1 Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS-IBSM-UPR9027, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France
2 Laboratoire de Biologie Moléculaire des Relations Plantes-Micro-organismes, INRA-CNRS, Castanet-Tolosan, France
Correspondence
Alain Filloux
filloux{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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Present address: Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
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INTRODUCTION |
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Lately, P. aeruginosa has been one of the most studied micro-organisms with respect to bacterial biofilm formation. It is a ubiquitous organism found in various environments and an opportunistic pathogen that can cause disease in a wide variety of organisms. In humans, it is mainly associated with nosocomial and cystic fibrosis infections (Costerton, 2001). Moreover, there are increasing numbers of reports suggesting that the biofilm lifestyle is predominant within sites of infection, such as in the lungs of cystic fibrosis patients (Singh et al., 2000
). Among the P. aeruginosa determinants that have been shown to be involved at various stages of biofilm formation, flagella and type IV pili (O'Toole & Kolter, 1998
), Cup fimbriae (Vallet et al., 2001
) or alginate (Davies & Geesey, 1995
) are the most frequently cited. In this study, we identified a P. aeruginosa gene cluster (pel) by random screening of non-adherent mutants on abiotic surfaces. The pel gene cluster was previously identified by Friedman & Kolter (2004a)
. They showed that pel mutations in the P. aeruginosa PA14 strain prevents the formation of a pellicle at the airliquid interface of bacterial cultures grown in static conditions. On the basis of this initial observation, they could show further that the pel mutants are not able to form robust and shear force-resistant biofilms on a solid surface of plastic or glass. They also suggested that the pel genes are involved in the production of a putative exopolysaccharide, which holds bacterial cells together within the mature biofilms and makes them resistant to thorough washing. Finally, they demonstrated that the PA14 matrix contains a glucose-rich structural component that is not found in the pel mutants. This component allows staining of the bacterial colony on Congo-red-containing medium. Congo red binds exopolysaccharides, including glucose-rich polymers such as cellulose. Friedman & Kolter suggested that five out of the seven pel gene products have sequence similarities with proteins involved in carbohydrate processing, which include PelA, PelC, PelD, PelE and PelF. Of these five proteins, only PelF could be recognized as a glycosyltransferase. In the present study, we further characterized the role of the pel gene cluster in the non-piliated PAK
pilA strain. Our systematic analysis of the seven pel mutants, which were engineered in this strain, revealed that they were defective for attachment on plastic and glass surfaces at very early stages of the biofilm development process. The pel genes and their genetic organization appeared well conserved in other Gram-negative bacteria, such as the plant pathogen Ralstonia solanacearum, and we were able to show that in R. solanacearum also, it serves a conserved and similar function.
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METHODS |
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Construction of P. aeruginosa and R. solanacearum mutant strains.
For P. aeruginosa, 500 bp DNA fragments contiguous to the 5' and 3' ends of the target gene were PCR amplified with the High Fidelity DNA polymerase (Roche) by using PAK chromosomal DNA as template and two pairs of oligonucleotides (ma01/ma02 and mb01/mb02, Table 2). In the first round of PCR, a product (ma) corresponding to the region upstream from the 5' end of the gene was generated using ma01/ma02 and a product (mb) corresponding to the region downstream from the 3' end of the gene was obtained using mb01/mb02. The oligonucleotide sequence of ma02 included the initiation codon, whereas mb01 contained the termination codon. The oligonucleotides ma02 and mb01 were designed in such a way that the resulting ma and mb fragments were overlapping PCR products. In a second round of PCR, the ma and mb fragments were used as template and joined by their overlapping ends when the external primers ma01 and mb02 were used for amplification. The resulting ma/mb DNA fragment contained the 5' and 3' regions of the target gene linked together. Within ma/mb, only the inititation and termination codons of the gene of interest, separated by a few codons, were left. The ma/mb DNA fragment was cloned into the pCR2.1 vector (TA-cloning kit, Invitrogen), and subcloned into pKNG101, which is non-replicative in P. aeruginosa. The recombinant plasmid, pKNGmamb, was maintained in the E. coli CC118
pir strain. The plasmid was mobilized into the appropriate P. aeruginosa strain. The mutants in which double recombination events occurred, resulting in the deletion of the gene of interest, were selected on sucrose plates as previously described (Kaniga et al., 1991
). Finally, the deletion event was checked by PCR, with appropriate primers. Following this strategy, the pelB, pelE and pelF genes were deleted in the PAK strain, whereas, the pelB, pelC, pelD, pelE, pelF and pelG genes were deleted in the PAK
pilA strain. For R. solanacearum, slight modifications in the strategy for the production of a joined DNA fragment containing the deleted gene of interest were introduced. Briefly, the 5' and 3' adjacent regions (named PCR1 and PCR2, approximately 1 kb in length) of the ragG gene were PCR amplified using the chromosomal DNA of strain GMI1000 as template and the Expand Long Template DNA polymerase. The oligonucleotides used included an EcoRI restriction site. The PCR products were digested with EcoRI and used in a ligation mix containing the pGEM-T (Promega) TA-cloning vector. A streptomycin/spectinomycin resistance cassette, extracted from the pHP45
plasmid, was cloned in the recombinant plasmid at the EcoRI site, localized at the junction between PCR1 and PCR2. The linear plasmid was transformed in R. solanacearum, as described previously (Boucher et al., 1985
), in order to delete the ragG gene after a double recombination event occurred. The ragG mutant was selected on BG plates containing antibiotic.
