate genes such as algD or algC occurs in mucoid Pseudomonas aeruginosa during its proliferation in the lungs of cystic fibrosis patients. These promoters have been shown to be responsive to environmental signals such as high osmolarity. The signaling is mediated by a so-called two-component signal transduction system, in which a soluble protein, AlgR2, undergoes autophosphorylation and transfers the phosphate to a DNA-binding response regulator protein, AlgR1. The phosphorylated form of AlgR1 has a high affinity for binding at upstream sequences of both the algC and algD promoters. Two AlgR1-binding sites (ABS) have been reported upstream of the algC gene. One of the two ABSs (algC-ABS1, located at -94 to -81) is critical for the algC activation process, while the second ABS (algC-ABS2, located at +161 to +174) is only weakly active. We now report the presence of a third ABS within the structural gene of algC, and this ABS (algC-ABS3) is also important for algC promoter activation. algC-ABS1 can be replaced functionally by algC-ABS2, algD-ABS1, or algD-ABS2 and somewhat weakly by algD-ABS3. Introduction of a half-integral turn in the DNA helix between the algC site of transcription initiation and algC-ABS1 allowed only slight reduction of promoter activity, suggesting that the binding site could be appreciably functional even when present in the opposite face of the helix. Activation of the algC promoter is independent of the relative location (upstream or downstream of the mRNA start site), the number of copies, or the orientation of algC-ABS1, suggesting that it behaves like a eukaryotic enhancer element in promoting transcription from the algC promoter.
Despite the availability of specific antibiotics, Pseudomonas aeruginosa bacteria still cause troublesome infections in patients with a variety of illnesses: extensive thermal injury, leukopenia from antineoplastic chemotherapy and other forms of immunosuppressive treatment, chronic pulmonary disease such as cystic fibrosis, or intravenous narcotic use. The use of antibiotics has improved the prognosis of pseudomonas infections considerably. However, patients with marginal or defective host immunity may need more extensive therapy to master the infection. By evaluating additional modalities of treatment such as granulocyte replacement, improved usage of antibiotics, and active (prophylaxis) or passive antibody administration, the optimal combination may be found.
Pseudomonas aeruginosa produces the exo–polysaccharide alginate almost exclusively in association with pulmonary infection in cystic fibrosis (CF). Transcriptional activation of the P. aeruginosa alginate genes appears to be affected by the growth environment. Two regulatory genes have been implicated in this environmental activation, one of which produces a gene product having significant amino acid homology with a class of regulatory proteins responsive to environmental stimuli. Understanding the mechanisms of environmental activation of the alginate genes in P. aeruginosa may lead to the development of novel treatment strategies for the eradication of this organism from the lungs of the CF patient.
Phosphomannomutase (PMM) activity was detected in the soluble cytoplasmic fraction of crude extracts of both mucoid (alginate-producing) and nonmucoid strains ofPseudomonas aeruginosa. The enzyme activity was concentrated and partially purified from cell extracts of mucoid strain V388 by precipitation with ammonium sulfate and by molecular exclusion chromatography. These preparations catalyzed the conversion of mannose 1-phosphate to mannose 6-phosphate in a coupled assay system that contained commercial phosphomannoisomerase, phosphoglucoisomerase, and glucose 6-phosphate dehydrogenase. Catalytic activity in this system was strictly dependent on the presence of glucose 1,6-diphosphate (apparent Km, 150 M) and exhibited a pH optimum of around 9 in Bicine-NaOH buffer. PMM exhibited an apparent Km of 60 M for mannose 1-phosphate, but concentrations greater than 150 M caused significant inhibition. Specific activities of PMM were consistently higher in the soluble fractions of mucoid strains (1.2–3.6 nmol/min/mg protein) than of nonmucoid strains (0.2–0.6 nmol/min/mg protein).
