Cynthia L. Pon

The oligomeric structure of nucleoid protein H‐NS is necessary for recognition of intrinsically curved DNA and for DNA bending

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.

Post-transcriptional regulation of CspA expression in Escherichia coli

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 λ PL promoter. After induction of transcription by thermal inactivation of the λ ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10°C, but poor expression was obtained if the cells were incubated at 37°C or 30°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°C compared to 10°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°C compared to 37°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°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.

Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy.

SummaryThe Escherichia coli hns gene, which encodes the nucleoid protein H-NS, was deprived of its natural promoter and placed under the control of the inducible lambda PL promoter. An hns mutant yielding a protein (H-NSΔ12) with a deletion of four amino acids (Gly112-Arg-Thr-Pro115) was also obtained. Overproduction of wild-type (wt) H-NS, but not of H-NSΔ12, resulted in a drastic loss of cell viability. The molecular events and the morphological alterations eventually leading to cell death were investigated. A strong and nearly immediate inhibition of both RNA and protein synthesis were among the main effects of overproduction of wt H-NS, while synthesis of DNA and cell wall material was inhibited to a lesser extent and at a later time. Upon cryofixation of the cells, part of the overproduced protein was found in inclusion bodies, while the rest was localized by immunoelectron microscopy to the nucleoids. The nucleoids appeared condensed in cells expressing both forms of H-NS, but the morphological alterations were particularly dramatic in those overproducing wt H-NS; their nucleoids appeared very dense, compact and almost perfectly spherical. These results provide direct evidence for involvement of H-NS in control of the organization and compaction of the bacterial nucleoid in vivo and suggest that it may function, either directly or indirectly, as transcriptional repressor and translational inhibitor.

Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions.

The most characteristic event of cold‐shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is ≥50 μM during early exponential growth at 37°C; therefore, its designation as a major cold‐shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid‐to‐late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold‐shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre‐existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post‐transcriptional controls regulate cspA expression under non‐stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA‐binding proteins Fis and H‐NS both in vivo and in vitro, while its decreased half‐life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5′ untranslated leader and is counteracted in vivo by the cold‐shock DeaD box RNA helicase (CsdA).

Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression

Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from −282 to +25 with two sites, closely flanking the DNA bend located at −150 from the transcriptional startpoint, partly overlapping the H‐NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H‐NS and the hns promoter region were studied by examining the effects of FIS and H‐NS on in vitro transcription of hns–cat fusions, as well as looking at the effect of FIS on preformed complexes containing H‐NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H‐NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns

Proteins from the prokaryotic nucleoid: primary and quaternary structure of the 15‐kD Escherichia coli DNA binding protein H‐NS

The primary sequence of H‐NS (136 amino acid residues, Mr = 15402), an abundant Escherichia coli DNA‐binding protein, has been elucidated and its quaternary structure has been investigated by protein‐protein cross‐linking reactions. It was found that H‐NS exists predominantly as a dimer, even at very low concentrations, but may form tetramers at higher concentrations and that the protein‐protein interaction responsible for the dimerization is chiefly hydrophobic.

The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor

Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3′,5′-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.

Translation initiation factor IF3: two domains, five functions, one mechanism?

Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N‐domain displayed neither affinity for ribosomes nor a detectable function, the isolated C‐domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S‐bound mRNA from ‘stand‐by’ to ‘P‐decoding’ site; (iii) dissociation of 30S–poly(U)–NacPhe‐tRNA pseudo‐ initiation complexes; (iv) dissociation of fMet‐tRNA from initiation complexes containing mRNA with the non‐canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C‐domain–30S interaction, the N‐domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S–IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem–loop of P‐site‐bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures.

Expression of the gene encoding the major bacterial nucleoid protein H‐NS is subject to transcriptional auto‐repression

Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at –313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild‐type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid‐log cultures and negligible in cells entering the stationary phase. The level of repression in early‐log phase was lower than in mid‐log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H‐NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H‐NS and Fis protein to transcriptional systems programmed with the same hns caf fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation get analyses revealed that H‐NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around –150. When these sites are filled by H‐NS, an additional site between approximately –20 and –65, which partly overlaps the promoter, is also occupied. Binding of H‐NS to this site is probably the ultimate cause of transcriptional auto‐repression.

Interaction of the main cold shock protein CS7.4 (CspA) of Escherichia coli with the promoter region of hns

Escherichia coli protein CS7.4 (CspA), homologous to the class of eukaryotic Y-box DNA-binding proteins, is a cold shock transcriptional activator of at least two genes, hns and gyrA. It was demonstrated that all or nearly all the elements necessary for the stimulation of hns transcription by CS7.4 protein are located in the proximal 110 bp DNA fragment of this gene with no additional elements being present in a longer fragment (660 bp) extending further upstream from the hns promoter. Protein CS7.4 bound strongly to the 110 bp segment of the hns promoter in crude extracts of cold shocked cells, but the purified protein displayed a weak interaction with the same DNA fragment. Purified CS7.4 protein also caused increased or decreased accessibility to DNase I at different sites of the 110 bp fragment of hns but the majority of these effects was seen only in the presence of RNA polymerase. Since gel shift experiments showed that protein CS7.4 stimulated the binding of RNA polymerase to the promoter of hns and since it is known that there are similarities between CS7.4 and ssDNA-binding proteins, we suggest that formation of the open complex by the RNA polymerase or protein-protein contacts between CS7.4 and the RNA polymerase are prerequisites for and/or the effects of the interaction of CS7.4 with its DNA target. The presence of a conserved CCAAT element in the hns promoter region, on the other hand, was found not to be stringently required for cold shock activation since expression of E coli of an hns-cat fusion containing the Proteus vulgaris hns promoter lacking a CCAAT box increased over four-fold after cold shock.