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For P. aeruginosa and for R. solanacearum, cells were grown in 5 ml M63 minimal medium supplemented with 0·2 % glucose, 0·5 % Casamino acids and 1 mM MgCl2, in a 50 ml Corning tube containing a semi-immersed glass cover slide. After a period of incubation of 6, 24 or 48 h, at 30 °C without shaking, the slide was removed and rinsed. The attached bacterial cells were visualized by phase-contrast microscopy using an Axiovert 200M microscope (magnification x100). Images were captured with a Hamamatshu type Orca ER camera.
Swimming, swarming and twitching motility assays.
For swimming and swarming assays, bacterial colonies were transferred with a sterile toothpick onto the surface of 0·3 % or 0·7 % agar plates, respectively. The plates were incubated at 37 °C for 24 h and the spreading of the colony on the agar could readily be observed. For twitching motility, plates containing 1·5 % agar were inoculated with a toothpick by stabbing the LB agar plates. After 24 h growth at 37 °C, the agar was removed and the plates were stained with 1 % crystal violet for 10 min and washed with water. Spreading of bacteria from the inoculation point could be observed by the size of the stained surface on the plastic.
Cell surface hydrophobicity.
Bacterial cells were grown overnight at 37 °C in LB medium. The cultures were centrifuged and the bacterial pellet was resuspended in PBS to adjust the OD600 to 0·8. Three millilitres of the suspension were collected in Corex tubes and mixed with 400 µl solvent (hexadecane, chloroform or ethyl acetate). After 15 min at 37 °C, the tubes were vortexed (twice for 30 s with a 5 s pause in between). The aqueous phase was collected and the OD546 determined (Rosenberg et al., 1980).
Congo red assay.
Tryptone (10 g l1) agar (1 %) plates without salt were supplemented with Congo red (40 µg ml1) and Coomassie brilliant blue (20 µg ml1). Bacteria were inoculated on the surface of the plates with a toothpick and grown at 30 °C. The colony morphology and colour were analysed (Romling et al., 1998).
LPS isolation and analysis.
Bacterial cultures for analysis of LPS were grown overnight in LB at 37 °C. A sample of 10 ml was collected and centrifuged. After washing with PBS, the cell pellet was resuspended in 1·5 ml Tris/EDTA buffer (50 mM Tris/HCl, 2 mM EDTA, pH 8·5). The bacteria were lysed by sonication. The cell envelopes were further collected by centrifugation and resuspended in 70 µl Tris buffer (2 mM Tris/HCl, pH 7·8) and 100 µl loading buffer (0·125 M Tris, 4 % SDS, 10 % -mercaptoethanol, 20 % glycerol, 0·01 % bromphenol blue). The samples were incubated at 95 °C for 10 min. A 30 µl aliquot of the sample was mixed with 30 µl Proteinase K (100 µg ml1) and incubated for 1 h at 60 °C. Ten microlitres of each sample was submitted to electrophoresis on a 15 % polyacrylamide gel containing SDS. LPS was visualized after silver staining. By using the cell envelope fraction, a colorimetric analysis of 2-keto-3-deoxyoctonate (KDO) content was performed as previously described (Weissbach & Hurwitz, 1959
).
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RESULTS |
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Involvement of the pelAG genes in biofilm formation
In order to assign a role for each of the pel genes in the biofilm formation process, the pelB, pelC, pelD, pelE, pelF and pelG genes were systematically deleted in the type IV pili-deficient PAKpilA strain. Upon introduction of the pel mutations in PAK
pilA, all strains presented a clear defect in their biofilm phenotype after 24 h incubation (Fig. 2
a). The level of attachment was quantified for each mutant, by measuring the crystal violet stain left in the microtitre dishes after washing and by comparing with the parental PAK
pilA strain (Fig. 2a
). The pel mutant strains attached with an efficiency of about 510 % relative to the PAK
pilA parental strain. This defect could be restored by the introduction of the corresponding pel genes in trans, as shown with pelF (pUCP3059) (Fig. 2a
). We concluded that all pel genes are individually involved in the biofilm phenotype analysed.
