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The transition from a planktonic (free-swimming) existence to growth attached to a surface in a biofilm occurs in response to environmental factors, including the availability of nutrients. We show that the catabolite repression control (Crc) protein, which plays a role in the regulation of carbon metabolism, is necessary for biofilm formation in Pseudomonas aeruginosa. Using phase-contrast microscopy, we found that a crc mutant only makes a dispersed monolayer of cells on a plastic surface but does not develop the dense monolayer punctuated by microcolonies typical of the wild-type strain. This is a phenotype identical to that observed in mutants defective in type IV pilus biogenesis. Consistent with this observation, crc mutants are defective in type IV pilus-mediated twitching motility. We show that this defect in type IV pilus function is due (at least in part) to a decrease in pilA (pilin) transcription. We propose that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development.
Biofilms are communities of microorganisms attached to a surface, and their formation occurs in response to a variety of environmental cues (6, 24, 25, 30, 36). In Pseudomonas aeruginosa, these environmental cues include an abundant supply of nutrients, the availability of oxygen, and an osmotically balanced growth medium (24, 26). Biofilm bacteria undergo a developmental program in response to environmental signals that leads to the expression of new phenotypes that distinguish these attached cells from their planktonically growing counterparts. For example, biofilm bacteria have been shown to be up to 1,000-fold more resistant to antibiotics than are planktonically grown cells (13) and gene and protein expression patterns are altered in planktonic versus biofilm-grown cells (6, 7, 8, 26).
A common theme in microbial development involves an input of environmental cues that results in an output of an altered physiological state or behavior. There are several examples of regulatory proteins and signalling pathways which play a central role in the transduction of environmental signals and precipitation of developmental changes that allow a bacterium to adapt to its environment. For example, Bacillus subtilis forms spores in response to a lack of nutrients and requires the central regulator Spo0A to integrate and respond to a number of intracellular and extracellular cues (12). In Myxococcus xanthus, environmental parameters such as nutrient deprivation, population density, and the presence of a solid surface trigger a series of social behaviors which culminate in the formation of multicellular fruiting bodies. The products of the asg genes act early in development to sense the appropriate nutritional signals and initiate the formation of fruiting bodies (33). As mentioned above, environmental signals play an important role in the transition of P. aeruginosa from an individual, planktonic existence to a life attached to a surface in a multicellular community. However, the molecular mechanism by which environmental signals trigger the regulatory events required for biofilm formation has not been elucidated.
P. aeruginosa uses tricarboxylic acid (TCA) cycle intermediates (which include organic acids such as succinate) as carbon sources preferentially over carbohydrates such as glucose or mannitol and appear do so in a cyclic-AMP-independent manner (29, 34). A locus was identified, designated crc, that when mutated results in a strain that can utilize glucose and mannitol even when TCA intermediates are also provided (37). However, the crc mutants do not constitutively express the enzymes and transporters required for glucose and mannitol utilization; these enzymes and transporters are only produced when the appropriate inducers are present. Therefore, Crc appears to be required for repression of carbohydrate metabolism in the presence of TCA intermediates (37). Crc has amino acid sequence similarity to a family of apurinic-apyrimidinic endonucleases which have been shown to be involved in DNA repair in Escherichia coli and other organisms (2, 9). However, Crc has none of the activities associated with these enzymes (19) and the mechanism by which Crc regulates expression of carbohydrate metabolism is not understood.
Here we present data showing that a P. aeruginosa PA14 crc mutant is defective in biofilm formation and that the inability to form a biofilm is due to a defect in type IV pilus-mediated twitching motility. Based on the role of Crc in the regulation of carbon metabolism and type IV pilus-mediated twitching motility, we propose that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development.
All of the bacterial strains and plasmids used in this study are shown in Table Table1.1. P. aeruginosa PA14 was grown on rich (Luria Bertani [LB]) medium or minimal medium (as indicated in each experiment) at 37°C. Unless otherwise stated, the minimal medium used was minimal M63 salts (28) supplemented with glucose (0.2%), MgSO4 (1 mM), and Casamino Acids (CAA; 0.5%). Antibiotics were added at the following concentrations: tetracycline, 150 μg/ml; ampicillin, 750 μg/ml; carbenicillin, 1 mg/ml. All enzymes for DNA manipulation were purchased from New England Biolabs (Beverly, Mass.). All plasmids were constructed in E. coli JM109 by using standard protocols (1) and transferred to P. aeruginosa by electroporation (3).
