Cell envelope mutants of <i>Pseudomonas putida</i>: physiological characterization and analysis of their ability to survive in soil

Rodríguez-Herva, José-Juan; Reniero, Daniela; Galli, E.; Ramos, Juan L.

doi:10.1046/j.1462-2920.1999.00058.x

Cited by 22 publications

(21 citation statements)

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“…In a recent review, O'Toole et al (30) further supported the idea that biofilm formation is a process of microbial development and is not unlike that observed in cell cycle-controlled swarmer-to-stalk cell transition in Caulobacter crescentus, sporulation in Bacillus subtilis, and fruiting-body formation by Myxococcus xanthus. This view is gaining increasing acceptance as studies on initial events in biofilm development reveal alterations in bacterial cell physiology that hint at changes that may occur throughout the developmental cycle (2,7,8,10,13,15,18,24,31,32,33,41,43,45,47).…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

et al. 2002

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Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.Recent investigations have been directed at determining the degree to which gene regulation during biofilm development controls the switch from planktonic to attached growth. Brözel and coworkers monitored changes in global gene expression patterns in attached Pseudomonas aeruginosa cells and found more than 11 proteins whose levels were altered during various stages of attachment (6). Genevaux and colleagues screened a library of Tn10 insertion mutants of Escherichia coli with altered adhesion abilities (18). Fifty adhesion-deficient mutants were isolated which showed less than 40% attachment compared to the wild type, and 22 mutants were found with an attachment of 40 to 75% compared to the wild type. The majority of these mutants were affected in motility. PrigentCombaret and coworkers (42) carried out a screen in E. coli K-12 similar to that of Genevaux and revealed major changes in the patterns of gene expression during the switch from planktonic to attached growth. They found attachment-dependent regulation of gene expression in 38% of the generated lacZ gene fusions (out of 446 clones). More recently, it has been shown that in Pseudomonas putida, more than 30 genes and 40 gene products were altered within 6 h following attachment (47).These results indicate that physiological changes in the transition from planktonic to attached cells are...

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Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

et al. 2002

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“…The mechanisms of bacterial adhesion to surfaces have become increasingly well characterized (4,18,19,20,22,25,31,32,34,40,42,45,49,50,52,56,57,67,68,71,72). However, little is known regarding the events following bacterial adhesion and during biofilm development.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, mutations in the lipopolysaccharide core (lipid A) biosynthesis genes of E. coli and P. fluorescens caused comparable reduction of bacterial adhesion (19,31,56). The requirement for increased adhesiveness is reflected in the surface-induced gene expression of two lipopolysaccharide biosynthesis genes, lpxD and wbpG, with lpxD encoding an enzyme that functions in lipid A biosynthesis and wbpG being essential for B-band lipopolysaccharide biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…Substantially reduced attachment to biotic and abiotic surfaces was observed in O-polysaccharidedeficient Pseudomonas spp. (17,19) and in E. coli strains with mutations in the lipopolysaccharide core biosynthesis genes rfaG, rfaP, and galU (19,31,56). The extracellular polysaccharide alginate was required for formation of thick, three-dimensional P. aeruginosa biofilms and was shown to be the intercellular material of P. aeruginosa microcolonies (45).…”

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confidence: 99%

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Characterization of Phenotypic Changes in Pseudomonas putida in Response to Surface-Associated Growth

2001

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The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface. A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria. In this study, a global analysis of physiological changes of the plant saprophyte Pseudomonas putida following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated. N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism. cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment of P. putida. Twenty-eight of these genes had known homologs. As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth. The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis. Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12-or 24-h biofilms. In contrast, PilA was observed in 12-h biofilms but not in planktonic culture. Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development. To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from P. putida and pure C 12 -HSL were added to 6-h planktonic cultures of P. putida, and cell extracts were analyzed by 2-D gel profiles. Differential expression of 16 proteins was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate that P. putida undergoes a global change in gene expression following initial attachment to a surface. Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes.

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“…On the basis of similarities to an O-antigen polymerase of Pseudomonas putida (43 %) (Rodriguez-Herva et al, 1999) and a repeating unit polymerase of Streptococcus agalactiae (42 %) (GenBank accession no. AF337958), Cps9vI was considered to be the CPS repeating unit polymerase.…”

Section: Cps9vi and Cps9vk Are Involved In Capsule Assemblymentioning

confidence: 99%

Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V The GenBank accession number for the sequence reported in this paper is AF402095.

Selm

Kolkman²,

Zeijst

et al. 2002

Microbiology

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The capsular polysaccharide (CPS) synthesis locus of Streptococcus pneumoniae serotype 9V was amplified by long-range PCR and sequenced. The locus was 17 368 bp in size and contained 15 ORFs. The genetic organization of the cluster shared many features with other S. pneumoniae capsule loci, including the presence of four putative regulatory genes at the 5' end. Comparative sequence analyses allowed putative functions to be assigned to each of the gene products. The ORFs appeared to encode, besides the four regulatory genes, five glycosyltransferases, two O-acetyltransferases, an Nacetylglucosamine 2-epimerase, a glucose 6-dehydrogenase, an oligosaccharide transporter protein and a polysaccharide repeating unit polymerase. These functions covered the steps proposed in the CPS biosynthesis of serotype 9V. TLC of carbohydrate intermediates formed after incubation of bacterial membrane preparations with14 C-labelled precursors demonstrated that the fifth ORF (cps9vE) encoded a UDP-glucosyl-1-phosphate transferase. This function was confirmed with the help of a cps9vE mutant that carried a deletion of a guanine residue located adjacent to a stretch of adenines. The identification and characterization of the serotype 9V locus is a major step in unravelling the 9V capsule biosynthesis pathway and broadens the insight into the genetic diversity of the S. pneumoniae capsule loci.

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Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil

Cited by 22 publications

References 59 publications

Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

Characterization of Phenotypic Changes in Pseudomonas putida in Response to Surface-Associated Growth

Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V The GenBank accession number for the sequence reported in this paper is AF402095.

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