Integrated regulatory responses of fimB to N-acetylneuraminic (sialic) acid and GlcNAc in Escherichia coli K-12

Abstract

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.

Fig. 1.

The fimB-yjhA intergenic region. Region (R)1 and R2 (small squares) lie close to 5′-GATC sequences protected from Dam methylation (21). The arrows and P mark the two known fimB promoters (23, 24). PS represents the size and position of the PCR product (Fim1-Fim2) used for EMSA and DNase I footprinting, and PB represents the PCR product used as a probe in Southern hybridization analysis. The length of DNA fragments (bp) generated after digestion with a combination of HpyCH4 IV and MboI are also shown.

Fig. 2.

The effect of ΔnanR, region 1 (Rm1) and region 2 (Rm5) mutations on the β-galactosidase produced by FimB-LacZ fusion in the absence (white bars) and presence (black bars) of sialic acid. The WT and mutant strains indicated were grown in RD glycerol medium to an OD₆₀₀ of ≈0.2 at 37°C with rapid aeration before sampling, and β-galactosidase activity was measured as described (38).

Fig. 3.

In vitro binding of NanR and NagC to regions 1 and 2. (A) EMSA with NanR was carried out with the protein concentrations indicated (3-24 nM). NanR was absent in F. (B) DNase I footprinting with NanR and NagC. DNA was labeled at Fim1 (lanes 1-10) or Fim2 (lanes 12-16) and incubated with the nanomolar concentrations of proteins indicated. Region 1 (NanR), region 2 (NagC1), and NagC2 are indicated. (C) Sequence of the DNA used. The position and orientation of primers Fim1 and Fim2 is highlighted and marked by arrows. The sequences in bold are the consensus sequence matches for NanR and NagC contained within the regions protected from DNase I digestion are shown. The position of the replacement mutations Rm1 (region 1 and NanR-binding site) and Rm5 (region 2 and NagC1-binding site), as well as the corresponding 5′-GATC sequences that are protected from Dam methylation, are underlined.

Fig. 4.

Methylation protection of regions 1 and 2 by Southern blot hybridization analysis. The analysis included WT (BGEC905, lanes 1 and 5), ΔnanR (KCEC357, lanes 2 and 6), ΔpdhR (KCEC231, lanes 3 and 7), and ΔnagC (KCEC505, lanes 4 and 8) strains grown at 37°C in RD glycerol medium (A) and RD glycerol containing 3 mM Neu₅Ac (B, lanes 1-4), or 3 mM GlcNAc (C, lanes 5-8). Chromosomal DNA was digested with HpyCH4 IV (A, lanes 1-4) or a combination of HpyCH4 IV and MboI (A, lanes 5-8 and B, lanes 1-8), and hybridized with a ³²P-labeled PCR product (Fig. 1; PB) as described (21).

Fig. 5.

The effect of GlcNAc on fimB expression. The β-galactosidase produced by strain BGEC905 (FimB-LacZ) in the presence of various concentrations of GlcNAc was measured as described (38). The bacteria were grown in RD glycerol medium to an OD₆₀₀ of ≈0.2 at 37°C with rapid aeration before sampling.