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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. 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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. 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.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. 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).

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Figure 1. Effect of (p)ppGpp on the biofilm formation and MSYA. A. Growth curve of J96 (squares) and J96 relA spoT (triangles) in LB media at 37°C. Growth was followed for 24 h by measuring OD600. The insert shows a close-up of the growth curve between 0 and 5 h. B and C. Biofilm formation observed after static overnight growth in LB media with 3% w/v mannosides (white bars) or without mannosides (black bars). B: with pathogenic isolates (J96 and 536, wt and relA spoT derivatives). C: with the non-pathogenic K12 strains (MG1655 and JKS132, and their relA spoT derivatives). Quantification of biofilms at OD545 is plotted for each strain pair. Qualitative observation of MSYA of the same strains mixed with yeast cells is given below the biofilms formation plots. Formation of aggregates were scored; – (absent) and ++ (strong).

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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).

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Figure 2. Transcriptional expression of type 1 fimbriae is regulated by (p)ppGpp. A. The effect of (p)ppGpp on fimA transcription was analysed using the fimA–lacZYA transcriptional reporter strain AAEC198A. Cultures of wt (AAEC198A, squares), relA (AAG40, circles) and relA spoT (AAG41, triangles) derivatives were grown in LB media at 37°C for 24 h. fimA transcription was monitored by β-galactosidase activity assays and plotted against time. B. The effect of amino acid starvation on fimA transcription. SHX (0.2 mM) was added to mid-log phase growing cells (OD600 of 0.4) and fimA transcription was analysed by monitoring β-galactosidase activity after the addition of SHX in a wt (AAEC198A, squares), relA strain (AAG40, circles) or without addition of SHX (AAEC198A, open squares). C. Effect on fimA transcription by IPTG induced expression of RelA’. Logarithmic growing cultures of AAEC198A harbouring pVI751 were diluted to OD600 of 0.05. 0.02 mM IPTG was used to induce the constitutive RelA’ protein (squares), while the (non-induced) control culture (circles) was supplemented with an equal volume of growth media.

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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.

Table 1. fimA expression in different genetic backgrounds.
Strain genotypeaβ-Gal activity (MU)bRatioc
WtppGpp0Wt/ppGpp0
  • a.

    Genotype in both cognate ppGpp+ (wt) and ppGpp0 strains, see Table 3 for strain names.

  • b.

    β-Galactosidase activity (Miller units, MU) measured after growth to mid-log phase (OD600 of 0.5) in LB media at 37°C.

  • c.

    The ratio was calculated by dividing the β-galactosidase values of cognate pairs of strains.

fimA–lacZYA  270 ± 14   31 ± 18.7
fimA–lacZYA, fimB, fimE5122 ± 2233585 ± 1171.4
fimA–lacZYA, Δhns trp::Tc  263 ± 10   42 ± 36.3
fimA–lacZYA, rpoS359::Tn10  302 ± 8   30 ± 28.2

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.

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Figure 3. The phase variation of type 1 fimbriae is modulated by (p)ppGpp. A. Schematic illustration of the PCR-based method used for quantifying the number of ON-cells in a population (i.e. cells that have the promoter directed in the productive orientation). The gel picture shows an example of the separation of the different fragments after 6% acrylamide gel electrophoresis and ethidium bromide staining. B. Quantification of the number of ON-cell in cultures of MG1655 (wt) and CF1693 (relA spoT). Cultures were grown in LB media at 37°C to mid-log phase (OD600 of 0.5) and treated as in (A). C. Quantification of the number of ON-cell in parental, relA and relA spoT derivatives of the pathogenic isolates; J96 and 536, grown in LB media overnight (16 h) at 37°C. Bar diagrams are the average numbers of ON-cells within the cell populations. Inserts show the two larger diagnostic ‘ON’ (484 bp) and ‘OFF’ (402 bp) fragments from one of three experiments used to obtain the standard deviations shown.

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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.

Table 2. fimE and fimB expression in different genetic backgrounds.
Strain genotypeaβ-Gal activity (MU)bRatioc
WtppGpp0Wt/ppGpp0
  • a. Genotype in both cognate ppGpp+ (wt) and ppGpp0 strains, see Table 3 for strain names.

  • b.

    β-Galactosidase activity (Miller units, MU) measured after growth to mid-log phase (OD600 of 0.5) in LB media at 37°C.

  • c.

    The ratio was calculated by dividing the β-galactosidase values of cognate pairs of strains.

fimE-lacZYA  53 ± 13  33 ± 11.6
fimB–lacZYA159 ± 16  49 ± 33.2
fimB–lacZYA, Δhns trp::Tc574 ± 18168 ± 83.4
fimB–lacZYA, rpoS359::Tn10151 ± 9  42 ± 43.6
fimB–lacZYA, ΔnanRΩsacB-Kanr  67 ± 2  24 ± 12.8

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).

