Present address: Departament de Microbiologia, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
(p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB
Article first published online: 4 MAY 2006
Volume 60, Issue 6, pages 1520–1533, June 2006
How to Cite
Åberg, A., Shingler, V. and Balsalobre, C. (2006), (p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB. Molecular Microbiology, 60: 1520–1533. doi: 10.1111/j.1365-2958.2006.05191.x
- Issue published online: 4 MAY 2006
- Article first published online: 4 MAY 2006
- Accepted 4 April, 2006.
- Top of page
- Experimental procedures
- Supporting Information
In this report we have examined the role of the regulatory alarmone (p)ppGpp on expression of virulence determinants of uropathogenic Escherichia coli strains. The ability to form biofilms is shown to be markedly diminished in (p)ppGpp-deficient strains. We present evidence (i) that (p)ppGpp tightly regulates expression of the type 1 fimbriae in both commensal and pathogenic E. coli isolates by increasing the subpopulation of cells that express the type 1 fimbriae; and (ii) that the effect of (p)ppGpp on the number of fimbrial expressing cells can ultimately be traced to its role in transcription of the fimB recombinase gene, whose product mediates inversion of the fim promoter to the productive (ON) orientation. Primer extension analysis suggests that the effect of (p)ppGpp on transcription of fimB occurs by altering the activity of only one of the two fimB promoters. Furthermore, spontaneous mutants with properties characteristic of ppGpp0 suppressors restore fimB transcription and consequent downstream effects in the absence of (p)ppGpp. Consistently, the rpoB3770 allele also fully restores transcription of fimB in a ppGpp0 strain and artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation. Our findings suggest that the (p)ppGpp-stimulated expression of type 1 fimbriae may be relevant during the interaction of pathogenic E. coli with the host.
- Top of page
- Experimental procedures
- Supporting Information
Bacteria rapidly adjust their cellular physiology in order to survive and thrive in changing environment. The adaptation of bacterial pathogens to the hostile microenvironment of their hosts requires precise regulatory control of gene expression. The effectors of the bacterial stringent response, guanosine tetra- and pentaphosphate [hereafter referred to as (p)ppGpp] are global regulatory signals that are rapidly produced in response to many environmental cues that result in unfavourable growth conditions (reviewed by Cashel et al., 1996; Magnusson et al., 2005). The synthesis and turnover of (p)ppGpp in Escherichia coli are dependent on two enzymes; the ribosome-associated RelA synthetase and the SpoT protein that has both synthetase and hydrolase activities (Cashel et al., 1996). Several studies have shown that in addition to metabolic processes, cellular functions involved in virulence of different pathogenic bacteria require (p)ppGpp for appropriate regulation and expression (Haralalka et al., 2003; Erickson et al., 2004; Lemos et al., 2004; Pizarro-Cerda and Tedin, 2004; Song et al., 2004; Magnusson et al., 2005).
For E. coli K12, (p)ppGpp has been suggested to be important for survival and biofilm formation (Balzer and McLean, 2002). The ability to form biofilm is considered a virulence factor important for the pathogenicity of several pathotypes of E. coli, including uropathogenic E. coli (Anderson et al., 2004; Kau et al., 2005). A number of bacterial components are important for the establishment of E. coli biofilms, among these are several types of adhesins (Reisner et al., 2003). Type 1 fimbriae are a key virulence factor of uropathogenic E. coli that mediate initial adhesion and invasion of bladder cells by binding to mannose-containing receptors (Hung et al., 2002; Mulvey, 2002; Snyder et al., 2004). In addition to playing a major role in the colonization of various host tissues, type 1 fimbriae have been shown to be important in the initial steps in biofilm formation (Schembri et al., 2001; 2003). Type 1 fimbriae expressing cells arise at several important steps during infection of a murine model of cystitis and are expressed as the bacteria form intracellular biofilm-like communities in epithelial cells (Anderson et al., 2003; Justice et al., 2004).
Type 1 fimbriae are encoded by the chromosomal fim determinant composed of a polycistronic operon comprising the seven structural genes (fimAICDFGH) and two monocistronic operons encoding two site-specific recombinases, FimB and FimE. Transcription of type 1 fimbriae genes is phase variable due to FimB and FimE mediated inversion of a 314 bp DNA fragment that contains the promoter for the polycistronic fim operon (Freitag et al., 1985; Klemm, 1986). Depending on the orientation of this invertible DNA fragment, the promoter is positioned to direct transcription of the structural fim genes (‘ON’ orientation) or not (‘OFF’ orientation). Several studies have provided evidence that the expression of type 1 fimbriae is altered in response to environmental stress conditions such as high osmolarity, pH and temperature, and that expression is induced upon entry into stationary phase (Gally et al., 1993; Dove et al., 1997; Schwan et al., 2002). In this study we demonstrate that the regulatory alarmone (p)ppGpp is involved in the expression of type 1 fimbriae and in the formation of biofilm in uropathogenic E. coli through its role in expression of the FimB recombinase. These results place type 1 fimbriation of E. coli within the stringent response modulon.
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- Experimental procedures
- Supporting Information
(p)ppGpp affect biofilm formation and mannose-sensitive yeast agglutination in both uropathogenic and non-pathogenic E. coli strains
To study the possible role of the regulatory alarmone (p)ppGpp in the virulence of uropathogenic E. coli, mutants incapable of (p)ppGpp synthesis (denoted ppGpp0) were generated using two extensively studied isolates, J96 and 536. The resulting ppGpp0 strains, obtained by transduction of the relA251 and the spoT207 alleles, are incapable of growth on minimal media, as has previously been described for the ppGpp0E. coli K12 strains (Xiao et al., 1991). When cultivated in rich media [Luria–Bertani (LB)], the generation times of the two pathogenic strains and their ppGpp0 derivatives were similar (22 ± 1 min; Fig. 1A, and data not shown).
The ability to form biofilms in the absence of (p)ppGpp was tested by static overnight growth in LB (see Experimental procedures). The ppGpp0 derivatives showed a substantial decrease in their comparative abilities to form biofilms (Fig. 1B, black bars). As expression of type 1 fimbriae is important for establishment of biofilms and these organelles bind to mannose-containing receptors (Anderson et al., 2003; Schembri et al., 2003; Justice et al., 2004), the ability to form biofilms was also tested under conditions where the type 1 adhesin was blocked by the presence of soluble mannosides. A clear reduction in biofilm formation was detected by the addition of mannosides in both wild-type (wt) and ppGpp0 strains (Fig. 1B, white bars). These results suggest that the expression of type 1 fimbriae is important for biofilm formation by these strains, and that the reduction in biofilm formation observed in the ppGpp0 derivatives could be due to low expression of type 1 fimbriae.