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pel mutants of a piliated PAK strain are impaired in formation of a mature biofilm
In the study by Friedman & Kolter (2004a), a
clear difference could be observed between a pel mutant and the parental PA14 strain only at a later stage: after 1824 h incubation. We further investigated whether the function of the pel genes, as observed in the early phase of biofilm formation, is linked to the strain (PA14 and PAK) or to the lack of type IV pili. We thus introduced pel mutations into the piliated PAK strain. PAK mutants deleted for the pelF, pelE and pelB genes were engineered and the biofilm phenotype tested and quantified with the crystal violet biofilm assay (Fig. 3
a). The mutant strains were incubated for 24 h in microtitre dishes, and the difference after staining was barely detectable. However, quantification experiments revealed that the biofilm formation efficiency of the PAK
pelB, PAK
pelE and PAK
pelF mutants was only about 6070 % relative to the parental PAK strain (Fig. 3a
). Furthermore, kinetic experiments were performed in which biofilm formation was evaluated after incubation periods ranging from 3 to 48 h (Fig. 3b
). The level of biofilm formed did not change significantly between the pel mutants and the PAK strain up to 12 h incubation. However, at 24 h the influence of the pel mutation could be observed and was strongly significant after 48 h with a 60 % reduction in biofilm formation efficiency. From these results we concluded that a pel mutation influences biofilm formation both in a piliated and a non-piliated PAK P. aeruginosa strain; however, the influence of the pel mutation at early stages of biofilm formation could only be observed in a non-piliated strain (PAK
pilA).
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DISCUSSION |
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One major novel aspect that we presented in our study on the pel genes is that they influence the initiation of the biofilm formation process and also, probably, irreversible attachment on abiotic surfaces. In contrast to other studies that searched for biofilm-deficient P. aeruginosa strains, we used a non-piliated strain, PAKpilA. In the conditions of our assay, this strain is able to form biofilms with an efficiency of 88 % relative to the PAK strain (Vallet et al., 2001
). In the present study, we performed a systematic mutagenesis of the seven pel genes in the non-piliated genetic background (transposon insertion for pelA and gene deletion for pelBG) and found that all these strains were totally deficient for initiation of attachment on plastic or glass. This was observed by quantifying the amount of crystal violet associated with the biofilm after staining (Fig. 2a
) or by direct observation in phase-contrast microscopy, which revealed that hardly any bacteria are attached to the abiotic surface (Fig. 4
). By using a similar screening procedure and a collection of mutants in the piliated P. aeruginosa PA14 strain, Friedman & Kolter were not able to identify pel mutants. However, they were able to identify pel mutants thanks to the observation of a pellicle at the airliquid interface of a statically grown bacterial culture. Finally, they showed that the phenotype associated with the pel mutation could be seen in the microtitre dish crystal violet-stained biofilm assay only after an incubation time of over 18 h, which might explain why they missed it in previous screens. More precisely, after 24 h, the biofilm formed by the parental PA14 strain becomes robust and resistant to washing procedures, whereas the biofilm formed by the pel mutants is not able to resist shear forces. To check whether the differences observed between the two studies is related to the strain used (PA14 and PAK) or the piliation status, we engineered pel mutations in a piliated PAK strain. We found that PAK
pel mutants do not have an obvious defect in biofilm formation using the crystal violet staining procedure (Fig. 3a
). Furthermore, we performed biofilm kinetics experiments and we revealed that the PAK
pel mutants formed less-robust biofilms after incubation periods of 24 h (Fig. 3b
), similar to the results observed by Friedman & Kolter. We thus concluded that the type IV pili, which are assembled and functional in the PAK
pel mutants, may mask the influence of the pel genes in the early stages of biofilm formation.