To generate cell material for enzyme assays, bacterial cultures were grown in 250 ml of minimal medium (18) with 20 mM mannitol as the sole carbon and energy source for at least 9 generations in log phase to fully induce the hex regulon (including glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and NAD-specific glyceraldehyde 3-phosphate dehydrogenase). An aliquot of cells was transferred to minimal medium containing 40 mM succinate and 20 mM mannitol, and growth was continued for 2 to 2.5 generations. Bacteria were chilled, centrifuged, and washed with 1/10 volume of cold minimal medium, and the cell pellets were frozen.
Extracts were prepared from frozen cell pellets by suspending the cells in 2 to 3 volumes of Tris-Cl (50 mM; pH 8)-dithiothreitol (1 mM). The suspended cells were broken by passage through a French press at 16,000 lb/in2 and clarified by centrifugation at 170,000 × g for 30 min (50,000 rpm in a Sorvall T-1270 rotor). Mannitol dehydrogenase was assayed on freshly prepared extracts, while the other assays were performed on thawed extracts. Glucose 6-phosphate dehydrogenase and mannitol dehydrogenase were assayed as previously described (14, 34). The NAD- and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase assays were similar to those of Tiwari and Campbell (35). Briefly, 1-ml reaction mixtures containing NAD or NADP at 0.5 mM; Tris-Cl at 100 mM (pH 8), sodium arsenate at 20 mM, l-cysteine at 3.3 mM, and sodium fluoride at 20 mM were incubated at 22°C. Reactions were started with the addition of glyceraldehyde 3-phosphate to 2 mM. For the determination of specific activities, extracts were assayed for protein by the method of Lowry et al. (17) with bovine serum albumin as the standard.
Twitching motility was assessed by stabbing cells into a very thin (2-mm) LB agar plate (1.5% agar) with a toothpick and incubating the plate for 24 to 48 h at 37°C. A haze of growth at the agar-plate interface and the characteristic flat, spreading colony morphology indicate strains that are proficient for twitching motility (21, 23).
The assay used to screen for biofilm formation-defective mutants is based on the ability of bacteria to initiate biofilm formation on polyvinyl chloride (PVC) plastic. The initiation of biofilm formation was assayed as previously described (25) by determining the ability of cells to adhere to the wells of 96-well microtiter dishes made of PVC (Falcon 3911 Microtest III Flexible Assay Plate; Becton Dickinson Labware, Oxnard, Calif.) using a modification of a previously reported protocol (11). The medium (100 μl/well) was inoculated with either (i) cells patched to LB agar plates using a multiprong device or (ii) a 1:100 dilution from an overnight LB culture. After inoculation, plates were incubated at 37°C for the indicated time and then 25 μl of a 1% crystal violet solution was added to each well (this dye stains the cells but not the PVC). The plates were incubated at room temperature for ~15 min, rinsed four times thoroughly and vigorously with water, blotted on paper towels, and scored for biofilm formation (see Fig. Fig.11).
Biofilm formation was quantified as previously described (25). Briefly, the crystal violet was solubilized in 95% ethanol and the A570 was determined.
Visualization of bacterial cells attached to PVC was performed by phase-contrast microscopy (400× magnification) using a Nikon Diaphot 200 inverted microscope (Nikon Corp., Tokyo, Japan). The images were captured with a black-and-white CCD72 camera integrated with a Power Macintosh 8600/300 computer with video capability. The images were processed with Scion Image software, a modification of NIH Image (National Institutes of Health, Bethesda, Md.) by the Scion Corporation (Frederick, Md.).
The DNA sequence flanking the transposon mutant insertion site was determined by using arbitrary primed PCR as previously reported (27). All other protocols were performed as previously reported (1, 25).
