Genetic Regulation Within E coli Biofilms

4.3.1 Gene Expression Within Biofilm: Cause or Consequence?

Planktonic and surface-attached growth modes are simple to distinguish pheno-typically. These two lifestyles are thought to require or involve a different gene expression setup, leading to the expression of some of the phenotypic characteristics of the biofilm phenotype. The existence of changes in gene expression within biofilm compared with a non-biofilm mode of growth was recognized early on. Gene fusion studies suggest that the expression of up to 38% of the E. coli genome is affected by biofilm formation (Prigent-Combaret et al. 1999). Such evidence for differential gene expression within bacterial biofilms has been provided by recent studies using DNA arrays. These studies indicate that, in fact, a lower proportion of the E. coli genome (5%-12%) is subject to differential expression in sessile vs planktonic life (Schembri et al. 2003b; Beloin et al. 2004; Ren et al. 2004a). While these studies (along with others conducted in other microorganisms) suggest the existence of a common pattern of gene expression in E. coli biofilms and have indeed identified some genes required for biofilm formation, detailed comparison of genes discovered reveal only a very modest overlap between the different studies. This underscores the difficulty in comparing analyses carried out with different strains, different experimental setups (biofilm device, medium, presence or absence of flow) and different time scales (i.e., with different E. coli biofilms), but it also raises the possibility that global analyses are not really appropriate for dealing with the extreme complexity in time and space that resides within a biofilm (Beloin and Ghigo 2005).

Moreover, it is important to distinguish between factors required for biofilm formation and factors induced by particular biofilm conditions. Indeed, although biofilm formation requires the expression of specific factors (see Sect. 4.3.1), major modifications in gene expression patterns within biofilms could also be induced by the drastic environmental changes occurring during biofilm formation. Biochemical and genetic evidence support the hypothesis that bacteria face different conditions within a biofilm as compared with planktonic growth (Huang et al. 1998; Prigent-Combaret et al. 1999, 2001). Indeed, biofilm bacteria are likely to be subjected to progressive microaerobic conditions, increased osmotic pressure, pH variation and decreased nutrient accessibility. These biofilm conditions often have strong similarities with conditions that prevail in stationary phase (planktonic) cultures, and when the stationary phase character of the bacterial lifestyle within biofilm has been investigated, it has generally been shown that a significant part of the E. coli K-12 biofilm response involves stationary-phase-induced genes (Schembri et al. 2003b; Beloin et al. 2004). Since many changes observed in biofilm gene expression are potentially a consequence rather than a cause of biofilm formation, the question as to whether these genes encode functions that are required by, or that are induced by, biofilm conditions remains to be determined.

4.3.2 Regulation of Biofilm Formation by Central Carbon Flux

Catabolite repression has recently been recognized to be a regulatory signal controlling E. coli biofilm formation (Jackson et al. 2002a). The presence of 0.2% glucose in rich medium appears to decrease biofilm biomass. However, this effect is more pronounced when glucose is added during the initial steps of biofilm formation rather than in later stages of biofilm maturation, suggesting that catabolite repression preferentially affects components required in the early stages of bacterial adhesion (Jackson et al. 2002a). Glucose repression is partially mediated by the cAMP receptor protein CRP. Indeed, a crp mutant displays decreased biofilm formation abilities compared with the wild type (Jackson et al. 2002a). Recently, Domka and co-workers identified two biofilm-induced genes, yceP and yliH (renamed bssS and bssR for regulator ofbiofilm through signal secretion) (Schembri et al. 2003b; Beloin et al. 2004; Ren et al. 2004a), which appear to be key regulators of several genes involved in catabolite repression and could participate in the negative effect of glucose on E. coli biofilm formation (Domka et al. 2006). Mutations in these two genes increased biofilm formation only in the presence of glucose and could possibly reduce both phosphorylation and transport of glucose (Domka et al. 2006). BssS (YceP) and BssR (YliH) could notably repress biofilm formation via different systems modulated by glucose, implicating regulators such as RpoS, CRP, CreC, and CsrA (Domka et al. 2006). These two genes were also recently identified as biofilm-induced when two asymptomatic uropathogenic E. coli strains (83972 and VR50) were grown in urine (Hancock and Klemm 2006). As opposed to results by Domka and co-workers (2006), the mutation of yceP in this study led to decreased biofilm formation, as previously observed in a study on biofilm formation of the F plasmid-bearing strain TG1 (Beloin et al. 2004). Therefore, these two genes, and especially yceP, appear to play differential roles in biofilm formation depending on the growth conditions and strains used.