Mucoidy in Pseudomonas aeruginosa is a critical virulence factor associated with chronic respiratory infections in cystic fibrosis. A cluster of three tightly linked genes. algU, mucA and mucB located at 67.5 min, controls development of mucoid phenotype. This locus is allelic with a group of mutations (muc) associated with conversion into constitutively mucoid forms. One of the genes previously characterized in this region, algU, is absolutely required for the transcriptional activation of algD, a critical event in the establishment of mucoidy. AlgU is homologous to the alternative sigma factor σ;H (Spo0H) controlling sporulation and competence in Bacillus. Two genes downstream of algU, mucA and mucB were further characterized in this study. Previous complementation studies have demonstrated that mucA is required for suppression of mucoidy in the muc-2 strain PAO568. In this work, complementation analysis indicated that, in addition. mucB was required for suppression of mucoidy in the muc-25 strain PAO581, and for enhanced complementation of the muc-2 mutation in PAO568. The complete nucleotide sequence of mucA and mucH was determined. Insertional inactivation of mucB on the chromosome of the standard genetic strain PAO resulted in mucoid phenotype, and in a strong transcriptional activation of algD. Thus, a loss of mucB function is sufficient to cause conversion of P. aeruginosa into the mucoid phenotype. Since the algU-mucA-mucB region is a general site where muc mutations have been mapped, it is likely that mucB participates in the emergence of mucoid forms. Both mucA and mucB play a regulatory role in concert with the sigma-like factor AlgU; all three genes, along with signal transduction and histone-like elements, control differentiation of P. aeruginosa into the mucoid phenotype.
Chronic respiratory complications in cystic fibrosis, compounded by recurring infections with mucoid Pseudomonas aeruginosa and the associated inflammation, are the primary cause of high mortality in this inheritable disease. Since the conversion of P. aeruginosa into the exopolysaccharide alginate overproducing strains plays a critical role in the establishment of chronic infection, studies are directed towards understanding the processes underlying this phenomenon. The genes (algU, mucA, and mucB) and genetic alterations responsible for conversion to mucoidy have been recently characterized. The mutations leading to the emergence of mucoid strains are superimposed on a regulatory system with elements that resemble those controlling other aspects of bacterial developmental physiology.
The nucleotide sequence of a 3.4-kb EcoRI-PstI DNA fragment of Xanthomonas campestris pv. campestris revealed two open reading frames, which were designated xanA and xanB. The genes xanA and xanB encode proteins of 448 amino acids (molecular weight of 48,919) and 466 amino acids (molecular weight of 50,873), respectively. These genes were identified by analyzing insertion mutants which were known to be involved in xanthan production. Specific tests for the activities of enzymes involved in the biosynthesis of UDP-glucose and GDP-mannose indicated that the xanA gene product was involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate. The deduced amino acid sequence of xanB showed a significant degree of homology (59%) to the phosphomannose isomerase of Pseudomonas aeruginosa, a key enzyme in the biosynthesis of alginate. Moreover, biochemical analysis and complementation experiments with the Escherichia coli manA fragment revealed that xanB encoded a bifunctional enzyme, phosphomannose isomerase-GDP-mannose pyrophosphorylase.
The pathogenesis of Pseudomonas aeruginosa infection in cystic fibrosis (CF) is a complex process attributed to specific characteristics of both the host and the infecting organism. In this study, the properties of the PAO1 neuraminidase were examined to determine its potential role in facilitating Pseudomonas colonization of the respiratory epithelium. The PAO1 neuraminidase was 1000-fold more active than the Clostridium perfringens enzyme in releasing sialic acid from respiratory epithelial cells. This effect correlated with increased adherence of PAO1 to epithelial cells after exposure to PAO1 neuraminidase and was consistent with in vitro studies demonstrating Pseudomonas adherence to asialoganglioside receptors. The regulation of the neuraminidase gene nanA was examined in Pseudomonas and as cloned and expressed in Escherichia coli. In hyperosmolar conditions neuraminidase expression was increased by 50% (P less than 0.0004), an effect which was OmpR dependent in E. coli. In Pseudomonas the osmotic regulation of neuraminidase production was dependent upon algR1 and algR2, genes involved in the transcriptional activation of algD, which is responsible for the mucoid phenotype of Pseudomonas and pathognomonic for chronic infection in CF. Under the hyperosmolar conditions postulated to exist in the CF lung, nanA is likely to be expressed to facilitate the initial adherence of Pseudomonas to the respiratory tract.