Plasmid pSMC31 (crc+ Cbr) carries a wild-type copy of the crc locus derived from P. aeruginosa PAO1. This plasmid was built by first amplifying crc+ from pPZ352 (19) by PCR using primers which generated a BamHI site at the 5′ end and an EcoRI site at the 3′ end of the crc gene. The PCR product was digested with EcoRI and BamHI and then ligated to pUCP18, which had also previously been digested with EcoRI and BamHI.
LB agar plates without NaCl were prepared and inoculated as described for the twitching motility assays above. Approximately 10 μl of distilled water (dH2O) was dropped on the edge of the colony, the bacteria were allowed to become suspended for ~30 s, and then the copper-coated grid was floated on the cell-containing dH2O drop for ~30 s, rinsed for 5 s with dH2O, stained for 45 s with phosphotungstate (2%), rinsed for 5 s with dH2O, and then gently blotted and air dried (D. Wozniak, personal communication). For each strain, at least 100 cells were examined. The pili are fragile structures; they are apparently easily lost during the preparation of electron microscopy (EM) grids. Samples were analyzed with a JEOL 1200 EX transmission electron microscope at 80 kV.
β-Galactosidase assays were performed as previously described without modification (22). The data presented are averages of two cultures assayed in duplicate (a total of four data points).
We previously performed a screen for mutants of P. aeruginosa PA14 defective in biofilm formation and identified mutations in genes required for flagellum-mediated swimming and type IV pilus-mediated twitching motility (24). In this search for biofilm formation mutants, we also obtained a sad-24 mutant strain with an insertion in the crc gene. The biofilm formation phenotype of this mutant is shown in Fig. Fig.1A.1A.
Comparison of the DNA sequence flanking the sad-24::Tn5 insertion to DNA sequences in the GenBank database revealed 100% identity over a 34-amino-acid region to the previously described crc locus of P. aeruginosa PAO1 (19), and this allele was therefore designated crc-24. The location of the insertion is near the 3′ end of the crc gene, and the mutated gene is predicted to code for a protein whose C-terminal 38 amino acids are deleted and replaced with 9 amino acids derived from a transposon sequence. We observed other phenotypes in the strain carrying the crc-24::Tn5 mutation, including overproduction of a pigment (presumably pyocyanin ) and a decrease in type IV pilus-mediated twitching motility.
It is important to note that the growth rate of the crc mutant is indistinguishable from that of the wild type in glucose-containing minimal medium supplemented with CAA (the same medium and growth conditions used in the screen to isolate this mutant; data not shown). Therefore, the defect in the crc mutant appears to be specific to biofilm formation and not a general defect in growth. Finally, in addition to its defect in biofilm formation on PVC plastic, this strain is also defective for biofilm formation on polystyrene, polycarbonate, and polypropylene (data not shown).
In order to confirm that the phenotypes of the crc-24 mutant strain were indeed caused by the mutation in the crc gene, we performed complementation analysis. The crc gene of P. aeruginosa PAO1 had been cloned previously (19), and using the cloned gene, we built a plasmid construct which carries only crc+ and no other complete open reading frame (pSMC31; see Methods and Materials for details of the plasmid construction). Figure Figure1B1B shows the results of the complementation assay performed on these strains. The plot in Fig. Fig.1B1B shows that based on quantitation of biofilm formation using the crystal violet assay, plasmid pSMC31 (crc+) fully rescued the biofilm formation defect of the crc-24 mutant strain to levels equivalent to those of the wild type (carrying either plasmid). As expected, the mutant carrying the vector control remained defective in biofilm formation.
The result above was confirmed and extended by directly visualizing biofilm formation by these strains using phase-contrast microscopy (Fig. (Fig.1C).1C). After incubation for 8 h, the wild type (carrying either plasmid) had formed a dense monolayer of cells punctuated with microcolonies on the surface of the PVC plastic. Examination of the crc-24 strain carrying pUCP18 (vector-only control) revealed a dispersed monolayer of cells markedly different from that of the wild-type strain. However, providing the crc-24 mutant with a wild-type copy of crc on pSMC31(crc+) resulted in a biofilm formation phenotype indistinguishable from that of the wild-type strain.