The CsrA protein has been extensively studied in recent years. This protein has been shown to repress biofilm formation (Jackson et al. 2002b). Until recently, CsrA was thought to affect biofilm formation only through repression of glycogen metabolism and its regulatory effect on the swimming-motility master regulator flhDC (Wei et al. 2001). Expression of csrA is indeed sharply decreased a few hours after initiation of growth on surfaces, a profile that is compatible with the decrease in flagellar gene expression upon attachment. On the other hand, csrA expression is reactivated after maturation of the biofilm (2-day-old biofilm). An increase in csrA expression in mature E. coli biofilm might also lead to resumption of swimming motility. Therefore, there may be a link between increased flagellar gene expression and biofilm detachment after reinitiation of swimming motility, a hypothesis found in early work on motility and biofilm (Pratt and Kolter 1998). In line with this, Jackson and co-workers showed that overexpression of csrA was responsible for biofilm dispersal (Jackson et al. 2002b). Lately, Wang and co-workers have shown that the biofilm effect exerted by CsrA is, in fact, results essentially from its effect on production of the polysaccha-ride adhesin PGA (pgaABCD), with a deletion of csrA having no effect on biofilm formation in a pgaC mutant (Wang et al. 2004, 2005). CsrA directly affects PGA production at a posttranscriptional level and may indirectly affect pgaABCD expression via its effect upon an as yet unidentified regulator of the pgaABCD operon (Wang et al. 2005). Two untranslated RNAs, CsrB and CsrC, antagonize CsrA activity by sequestering this protein; consequently, deletion of either csrB or csrC represses biofilm formation (Wang et al. 2005). The Csr (carbon storage regulatory) system also involves the UvrY protein, the cognate regulator of the two-component system BarA/UvrY (Suzuki et al. 2002; Sahu et al. 2003). Whereas CsrA is necessary for UvrY activity, UvrY in turn activates csrB expression, thus implementing expression of the CsrA/CsrB/CsrC negative regulatory loop (Suzuki et al. 2002). Mutation of either barA or uvrY attenuates biofilm formation, suggesting that BarA and UvrY are necessary for development of a biofilm (Suzuki et al. 2002; Sahu et al. 2003). Besides the high number of genes regulated by UvrY (Oshima et al. 2002), the effect of UvrY on biofilm formation is directly linked to its role on CsrB/CsrC and disappears in a mutant that is unable to synthesize PGA (Wang et al. 2005). Recently, a new activator of pgaABCD, and thus biofilm formation, was discovered: the LysR-type-positive regulator NhaR that seems to activate this operon specifically in response to increased Na+ concentration or pH (Goller et al. 2006).

  1. 3.3 Quorum-Sensing Molecules Regulate E. coli Biofilm Formation
  2. 3.3.1 SdiA, a Homoserine Lactone, Activates UvrY and Biofilm Formation

While E. coli is not known to synthesize N-acylated homoserine lactones (AHL) (Ahmer 2004) and has no apparent AHL synthase in its genome, it contains the sdiA

gene that encodes a protein of the LuxR family. LuxR proteins possess one domain for binding N-acylated homoserine lactones and a second domain for binding DNA. An sdiA mutant has been shown to produce threefold less biofilm than a wild type E. coli strain (Suzuki et al. 2002). This effect appears to be mediated by SdiA activation of the uvrY gene (Suzuki et al. 2002), and consequently, predominantly by the CsrB/CsrC untranslated RNA effect on CsrA. While the environmental signal that permits SdiA of E. coli to regulate uvrY expression remains to be determined, a study by van Houdt and co-workers showed that, at 30°C, E. coli responds in an SdiA-dependent manner to the addition of AHL by modification of expression of 15 genes, including upregulation of uvrY (Van Houdt et al. 2006). Also consistent with these results is the increased expression of sdiA in mutants of either yceP or yliH, whose deletion caused an increase in E. coli biofilm formation (Domka et al. 2006). This leaves us with the possibility that the biofilm formation abilities of E. coli can potentially be modulated by quorum-sensing AHL signaling molecules from other species, eventually interacting with E. coli in natural environments.

