The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C. 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 of the 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 cspA. This purified protein, which is homologous to human DNA-binding 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.

The expression of plasmid‐borne virF of Shigella encoding a transcriptional regulator of the AraC family, is required to initiate a cascade of events resulting in activation of several operons encoding invasion functions. H‐NS, one of the main nucleoid‐associated proteins, controls the temperature‐dependent expression of the virulence genes by repressing the in vivo transcription of virF only below a critical temperature (∼32°C). This temperature‐dependent transcriptional regulation has been reproduced in vitro and the targets of H‐NS on the virF promoter were identified as two sites centred around −250 and −1 separated by an intrinsic DNA curvature. H‐NS bound cooperatively to these two sites below 32°C, but not at 37°C. DNA supercoiling within the virF promoter region did not influence H‐NS binding but was necessary for the H‐NS‐mediated transcriptional repression. Electrophoretic analysis between 4 and 60°C showed that the virF promoter fragment, comprising the two H‐NS sites, undergoes a specific and temperature‐dependent conformational transition at ∼32°C. Our results suggest that this modification of the DNA target may modulate a cooperative interaction between H‐NS molecules bound at two distant sites in the virF promoter region and thus represents the physical basis for the H‐NS‐dependent thermoregulation of virulence gene expression.

Escherichia coli hns , encoding the abundant nucleoid protein H‐NS, was subjected to site‐directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild‐type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase‐mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non‐curved DNA, but lost the wild‐type capacity to recognize preferentially curved DNA and to actively bend non‐curved DNA, a property of wild‐type H‐NS demonstrated here for the first time. DNase I footprinting and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H‐NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non‐curved DNA can be attributed to a defect in protein–protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H‐NS of the oligomerization domain (C‐domain) of bacteriophage λ cI repressor.

TheEscherichia colichromosome contains two distantly located genes,gadAandgadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad). The Gad reaction contributes to pH homeostasis by consuming intracellular H⁺and producing γ-aminobutyric acid. This compound is exported via the protein product of thegadCgene, which is cotranscribed withgadB. Here we demonstrate that transcription of bothgadAandgadBCis positively controlled bygadX, a gene downstream ofgadA, encoding a transcriptional regulator belonging to the AraC/XylS family. ThegadXpromoter encompasses the 67-bp region preceding thegadXtranscription start site and contains both RpoD and RpoS putative recognition sites. Transcription ofgadXoccurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of thegadstructural genes. However, P_T5lacO-controlledgadXexpression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when thegadsystem is normally repressed. Autoregulation of the wholegadsystem is inferred by the positive effect of GadX on thegadApromoter andgadAXcotranscription. Transcription ofgadXis derepressed in anhnsmutant and strongly reduced in bothrpoSandhns rpoSmutants, consistent with the expression profile ofgadstructural genes in these genetic backgrounds. Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX bindsgadAandgadBCpromoters at different sites and with different binding affinities.