Type 1 fimbriae expressing cells rapidly cause agglutination of yeast cells that can also be inhibited by the addition of mannosides. We tested the ability of the different pathogenic strains to cause mannose-sensitive yeast agglutination (MSYA). Consistent with the proposed role for (p)ppGpp in expression of type 1 fimbriae, we found a clear difference between the abilities of the uropathogenic strains and their ppGpp0 counterparts to cause agglutination of yeast. The ppGpp0 strains did not show any agglutination, whereas agglutination was clearly apparent with the wt strains (bottom of Fig. 1B). The agglutination observed with the wt strains could be effectively blocked by the addition of mannosides (data not shown). Similar experiments were performed using relA mutant strains; however, no difference was observed when compared with wt, suggesting that complete loss of (p)ppGpp is required for the effect observed by the MSYA under the conditions used (data not shown).
Biofilm formation and MSYA were also assayed using the non-pathogenic E. coli K12 strain MG1655 and its ppGpp0 counterpart CF1693. The experimental results are similar to those of the pathogenic strains, with a clear decrease in biofilm formation and type 1 fimbriae expression, as observed by MSYA, in the ppGpp0 strain (Fig. 1C). Using a semi-quantitative method for the MSYA, we detected a fourfold reduction of type 1 fimbriae expressing cells in the ppGpp0 strain. When a MG1655 mutant lacking the specific adhesin of type 1 fimbriae was used (ΔfimH, JKS132), very low level of biofilm was observed in both the fimH mutant strain and its ppGpp0 counterpart. Furthermore, both strains were negative when tested for MSYA (Fig. 1C). Consistent with previous work by others (Beloin et al., 2006), the level of biofilm formation is strain-dependent, with the commensal strain MG1655 exhibiting a better ability to produce biofilm than the pathogenic isolates (compare Fig. 1B and C).
Transcription of type 1 fimbriae is regulated by (p)ppGpp
To further characterize the (p)ppGpp-mediated regulation of the type 1 fimbriae, transcriptional analyses were performed. The expression of E. coli type 1 fimbriae was monitored using a MG1655-derived strain (AAEC198A). This strain contains a chromosomal lacZYA transcriptional fusion to the fimA gene that encodes the major fimbrial subunit (fimA–lacZYA). Transcriptional profiles of fimA were monitored through the growth curve for the wt, the relA, and the ppGpp0 derivative. As shown in Fig. 2A (squares) transcription of fimA in the wt strain increases and reaches the maximal level as the cells enter stationary phase. Notably, transcription of fimA is not stimulated upon entry into stationary phase in the ppGpp0 strain, and the level of transcription is reduced about eight to 10-fold (Fig. 2A, triangles). As with the assays described in the preceding section, the relA strain shows a similar profile as the wt strain, although a slightly lower level of β-galactosidase was detected in stationary phase (Fig. 2A, circles).
To confirm that the expression of the fimA gene is affected by alterations in the intracellular levels of the alarmone (p)ppGpp, the effect of induction of (p)ppGpp was tested by two independent strategies. As a first approach, the levels of (p)ppGpp were raised by inducing amino acid starvation in exponentially growing cultures. This was accomplished by addition of serine hydroxamate (SHX) to the growth media, which leads to a very rapid accumulation of (p)ppGpp in the cell (Shand et al., 1989). Accordingly, non-growth inhibiting levels of SHX was added to logarithmically growing cultures of AAEC198A (wt) and AAG40 (relA), and activity from the fimA–lacZYA fusion was followed for 30 min. Transcription of the fimA–lacZYA fusion rapidly increases after the addition of SHX to the wt strain (Fig. 2B). No induction occurs when SHX was added to a culture of the relA strain (Fig. 2B, circles) or when no SHX was added to wt (Fig. 2B, open squares). As a second approach, a plasmid containing a truncated version of the relA gene (pVI751) was introduced into the AAEC198A strain. The 455 aa truncated RelA’ protein is catalytically active independent of association with ribosomes, hence, its overexpression results in a rapid increase in intracellular (p)ppGpp levels (Schreiber et al., 1991; Svitil et al., 1993). Upon IPTG (isopropyl β-D-1-thiogalactopyranoside) induction of the pVI751-encoded RelA’, we observed increased transcription of fimA–lacZYA fusion, which was not observed with a vector control (data not shown) or without addition of IPTG (Fig. 2C).
(p)ppGpp regulates the inversion of the DNA fragment containing the main fim promoter
In vivo transcription of the fimA–lacZYA fusion is > eightfold reduced in the absence of (p)ppGpp (Table 1 and Fig. 2A). Both promoter activity and phase variation contribute to expression of the type 1 fimbriae. To define at which of these two levels (p)ppGpp regulation occurs, strains carrying the fimA–lacZYA fusion but unable to invert the promoter DNA fragment were used (AAEC374A derivatives). These strains have the promoter locked in the ON orientation due to inactivation of both recombinases. In contrast to the phase variable proficient strain that exhibits > eightfold reduction in the absence of (p)ppGpp, only a 1.4-fold reduction attributable to lack of (p)ppGpp was observed in the locked ON strain (Table 1). These results suggest a role of (p)ppGpp in regulating the inversion of the DNA fragment containing the main fim promoter.
|Strain genotypea||β-Gal activity (MU)b||Ratioc|
|fimA–lacZYA||270 ± 14||31 ± 1||8.7|
|fimA–lacZYA, fimB, fimE||5122 ± 223||3585 ± 117||1.4|
|fimA–lacZYA, Δhns trp::Tc||263 ± 10||42 ± 3||6.3|
|fimA–lacZYA, rpoS359::Tn10||302 ± 8||30 ± 2||8.2|
The population of cells with the promoter in the ON orientation (ON-cells) was measured by a PCR-based approach using mid-log phase grown cultures of MG1655 and its ppGpp0 counterpart CF1693 as templates (see Experimental procedures, Fig. 3A). Barely any ON-cells were detected in the ppGpp0 strain and when the percentage of ON-cells was estimated, a sixfold reduction was detected (Fig. 3B). To corroborate these data, we analysed the amount of ON-cells in the uropathogenic strains by taking samples from overnight cultures of the two parental strains (J96 and 536) and their relA and relA spoT derivatives. Depending on the strain background, the reduction in the ppGpp0 strains was between 13- and 20-fold. No significant decrease in the percentage of ON-cells was detected in the relA mutant derivative strains (Fig. 3C). Together, our results suggest that (p)ppGpp primarily regulates the expression of type 1 fimbriae by altering the inversion process, and has little or no direct effect on the fimA promoter activity per se.
The expression of FimB recombinase is regulated by (p)ppGpp
The inversion of the fim promoter region is catalysed by the FimB and FimE recombinases (Klemm, 1986; Gally et al., 1996). To investigate if (p)ppGpp regulates the expression of one or both of the recombinases, the relA251 and spoT207 alleles were introduced into strains with chromosomal transcriptional lacZYA fusions to either fimB (AAEC261A) or fimE (AAEC200). Lack of (p)ppGpp has little effect on fimE transcription, as measured by comparing the β-galactosidase activity of the reporter in the wt and ppGpp0 strains (Table 2). However, > threefold reduction in transcription of fimB was detected in the ppGpp0 strain (Table 2). FimB promotes phase variation of the fim promoter region from the OFF to the ON state and even small variations in FimB levels may cause major changes in the frequency of recombination (Klemm, 1986; Sohanpal et al., 2004). Therefore, reduced expression of FimB in the absence of (p)ppGpp could readily explain the effect of lack of (p)ppGpp on transcription of fimA and subsequent effects on the biofilm formation.
|Strain genotypea||β-Gal activity (MU)b||Ratioc|
|fimE-lacZYA||53 ± 13||33 ± 1||1.6|
|fimB–lacZYA||159 ± 16||49 ± 3||3.2|
|fimB–lacZYA, Δhns trp::Tc||574 ± 18||168 ± 8||3.4|
|fimB–lacZYA, rpoS359::Tn10||151 ± 9||42 ± 4||3.6|
|fimB–lacZYA, ΔnanRΩsacB-Kanr||67 ± 2||24 ± 1||2.8|
To further test this idea, transcription of the fimB–lacZYA fusion was monitored (Fig. 4A–C) in the same series of experiments as described for the fimA–lacZYA fusion (Fig. 2A–C). The fimB transcriptional fusion shows very similar profiles to those of the fimA fusion. First, transcription of fimB is induced as the cells approach stationary phase, with transcription being reduced fourfold in the absence of (p)ppGpp, while a relA mutant strain shows a similar profile as the wt but with a slightly lower level in the stationary phase (Fig. 4A). Second, elevation of (p)ppGpp levels by SHX-induced amino acid starvation in logarithmic growing wt cultures results in a clear induction of fimB transcription after 30 min, with little or no increase in the control culture or the relA mutant (Fig. 4B). Third, induction of the constitutively active RelA’ protein also increases transcription from the fimB promoter 3.5-fold (Fig. 4C).
The ppGpp0 derivatives of transcriptional reporter strains were generated by P1 transduction of the relA251::Kmr, spoT207::Cmr alleles from CF1693. Therefore, although unlikely, the possibility exists that effects attributed to loss of ppGpp could partly be due to unknown co-transduced alterations in adjacent loci, or backbone differences between MG1655 and the uropathogenic strains. To test this possibility, ppGpp0 derivatives of MG1655 and J96 were constructed by directed inactivation of the relA and the spoT genes by gene replacement (see Experimental procedures). Analysis of fimB and fimA transcription in MG1655 and determination of type 1 expression in J96 (MSYA and the percentage of ON-cells) gave essentially identical results as those generated by P1 transduction (data not shown).
We also analysed the effect of (p)ppGpp on the transcription of fimB by primer extension analysis (Fig. 4D). In our experiments, we could detect two transcriptional start points for the fimB gene, a minor start site denoted P1, and a predominant start site denoted P2. These transcriptional start sites are located very close to two promoters previously identified by others (Olsen and Klemm, 1994; Schwan et al., 1994). Consistent with the results obtained with the chromosomal fimB–lacZYA fusion that monitor the activity of both promoters (Fig. 4A), the level of transcripts was clearly reduced in the absence of (p)ppGpp (Fig. 4D). Interestingly, we only observed downregulation (approximately threefold) for the predominant start site associated with the P2 promoter in the ppGpp0 strain, whereas no apparent effect was observed on the P1 promoter (Fig. 4D). Similar transcriptional analysis of fimB expression in the pathogenic J96-derived strains likewise revealed two transcriptional start sites, with approximately threefold lower fimB expression from the P2 promoter in the ppGpp0 strain (Fig. 4E). The reduction in transcript levels detected by primer extension analysis in both MG1655 and pathogenic J96 was similar to the reduction detected using the chromosomal fimB–lacZYA fusion.
As the FimB recombinase regulates the percentage of cells with the fim promoter in the ON orientation, we determined if the percentage of ON-cells increased concomitantly with increased transcription of the fimB gene mediated by induction of the RelA’ protein. Samples were taken after RelA’ induction in AAEC198A at the same time points as in Fig. 2C. A clear increase in the percentage of ON-cells was detectable after 2–3 h of IPTG induction of RelA’ (Fig. 4F, insert), with the ON-cell population increasing to 15% after 3 h (Fig. 4F, squares). This increase did not occur in control cultures without IPTG (Fig. 4F, circles). Likewise, when similar experiments were performed with uropathogenic J96, a 3.5-fold increase in the percentage of ON-cells was observed after induction of the RelA’ protein. These results lead us to conclude that the (p)ppGpp-dependent induction of fimB transcription results in an increase in the percentage of cells having the main fim promoter in the productive orientation (ON-cells).
To further test the idea that ppGpp-deficient strains had reduced type 1 fimbriation is because the decreased levels of FimB, we manipulated FimB levels by introducing plasmid pPKL9, a pBR322-based plasmid that carries the fimB gene under the tet promoter. As it has been previously described (Klemm, 1986; Schembri et al., 2002), elevated levels of FimB increase the levels of ON-cells in the wt strain, and are shown here to also alleviate any detectable difference between the wt and the ppGpp0 derivative (Fig. 4G). Consistent with this result, the presence of plasmid pPKL9 also complement the lack of (p)ppGpp when phenotypic expression of type 1 fimbriae was tested by MSYA, biofilm formation and fimA–lacZYA expression (Fig. 4G and data not shown).