The Pseudomonas aeruginosa exopolysaccharide alginate is an important virulence factor in chronic pulmonary infections of cystic fibrosis patients. We determined the nucleotide sequence of the gene, algB, which regulates the level of exopolysaccharide produced by mucoid P. aeruginosa. The predicted amino acid sequence of AlgB revealed a high degree of similarity to the regulatory proteins in the NtrC subclass of 'two-component regulatory systems'. AlgB expression in Escherichia coli minicells showed a molecular weight of approximately 50,000 Da, comparable to that of the inferred amino acid sequence (49,318 Da). We show that algB is transcriptionally active in mucoid strains of P. aeruginosa and regulates the expression of the alginate biosynthetic gene, algD, thereby resulting in increased expression of alginate in mucoid P. aeruginosa.
Alginate (Alg), a random polymer of mannuronic acid and glucuronic acid residues, is synthesized and secreted by Pseudomonas aeruginosa primarily during its infection of the lungs of cystic fibrosis patients. The molecular biology and biochemistry of the enzymatic steps leading to the production of the Alg precursor GDP-mannuronic acid have been elucidated, but the mechanism of polymer formation and export of Alg are not understood. We report the nucleotide sequence of a 2.4-kb DNA fragment containing the algE gene, previously designated alg76, encoding the AlgE protein (Mr 54,361) that is believed to be involved in these late steps of Alg biosynthesis. Expression of algE appears to occur from its own promoter. The promoter region contains several direct and inverted repeat sequences and shares structural similarity with promoters of several other alg genes from P. aeruginosa. In addition, the AlgE protein was overproduced from the tac promoter in P. aeruginosa. N-terminal amino acid sequence analysis showed that the polypeptide contains a signal peptide which is cleaved to form the mature protein during AlgE export from the cell cytoplasm.
Most strains of Pseudomonas aeruginosa isolated from the respiratory tracts of cystic fibrosis patients have a mucoid colony morphology due to the synthesis of an expolysaccharide called alginate. The algB gene product (AlgB) is necessary for the high-level production of alginate in mucoid P. aeruginosa. In this study, AlgB was shown to be involved in the transcription of algD, a gene previously demonstrated to be activated in mucoid P. aeruginosa. In vitro and in vivo expression studies reveal that algB encodes a protein with a molecular size of 49 kDa. The DNA sequence of a 2.2-kb P. aeruginosa fragment containing algB was also determined. The amino-terminal domain of AlgB was found to be conserved with the amino-terminal domains of the response regulator class of two-component regulatory proteins. The central domain of AlgB has sequences highly conserved with those in the NtrC subfamily of transcriptional activators (NtrC, NifA, HydG, DctD, FlbD, TyrR, and PgtA). The central domain of AlgB also contains a potential nucleotide binding site. AlgB is the first NtrC homolog described from P. aeruginosa. At the carboxy terminus of AlgB, a helix-turn-helix motif was observed, suggesting that AlgB is a DNA-binding protein. The strongly conserved NtrC-like central domain of AlgB is not present in AlgR, another alginate response regulator. This study therefore identifies and characterizes the second of at least two unique response regulators used by P. aeruginosa to control alginate gene expression.
SDS-polyacrylamide gel electrophoresis of outer membrane (OM) proteins of different mucoid strains of P. aeruginosa revealed a protein of about 54 kDa that was absent in nonmucoid strains. This 54 kDa protein was expressed under iron-restricted and iron sufficient growth conditions. Electrophoretic mobility of the 54 kDa protein was modified by the solubilization temperature as well as by the addition of lipopolysaccharide and alginate prior to electrophoresis. Treatment of OMs with octylglucoside/KCl or SDS completely extracted the 54 kDa protein at low temperatures. The possible role of this protein in biosynthesis and/or excretion of bacterial alginate is discussed.