Whereas the involvement of the pel gene cluster in attachment and biofilm formation seems to be established, the exact role of the pel gene products remains to be elucidated. Friedman & Kolter have suggested that the pel genes are involved in the production of a novel glucose-rich polysaccharide component that contributes to the formation of a matrix material that encases the cells and holds them together in a resistant biofilm. We also confirmed that in the PAK strains, expression of the pel genes is associated with the production of a matrix component that allows binding of Congo red. Since the pel mutations in PAKpilA strongly alter the association of the bacteria with the surface, we suggest that the component synthesized by the pel system is responsible for initial cell-surface contact and is not simply required for cellcell contact at later stages in the biofilm development process. However, the cell-surface interaction process involving the pel genes appears to be dispensable when type IV pili are present. In this case, the function of the pili is thus sufficient to allow an irreversible attachment of the bacteria to the surface. The role of expolysaccharide at early and late stages of the biofilm formation process has already been studied. It has been described for vps mutants in V. cholerae that, using the standard biofilm assay in microtitre dishes, no crystal violet staining can be observed even after 80 h incubation, whereas the parental strain reaches a maximum at 29 h (Watnick et al., 1999
). This suggests that vps-encoded exopolysaccharide might have a role at quite early stages of biofilm development. In contrast, with an E. coli mutant defective in the production of colanic acid, even though the crystal violet staining observed with the mutant is significantly less intense than that observed for the wild-type strain, it increases over time, and ultimately closely approximates that of the parent. In this case, colanic acid has been suggested to be critical for the formation of the complex three-dimensional structure and depth of E. coli biofilms (Danese et al., 2000
).
It was recently shown that the exopolysaccharide alginate is not a major component of the the biofilm matrix of the non-mucoid P. aeruginosa strains PA14 or PAO1, suggesting that some other exopolysaccharides might be the predominant biofilm scaffolding in these strains (Wozniak et al., 2003). Whereas the pel gene cluster may play a crucial role in the biogenesis of this yet-uncharacterized polysaccharide, an additional cluster, named psl, has also been proposed to participate in the production of exopolysaccharides that may be minor constituents of the biofilm matrix (Friedman & Kolter, 2004b
; Jackson et al., 2004
; Matsukawa & Greenberg, 2004
). With respect to the pel cluster, Friedman & Kolter (2004a)
suggested that five out of the seven pel genes encode proteins with similarities to components involved in carbohydrate processing. We agree that PelF is probably a glycosyltransferase and suggest that it belongs to the GT4 family (Coutinho et al., 2003
), which might be involved in a process of glycosylation required for polysaccharide biogenesis and/or glycoprotein synthesis. Among the pel gene products, which were not assigned a homologue, we showed here that PelG shows weak sequence but strong structural similarities with members of the PST family. PSTs are integral membrane proteins that mediate export of polysaccharides across the cytoplasmic membrane. Transmembrane transporters have recently been classified, and PST members, such as PelG, will fall in the TC #2.A.66 exporter superfamily (Hvorup et al., 2003
). PSTs may function together with auxiliary proteins, located within the bacterial cell envelope, that allow passage of complex carbohydrate across both membranes of the Gram-negative bacterial envelope (Paulsen et al., 1997
). The putative flipping role of PST members is commonly required for LPS O-antigen repeat unit export or for extracellular (EPS) and capsular polysaccharide (CPS) export. Interestingly, three Pel proteins have transmembrane domains (PelB, PelD and PelE) and are predicted to be located within the cytoplasmic membrane, with PelB and PelE, which carry tetratricopeptide repeats (TPRs) involved in proteinprotein interactions (Smith et al., 1995
). More interestingly, we have identified a putative lipoprotein signal peptide in the deduced amino acid sequence of the pelC gene. The amino acid residue that follows the cysteine residue, which will be the first residue of the mature lipoprotein, is not an aspartate residue. The +2 position in the amino acid sequence of mature lipoproteins has been shown to be crucial for subcellular localization (Yamaguchi et al., 1988
). In the case where the +2 residue is an aspartate, the lipoprotein is retained within the inner membrane, whereas any other residues, mostly serine, as found in PelC, direct the protein to the outer membrane. This observation strongly suggests that the putative PelC lipoprotein is targeted to the outer membrane. It is attractive to suggest that Pel proteins may constitute a novel type of multi-component polysaccharide transport system. PelG could be the PST, PelCDE the auxiliary transporter proteins, with PelC in the outer membrane and PelDE in the inner membrane. Enzymes more directly involved in carbohydrate processing could be PelF, which is the putative glycosyltransferase of the system, whereas PelA and PelB contain regions with weak similarities to domains found in glycosylhydrolase and cellulase synthase, respectively. It should be noticed that the lack of any one of the Pel components alters the function of the whole system, resulting in the associated biofilm-deficient phenotype.
Our future studies will aim to identify more precisely the nature of the components produced in a pel-dependent manner and more particularly the nature of the glycosylation event that could be supported by PelF. Moreover, the role of the P. aeruginosa pel cluster appears to be functionally conserved in other bacteria, such as the plant pathogen R. solanacearum. The characterization of the molecular targets for the pel glycosylation gene cluster will certainly reveal novel features associated with P. aeruginosa biofilms, but more significantly features associated with the virulence of bacterial animal and plant pathogens.
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ACKNOWLEDGEMENTS |
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Received 16 June 2004;
revised 9 November 2004;
accepted 9 November 2004.
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