The (p)ppGpp-mediated regulation of FimB recombinase does not involve RpoS, H-NS or NanR
The expression of fimB has been previously described to be regulated by the alternative σ-factor RpoS (the stress/stationary sigma factor) in E. coli K12 W3110 (Dove et al., 1997), the global regulator heat-stable nucleoid-structuring protein (H-NS) (Donato et al., 1997) in E. coli K12 MG1655, and the sialic acid response regulator NanR (Sohanpal et al., 2004) in E. coli K12 MG1655. To determine if any of these regulators are involved in the (p)ppGpp-dependent regulation of fimB, null mutations of each gene were introduced into the wt (AAEC261A) and ppGpp0 (AAG51) fimB–lacZYA reporter strains. The increased level of transcription of fimB observed in the hns mutant (Table 2) as compared with the isogenic wt, is consistent with previously described data (Donato et al., 1997). However, we observed a similar fold increase with the hns ppGpp0 double mutant strain compared with its hns+ ppGpp0 counterpart (Table 2). Thus, the ratio of transcription of fimB in wt and ppGpp0 strains is unaffected by lack of H-NS. Similarly, lack of RpoS or NanR had little effect on relative fimB transcription in the wt as compared with the ppGpp0 strains (Table 2). Thus, taken together, these results suggest that RpoS, H-NS and NanR are not involved in the (p)ppGpp-dependent regulation of fimB. In a comparable series of experiments, we also analysed the effect of rpoS and hns on transcription of fimA in the presence or absence of (p)ppGpp (Table 1). As with fimB, no major differences were observed when comparing the parental and mutant strains.
(p)ppGpp suppressor mutants restore fimB and fim expression
When plated on LB agar plates containing Xgal (5-bromo-4-cloro-3-indolyl-β-D-galactosidase), colonies of the ppGpp0 strain carrying the fimB–lacZYA gene fusion (AAG51) are white, consistent with the very low levels of β-galactosidase generated from the transcriptional fimB–lacZYA fusion of this strain (Fig. 5A and Table 2). However, we occasionally observed blue colonies of AAG51, resembling those of the ppGpp+ parent and indicative of higher transcription from the fimB–lacZYA. To study these compensatory mutations, 88 ppGpp0 derivatives with apparent restored expression (blue colony phenotype) and 88 ppGpp0 clones with a white colony phenotype were isolated and analysed. Transcription from the fimB–lacZYA reporter was analysed by β-galactosidase activity assays using 10 randomly chosen colonies of each phenotype. We found that the white clones showed a similar level of fimB transcription as the parental ppGpp0 strain, while the blue clones showed a similar or even higher level of fimB transcription than the ppGpp+ strain (Fig. 5A).
Failure to grow on minimal media due to the inability to induce the expression of several amino acid biosynthetic operons is a characteristic phenotype of the ppGpp0E. coli (Xiao et al., 1991; Cashel et al., 1996). When we compared the abilities of the 88 clones of both phenotypes to grow on minimal media we found that a very low percentage (4.5%) of the white clones was capable of growth on minimal media, while the majority (91%) of the blue clones could grow on minimal media (Fig. 5A). The results described above suggest that the restoration of transcription of fimB is achieved by suppressor mutations that compensate for the lack of (p)ppGpp. Prototrophy restoring suppressor mutations of ppGpp0 strains are frequently located within rpoB and rpoC genes encoding the β and β′ subunits of RNA polymerase (Cashel et al., 1996; Zhou and Jin, 1998; Murphy and Cashel, 2003). Some of these mutations also mediate resistance to the drug rifampicin (Jin and Gross, 1988; 1989), and usually harbour amino acid substitutions within the known Rif-clusters of the rpoB gene (Murphy and Cashel, 2003; see Fig. 5C). Therefore, we tested all the different clones for resistance to 50 µg ml−1 of rifampicin. As summarized in Fig. 5A, clones with restored transcription of fimB–lacZYA (blue colony phenotype) showed a higher frequency of rifampicin resistance than the white colony phenotype clones (63% compared with 30%). DNA sequencing of the rpoB Rif-clusters of blue colony phenotype clones was performed to identify potential suppressor mutations (see Experimental procedures). Of the 14 tested, 12 clones have single amino acid substitutions of RpoB; Q159P (10/12), G181V (1/12) and G570A (1/12) (Fig. 5C), while the mutant alleles of the remaining two clones tested have not been identified.
To determine if similar results could be obtained with the pathogenic strains, ppGpp0 derivatives of J96 and 536 were grown for 48 h and then plated on minimal media plates and on rich LB agar. Figure 5B summarizes the frequency of minimal media growth as compared with control cultures grown for 4 h only. In all control cultures, the number of minimal media growing colonies was less than 0.3% of those that grew on rich media, while 48 h of growth resulted in a much higher percentage of the cells able to grow on minimal media (ranging from 69% to 73% depending on the strain background). When testing 200 prototrophic clones derived from the 48 h growth conditions for each strain, we found that 12–35% also exhibit resistance to 50 µg ml−1 rifampicin (Fig. 5B). In order to test if the prototrophic rifr clones also exhibit restored type 1 fimbriae expression, we determined the ON-cell population in six clones derived from the ppGpp0 derivatives of J96. The average value determined for these clones shows increased number of ON-cells as compared with the parental ppGpp0 strain, and similar value as J96 (Figs 5B and 3A). The Rif-clusters of the rpoB gene of three of these suppressor clones were sequenced and found to contain the amino acid substitution S788F (Fig. 5C).
To corroborate the apparent genetic link between the expression of fimB and suppressor mutations of the ppGpp0 phenotype, the transcription of the fimB–lacZYA fusion was analysed with an extensively studied rpoB mutant allele. The rpoB3370 (T563P) allele has been isolated independently by several research groups when looking for suppression of ppGpp0 phenotypes and/or resistance to rifampicin (Zhou and Jin, 1998; Murphy and Cashel, 2003). The rpoB3370 allele was introduced into AAEC261A (wt) and AAG51 (ppGpp0), and the transcription of the fimB–lacZYA fusion in mid-log phase cultures (OD600 of 0.5) was assessed by β-galactosidase assays. We found that the rpoB3370 allele restores fimB transcription in the ppGpp0 strain to the same levels as observed in the ppGpp+ wt strain, which is itself slightly stimulated by this rpoB mutation (Fig. 5D). Similar results were obtained with cultures at late log phase (OD600 of 1.5; data not shown). Thus, these genetic experiments corroborate and underscore the physiological significance of the data suggested from the results of manipulation of ppGpp levels via expression of RelA’ and by SHX treatment.