  1. 3.3.2 The AI-2 Signaling Molecule Modulates E. coli Biofilm Formation
  2. coli strains do secrete the autoinducer-2 (AI-2) quorum-signaling molecule that is encoded by genes of the luxS family and that has been regarded as a universal cell-cell communication signal (Xavier and Bassler 2003). An lsr-like transporter system has been described recently in E. coli K-12 and this could serve to internalize AI-2 molecules (Xavier and Bassler 2005). Nor does disruption of the AI-2 signaling system of E. coli appear to modify biofilm maturation mediated by dere-pressed IncF plasmids in a flow-cell system (Reisner et al. 2003). A luxS mutation either did not affect or only moderately reduced the initial adhesion steps in biofilm formation on a microtiter plate (Colon-Gonzalez et al. 2004; Gonzalez Barrios et al. 2006). However, a furanone-based molecule that inhibits the E. coli AI-2 signaling system has been shown to decrease the thickness of E. coli biofilm formed on steel coupons or on air-liquid interfaces, and to increase the percentage of dead cells within the same biofilm (Ren et al. 2001, 2004b). Consistently, Gonzalez Barrios and co-workers showed that the addition of AI-2 activated E. coli biofilm formation through a complex regulatory cascade where MqsR (motil-ity quorum-sensing regulator), encoded by a biofilm-induced gene (Ren et al. 2004a), induced the flagellar operon activator two-component system QseBC (Sperandio et al. 2002) that in turn activated E. coli swimming motility (Gonzalez Barrios et al. 2006). In the presence of glucose, YdgG, another protein encoded by a biofilm-induced gene (Ren et al. 2004a) and renamed TqsA (transport quorum-sensing A), may participate in this regulatory cascade by exporting AI-2 molecules outside the cells, as well as the two genes bssS (yceP) and bssR (yliH) that are implicated in catabolite repression of the lsr operon that imports AI-2 into the cells (Domka et al. 2006). Indeed, a deletion of ydgG leads, in LB + glucose, to increased biofilm formation and, in different media, to an increase in intracellular levels of AI-2 (Herzberg et al. 2006). Furthermore, YdgG was found to repress cell surface determinants (genes related to flagellum, type 1 fimbriae, Ag43, curli, and polysaccharide production), as well as 10 genes newly recognized as important for E. coli biofilm formation (yfjR, bioF, yccW, yjbE, yceO, ttdA, fumB, yjiP, gutQ, and yihR), and it appears to control these genes through AI-2 transport (Herzberg et al. 2006).
  3. 3.3.3 Indole

Indole production is a phenotypic trait displayed by several Gram-negative bacteria including E. coli. Indole is produced by the degradation of tryptophane, a reaction performed by tryptophanase encoded by the tnaA gene (Newton and Snell 1964). Indole has been described as a potential extracellular signal (Wang et al. 2001). Genes necessary for indole production (including tnaA) have been shown to be induced by addition of E. coli stationary-phase supernatant (Ren et al. 2004c), suggesting the existence of complex cross-talk between different extracellular signaling pathways. A mutant of E. coli K-12 S17-1 for gene tnaA is unable to produce a biofilm in 96-well polystyrene microtiter plates in LB medium (Di Martino et al. 2002). Addition of exogenous indole has no effect on biofilm formation of S17-1 itself (as shown also for E. coli K-12 MG1655 strains; Bianco et al. 2006), but restores normal biofilm formation in S17-1 tnaA (Di Martino et al. 2003). Moreover, whereas oxindolyl-L-alanine, a specific inhibitor of tryptophanase, has no effect on biofilm development of Klebsiella pneumoniae, an indole nonproducing species, it has a dose-dependent inhibitory effect on biofilm development of S17-1 and also of other indole-producing species such as urinary isolates of E. coli, Klebsiella oxy-toca, Citrobacter koseri, Providencia stuartii, and Morganella morganii grown in LB or synthetic urine (Di Martino et al. 2003). A link between indole and AI-2 pathways has been recently pointed out by Herzberg and co-workers and Domka and co-workers (Domka et al. 2006; Herzberg et al. 2006). In LB + glucose, a mutation of tqsA (ydgG) that exports AI-2 seems to activate the expression of the AcrEF multidrug efflux pump that exports indole outside the cells (Herzberg et al. 2006). Moreover, in LB + glucose and not in LB, mutation of bssS (yceP) or bssR (yliH), which also regulates AI-2 concentration, strongly reduces both intra- and extracellular concentrations of indole in E. coli K-12 BW25113, and at the same time is responsible for induction of expression of acrE and acrF and for repression of mtr, encoding pumps that both export and import indole (Domka et al. 2006). These authors conclude that bssR (yliH) and bssS (yceP) mutants increase biofilm formation by repressing indole concentrations through a catabolite repression-related process; they infer that indole represses E. coli biofilm formation, a conclusion that appears to be in conflict with results of Di Martino and co-workers (Di Martino et al. 2002, 2003). Given the differences in the media (LB or synthetic urine versus LB + glucose) and strains used (S17-1 and other Gram-negative bacteria vs BW25113), as well as the types of mutants analyzed (tnaA vs bssR and bssS), the role of indole in biofilm development appears to depend considerably on the conditions used (as does the role of yceP and yliH), and therefore remains to be elucidated. O-Acetyl-l-Serine, Another Extracellular Signal, Regulates E. coli Biofilm Formation

