Synthesis of the A‐band polysaccharide sugar <scp>D</scp>‐rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in <i>Pseudomonas aeruginosa</i>

Rocchetta, Heather L.; Pacan, Jennifer C.; Lam, Joseph S.

doi:10.1046/j.1365-2958.1999.0erat.x

Cited by 24 publications

(73 citation statements)

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“…In P. aeruginosa, there can be an overlap between EPS synthesis gene functions and LPS functions (23). Thus, pslA could be involved in LPS synthesis and an LPS modification could be responsible for the abnormal biofilm phenotype of pslA mutants.…”

Section: Resultsmentioning

confidence: 99%

“…We know that motility is involved in biofilm formation, but both the twitching and swimming motilities of a pslA mutant were comparable to those of the parent (Table 2). There is evidence that LPS influences biofilm architecture (23). One might imagine that the psl gene cluster affects LPS synthesis, but at least by comparing LPSs in silver-stained sodium dodecyl sulfatepolyacrylamide gels, the mutant and the parent were indistinguishable.…”

Section: Discussionmentioning

confidence: 99%

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Putative Exopolysaccharide Synthesis Genes Influence Pseudomonas aeruginosa Biofilm Development

2004

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An analysis of the Pseudomonas aeruginosa genomic sequence revealed three gene clusters, PA1381-1393, PA2231-2240, and PA3552-3558, in addition to the alginate biosynthesis gene cluster, which appeared to encode functions for exopolysaccharide (EPS) biosynthesis. Recent evidence indicates that alginate is not a significant component of the extracellular matrix in biofilms of the sequenced P. aeruginosa strain PAO1. We hypothesized that at least one of the three potential EPS gene clusters revealed by genomic sequencing is an important component of P. aeruginosa PAO1 biofilms. Thus, we constructed mutants with chromosomal insertions in PA1383, PA2231, and PA3552. The mutant with a PA2231 defect formed thin unstructured abnormal biofilms. The PA3552 mutant formed structured biofilms that appeared different from those formed by the parent, and the PA1383 mutant formed structured biofilms that were indistinguishable from those formed by the parent. Consistent with a previous report, we found that polysaccharides were one component of the extracellular matrix, which also contained DNA. We suggest that the genes that were inactivated in our PA2231 mutant are required for the production of an EPS, which, although it may be a minor constituent of the matrix, is critical for the formation of P. aeruginosa PAO1 biofilms.Pseudomonas aeruginosa is an important opportunistic pathogen that is capable of causing several different types of infections, including chronic biofilm infections. For example, P. aeruginosa infects the lungs of people with the genetic disease cystic fibrosis (CF), and chronic CF lung infections are considered biofilm infections (5, 12, 30). Mature P. aeruginosa biofilms consist of groups of cells growing together in clusters embedded in an extracellular polymeric matrix (4, 5). Quite often, P. aeruginosa isolated from the lungs of CF patients exhibits a mucoid phenotype resulting from the overproduction of the exopolysaccharide (EPS) alginate (12, 34). However, strains that do not produce much alginate are capable of forming mature biofilms in vitro (18). In fact, the commonly studied laboratory strain PAO1 makes little or no alginate (17,36).If alginate is not the extracellular matrix for biofilms of strain PAO1, what is? A recent report showed that a major polymer in the extracellular matrix of P. aeruginosa PAO1 biofilms is DNA (35). However, this does not rule out the possibility that P. aeruginosa PAO1 makes an EPS other than alginate and that this polysaccharide is an important constituent of the extracellular polymeric matrix.A clue that polysaccharides other than alginate might be involved in P. aeruginosa biofilms came from the P. aeruginosa genome sequencing project. The annotation of the genome suggested that there are gene clusters in addition to the alginate biosynthesis gene cluster that may be involved in the synthesis of EPSs. One of these clusters is PA1381-1393, a second is PA2231-2245, and a third is PA3552-3558 (31). The PA1381-1393 cluster has an anomalously low GϩC content, and the...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Putative Exopolysaccharide Synthesis Genes Influence Pseudomonas aeruginosa Biofilm Development

2004

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“…1; Table 2), psl may regulate or constitute a portion of the LPS synthesis pathway. LPS has been implicated in the architecture of P. aeruginosa biofilms (44), and a prior study suggested that pslB (designated ORF 488) may be involved in A-band LPS synthesis (45). To determine if the psl-dependent effects on biofilm formation are LPS related, we compared the LPS profiles of PAO1, WFPA60, a PAO1 algC mutant, and the corresponding algC complemented strain.…”

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confidence: 99%

Identification ofpsl, a Locus Encoding a Potential Exopolysaccharide That Is Essential forPseudomonas aeruginosaPAO1 Biofilm Formation

et al. 2004

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Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.