- Top of page
- Experimental procedures
- Supporting Information
The ability to form biofilm is a key feature during the pathogenesis of several microbes (Donlan and Costerton, 2002; Kau et al., 2005). Recently, it has been shown that uropathogenic E. coli form complex bacterial communities with biofilm-like traits during intracellular growth in umbrella cells of the bladder (Anderson et al., 2003; Justice et al., 2004). The establishment of biofilm communities is a multifactor process in which commitment to growth as a biofilm can be instigated in response to several environmental inputs and physiological stresses (Stanley and Lazazzera, 2004). However, which regulatory factors are involved in transduction of these signals is not fully understood. In this report, we show that the biofilm forming abilities of two uropathogenic and a non-pathogenic E. coli K12 strain are regulated by the stringent response alarmone (p)ppGpp, as evidenced by reduced biofilm formation by strains lacking (p)ppGpp (Fig. 1). Type 1 fimbriae are involved in the initial steps of biofilm formation by E. coli (Schembri et al., 2003; Justice et al., 2004). We show here that (p)ppGpp-mediated regulation of biofilm formation is associated with expression of type 1 fimbriae (Fig. 1), and that transcription of the fim genes is itself regulated by (p)ppGpp (Fig. 2), through increased numbers of cells in the population that have the promoter in the productive ON orientation (Fig. 3). This increase in the number of ON-cells can be traced to a role for (p)ppGpp in efficient transcription of the fimB gene whose product stimulates inversion of the DNA fragment containing the fim promoter to the ON orientation (Fig. 4). Metabolic stress and many environmental conditions that reduce bacterial growth are cues for rapid induction of the intracellular levels of (p)ppGpp (Cashel et al., 1996). It has been shown that intracellularly growing uropathogenic E. coli pass through different developmental stages. After an initial stage of rapid growth, the bacteria mature to a slower growing population that express biofilm traits, such as the expression of type 1 fimbriae and another adhesin Ag43 (Anderson et al., 2003; Justice et al., 2004). Our results provide a possible regulatory mechanism for the changes in expression patterns upon slower growth of intracellular bacteria. Under this scenario, reduced growth rate would presumably induce elevated levels of (p)ppGpp, which in turn would stimulate the expression of type 1 fimbriae, to trigger successful establishment of an intracellular biofilm-like community.
The effect of (p)ppGpp on the expression of type 1 fimbriae appears to operate almost exclusively through modification of phase variation of the fim promoter region to the ON orientation. This conclusion is based on the following findings. First, in the absence of phase variation (p)ppGpp has little effect on transcription of the fim promoter per se, but phase variable strains that lack (p)ppGpp have a > eightfold reduced expression of a fimA–lacZYA transcription reporter (Fig. 2, Table 1). Second, a phase variable strain lacking (p)ppGpp have a reduced population of cells with the fim promoter in the ON orientation, while manipulations that increase (p)ppGpp levels increases the ON-cell population size (Figs 3 and 4). Third, transcription of the fimB gene that encodes the OFF-to-ON FimB recombinase is > threefold higher in ppGpp+ strains than in ppGpp0 strains, while that of the ON-to-OFF FimE recombinase is only mildly affected (Table 2). Fourth, artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation (Fig. 4G). Hence, we conclude that it is ultimately the (p)ppGpp-mediated enhancement of the FimB recombinase expression that results in (p)ppGpp-dependent increases of E. coli type 1 fimbriation.
None of three proteins previously implicated in transcriptional regulation of fimB (H-NS, RpoS and NanR) appear to contribute to the (p)ppGpp-mediated regulation of fimB transcription (Table 2). Moreover, as (p)ppGpp deficiency cause similar effect in both uropathogenic and the K12 strain that lacks the PapB regulator, the cross-talk between PapB and type 1 fimbriae (Xia et al., 2000) cannot account for the (p)ppGpp-mediated regulation. Interestingly, lack of (p)ppGpp results in a decrease in the number of transcripts from only one of the two fimB promoters, namely the P2 promoter (Fig. 4D; Fig. S1). Spontaneous mutants that restore fimB transcription in ppGpp0 strains, and have phenotypes of known RNA polymerase suppressor that compensate for lack of cellular (p)ppGpp (Zhou and Jin, 1998; Murphy and Cashel, 2003) can be readily isolated (Fig. 5A–C). In addition, the defined RpoB-T563P mutant allele restores transcription of fimB in a ppGpp0 strain (Fig. 5D). These results provide strong genetic evidence that the action of (p)ppGpp on fimB transcription is mediated through its effects on the transcriptional apparatus. Taken together, the results suggest that (p)ppGpp acts directly by modulating initiation of transcription at the P2 fimB promoter. However, we cannot exclude the possibility that some unknown activator of fimB transcription, that is dependent on (p)ppGpp for its expression, underlies the (p)ppGpp-mediated regulation of this promoter. Distinguishing between these two possibilities is a focus of ongoing research.
The mechanism of positive regulation of genes by (p)ppGpp has not been as extensively studied as for negatively regulated genes. Paul et al. have shown the importance of the critical cofactor DksA in both ppGpp-mediated positive regulation of E. coli amino acid biosynthesis promoters (Paul et al., 2005), and negative regulation of an rRNA promoter (Paul et al., 2004a). Although in many cases the phenotypes of a DksA null strain resemble those of ppGpp0 strains, the phenotypes do not completely overlap (Magnusson et al., 2005 and references therein). Transcription of fimB and fimA in a DksA null strain has been studied, but in neither case was transcription observed to be downregulated, as it is in (p)ppGpp0 strains (A. Åberg et al., unpubl. data).
Growth as biofilms can be considered a survival strategy, which is shown here to be under the control of (p)ppGpp. Production of (p)ppGpp elicits the classic stringent response in which the translational capacity of the cell is downregulated to adjust to the reduced demand under slow growth conditions (reviewed in Paul et al., 2004b). As such, the involvement of (p)ppGpp in eliciting increased expression of the protein-rich extracellular type 1 fimbriae, may appear rather unexpected. However, (p)ppGpp is involved in more than metabolic regulation and, as a global regulator of the bacterial expression (p)ppGpp controls a number of cellular processes associated with survival in the face of reduced growth and stress (Magnusson et al., 2005 and references therein). Thus, induction of intracellular (p)ppGpp levels can be considered as an alert signal that promotes expression of genes, including those for type 1 fimbriae, which could increase the probability of surviving adverse conditions. Research during the last years has suggested that ubiquitous regulatory networks that control household metabolism have been adopted to regulate cellular functions that are present and/or relevant only for a subset of strains (Finlay and Falkow, 1997). Regulation of virulence functions of certain pathogenic bacteria strains is a case in point. This study provides such an example, namely the involvement of the effectors of the stringent response in regulating expression of an adhesive organelle that is important in the first steps of establishment of an infectious process of uropathogenic E. coli. The alarmone (p)ppGpp has also previously been shown to be involved in regulation of different virulence factors in a range of non-E. coli pathogenic bacteria (Godfrey et al., 2002; Haralalka et al., 2003; Erickson et al., 2004; Lemos et al., 2004; Pizarro-Cerda and Tedin, 2004; Song et al., 2004). The levels of (p)ppGpp are likely to be rapidly induced in bacteria in response to almost any environment that would result in slow proliferation. This appears to make (p)ppGpp an ideal signal to couple to regulation of virulence factors that need to be expressed during growth and survival inside the host and/or in response to eukaryotic defence systems.