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Figure 4. Transcription of the fimB gene is regulated by (p)ppGpp. A. The effect of (p)ppGpp on fimB transcription was analysed using the derivatives of the transcriptional fimB–lacZYA reporter strain AAEC261A. Cultures of wt (AAEC261A, squares), relA (AAG50, circles) and relA spoT (AAG51, triangles) derivatives were grown in LB media at 37°C for 24 h. fimB transcription was monitored by β-galactosidase activity assays and plotted against time. B. The effect of SHX induced amino acid starvation on mid-log phase growing cells (OD600 of 0.4). Closed squares AAEC261A (wt) with SHX; open squares AAEC261A (wt) without SHX; circles AAG50 (relA) with SHX. C. Effect on transcription of fimB by induction of RelA’ expression. Logarithmic growing cultures of AAEC261A (wt) harbouring pVI751 were diluted and treated as described under Fig. 2C. Squares, culture induced by IPTG; circles, control culture with no IPTG. D. Primer extension analysis of total RNA samples from MG1655 (wt) and CF1693 (relA spoT) harbouring the plasmid pAAG25 using the primer pTE-1. The transcriptional start sites from the P1 and P2 promoters of the fimB gene are indicated. Size markers provided by sequencing reactions using the same primer and plasmid pAAG25 as the template are shown to the left. Additional sequence information and precise locations of the P1 and P2 fimB promoters are provided as a supplementary material (Fig. S1). E. Primer extension analysis of total RNA samples from J96 (wt) and J96 relA spoT using the primer fimB-2. The transcriptional start site from the P2 promoter of the fimB gene is shown. F. Effect of induced RelA’ expression on phase variation in the AAEC198A strain harbouring pVI751. At the same time points as in Fig. 2C, samples were taken for analysis of the ON-cell population. The insert picture shows the separation of the diagnostic DNA fragments from the induced culture. The ON-cells in the induced culture (squares) and the control culture (circles) were quantified and are plotted against time. Standard errors from three independent experiments were included in all figures. G. Effect of the plasmids pBR322 (control) and pPKL9, harbouring the fimB gene under the tet promoter in MSYA (top panel) and in the number of ON-cells (lower panel) in the parental MG1655 strain and the ppGpp0 derivative CF1693. Cultures were grown in LB media at 37°C to mid-log phase (OD600 of 0.5) and treated as in Fig. 3A. Formation of aggregates were scored; – (absent), ++ (strong) and +++ (very strong).

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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).

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Figure 5. Analysis of the suppressor mutants with restored expression of the fim genes. A. Analysis of wt (AAEC261A) and spontaneous suppressor clones of the relA spoT strain (AAG51). Clones from AAG51 exhibiting a white colony phenotype and clones from AAG51 with blue phenotype clones (fimB restored expression) isolated under the same conditions were analysed (see table footnotes and Experimental procedure). B. Analysis of clones derived from ppGpp0 pathogenic strains as described in the text. C. Schematic illustration of the known Rif-clusters (black boxes) within RpoB. Solid arrows indicate the location of single amino acid substitutions identified during this study. The most frequently observed mutation is marked in bold. The dotted arrow indicates the location of the rpoB3770 (T563P) allele. D. Effect of the rpoB3770 allele on fimB–lacZYA transcription. The expression of fimB was analysed after introducing the rpoB3770 allele in both wt (AAEC261A) and relA spoT (AAG51) strains. Cultures were grown to mid-log phase (OD600 of 0.5) at 37°C in LB media. Relative transcription was determined setting the β-galactosidase values of the wt strain (AAEC261A) to 1. Standard deviation from two independent cultures of each strain is shown.

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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.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. 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.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. 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.

Table 3.  Bacterial strains and plasmids used in this study.
StrainsRelevant characteristics/descriptionReference/source
J96Pathogenic isolate 
J96 relAJ96 relA251::KmrThis study
J96 relA spoTJ96 relA251::Kmr, spoT207::CmrThis study
536Pathogenic isolate 
536 relA536 relA251::KmrThis study
536 relA spoT536 relA251::Kmr, spoT207::CmrThis study
MG1655F-, ilvG, rph1Guyer et al. (1981)
CF1652MG1655 relA251::KmrXiao et al. (1991)
CF1693MG1655 relA251::Kmr, spoT207::CmrXiao et al. (1991)
JKS132MG1655 ΔfimHSchembri et al. (2002)
AAG30JKS132 relA251::Kmr, spoT207::CmrThis study
AAEC198AMG1655 (ΔlacZYA fimA–lacZYA)Blomfield et al. (1991)
AAG40AAEC198A relA251::KmrThis study
AAG41AAEC198A relA251::Kmr, spoT207::CmrThis study
AAEC374AMG1655 (ΔlacZYA fimA–lacZYA fimB-amb6 fimE-am18, phase locked on)Blomfield et al. (1993)
AAG35AAEC374A relA251::Kmr, spoT207::CmrThis study
AAEC261AMG1655 (ΔlacZYA fimB–lacZYA)Blomfield et al. (1993)
AAG50AAEC261A relA251::KmrThis study
AAG51AAEC261A relA251::Kmr, spoT207::CmrThis study
AAEC200MG1655 (ΔlacZYA fimE–lacZYA)Blomfield et al. (1993)
AAG39AAEC200 relA251::Kmr, spoT207::CmrThis study
AES7AAEC198A Δhns trp::TcrA. Sjöström
AAG49AES7 relA251::Kmr, spoT207::CmrThis study
AES10AAEC261A Δhns trp::TcrA. Sjöström
AAG59AES10 relA251::Kmr, spoT207::CmrThis study
RH90MC4100 rpoS359::Tn10Hengge-Aronis and Fischer (1992)
AAG47AAEC198A rpoS359::Tn10This study
AAG48AAG41 rpoS359::Tn10This study
AAG55AAEC261A rpoS359::Tn10This study
AAG57AAG51 rpoS359::Tn10This study
KCEC341MG1655 ΔnanRΩsacB-KanrSohanpal et al. (2004)
AAG69AAEC261A ΔnanRΩsacB-KanrThis study
AAG70AAG51 ΔnanRΩsacB-KanrThis study
EC3954MG1655 rpoB3370 thi::Tn10V. Shingler
AAG66AAEC261A rpoB3370 thi::Tn10This study
AAG67AAG51 rpoB3370 thi::Tn10This study

Genetic techniques

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.

Biofilm-formation assay

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 assay

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).

Acknowledgement

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. 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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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. 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.

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MMI5191FigandTables.pdf63KSupporting info item

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