RESUMO
We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (O(SA1) and O(SA2)), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only. H-NS binds to multiple sites in the promoter region, including two sites (H-NS2 and H-NS3) that overlap O(SA1) and O(SA2), respectively. Mutations that disrupt either O(SA1) or O(SA2) eliminate or reduce the activating effect of SlyA but have different effects on the level of expression. We interpret these results as reflecting the relative competition between SlyA and H-NS binding. Moreover we show that SlyA is capable of displacing H-NS from its binding sites in vitro. We suggest SlyA binding prevents H-NS binding to H-NS2 and H-NS3 and the subsequent oligomerization of H-NS necessary for full inhibition of fimB expression. In addition, we show that SlyA activates fimB expression independently of two other known regulators of fimB expression, NanR and NagC. It is demonstrated that the rarely used UUG initiation codon limits slyA expression and that low SlyA levels limit fimB expression. Furthermore, Western blot analysis shows that cells grown in rich-defined medium contain ~1000 SlyA dimers per cell whereas those grown in minimal medium contain >20% more SlyA. This study extends our understanding of the role that SlyA plays in the host-bacterial relationship.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/biossíntese , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/biossíntese , Regulação Bacteriana da Expressão Gênica , Integrases/biossíntese , Fatores de Transcrição/metabolismo , Fatores de Virulência/biossíntese , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Interações Hospedeiro-Patógeno/genética , Integrases/genética , Mutação , Regiões Operadoras Genéticas/genética , Ligação Proteica/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Virulência/genéticaRESUMO
Expression of the FimB recombinase, and hence the OFF-to-ON switching of type 1 fimbriation in Escherichia coli, is inhibited by sialic acid (Neu(5)Ac) and by GlcNAc. NanR (Neu(5)Ac-responsive) and NagC (GlcNAc-6P-responsive) activate fimB expression by binding to operators (O(NR) and O(NC1) respectively) located more than 600 bp upstream of the fimB promoter within the large (1.4 kb) nanC-fimB intergenic region. Here it is demonstrated that NagC binding to a second site (O(NC2)), located 212 bp closer to fimB, also controls fimB expression, and that integration host factor (IHF), which binds midway between O(NC1) and O(NC2), facilitates NagC binding to its two operator sites. In contrast, IHF does not enhance the ability of NanR to activate fimB expression in the wild-type background. Neither sequences up to 820 bp upstream of O(NR), nor those 270 bp downstream of O(NC2), are required for activation by NanR and NagC. However, placing the NanR, IHF and NagC binding sites closer to the fimB promoter enhances the ability of the regulators to activate fimB expression. These results support a refined model for how two potentially key indicators of host inflammation, Neu(5)Ac and GlcNAc, regulate type 1 fimbriation.
Assuntos
Acetilglucosamina/farmacologia , Proteínas de Ligação a DNA/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Integrases/genética , Fatores Hospedeiros de Integração/metabolismo , Ácido N-Acetilneuramínico/farmacologia , Sítios de Ligação , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/fisiologia , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Integrases/efeitos dos fármacos , Integrases/metabolismo , Fatores Hospedeiros de Integração/efeitos dos fármacos , Fatores Hospedeiros de Integração/genética , Metilação , Mutagênese , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Bacterial-host attachment by means of bacterial adhesins is a key step in host colonization. Phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (mainly on-to-off switching). fimB is separated from the divergent yjhATS operon by a large (1.4 kbp) intergenic region. Short ( approximately 28 bp) cis-active elements (regions 1 and 2) close to yjhA stimulate fimB expression and are required for sialic acid (Neu(5)Ac) sensitivity of its expression [El-Labany, S., Sohanpal, B. K., Lahooti, M., Akerman, R. & Blomfield, I. C. (2003) Mol. Microbiol. 49, 1109-1118]. Here, we show that whereas NanR, a sialic acid-response regulator, binds to region 1, NagC, a GlcNAc-6P-responsive protein, binds to region 2 instead. The NanR- and NagC-binding sites lie adjacent to deoxyadenosine methylase (Dam) methylation sites (5'-GATC) that are protected from modification, and the two regulators are shown to be required for methylation protection at regions 1 and 2, respectively. Mutations in nanR and nagC diminish fimB expression, and both fimB expression and FimB recombination are inhibited by GlcNAc (3- and >35-fold, respectively). Sialic acid catabolism generates GlcNAc-6-P, and whereas GlcNAc disrupts methylation protection by NagC alone, Neu(5)Ac inhibits the protection mediated by both NanR and NagC as expected. Type 1 fimbriae are proinflammatory, and host defenses enhance the release of both Neu(5)Ac and GlcNAc by a variety of mechanisms. Inhibition of type 1 fimbriation by these amino sugars may thus help balance the interaction between E. coli and its hosts.
Assuntos
Acetilglucosamina/farmacologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Integrases/fisiologia , Ácido N-Acetilneuramínico/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Sequência de Bases , Metilação de DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Integrases/genética , Dados de Sequência Molecular , Recombinação Genética/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
The phase variation of type 1 fimbriation in Escherichia coli is controlled by the inversion of a 314 bp element of DNA, determined by FimB (switching in both directions) or FimE (switching from the ON-to-OFF orientation predominantly), and influenced by auxiliary factors IHF, Lrp and H-NS. The fimB gene is separated from the divergently transcribed yjhATS operon by a large (1.4 kbp) intergenic region of unknown function. Here, we show that fimB expression is regulated by multiple cis-active sequences that lie far upstream (>600 bp) of the transcription start sites for the recombinase gene. Two regions characterized further (regions 1 and 2) show sequence identity, and each coincides with a methylation-protected Dam (5'-GATC) site. Regions 1 and 2 apparently control fimB expression by an antirepression mechanism that involves additional sequences proximal to yjhA. Region 1 encompasses a 27 bp DNA sequence conserved upstream of genes known (nanAT ) or suspected (yjhBC) to be involved in sialic acid metabolism, and we show that FimB expression and recombination are suppressed by N-acetylneuraminic acid. We propose that E. coli recognizes the amino sugars as a harbinger of potential host defence activation, and suppresses the expression of type 1 fimbriae in response.