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“…In addition to LPS biosynthesis, rml and xanB orthologues in Pseudomonas aeruginosa, Escherichia coli, and X. campestris pv. campestris are required for the biosynthesis of exopolysaccharides (19,32). The xanB, rmlA, and rmlC mutants all produced lower amounts of EPS when assayed by the phenol-sulfuric acid method for total carbohydrates (13) (not shown), suggesting that the three genes are also involved in EPS biosynthesis in S. maltophilia WR-C.…”

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Differential Biofilm Formation and Motility Associated with Lipopolysaccharide/Exopolysaccharide-Coupled Biosynthetic Genes inStenotrophomonas maltophilia

2006

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Stenotrophomonas maltophilia WR-C is capable of forming biofilm on polystyrene and glass. The lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes rmlA, rmlC, and xanB are necessary for biofilm formation and twitching motility. Mutants with mutations in rmlAC and xanB display contrasting biofilm phenotypes on polystyrene and glass and differ in swimming motility.Microorganisms can develop biofilms or clogging mats, causing the failure of septic tanks, systems for on-site wastewater disposal. If water in these clogged systems were contaminated by pathogens, it would pose a threat to human health. We isolated Stenotrophomonas maltophilia strain WR-C from a clogged septic tank system that consistently formed biofilms on sand grains, produced exopolysaccharides (EPS), and caused clogging in sand columns (33). S. maltophilia is a gram-negative, rod-shaped, and obligate aerobic bacterium with polar flagella in the ␥-␤ subdivision of Proteobacteria (3,14). It is found in various environments and recently has emerged as an important human pathogen. Very little is known about the mechanisms of biofilm formation by S. maltophilia. It has been shown to adhere to HEp-2 cells as well as abiotic surfaces, such as plastic, glass, and Teflon (7-10, 16).To identify genes that are involved in biofilm formation by S. maltophilia WR-C, about 4,500 transposon mutants generated with the EZ::TN Ͻ R6K␥ori/KAN-2 Ͼ Tnp transposome (Epicenter, Madison, Wis.) were screened for defects in biofilm formation in 96-well polystyrene plates (Becton-Dickinson Labware, Franklin Lakes, N.J.) by using a modified microtiter plate assay (11,29). Briefly, wells containing 200 l Trypticase soy (TS) broth were inoculated with overnight cultures and incubated at 30°C, 50 rpm for 2 days. Biofilm cells were stained with 0.1% crystal violet and washed, the stain remaining in the cells was solubilized with 70% ethanol, and the optical density at 590 nm was determined. Three mutants, TPH7, TPH11, and TPH13, whose growth was similar to that of the wild type but which were deficient in biofilm formation, were selected for further characterization.The transposon flanking regions were rescued by "rescue cloning" as described by the manufacturer and sequenced by using the primers KAN-2 FP-1 and R6KAN-2 RP-1 (Table 1). The transposon-inserted genes are homologous to three genes involved in the biosynthesis of nucleotide sugar precursors of lipopolysaccharide (LPS) and EPS in Xanthomonas campestris pv. campestris ATCC 33913 (GenBank accession no. AE008922) (6). The genes are rmlC (for TPH7, 74% identity), rmlA (for TPH11, 78% identity), and xanB (for TPH13, 80% identity), which are located in the rml and xan operons of X. campestris pv. campestris (19,20). As no genome sequence is available and the gene cluster involved in LPS/EPS biosynthesis has not been reported for S. maltophilia, the DNA segments flanking the transposon in TPH7, TPH11, and TPH13 were sequenced. The complete sequences of the rmlBACD and xanAB operons and their flanking genes were deter...

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Synthesis of the A‐band polysaccharide sugar D‐rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa

Abstract: (1994) Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5Ј-diphospho-D-mannose pyrophosphorylase, a bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.

Cited by 24 publications

References 61 publications

Putative Exopolysaccharide Synthesis Genes Influence Pseudomonas aeruginosa Biofilm Development

Putative Exopolysaccharide Synthesis Genes Influence Pseudomonas aeruginosa Biofilm Development

Identification ofpsl, a Locus Encoding a Potential Exopolysaccharide That Is Essential forPseudomonas aeruginosaPAO1 Biofilm Formation

Differential Biofilm Formation and Motility Associated with Lipopolysaccharide/Exopolysaccharide-Coupled Biosynthetic Genes inStenotrophomonas maltophilia

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