The biochemical mechanism by which alpha-L-guluronate (G) residues are incorporated into alginate by Pseudomonas aeruginosa is not understood. P. aeruginosa first synthesizes GDP-mannuronate, which is used to incorporate beta-D-mannuronate residues into the polymer. It is likely that the conversion of some beta-D-mannuronate residues to G occurs by the action of a C-5 epimerase at either the monomer (e.g., sugar-nucleotide) or the polymer level. This study describes the results of a molecular genetic approach to identify a gene involved in the formation or incorporation of G residues into alginate by P. aeruginosa. Mucoid P. aeruginosa FRD1 was chemically mutagenized, and mutants FRD462 and FRD465, which were incapable of incorporating G residues into alginate, were independently isolated. Assays using a G-specific alginate lyase from Klebsiella aerogenes and 1H-nuclear magnetic resonance analyses showed that G residues were absent in the alginates secreted by these mutants. 1H-nuclear magnetic resonance analyses also showed that alginate from wild-type P. aeruginosa contained no detectable blocks of G. The mutations responsible for defective incorporation of G residues into alginate in the mutants FRD462 and FRD465 were designated algG4 and algG7, respectively. Genetic mapping experiments revealed that algG was closely linked (greater than 90%) to argF, which lies at 34 min on the P. aeruginosa chromosome and is adjacent to a cluster of genes required for alginate biosynthesis. The clone pALG2, which contained 35 kilobases of P. aeruginosa DNA that included the algG and argF wild-type alleles, was identified from a P. aeruginosa gene bank by a screening method that involved gene replacement. A DNA fragment carrying algG was shown to complement algG4 and algG7 in trans. The algG gene was physically mapped on the alginate gene cluster by subcloning and Tn501 mutagenesis.
Mortality among cystic fibrosis (CF) patients is most commonly attributed to pulmonary infection by mucoid, alginate-producing Pseudomonas aeruginosa. The initial infecting P. aeruginosa are typically non-mucoid; however, upon continued exposure to the CF lung environment, they become highly mucoid. The CF lung is an osmotically high environment because of the presence of substantial concentrations of electrolytes and dehydrated mucus. In this report we demonstrate that ethanol (a commonly used dehydrating agent) activates transcription from a critical alginate promoter, algD, and show that prolonged exposure to ethanol allows switching to the mucoid form. This activation appears to be dependent on DNA gyrase. Analysis of alginate gene activation, and the subsequent reversal of the activation process by bacterial DNA gyrase inhibitors, should aid the development of treatment strategies for CF patients infected with this organism.
Alginate (Alg), an exopolysaccharide with strong gelling properties, is produced by Pseudomonas aeruginosa primarily during its infection of the cystic fibrosis (CF) lung. The alg genes are normally not expressed in other environments. The promoter for a critical Alg biosynthetic gene, algD, encoding GDP-mannose dehydrogenase, is activated only under conditions reminiscent of the CF lung (i.e., under high osmolarity), and at least two regulatory genes, algR1 and algR2, have been implicated in this activation process. The physical mapping of a 4.4-kb region harboring algR2 has been accomplished and the complete nucleotide sequence of this fragment, including that of algR2, is presented. The cloning and complementation experiments also demonstrate the presence, on this fragment, of regulatory gene(s) different from algR1 and algR2. The expression of the algR2 gene allows a high level of activation of the algD promoter in Escherichia coli, in the presence of algR1 in a high osmotic environment, suggesting that the AlgR2 and AlgR1 proteins act cooperatively to activate the algD promoter. Hyperexpression of the algR2 gene from the tac promoter also allows the conversion of nonmucoid cells of strain 8822, a spontaneous revertant of the mucoid CF isolate strain 8821, back to mucoidy, but not that of the clinical isolate, strain PAO1.
The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome.
Phosphomannose isomerase (PMI) has been proposed to catalyze the first step of the alginic acid biosynthetic pathway in Pseudomonas aeruginosa. The nucleotide sequence of the P. aeruginosa pmi gene contained on a 2.0-kb BamHI-SstI DNA fragment has been determined. The gene was defined by the start and stop codons and by in vitro disruption of an open reading frame of 1440 bp corresponding to a polypeptide product with a predicted Mr of 52 860. This polypeptide displayed an apparent Mr of approx. 56 000 upon electrophoresis of a maxicell extract on sodium dodecyl sulfate-polyacrylamide gels. The codon utilization of the pmi gene was distinct in the wobble base preference and influenced by the high G + C content (66 mol%) of the P. aeruginosa DNA. Computer assisted matching analysis failed to demonstrate any significant homology at the nucleotide level between the P. aeruginosa pmi and Escherichia coli manA (pmi) genes. However, sequences homologous to the P. aeruginosa pmi gene were found in other Pseudomonas species, such as P. putida and P. mendocina, and in Azotobacter vinelandii, all capable of producing alginic acid.
