No abstract

The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

No abstract

The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon ofEscherichia coli, its expression being enhanced 3-to 4-fold during the growth lag that follows a shift from 37C to 100C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift ofthe 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of espA. This purified protein, which is homologous to human DNAbinding protein YB-1, recognizes some feature of the 110-bp promoter region of hns and acts as a cold shock transcriptional activator of this gene since it stimulates the expression of CAT activity and of cat transcription in in vitro systems programmed with plasmid DNA carrying the hns-cat fusion.Several bacterial proteins with DNA-binding property have been implicated in condensation of the chromosome and in organization of the prokaryotic nucleoid. The most abundant and best characterized of these are HU (NS) and H-NS (Hla) proteins (for reviews, see refs. 1-4). H-NS (136 residues) is a neutral, heat-stable, dimeric protein (5) that displays high affinity for curved DNA (6) and has been localized primarily in the nucleoid by immunoelectron microscopy (7). H-NS is encoded by hns, a gene that has been cloned and characterized in Escherichia coli (8) as well as in other Enterobacteriaceae (9) and that has been ultimately mapped at 27 min on the E. coli chromosome (4). Mutations in hns were found to increase bacteriophage Mu-specific transcription and to increase dramatically the mini-Mu transposition rate (10). Several mutations causing a number of apparently unrelated phenotypes have been found to be allelic with hns. These are bglY, which activates expression of the cryptic bgl operon (11) and causes large chromosomal deletions (12); pilG, which greatly increases the site-specific DNA inversion responsible for fimbrial phase variation (13); drdX, which induces expression of the pilus adhesin (pap) genes at low temperature in uropathogenic strains (14); cur-], causing a conditional uracil requirement (15); osmZ, altering the osmoregulated expression of proU operon (16, 17); and virR, which affects the temperature-regulated expression of plasmid-borne virulence genes in Shigella flexneri (18).A common basis for these pleiotropic effects could be an altered compaction and fluidity of the genome (12) leading to (or coupled with) a transcriptional derepression of some genes.An important role of H-NS in controlling the compaction of the nucleoid is also suggested by the observation that the nucleoids undergo a dramatic conden...

The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible lambda PL promoter. After induction of transcription by thermal inactivation of the lambda ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10 degrees C, but poor expression was obtained if the cells were incubated at 37 degrees C or 30 degrees C. The reason for this differential temperature-dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37 degrees C compared to 10 degrees C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half-lives of the cspA transcript were substantially longer at 10 degrees C compared to 37 degrees C; (iii) S30 extracts as well as 70S ribosomes prepared from cold-shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37 degrees C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold-shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock.