1 Swiss Federal Institute of Environmental Technology (EAWAG), Überlandstrasse 133, CH-8600 Dübendorf, Switzerland
2 Unité de Microbiologie et Génétique (CNRS UMR 5122), Institut National des Sciences Appliquées de Lyon, 10 rue Dubois, 69622 Villeurbanne Cedex, France
Correspondence
Paolo Landini
landini{at}eawag.ch
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ABSTRACT |
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INTRODUCTION |
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Several extracellular structures including lipopolysaccharide (Jucker et al., 1998; Williams & Fletcher, 1996
), flagella and pili (Pratt & Kolter, 1998
) and curli (Olsen et al., 1989
; Vidal et al., 1998
) are involved in initial adhesion of bacterial cells to a solid surface and/or in subsequent steps of biofilm formation. Curli are fibrils made of protein present in Escherichia coli and Salmonella spp., where they are known as thin aggregative fimbriae (Romling et al., 1998b
). In E. coli, curli promote both initial adhesion and cellcell interaction (Prigent-Combaret et al., 2000
). Genes involved in curli production are clustered in two divergent operons: the csgBA operon, encoding the structural components of curli; and the csgDEFG operon, encoding genes necessary for export of the curli subunit and stabilization of the fibres (the csgEG genes; Chapman et al., 2002
) and csgD, a transcription factor belonging to the luxR family necessary for csgBA expression (Hammar et al., 1995
). Despite this gene organization being extremely conserved among E. coli and Salmonella enterica strains (Romling et al., 1998a
), the curli-encoding genes are not expressed in many laboratory strains of E. coli, due to silencing of the csgD promoter (Hammar et al., 1995
). Expression of the csgD promoter is affected, either positively or negatively, by several transcriptional regulators, including rpoS, crl, hns and ompR (Romling et al., 1998b
; Prigent-Combaret et al., 2001
). Mutations in these regulatory genes, such as the ompR234 mutation, which results in more efficient ompR-dependent activation of the csgD promoter, can stimulate curli production and biofilm formation in laboratory strains (Vidal et al., 1998
; Prigent-Combaret et al., 2001
). In this report, we investigated the effects of the ompR234 mutation on global transcription regulation in the E. coli MG1655 laboratory strain and on the ability of this strain to attach to solid surfaces. We show that ompR234-dependent activation of csgD results in activation of the csgBA and yaiC operons in the stationary phase of growth. An 11 bp sequence conserved in the two promoters appears to be necessary for csgD activation. This putative binding site is conserved in other promoters, two of which, pepD and yagS, are negatively regulated by csgD. Thus we conclude that ompR-dependent activation of csgD results in a co-ordinated response leading to regulation of at least four independent genes or operons.
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METHODS |
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Biofilm and adhesion assays.
Determination of biofilm formation in microtitre plates was carried out as in Dorel et al. (1999). The ratio between surface-attached and unattached bacteria was estimated by measuring the OD600. At least three independent assays were performed and means were calculated. To determine initial attachment to a solid surface, we used the sand column assay described in Simoni et al. (1998)
. Bacteria were grown to either mid-exponential or stationary phase in M9/Glucose supplemented with 5 % L broth, harvested, washed and resuspended in PBS to an OD280=0·8 (initial absorbance, A0). The bacterial suspension was loaded onto a column filled with fine sea sand grains (9 g sand) and fractions were collected at the outlet. The ratio between the number of bacteria in the flow through and in the initial suspension (A/A0, where A is the absorbance of the bacterial suspension at the output of the column) was determined spectrophotometrically. The percentage of cells adhering to the column is calculated as (1-A/A0)x100. Microscopical analysis of the column sand grains shows that bacteria attach as single cells in the conditions used in our experiments (data not shown).
Global transcription experiments.
The Panorama gene array system (Sigma) was used to compare global gene expression in the MG1655 (wild-type) and the PHL628 (ompR234) strains. Both strains were grown for 14 h in M9/Glucose supplemented with 5 % L broth at 28 °C. Total RNA was isolated as described by Sambrook et al. (1989) and 2 µg was subjected to RT-PCR according to the manufacturer's instructions in the presence of 20 µCi (740 kBq) per reaction of [
-33P]ATP in a final volume of 50 µl. The mixture was loaded onto Sephadex G-50 to remove the unincorporated nucleotides; the products of the RT-PCR were eluted in 200 µl TE buffer (10 mM Tris/HCl, pH 8·0; 1 mM EDTA) and directly used for hybridization. The gene arrays were exposed to a phosphorimager (Molecular Dynamics) and analysed as described in Tao et al. (1999)
. The intensity of each spot was quantified and the local background was subtracted using the Array Vision software (Research Imaging). The duplicate spots were averaged and expressed as a percentage of the total of intensities of all the spots on the DNA array. This value was used to calculate the ratio of mRNA levels of PHL628 and MG1655. The correlation coefficients of the percentage intensities determined individually for the duplicate spots on a single blot ranged from 0·953 to 0·990. Two independent experiments were performed; we considered as significant differences in expression between MG1655 and PHL628 higher than 2·5-fold. Sequence analysis and searches were carried out using the Colibri Web Server (http://genolist.pasteur.fr/Colibri/genome.cgi).