In addition to assessing biofilm formation, we also investigated whether pSMC31 (crc+) could complement the defect in twitching motility of the crc-24 mutant (Fig. (Fig.2).2). The hazy zone of growth around the wild-type colonies is the “twitching zone”; that is, the area containing cells which have used type IV pilus-mediated twitching motility to move away from the point of inoculation. The wild-type strain (carrying either plasmid) and the crc-24 mutant carrying pSMC31 (crc+) both formed an obvious twitching zone with an average size of 9.5 mm (measured as the distance from the colony edge to the edge of the twitching zone). In contrast, the crc-24 mutant carrying pUCP18 (vector control) produces a twitching zone about sixfold smaller (1.5 mm) than that produced by the wild-type strain. Furthermore, the crc-24 mutant carrying pSMC31 (crc+) no longer overproduced pyocyanin (data not shown). Based on these results, we conclude that the crc-24 mutation caused the observed phenotypes.
The crc-24::Tn5 mutant of P. aeruginosa PA14 is defective for twitching motility. This observation suggested that crc is required for the synthesis or function of type IV pili. As assessed by phase-contrast microscopy, the crc-24 mutant showed a pattern of dispersed cells on the PVC plastic surface after incubation for up to 8 h (Fig. (Fig.1C),1C), a phenotype identical to that observed previously for mutants lacking type IV pili (24). Therefore, the crc mutant has a biofilm formation defect indistinguishable from that of a strain which lacks type IV pili.
Three lines of evidence demonstrated that although the crc-24 mutant is defective in twitching motility, this strain still has at least some functional type IV pili. The first line of evidence is that the crc-24 mutant retains residual twitching motility, as shown in Fig. Fig.2.2. Second, the crc-24 mutant remains sensitive to infection by bacteriophage SN-T (15), which utilizes type IV pili as a receptor (26). The abilities of phage SN-T to form plaques on the wild-type strain and the crc-24 mutant are indistinguishable (data not shown). It is interesting that although the crc-24 mutant has decreased twitching motility, it is still sensitive to phage SN-T infection. This observation may reflect the sensitivity of the phage infection assays, that is, the possibility that only a single phage or a few phage are sufficient for infection and lysis. The wild-type strain may be bound by many more phage particles than are required for cell lysis. No plaques were formed on other type IV pilus mutants (such as pilB and pilC mutants), even using a phage lysate at a titer of ~3 × 107 PFU/ml. Finally, we directly visualized type IV pili by using EM. Type IV pili are polar structures found at the same end of the cell as the flagellum. As shown in Fig. Fig.3,3, in the wild-type strain the type IV pili are readily visible as multiple, polarly localized rods which are somewhat thinner than the single polar flagellum. These pili are also visible in the crc mutant, consistent with the results of the phage susceptibility tests and twitching motility assays. However, in the pilB strain, which is not susceptible to lysis by phage SN-T and retains no detectable twitching motility, no pili were visible.
As mentioned above, mutations in crc were originally isolated in P. aeruginosa PAO1 based on their defects in the regulation of carbon metabolism. However, these mutations were not tested for defects in biofilm formation. The constructed crc::Cbr mutation in P. aeruginosa PAO1, like the crc::Tn5 mutation in P. aeruginosa PA14, results in a strain defective in biofilm formation (data not shown). We have also shown that a strain carrying the crc::Cbr allele is also defective for twitching motility and remains sensitive to lysis by type IV pilus-specific bacteriophage SN-T. Prince and coworkers had also indicated that a crc mutant of P. aeruginosa PAO1 is defective in twitching motility (4). These data further confirm a role for crc in biofilm formation and demonstrate that the defect in biofilm formation is not specific to P. aeruginosa PA14.
