Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth - PubMed (original) (raw)

Comparative Study

Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth

K Sauer et al. J Bacteriol. 2001 Nov.

Abstract

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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Figures

FIG. 1

Protein yield from tubing after different attachment times. For each attachment time point, the cell suspensions of four silicone tubes were combined and harvested by centrifugation.

FIG. 2

2-D images of crude protein extracts of planktonic P. putida grown in a chemostat. The crude protein extracts (500 μg) were extracted and separated on nonlinear Immobiline Dry-Strips (pH 3 to 10), followed by SDS–11% polyacrylamide gels. The gels were stained with Coomassie brilliant blue. The boxes A to F indicate areas that are enlarged in Fig. 3 and 5.

FIG. 3

Enlarged partial 2-D gels showing crude protein extract of planktonic P. putida grown in a chemostat (A1 to E1) and after a period of 6 h of attachment time (A2 to E2). The sections A1 to E1 show an enlarged view of the 2-D image in Fig. 2. The sections A2 to E2 are the corresponding sections in the 2-D gel of crude protein extracts obtained under attached growth conditions. Open arrows indicate protein spots, which are up-regulated in attached cells, while solid arrows mark those protein spots that are up-regulated in planktonic cells.

FIG. 4

Immunoblot of b-type flagella (A) and type IV pili (B) of whole P. putida cells grown in minimal medium in a chemostat or attached to silicone surface during biofilm development. Whole cells were analyzed by SDS-PAGE, and the proteins were electroblotted onto nitrocellulose membranes (3). The membranes were probed with polyclonal b-type flagella antibodies (A) or monoclonal type IV pilus antibodies (B). Goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase was used as the secondary antibody. Antibody binding was detected by colorimetric analysis (3). M, marker; PilA−, type-IV-pilus-deficient P. aeruginosa PA416 (49); P, planktonic, chemostat-grown P. putida cells; 12 h and 1 d, attached P. putida cells after 12 h and 1 day of attachment time, respectively.

FIG. 5

Enlarged partial 2-D gels of crude protein extracts of P. putida in the absence (A1 to F1) and presence (A2 to F2) of the 3OC12-HSL signal molecule. The sections A1 to F1 and A2 to F2 correspond to the boxes A to F shown in Fig. 2. Arrows indicate differences in the 2-D protein pattern of chemostat-grown cells in the absence and presence (10 μM) of the C12-HSL signal molecule.

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Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth - PubMed (original) (raw)