Pseudomonas aeruginosa is an adaptable, saprophytic bacterium with the potential to cause a variety of opportunistic infections in compromised hosts. In patients with cystic fibrosis, chronic pulmonary colonization with mucoid alginate-producing mutants of P. aeruginosa is a major cause of morbidity and mortality and is an interesting example of microbial adaptation and host-bacterium interaction.
Gene(s) conferring the ability of Rhizobium leguminosarum biovar viciae strain TOM to nodulate primitive peas (cultivar Afghanistan) had been located in a 2.0 kb region of its sym plasmid, pRL5JI. In this DNA, a single open reading frame of 1101 bp, corresponding to a gene, nodX was found. nodX is downstream of nodJ which is present in strain TOM and also in the sym plasmid of a typical strain of this biovar. nodX specifies a hydrophobic protein (of Mr 41,036) with no clear similarity to other proteins in data bases. Mutations in nodX abolished nodulation of Afghanistan peas but not nodulation of commercial peas. nodX-lacZ fusions were used to show that transcription of nodX was activated by root exudates from both commercial and Afghanistan peas and by defined flavonoids. Exudate from Afghanistan peas activated nod genes of typical strains of R. leguminosarum biovar viciae which fail to nodulate these peas; thus, their failure to nodulate these primitive peas is not due to a lack of activation of their nod genes by exudate from Afghanistan peas. A homologue of nodX exists in R. leguminosarum biovar trifolii (which nodulates clover) but not in typical strains of R. leguminosarum biovar viciae.
The slime polysaccharides produced by Pseudomonas aeruginosa isolated from a variety of human infections were investigated. Slime production in culture seemed optimal when adequate amounts of carbohydrate were present and under conditions of either high osmotic pressure or inadequate protein supply. The polysaccharides produced by the organisms were similar to each other, to the slime of Azotobacter vinelandii, and to seaweed alginic acids. They were composed of beta-1,4-linked d-mannuronic acid residues and variable amounts of its 5-epimer l-guluronic acid. All bacterial polymers contained o-acetyl groups which are absent in the alginates. The polysaccharides differed considerably in the ratio of mannuronic to guluronic acid content and in the number of o-acetyl groups. The particular composition of the slime was not found to be characteristic for the disease process from which the mucoid variants of P. aeruginosa were obtained.
An enzyme preparation was isolated from liquid cultures of Azotobacter vinelandii by precipitation with ammonium sulphate after removal of cells by centrifugation. When incubated in the presence of calcium ions with alginate prepared from brown algae, the enzyme epimerizes D-mannuronic acid residues to L-guluronic acid residues in the polymer chain. An assay is described, based on the difference in colour intensity of the two uronic acids in the Dische carbazole reaction. The influence of calcium, strontium, magnesium, and sodium ions, and of pH and temperature on the activity was determined. The energy required for the transformation of D-mannuronic to L-guluronic acid residues is thought to be supplied by the stronger binding of calcium ions to the latter type of monomer. By using polymannuronic acid as a substrate, attempts were made to determine the end-point of the reaction and to follow the formation of the two different types of L-guluronic acid-containing blocks during the epimerization reaction. The results demonstrate that both homopolymeric blocks of L-guluronic acid and blocks having an alternating sequence are formed. No transformation of the latter to the former type could be demonstrated, and both block types thus appear to be end-products of the enzymic reaction.