The crc locus was originally identified in P. aeruginosa PAO1 during a screen for genes that participate in catabolite repression control of enzymes involved in mannitol utilization (37). In order to determine if a P. aeruginosa PA14 crc mutant is also defective in the regulation of carbon metabolism (in addition to the other observed phenotypes), we assessed the crc-24 mutant isolated in the biofilm mutant screen for its effects on catabolite repression in P. aeruginosa PA14. These data are summarized in Table Table2.2. In wild-type P. aeruginosa PAO1, the levels of enzyme activities required for glucose and mannitol utilization are repressed in the presence of succinate (29, 34). Similarly, levels of representative enzymes involved in mannitol and glucose catabolism in P. aeruginosa PA14 (mannitol dehydrogenase, glucose 6-phosphate dehydrogenase, and NAD-specific glyceraldehyde 3-phosphate dehydrogenase) are up to eightfold higher when cells are grown in the absence versus the presence of succinate. The level of these enzymes in P. aeruginosa PA14 carrying the crc-24 allele is no longer as strongly repressed (or not repressed at all) when cells are grown in the presence of succinate. This is the phenotype that originally defined the crc locus (37). The NADP-specific glyceraldehyde 3-phosphate dehydrogenase (as opposed to the NAD-specific enzyme) is not regulated by Crc, and the enzyme level shows a less-than-twofold change when cells are grown in the presence versus the absence of succinate.
Strains which express amidase are sensitive to the amide analog fluoroacetamide (FAA), which is converted to the toxic metabolite fluoroacetate by this enzyme (5). Expression of amidase is normally repressed when cells are grown on succinate, and this repression is dependent on Crc. Strains without functional Crc express amidase even in the presence of succinate and die when FAA is included in the medium. Thus, FAA sensitivity acts as an indirect assay of Crc function. Figure Figure44 shows the growth of the wild type, the crc-24 mutant, and the crc-24 mutant carrying pSMC31 (crc+) on minimal medium containing succinate with (A) or without (B) FAA. All of the strains grew equally well on minimal medium containing succinate without FAA (Fig. (Fig.4B).4B). In contrast, the crc-24 mutant showed no growth on the FAA-containing medium (Fig. (Fig.4A).4A). The wild type and the crc-24 mutant carrying the wild-type copy of crc on a plasmid grew well on this medium. Therefore, although the strain carrying the crc-24 mutation was isolated in a screen for mutants unable to form a biofilm, this strain is also defective in the Crc-mediated, succinate-dependent catabolite repression of enzymes required for carbohydrate catabolism.
Phibbs and coworkers reported that P. aeruginosa PAO1 carrying mutations in crc had increased levels of enzymes and transporters involved in carbohydrate metabolism even under conditions in which expression of these functions should have been repressed (i.e., in the presence of TCA intermediates such as succinate) (20, 37). However, the mechanism by which Crc could alter gene expression has not yet been determined.
We have obtained evidence that Crc can function to regulate gene expression at the level of transcription. Specifically, strains with mutations in crc have markedly lower levels of expression of pilA, which codes for the major pilin subunit, and pilB, which codes for an accessory factor required for pilus assembly. These genes are positioned adjacent to each other on the chromosome but are transcribed divergently (21, 23). We introduced plasmid-borne pilA-lacZ (pMSZ25) and pilB-lacZ (pPB18) transcriptional fusions (along with a vector control, pDN19) into wild-type P. aeruginosa PAO1 and its crc::Cbr derivative. The results of β-galactosidase assays performed on these strains are summarized in Fig. Fig.5.5. Expression of the pilA-lacZ transcriptional fusion activity was 132-fold higher in the wild type than in the crc mutant strain (crc+, 3,965 miller units [MU]; crc mutant, 30 MU). Similarly, the expression of the pilB-lacZ transcriptional fusion in the wild type was 641 MU, compared to 98 MU from this same fusion in the crc::Cbr mutant (a decrease of 6.5-fold). As expected, the vector control showed only minimal activity in both genetic backgrounds.
Environmental signals play an important role in regulating the formation of biofilms; however, very little is known about the molecular mechanisms necessary to transduce the environmental signals that trigger biofilm development. We report here the isolation of a strain defective in biofilm formation which has a mutation in crc, a locus which may participate in the linking of environmental signals with biofilm development. It has been shown previously that the crc gene is involved in the regulation of carbon metabolism in P. aeruginosa (20, 37). We show here that Crc has a role in the formation of biofilms on abiotic surfaces and that P. aeruginosa PA14 crc mutants not only are defective in type IV pilus-mediated twitching motility (Fig. (Fig.11 and and2)2) but also have all of the carbon source utilization phenotypes described for the crc mutants of P. aeruginosa PAO1 (Table (Table22 and Fig. Fig.44).