Other in vivo assays.
For luciferase assays, bacterial strains MG1655 and PHL628 containing the different reporter plasmids were grown overnight. These cells were either directly assessed for reporter gene activity, or diluted 1 : 200 in fresh medium for the time-course experiments, where samples were taken at different time points, starting at OD600=0·1. The samples were adjusted to an OD600 of 0·050·1 in PBS buffer. Twenty microlitres of this solution was tested for luciferase activity by adding 200 µl PBS containing n-decanal to a final concentration of 2 nM. Measurement of relative light units (RLU) was conducted by a 2 s pre-measurement delay followed by a 3 s measurement after addition of the substrate in a MicroLumat LB 96 P luminometer (Berthold Technologies). Results are expressed as RLU per OD600 of the tested bacterial samples.
Primer extension was carried out as described in Prigent-Combaret et al. (2001). RNA from stationary phase cultures of either MG1655 or PHL628 (wherever expression of the gene of interest was higher) transformed with pJPcsgB, pJPyaiC, pJPpepD or pJPyagS was used for transcript analysis. We used the 5'-GATAAGTGAGAAGGAAGTTTC-3' primer, which anneals to the coding strand between 147 and 167 nucleotides downstream of the luxAB gene transcription start. The primer was labelled at the 5' end with fluorescent dye IRD-800 (MWG Biotech). Twenty micrograms of total RNA (extracted as for the gene array experiments) was used for each assay. The start site was determined using a sequencing ladder of the corresponding gene as molecular mass marker.
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RESULTS |
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Location of the putative CsgD-binding site at different promoters
The results of the previous experiments suggest that CsgD might function as either an activator (at the csgB and yaiC promoters) or a repressor (at the pepD and yagS promoters). Since the effect of a regulatory protein is determined by the location of its binding site relative to the promoter elements (Lloyd et al., 2001), we determined the position of the conserved 11 bp sequence necessary for CsgD-dependent regulation (Fig. 2b
). The transcription start points for the various promoters were identified using primer extension; the transcription start points for the csgB, yaiC and pepD promoters are shown in Fig. 3
. The yagS is the second gene in an operon controlled by the yagT promoter (Colibri Web Server, http://genolist.pasteur.fr/Colibri/). Despite the presence of possible promoter elements in the DNA region immediately upstream of the yagS ORF, we were not able to find any specific transcription start for the yagS gene (Fig. 3b
). Our experiments confirmed the already reported transcription start site for csgB (Arnqvist et al., 1994
); the conserved sequence necessary for activation by CsgD overlaps the -35 region (from -42 to -32; Fig. 3b
), consistent with the location for an activator binding site (Busby & Ebright, 1994
). The yaiC promoter possesses a -10 sequence perfectly matching the consensus for RNA polymerase, although in an unusual location relative to the transcription start point. We could identify no -35 sequence for yaiC, consistent with its role of positively controlled promoter (Fig. 3b
). Unlike at the csgB promoter, the 11 bp sequence necessary for CsgD-dependent regulation is positioned between -70 and -60 relative to the transcription start site. This location would still be consistent with the possible role of CsgD as an activator (Busby & Ebright, 1994
).
According to a previous study, pepD transcription in the exponential phase of growth is directed by two distinct promoters, called pepD1 and pepD2 (Henrich et al., 1990). Although we could confirm the presence of two different transcription start sites in the stationary phase of growth (Fig. 3
), only the start site corresponding to pepD1 matched the one previously reported. In contrast, we find a second transcription start 71 bp downstream of pepD1 (39 bp downstream of the previously detected pepD2 start site). Thus, according to our results, the start point for pepD2 would be located within the conserved 11 bp sequence, consistent with a possible role of CsgD as a repressor at the pepD promoter.
Effects of csgD-regulated genes on biofilm formation
The csgD gene controls the expression of factors involved in biofilm formation, such as the curli operon and the adrA gene in Salmonella. Thus we investigated the possible involvement in biofilm formation by the other csgD-dependent genes identified in this study. Either pepD or yagS was cloned into the multicopy pGEM-T Easy plasmid to obtain low-level expression, independent of either CsgD or OmpR234. We transformed the MG1655 strain, which does not express csgD, with the plasmids carrying either pepD or yagS and tested their effects on biofilm formation (Fig. 4). As a control, we also expressed in a csgD-independent fashion known determinants for biofilm formation (csgB, csgG, yaiC and csgD itself). Expression of none of these genes resulted in significant effects on MG1655 growth rate (data not shown). As expected, expression of CsgD from pGEM-T Easy resulted in increased formation of biofilm by MG1655, while csgD-independent expression of either csgB or csgG had little or no effect on biofilm formation. This result was also expected, since production of curli requires concomitant expression of both the csgBA and the csgDEFG operons. In contrast, low-level csgD-independent expression of yaiC resulted in a significant increase of attached cells (from 30 to 52 % of total cells). Expression of either the yagS or the pepD genes resulted in the opposite effect, with a significant reduction in the number of attached cells (down to 19 and 17 %, respectively).