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- Experimental procedures
- Supporting Information
Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used are shown in Table 3 and Table S1 respectively. Strains were grown in LB media (Bertani, 1951) at 37°C with vigorous shaking (200 rpm), unless otherwise stated. When necessary, antibiotics were used at the following concentrations; carbenicillin 50 µg ml−1, chloramphenicol 15 µg ml−1, kanamycin 25 µg ml−1, tetracycline 12.5 µg ml−1 and rifampicin 50 µg ml−1. Bacterial growth was monitored by measuring OD600 in Beckman DU®-68 Spectrophotometer, where 0.5 unit of OD600 corresponds to mid-log phase growth and 5 × 108 bacteria ml−1. When required, LB agar plates (Bertani, 1951) were supplemented with 40 µg ml−1 of Xgal. Minimal media plates with the following composition were used; 1 × M9 salts (Sambrook and Russell, 2001), 0.4 mM glucose, 10 µM thiamine and 1.5% bactoagar. For relA’ induction from the pVI751 plasmid, IPTG (Sigma) was added to the media to a final concentration of 0.02 mM.
|J96 relA||J96 relA251::Kmr||This study|
|J96 relA spoT||J96 relA251::Kmr, spoT207::Cmr||This study|
|536 relA||536 relA251::Kmr||This study|
|536 relA spoT||536 relA251::Kmr, spoT207::Cmr||This study|
|MG1655||F-, ilvG, rph1||Guyer et al. (1981)|
|CF1652||MG1655 relA251::Kmr||Xiao et al. (1991)|
|CF1693||MG1655 relA251::Kmr, spoT207::Cmr||Xiao et al. (1991)|
|JKS132||MG1655 ΔfimH||Schembri et al. (2002)|
|AAG30||JKS132 relA251::Kmr, spoT207::Cmr||This study|
|AAEC198A||MG1655 (ΔlacZYA fimA–lacZYA)||Blomfield et al. (1991)|
|AAG40||AAEC198A relA251::Kmr||This study|
|AAG41||AAEC198A relA251::Kmr, spoT207::Cmr||This study|
|AAEC374A||MG1655 (ΔlacZYA fimA–lacZYA fimB-amb6 fimE-am18, phase locked on)||Blomfield et al. (1993)|
|AAG35||AAEC374A relA251::Kmr, spoT207::Cmr||This study|
|AAEC261A||MG1655 (ΔlacZYA fimB–lacZYA)||Blomfield et al. (1993)|
|AAG50||AAEC261A relA251::Kmr||This study|
|AAG51||AAEC261A relA251::Kmr, spoT207::Cmr||This study|
|AAEC200||MG1655 (ΔlacZYA fimE–lacZYA)||Blomfield et al. (1993)|
|AAG39||AAEC200 relA251::Kmr, spoT207::Cmr||This study|
|AES7||AAEC198A Δhns trp::Tcr||A. Sjöström|
|AAG49||AES7 relA251::Kmr, spoT207::Cmr||This study|
|AES10||AAEC261A Δhns trp::Tcr||A. Sjöström|
|AAG59||AES10 relA251::Kmr, spoT207::Cmr||This study|
|RH90||MC4100 rpoS359::Tn10||Hengge-Aronis and Fischer (1992)|
|AAG47||AAEC198A rpoS359::Tn10||This study|
|AAG48||AAG41 rpoS359::Tn10||This study|
|AAG55||AAEC261A rpoS359::Tn10||This study|
|AAG57||AAG51 rpoS359::Tn10||This study|
|KCEC341||MG1655 ΔnanRΩsacB-Kanr||Sohanpal et al. (2004)|
|AAG69||AAEC261A ΔnanRΩsacB-Kanr||This study|
|AAG70||AAG51 ΔnanRΩsacB-Kanr||This study|
|EC3954||MG1655 rpoB3370 thi::Tn10||V. Shingler|
|AAG66||AAEC261A rpoB3370 thi::Tn10||This study|
|AAG67||AAG51 rpoB3370 thi::Tn10||This study|
Basic molecular genetic manipulations were performed essentially as described previously (Sambrook and Russell, 2001). All DNA primers used in this work are specified in Table S2. DNA sequencing was performed using the DYEnamic ET Terminator Cycle Sequencing Kit according to the manufacturer’s protocol (Amersham Biosciences). PCR reactions used a MJ PTC-100™ thermal cycler (MJ Research). Different gene mutations were introduced by P1 transductions (Willetts et al., 1969); relA251::Kmr from CF1652, spoT207::Cmr allele from CF1693, rpoS359::Tn10 from RH90, rpoB3370 thi::Tn10 from EC3954 and ΔnanRΩsacB-Kanr from KCEC341. The pAAG25 plasmid was constructed by cloning the PCR products of fimB-1 and fimB-2 primers between the EcoRI and BamHI sites of pTE103. The fidelity of the PCR amplified DNA was confirmed by DNA sequencing. Plasmid pVI751 was constructed by cloning the relA’-containing EcoRI to PstI fragment of pALS13 between these sites of pMMB66EH. Gene disruption was carried out by allelic exchange using the suicide plasmid pKO3 (in MG1655 derivatives) as described (Link et al., 1997; Merlin et al., 2002), or by using lambda Red-mediated recombination of linear DNA fragments (in J96) as described (Datsenko and Wanner, 2000; Murphy and Campellone, 2003). The relA and spoT alleles were created as follows. relA alleles: in MG1655 deletions from amino acid 6 to 743 and in J96 deletions from amino acid 4 to 733; spoT alleles: in MG1655 deletions from amino acid 7 to 699 and in J96 deletions from amino acid 3 to 699. relA spoT derivatives strains from MG1655 and J96 carrying the corresponding new alleles showed auxotrophy.