Pseudomonas aeruginosa can express two distinct forms of lipopolysaccharide (LPS), called A-band and B-band. As an attempt to understand the molecular biology of the synthesis and regulation of these LPS antigens, a recombinant plasmid, pFV3, containing genes for A-band expression was isolated previously. In the present study, P. aeruginosa strain PAO1 was mutagenized with transposon Tn5-751 and yielded a B-band-deficient mutant, called ge6. This mutant was mated with a PAO1 genomic library, and transconjugants were screened for complementation of B-band using B-band-specific monoclonal antibody MF15-4. Recombinant plasmid pFV100 was subsequently isolated by its ability to complement B-band expression in ge6. SDS-PAGE analysis of LPS from ge6 and ge6(pFV100) revealed that ge6 was deficient in expression of B-band, while ge6(pFV100) had an LPS profile similar to that of the parent strain PAO1. With A-band and B-band genes cloned in separate plasmids, pFV3 and pFV100 respectively, we were able to determine the map location of these LPS genes on the P. aeruginosa PAO1 chromosome using pulsed-field gel electrophoresis. A-band genes mapped at 5.75 to 5.89 Mbp (SpeI fragment SpK; DpnI fragment DpF2), while genes involved with expression of B-band LPS mapped at 1.9 Mbp (SpeI fragments SpC, SpI and SpAI; DpnI fragment DpD) on the 5.9 Mbp chromosome. We also performed initial characterization of a gene involved with synthesis of A-band present on pFV3. We previously reported that recombinant plasmid pFV3 and subcloned plasmid pFV36 complemented A-band synthesis in rd7513, an A- mutant derived from A+ strain AK1401. pFV36 was mutagenized with transposon Tn1000 to reveal a one-kilobase region capable of complementing the expression of A-band in the A- strain rd7513. This region was subcloned as a 1.6 kb KpnI fragment into plasmid vector pAK1900 and the resulting clone named pFV39. Labelling of proteins encoded by pAK1900 and pFV39 in Escherichia coli maxicells revealed a single unique polypeptide of approximately 37 kDa expressed by pFV39. Supernatants from disrupted cells of rd7513(pFV39) and AK1401 converted 14C-labelled-guanosine diphospho (GDP)-D-mannose to GDP-rhamnose, while supernatants from rd7513 did not show synthesis of GDP-rhamnose. The data therefore suggest that conversion of GDP-D-mannose to GDP-rhamnose is required for synthesis of A-band LPS, and that a 37 kDa protein is involved in this conversion.
Strains of Pseudomonas aeruginosa causing pulmonary infections in cystic fibrosis patients have an unusual mucoid phenotype because of production of the capsule-like exopolysaccharide, alginate. Transcriptional activation of algD, the first gene of a large alginate biosynthetic gene cluster, is associated with conversion to the alginate-producing (Alg+) phenotype. In this study, we examined the regulation of alginate genes immediately downstream of algD. Mutants of the Alg+ strain FRD1 were constructed by gene replacement with defined Tn501 (8.2kb) insertions in the alginate biosynthetic gene cluster, resulting in an Alg- phenotype. The Alg+ phenotype of these mutants was restored by integration of narrow-host-range plasmids containing DNA fragments from P. aeruginosa that reconstructed a continuous alginate gene cluster. A broad-host-range plasmid containing the entire alginate gene cluster except for the terminal gene, algA, was unable to complement an alG::Tn501 mutant unless algA was transcribed from a second plasmid. This indicated that any Tn501 insertion in the cluster was polar on downstream alginate genes. Northern blot hybridization experiments also showed that a transposon insertion downstream of algD adversely affected algG and algA transcription. These results provided evidence that the alginate biosynthetic gene cluster has an operonic structure and is cotranscribed from the algD promoter.
Chronic respiratory complications in cystic fibrosis, compounded by recurring infections with mucoid Pseudomonas aeruginosa and the associated inflammation, are the primary cause of high mortality in this inheritable disease. Since the conversion of P. aeruginosa into the exopolysaccharide alginate overproducing strains plays a critical role in the establishment of chronic infection, studies are directed towards understanding the processes underlying this phenomenon. The genes (algU, mucA, and mucB) and genetic alterations responsible for conversion to mucoidy have been recently characterized. The mutations leading to the emergence of mucoid strains are superimposed on a regulatory system with elements that resemble those controlling other aspects of bacterial developmental physiology.