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DISCUSSION |
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Similar to its Salmonella adrA homologue, the yaiC gene is regulated by csgD, and could be the main determinant for adhesion in the PHL857 (csgA ompR234 double mutant) strain. Both csgB and yaiC require an 11 bp conserved sequence for activation by CsgD (Fig. 2). This sequence was also found in two other genes negatively regulated in the ompR234 mutant strain, pepD and yagS (Table 3
, Fig. 3
). The conserved sequence (CGGGKGAKNKA) is totally unrelated to any so far reported OmpR-binding sites, and we propose that it might be the target sequence for CsgD. Thus CsgD might be able to act as both a positive (at the csgBA and yaiC promoters) and a negative (at the pepD and yagS promoters) transcription regulator. Interestingly, the locations of the putative CsgD-binding site differ in the csgB and yaiC promoter regions, suggesting that CsgD might activate transcription at these two promoters with different mechanisms. At csgB, the CsgD-binding site overlaps the -35 sequence, typical of an activator that contacts the
subunit of RNA polymerase (Busby & Ebright, 1994
), while at yaiC its target sequence is located at -70. The binding site is present as an inverted repeat in the yaiC promoter region, possibly suggesting that CsgD might bind this promoter as a dimer. The different location and the presence of the inverted repeat strongly suggest that CsgD might activate transcription at the csgB and at the yaiC promoter regions with different mechanisms. We could not identify any other sites for known regulators common to the csgD-dependent promoters.
Since CsgD appears to regulate genes involved in biofilm formation, we tested the possibility that the newly identified pepD and yagS genes might also play a role in this process. Low-level, csgD-independent expression of either pepD or yagS negatively affects biofilm formation (Fig. 4). Thus our results strongly suggest that in addition to the positive regulation of the csgBA and yaiC promoters, CsgD can repress the expression of negative determinants for biofilm formation such as pepD and yagS (Table 3
). While the functions of the csgBA operon (encoding curli) and of the yaiC gene (regulator of cellulose biosynthesis) are strictly related to production of extracellular polymers and to biofilm formation, neither pepD nor yagS appears to encode extracellular proteins or to be directly involved in the biosynthesis of adhesion determinants. The product of the pepD gene, dipeptidase D, cleaves the unusual dipeptide carnosine, and is induced by phosphate starvation (Klein et al., 1986
; Henrich et al., 1992
). Interestingly, pepD is up-regulated in luxS-deficient mutants of the enterohaemorrhagic E. coli O157 : H7, suggesting that quorum sensing negatively controls dipeptidase D expression (Sperandio et al., 2001
). Quorum sensing is necessary for efficient biofilm formation in several Gram-negative species (Davies et al., 1998
; Miller & Bassler, 2001
). This suggests that carnosine might act as a signal molecule and that repression of the pepD gene might allow its accumulation as part of a switch to biofilm growth.
The yagS gene appears to be controlled by the yagT promoter, and we could not map any yagS-specific transcription start. However, yagS was down-regulated in the PHL628 strain, as determined by global gene expression (Table 3) and RT-PCR assays (data not shown). It is possible that CsgD might repress yagS transcription by preventing elongation by RNA polymerase, rather than controlling the expression of a specific yagS promoter. Low-level csgD-independent expression of yagS negatively affects biofilm formation (Fig. 4
), suggesting a role for this gene as a negative determinant for biofilm formation. The yagS gene encodes a putative FAD-binding subunit of xanthine dehydrogenase, an enzyme involved in purine catabolism; thus yagS, as pepD, might be involved in the synthesis or in the degradation of a signal molecule important for biofilm formation.
In addition to the genes that show differential expression in the ompR234 mutant, a search for the putative CsgD-binding sequence (CGGGKGAKNKA) performed using the Colibri Web Server (http://genolist.pasteur.fr/Colibri/genome.cgi) reveals that this sequence is present in only one more gene in the E. coli chromosome, the putative aldehyde dehydrogenase gene (aldH). Despite the presence of the putative CsgD-binding site, aldH is expressed at similar levels in both the MG1655 and PHL628 strains in the growth conditions we tested, suggesting that CsgD might only regulate aldH in response to specific growth or environmental conditions, possibly in concert with additional transcriptional regulators. Thus our results show that csgD regulates a limited set of genes, suggesting that the central role of this regulator is the establishment of the biofilm phenotype in E. coli.
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ACKNOWLEDGEMENTS |
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Received 18 February 2003;
revised 26 May 2003;
accepted 28 May 2003.