Another diffusible molecule, O-acetyl-l-serine (OAS), appears to modulate E. coli biofilm formation. A mutation in the gene coding for a serine acetyltransferase cysE, which catalyzes the conversion of serine to O-acetyl-l-serine, was shown to enhance biofilm formation through reduction of the amount of an extracellular signal molecule. The authors suggest that OAS or other cysteine metabolites may play a physiological role, possibly by activating genes whose expression leads to inhibition of biofilm formation (Sturgill et al. 2004).

4.3.4 Regulation of Biofilm and Virulence in E. coli

As stated earlier in this chapter, another regulator, the virulence activator RfaH, has recently been linked to biofilm formation in E. coli (Beloin et al. 2006). A mutation in rfaH was shown to derepress biofilm formation in several E. coli strains, including uropathogenic strain 536. Since expression of several E. coli virulence-associated genes depends on RfaH, the increased biofilm phenotype of the nonvirulent rfaH mutant of strain 536 (Nagy et al. 2002) indicates that RfaH-dependent biofilm formation and virulence gene expression are mutually exclusive processes and that biofilm formation may not be regarded as a virulence trait per se. This idea is currently reinforced by other studies also showing inverse regulation of virulence and biofilm-promoting factors in bacteria such as P. aeruginosa, Xanthomonas campes-tris, and Bordetella bronchiseptica (Dow et al. 2003; Goodman et al. 2004; Irie et al. 2004; Kuchma et al. 2005). Recent data consistently indicate that biofilm of uropathogenic E. coli must be formed at the right place under appropriate conditions, and that this may also promote virulence under certain growth conditions (Anderson et al. 2003; Justice et al. 2004). These results suggest that biofilm formation definitely plays a role in the persistence of bacteria rather than being directly implicated in the infective mechanism itself. However, biofilms constitute reservoirs of bacteria that are potentially virulent, and the switch from biofilm to virulence and vice-versa might well be controlled by several regulators such as RfaH in E. coli.

4.3.5 Influence of Other Global Regulators on E. coli Biofilm Formation

Two regulators, H-NS and RpoS, associated with responses to environmental conditions, also play a role in modulating biofilm formation. H-NS is a nucleoid-associated protein that has been shown to regulate a large number of genes in E. coli (approximately 5% of the E. coli K-12 genome), including numerous cell envelope components such as flagella, type 1 fimbriae, LPS, and colanic acid, most of them linked to environmental stimuli including pH, oxygen, temperature, and osmolarity (Dorman and Bhriain 1992; Sledjeski and Gottesman 1995; Olsen et al.

1998; Soutourina et al. 1999; Hommais et al. 2001; Soutourina and Bertin 2003; Dorman 2004). The pleiotropic nature of the H-NS effect within the cells, as well as the fact that a mutation in the hns gene results in a reduction in the growth rate (Barth et al. 1995), is hardly compatible with a clear definition of its role in E. coli biofilm formation. Nevertheless, H-NS appears to be necessary for E. coli to attach to sand columns when it is grown under oxygen-limited conditions (Landini and Zehnder 2002). H-NS often interferes with the expression of genes that depend on the RpoS sigma factor. This interference occurs both by competing with RpoS for binding to the promoter of these genes and by indirectly repressing rpoS translation and stimulating RpoS turnover (Hengge-Aronis 1996, 2002). Whereas rpoS expression in E. coli seems unchanged between cells grown as a planktonic culture in a chemostat and as a biofilm (Adams and McLean 1999; Schembri et al. 2003b; Beloin et al. 2004), the role of RpoS in biofilm formation remains controversial. Depending on the experimental setup, an rpoS mutation has different effects on E. coli biofilm development, ranging from a strong negative effect to a positive effect (Adams and McLean 1999; Corona-Izquierdo and Membrillo-Hernandez 2002; Jackson et al. 2002b; Schembri et al. 2003b). These results pinpoint the difficulty of comparing studies performed using different experimental protocols, and consequently definitive conclusions cannot be drawn concerning the role of RpoS in the formation of E. coli biofilms.

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