Colonization of the cystic fibrosis lung by Pseudomonas aeruginosa is greatly facilitated by the production of an exopolysaccharide called alginate. In this study we determined the nucleotide sequence of an alginate modification gene, algF, which controls the addition of acetyl groups to alginate. Expression of algF using a T7 promoter-expression system showed that algF codes for a 24.5 kDa polypeptide (predicted size 22,832 Da) that is processed to 19.5 kDa. The N-terminus of the processed polypeptide matched the predicted amino acid sequence of AlgF starting at Asp-29. An algF mutant failed to produce alginate owing to a polar effect on the downstream algA gene. Although the algA gene, provided in trans, restored synthesis of alginate, the alginate was non-acetylated. We show that a plasmid containing both the algF and algA gene complements the alginate acetylation defect of the algF mutant strain.
The alg8 and alg44 genes, which are required for alginate biosynthesis by Pseudomonas aeruginosa, are located in the alginate biosynthetic gene cluster between the algD and algE genes. The nucleotide sequence of these two genes is presented. Although the functions of the Alg8 and Alg44 proteins are not known, we believe that they may be involved in the polymerization of mannuronic acid residues to form alginate.
The alginate lyase-encoding gene (algL) of Pseudomonas aeruginosa was localized to a 1.7-kb EcoRI-XbaI fragment within the alginate biosynthetic gene cluster at 34 minutes on the chromosome. The nucleotide sequence of this DNA fragment revealed an ORF encoding a protein of M(r) 40,885 which is transcribed in the same orientation as the other alg genes within the biosynthetic gene cluster. The predicted protein has a potential N-terminal signal peptide which is consistent with its proposed periplasmic location. The AlgL protein was overproduced in Escherichia coli and purified. The purified protein was shown to have alginate lyase activity. In addition, an algL insertion mutant of the mucoid P. aeruginosa 8830 was constructed. This mutant (alm1) had a nonmucoid phenotype due to a polar effect on the transcription of an essential alg gene, algA. Thus, the algL gene is located within a region of the alginate biosynthetic gene cluster that appears to be non-essential for alginate production.
Mucoid strains of Pseudomonas aeruginosa produce a high-molecular-weight exopolysaccharide called alginate that is modified by the addition of O-acetyl groups. To better understand the acetylation process, a gene involved in alginate acetylation called algF was identified in this study. We hypothesized that a gene involved in alginate acetylation would be located within the alginate biosynthetic gene cluster at 34 min on the P. aeruginosa chromosome. To isolate algF mutants, a procedure for localized mutagenesis was developed to introduce random chemical mutations into the P. aeruginosa alginate biosynthetic operon on the chromosome. For this, a DNA fragment containing the alginate biosynthetic operon and adjacent argF gene in a gene replacement cosmid vector was utilized. The plasmid was packaged in vivo into lambda phage particles, mutagenized in vitro with hydroxylamine, transduced into Escherichia coli, and mobilized to an argF auxotroph of P. aeruginosa FRD. Arg+ recombinants coinherited the mutagenized alginate gene cluster and were screened for defects in alginate acetylation by testing for increased sensitivity to an alginate lyase produced by Klebsiella aerogenes. Alginates from recombinants which showed increased sensitivity to alginate lyase were tested for acetylation by a colorimetric assay and infrared spectroscopy. Two algF mutants that produced alginates reduced more than sixfold in acetyl groups were obtained. The acetylation defect was complemented in trans by a 3.8-kb XbaI-BamHI fragment from the alginate gene cluster when placed in the correct orientation under a trc promoter. By a merodiploid analysis, the algF gene was further mapped to a region directly upstream of algA by examining the polar effect of Tn501 insertions. By gene replacement, DNA with a Tn501 insertion directly upstream of algA was recombined with the chromosome of mucoid strain FRD1. The resulting strain, FRD1003, was nonmucoid because of the polar effect of the transposon on the downstream algA gene. By providing algA in trans under the tac promoter, FRD1003 produced nonacetylated alginate, indicating that the transposon was within or just upstream of algF. These results demonstrated that algF, a gene involved in alginate acetylation, is located directly upstream of algA.