Briefly, 10 µl of bacterial culture (OD600 of 0.4) was inoculated into 190 µl of LB media containing appropriate antibiotics, either with or without 3% w/v mannose (Methyl α-D-mannopyranoside, Sigma). Cultures in wells of a non-tissue culture treated U-bottom 96-well plastic plate were incubated statically at 37°C for 15 h, thereafter the medium was discarded and the wells were washed with phosphate-buffered saline (PBS). Quantification of biofilm formation was performed as previously described by Stepanovic et al. (2000).
Mannose-sensitive yeast agglutination assay
Bacterial cultures were grown for 16 h under shaking conditions, washed in PBS, and resuspended to a concentration of 5 × 109 bacteria ml−1. Yeast cells (Saccharomyces cerevisiae) were washed and resuspended in PBS with or without 3% w/v mannose (Methyl α-D-mannopyranoside, Sigma) to an OD600 of 5. Bacteria and yeast cells were mixed in proportion 1:1 (v/v) on a glass-slide and incubated on ice for 30 min. The presence of aggregates was considered as agglutination positive and is reported as – (absent), ++ (strong) and +++ (very strong).
Detection and quantification of the ON/OFF state
Detection and quantification of the percentage of cells with the fim promoter in the ON orientation were performed by a PCR amplification-based method as described earlier (Xia et al., 2000) using primers 2535 and 3137. These primers amplify the promoter region, and the resulting fragments were digested with HinfI and separated in a 6.5% TBE-acrylamide-gel (see illustration in Fig. 3A). Ethidium bromide stained gels were quantified using the FlourS Multi-Imager system equipped with the QuantityOne analysis software (Bio-Rad). The images were electronically inverted to facilitate visualization of the bands. The intensities of the bands corresponding to the 118 bp (ON) and 200 bp (OFF) fragments were measured. As the intensity directly correlates with the sizes of the fragment, a normalization procedure was applied for the 118 bp fragment band using the following formula: Intensity(ONcorr) = (intensityON)/fragment size(ON)) × fragment size(OFF). In order to estimate the percentage of ON-cells in a specific sample, the corrected intensity values of the ON-band were compared with the total intensity of both the ON- and the OFF-bands. For each sample, at least two PCR reactions and two gel analyses of each PCR reaction were performed.
β-Galactosidase activity measurements were performed as described by Miller (1992). Data are mean values of duplicate determinations in at least three independent experiments plotted with standard errors. Control primer extension analysis from the lacZ reporter in wt and ppGpp0 K12 strains gave essentially identical results (data not shown), indicating that activity assays accurately reflect the lacZ mRNA levels.
Amino acid starvation experiment
Amino acid starvation was induced by addition of SHX (Sigma) to logarithmic growing cells (OD600 of 0.4). Experiments using a range of SHX from 0.2 to 4 mM showed that the chosen concentration of 0.2 mM of SHX had no discernable effect on growth rate of wt strains. Samples, taken at different time points after the addition of SHX, were used for both β-galactosidase activity assays and determination of the percentage of ON-cells. As controls, replica cultures were treated identically but without added SHX.
Primer extension analysis
The primer extension reactions were performed as described by Balsalobre et al. (2003). RNA was isolated using the Total RNA Midi isolation kit from VIOGENE and 5 ml cultures grown to the beginning of stationary phase (OD600 of 1.5) in LB media at 37°C. RNA samples from strains containing fimB promoter plasmid pAAG25 were analysed using [γ-32P]-ATP kinase-labelled oligonucleotide pTE-1. RNA samples from J96 and J96 relA spoT were analysed using fimB-2. Control sequencing reactions were performed using the T7 sequencing kit (Amersham Biosciences) according to the manufacturer’s instructions.
Isolation of suppressor mutants
For the isolation of suppressor mutants with restored transcription of fimB, the ppGpp0 strain AAG51 that harbours a fimB–lacZYA chromosomal fusion was used. This strain has a white phenotype on LB agar plates containing Xgal due to the low expression of fimB. After 48 h growth in rich media, AAG51 was plated on plates containing Xgal and the appearance of suppressor mutants with restored fimB expression (blue colony phenotype) was assessed. A collection of 88 colonies of each phenotype (blue and white colonies) was isolated from five independent cultures. To isolate suppressor mutants from the pathogenic J96 and 536 strains, cultures were grown for 4 h (control) or 48 h in LB. Different dilutions were plated on minimal media and LB agar plates.
Mapping of the suppressor mutants
Mapping of rifr suppressor mutants within the rpoB gene was done by PCR amplification of DNA spanning the known Rif-clusters (Fig. 5C) and subsequent sequencing of the PCR products. Fragments amplified from AAG51 or the pathogenic derivative strains were sequenced as controls. Each clone was sequenced in both directions at least three times. Primers rpoB-1 and rpoB-2 amplify a DNA fragment spanning Rif regions I and II (amino acid 450–626); primers rpoB-3 and rpoB-4 amplify a DNA fragment spanning Rif region IV (amino acid 91–261); and primers rpoB-5 and rpoB-6 amplify a DNA fragment spanning Rif region III (amino acid 617–804).
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- Supporting Information
We thank Anna Bergelin for her contribution to the work with the pathogenic strains, Dr Chun Chau Sze for construction of pVI751, Annika Sjöström for providing us with the hns mutant strains, Dr Per Klemm for plasmid pPKL9, Dr Mark A. Schembri for the fimH mutant strain and Dr Ian C. Blomfield for the nanR mutant. This work was supported by grants from the Swedish Research Council, the Faculty of Medicine at Umeå University and the Program Ramón y Cajal of the Spanish Ministry of Education and Sciences.
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- Experimental procedures
- Supporting Information
- 2003) Intracellular bacterial biofilm-like pods in urinary tract infections. Science 301: 105–107. , , , , , and (
- 2004) Intracellular bacterial communities of uropathogenic Escherichia coli in urinary tract pathogenesis. Trends Microbiol 12: 424–430. , , , and (
- 2003) Transcriptional analysis of the sfa determinant revealing mRNA processing events in the biogenesis of S fimbriae in pathogenic Escherichia coli. J Bacteriol 185: 620–629. , , , , and (
- 2002) The stringent response genes relA and spoT are important for Escherichia coli biofilms under slow-growth conditions. Can J Microbiol 48: 675–680. , and (
- 2006) The transcriptional antiterminator RfaH represses biofilm formation in Escherichia coli. J Bacteriol 188: 1316–1331. , , , , , , and (
- 1951) Studies on lysogenesis I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 63: 293–300. (
- 1991) Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants. Mol Microbiol 5: 1439–1445. , , and (
- 1993) Lrp stimulates phase variation of type 1 fimbriation in Escherichia coli K-12. J Bacteriol 175: 27–36. , , , , and (
- 1996) The stringent response. In Escherichia coli and Salmonella Cellular and Molecular Biology. Vol. 1. Neidhardt, F.C., CurtissR., III, Ingraham, J.L., Lin, E.C.C., Low, K.B., Magasanik, B., et al. (eds). Washington, DC: ASM Press, pp. 1458–1496. , , , and (
- 2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97: 6640–6645. , and (
- 1997) Promoter-specific repression of fimB expression by the Escherichia coli nucleoid-associated protein H-NS. J Bacteriol 179: 6618–6625. , , and (
- 2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15: 167–193. , and (
- 1997) Control of Escherichia coli type 1 fimbrial gene expression in stationary phase: a negative role for RpoS. Mol Gen Genet 254: 13–20. , , and (
- 2004) Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster. Infect Immun 72: 5638–5645. , , , , and (
- 1997) Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61: 136–169. , and (
- 1985) Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli. J Bacteriol 162: 668–675. , , , and (
- 1993) Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media. J Bacteriol 175: 6186–6193. , , , and (
- 1996) Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K-12. Mol Microbiol 21: 725–738. , , and (
- 2002) The role of the stringent response in the pathogenesis of bacterial infections. Trends Microbiol 10: 349–351. , , and (
- 1981) Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol 45: 135–140. , , , and (
- 2003) Mutation in the relA gene of Vibrio cholerae affects in vitro and in vivo expression of virulence factors. J Bacteriol 185: 4672–4682. , , and (
- 1992) Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli. Mol Microbiol 6: 1877–1886. , and (
- 2002) Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection. Mol Microbiol 44: 903–915. , , , , , , et al. (
- 1988) Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J Mol Biol 202: 45–58. , and (
- 1989) Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli. J Bacteriol 171: 5229–5231. , and (
- 2004) Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc Natl Acad Sci USA 101: 1333–1338. , , , , , , and (
- 2005) Interaction of uropathogenic Escherichia coli with host uroepithelium. Curr Opin Microbiol 8: 54–59. , , and (
- 1986) Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli. EMBO J 5: 1389–1393. (
- 2004) Effects of RelA on key virulence properties of planktonic and biofilm populations of Streptococcus mutans. Infect Immun 72: 1431–1440. , and (
- 1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol 179: 6228–6237. , , and (
- 2005) ppGpp: a global regulator in Escherichia coli. Trends Microbiol 13: 236–242. , , and (
- 2002) Tools for characterization of Escherichia coli genes of unknown function. J Bacteriol 184: 4573–4581. , , and (
- 1992) A Short Course in Bacterial Genetics – Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. (
- 2002) Adhesion and entry of uropathogenic Escherichia coli. Cell Microbiol 4: 257–271. (
- 2003) Isolation of RNA polymerase suppressors of a (p)ppGpp deficiency. Methods Enzymol 371: 596–601. , and (
- 2003) Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol 4: 11. , and (
- 1994) Localization of promoters in the fim gene cluster and the effect of H-NS on the transcription of fimB and fimE. FEMS Microbiol Lett 116: 95–100. , and (
- 2004a) DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell 118: 311–322. , , , , , , and (
- 2004b) rRNA transcription in Escherichia coli. Annu Rev Genet 38: 749–770. , , , and (
- 2005) DksA potentiates direct activation of amino acid promoters by ppGpp. Proc Natl Acad Sci USA 102: 7823–7828. , , and (
- 2004) The bacterial signal molecule, ppGpp, regulates Salmonella virulence gene expression. Mol Microbiol 52: 1827–1844. , and (
- 2003) Development and maturation of Escherichia coli K-12 biofilms. Mol Microbiol 48: 933–946. , , , , and (
- 2001) Molecular Cloning – A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. , and (
- 2001) FimH-mediated autoaggregation of Escherichia coli. Mol Microbiol 41: 1419–1430. , , and (
- 2002) DNA microarray analysis of fim mutations in Escherichia coli. Mol Genet Genomics 267: 721–729. , , , , and (
- 2003) Global gene expression in Escherichia coli biofilms. Mol Microbiol 48: 253–267. , , and (
- 1991) Overexpression of the relA gene in Escherichia coli. J Biol Chem 266: 3760–3767. , , , , , and (
- 1994) Analysis of the fimB promoter region involved in type 1 pilus phase variation in Escherichia coli. Mol Gen Genet 242: 623–630. , , and (
- 2002) Osmolarity and pH growth conditions regulate fim gene transcription and type 1 pilus expression in uropathogenic Escherichia coli. Infect Immun 70: 1391–1402. , , , , and (
- 1989) Correlation between histidine operon expression and guanosine 5′-diphosphate-3′-diphosphate levels during amino acid downshift in stringent and relaxed strains of Salmonella typhimurium. J Bacteriol 171: 737–743. , , , , and (
- 2004) Transcriptome of uropathogenic Escherichia coli during urinary tract infection. Infect Immun 72: 6373–6381. , , , , , , et al. (
- 2004) Integrated regulatory responses of fimB to N-acetylneuraminic (sialic) acid and GlcNAc in Escherichia coli K-12. Proc Natl Acad Sci USA 101: 16322–16327. , , , , and (
- 2004) ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1. J Biol Chem 279: 34183–34190. , , , , , , et al. (
- 2004) Environmental signals and regulatory pathways that influence biofilm formation. Mol Microbiol 52: 917–924. , and (
- 2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40: 175–179. , , , , and (
- 1993) Guanosine tetraphosphate inhibits protein synthesis in vivo. A possible protective mechanism for starvation stress in Escherichia coli. J Biol Chem 268: 2307–2311. , , and (
- 1969) Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J Bacteriol 97: 244–249. , , and (
- 2000) Regulatory cross-talk between adhesin operons in Escherichia coli: inhibition of type 1 fimbriae expression by the PapB protein. EMBO J 19: 1450–1457. , , , and (
- 1991) Residual guanosine 3′,5′-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem 266: 5980–5990. , , , , , and (
- 1998) The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like ‘stringent’ RNA polymerases in Escherichia coli. Proc Natl Acad Sci USA 95: 2908–2913. , and (
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- Experimental procedures
- Supporting Information
Fig. S1. Schematic representation of the promoter region of fimB. Table S1. Plasmids used in this study. Table S2. Oligonucleotides used in this study.
|MMI5191FigandTables.pdf||63K||Supporting info item|
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