We propose that the crc mutant is defective in biofilm formation because it lacks fully functional type IV pili. Two lines of evidence support this conclusion. First, we have shown previously that type IV pili and type IV pilus-mediated twitching motility are necessary for the development of biofilms in P. aeruginosa PA14 (24). In fact, the observation that the crc-24 mutant has a defect in twitching motility but still retains at least some type IV pili on its surface (based on phage sensitivity and EM studies) further supports our previous hypothesis that twitching motility (and not just the presence of the pili on the cell surface) plays an important role in biofilm development (24). The direct observation of the biofilm formed by a crc mutant on PVC plastic using phase-contrast microscopy also supports the contention that it is the defect in type IV pilus assembly and/or function that leads to the biofilm formation defect. The phase-contrast micrograph in Fig. Fig.1C1C shows that the phenotype of the crc-24 mutant is indistinguishable from that reported for a pilB mutant; strains carrying mutations in pilB have been shown previously to make no detectable type IV pili (23) and to be defective in biofilm formation (24). Therefore, the biofilm formation phenotype of the crc-24 mutant can be accounted for by its defect in type IV pilus assembly and/or function.
The results of twitching motility assays suggested that crc mutants have a defect in the synthesis, assembly, and/or function of type IV pili. Based on previous studies of the role of Crc in the regulation of carbon metabolism (20, 37), it was possible that Crc exerts its effect via gene expression at some level. As shown in Fig. Fig.5,5, the expression of genes whose products are essential for type IV pilus biogenesis (pilA and pilB) is markedly reduced in crc mutant backgrounds compared to that in the wild-type strain. This is the first report of Crc regulation of a non-carbohydrate metabolism target and the first demonstration that Crc can affect regulation at the transcriptional level. It is possible, therefore, that decreased levels of the pilin subunit, accessory factors required for assembly of the pilin subunit into functional pili, and/or expression of gene products required for extension-retraction of pili cause the defect in twitching motility and biofilm formation observed in P. aeruginosa PA14 and PAO1 crc mutants.
It is clear from work in many laboratories that biofilm formation occurs in response to various environmental cues (6, 24, 25, 30, 36). However, how these environmental signals are sensed and transduced by the biofilm-forming bacteria and the molecular mechanism(s) utilized to initiate the development of a biofilm in response to these cues are not understood.
We propose that Crc may play a role in the transduction of nutritional signals that trigger biofilm development by P. aeruginosa. Crc has been shown to be involved in the regulation of carbon utilization by preventing the utilization of sugars such as glucose when P. aeruginosa PAO1 cells are also provided with TCA cycle intermediates (20, 37). We have shown that a crc mutant of P. aeruginosa PA14, like its P. aeruginosa PAO1 counterpart, is defective in the succinate-dependent catabolite repression of enzymes required for carbohydrate catabolism. We have also shown here that Crc is necessary for biofilm formation, possibly by controlling the transcription of genes required for type IV pilus biogenesis. Therefore, Crc may be part of a signal transduction pathway that can sense and respond to nutritional signals and thereby play a role in the bacterium's transition from planktonic to biofilm growth in response to these nutritional signals. Additional work is needed to determine the precise mechanism by which Crc regulates its various targets and to identify other members of this signalling pathway. Finally, the fact that crc cross-hybridizing sequences are also found in a variety of fluorescent pseudomonads (19) suggests that Crc plays a similar role in biofilm formation in these related organisms.
This work was supported by grants from the National Institutes of Health (GM 58213) and the Cystic Fibrosis Foundation to R.K. and Fellowship DRG of the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation and a Medical Foundation Charles A. Hood postdoctoral fellowship to G.A.O.
We thank Tyler Kokjohn for providing phage SN-T and advice on working with this phage, Steve Lory for plasmids, and S. E. Finkel for his reading of the manuscript. We also thank Maria Ericksson for her invaluable assistance with EM, and we thank Elizabeth Batten for technical assistance.