A molecular mechanism for the repression of transcription by the H-NS protein.

The H-NS protein is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Although H-NS does not exhibit a high DNA sequence specificity, a number of H-NS-responsive promoters have been shown to contain regions of intrinsic DNA curvature located either upstream or downstream of the transcription start point. We have studied H-NS binding to DNA and in vitro transcriptional regulation by H-NS at several synthetic promoters with or without curved sequences inserted upstream of the Pribnow box. We show how such inserts determine the final organization of H-NS-containing nucleoprotein complexes and how this affects transcription. We refine a two-step mechanism for the constitution of H-NS assemblies that are efficient in regulation.

The ChiA (YheB) protein of Escherichia coli K-12 is an endochitinase whose gene is negatively controlled by the nucleoid-structuring protein H-NS.

The chromosome of Escherichia coli K-12 contains a putative gene, yheB (chiA), at centisome 74.7, whose product shows sequence similarity with chitinases of bacterial and viral origin. We cloned the chiA (yheB) gene and demonstrated that it codes for a 94.5 kDa periplasmic protein with endochitinase/lysozyme activity. Under standard laboratory growth conditions, chiA expression is very low, as shown by the Lac- phenotype of a chiA transcriptional fusion to a promoterless lacZ reporter. To identify factors that control chitinase gene expression, we generated random Tn10 insertions in the chromosome of the fusion-containing strain, selecting for a Lac+ phenotype. The majority of the mutations that caused a Lac+ phenotype mapped to the hns gene, encoding the nucleoid-structuring protein H-NS. Transcription of chiA in vivo is driven by a single sigma70 promoter and is derepressed in an hns mutant. Using a competitive gel retardation assay, we demonstrated that H-NS binds directly and with high affinity to the chiA promoter region. In addition to hns, other E. coli mutations causing defects in global regulatory proteins, such as fis, crp or stpA in combination with hns, increased chiA expression to different extents, as did decreasing the growth temperature from 37 degrees C to 30 degrees C. A possible physiological function of ChiA (YheB) endochitinase in E. coli K-12 is discussed.

Isolation and characterization of vicH, encoding a new pleiotropic regulator in Vibrio cholerae.

During the last decade, the hns gene and its product, the H-NS protein, have been extensively studied in Escherichia coli. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in E. coli and in Salmonella enterica serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of vicH, an hns-like gene in Vibrio cholerae, the etiological agent of cholera. This gene was isolated from a V. cholerae genomic library by complementation of different phenotypes associated with an hns mutation in E. coli. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in E. coli. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. The vicH gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A V. cholerae wild-type strain expressing a vicHDelta92 gene lacking its 3' end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the vicH wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in V. cholerae, in particular by affecting flagellum biosynthesis.

Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon.

Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility. In the present paper, we show that crp and hns mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a reduced expression in vivo of fliA and fliC, which encode the specific flagellar sigma factor and flagellin, respectively. Overexpression of the flhDC master operon restored, at least in part, motility in crp and hns mutant strains, suggesting that this operon is the main target for both regulators. Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays. In vitro transcription experiments showed that CAP activates flhDC expression while H-NS represses it. In agreement with this observation, the activity of a transcriptional fusion carrying the flhDC promoter was decreased in the crp strain and increased in the hns mutant. In contrast, the activity of a transcriptional fusion encompassing the entire flhDC regulatory region extending to the ATG translational start codon was strongly reduced in both hns and crp mutants. These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complementation of the nonmotile phenotype in a crp mutant by activation-deficient CAP mutated proteins and characterization of cfs, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activates flhDC transcription by binding to its promoter and interacting with RNA polymerase.

Molecular aspects of the E. coli nucleoid protein, H-NS: a central controller of gene regulatory networks.

The nucleoid-associated protein H-NS has a central role in the structuring and control of the enteric bacterial chromosome. This protein has been demonstrated to contribute to the regulation of expression for approximately thirty genes. In this article, the molecular aspects of H-NS structure and function are briefly reviewed. H-NS contains at least two independent structural domains: a C-terminal domain, involved in the DNA-protein interactions, and a N-terminal domain, likely involved in protein-protein interactions. Recent reports have revealed that H-NS is a key factor in a multi-component gene regulatory system. Factors have now been discovered which can backup or antagonise H-NS action at certain promoters. These recent findings are summarised and discussed in relationship to the role of H-NS in DNA packaging and nucleoid structure.

Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives.

Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.

DNA-stacking interactions determine the sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion.

Bis(1,10-phenanthroline)-copper(I) ion (OP2Cu+) binds reversibly to B-DNA and makes single-stranded cuts by oxidative attack on the deoxyribose moiety. The deoxyribonuclease activity is sequence-dependent yet not nucleotide-specific at the cutting site. OP2Cu+ sequence specificity was analysed in terms of local variations of DNA stability. Kinetic constants of strand cleavage were measured at sequence positions on the two strands and converted into activation free energies of the cleavage reaction. DNA unwinding free energies were calculated from the base sequence using B-DNA stacking parameters for calculations. The two free-energy variations were statistically compared for a series of DNA restriction fragments bearing the binding sites of regulatory proteins and representing a total of 345 DNA base positions. This study shows that the mean activation free energy of strand cleavage at a pair of opposing sugars across the DNA minor groove varies like the unwinding free energy of the DNA sequence delimited by opposing sugars (3 to 4 bp). A statistical equality between the two free-energy variations is demonstrated when considering the sum of the two cleavage events at the opposing sugars. Systematic deviations between the two free-energy distributions were observed at specific sequences, including polypurine-polypyrimidine tracts (AnTm/AmTn, CnTmCp/GpAmGn), alternating purine-pyrimidine tracts ((TA)n/(TA)n, (TG)n/(CA)n) and at certain G+C-rich triplets (GGC, GCC and CGC). The physical significance of these observations is discussed and a model of OP2Cu+ binding and cleavage specificity based on the free-energy equality is proposed.

Escherichia coli protein analogs StpA and H-NS: regulatory loops, similar and disparate effects on nucleic acid dynamics.

Expression of the Escherichia coli StpA protein was investigated and a functional comparison undertaken with the structurally analogous nucleoid protein H-NS. Analysis of stpA and hns expression indicated that although stpA transcript levels are much lower than those of hns, the two gene products are capable of both negative autogenous control and cross-regulation. Examination of cellular proteins in stpA, hns, or stpA-hns backgrounds revealed that StpA can repress and activate a subset of H-NS-regulated genes. Mechanistic parallels in regulation of gene expression are indicated by the ability of both proteins to inhibit transcription from promoters containing curved DNA sequences, and to form nucleoprotein structures that constrain DNA supercoils. Despite their functional similarities, each molecule is capable of independent activities. Thus, H-NS regulates a class of genes that are unaffected by StpA in vivo, whereas StpA has much stronger RNA chaperone activity in vitro. We therefore propose that in addition to its role as a molecular back-up of H-NS, StpA's superior effect on RNA may be exploited under some specific cellular conditions to promote differential gene expression.

Stimulation of RNA polymerase II elongation by chromosomal protein HMG-14.

The high-mobility group protein 14 (HMG-14) is a non-histone chromosomal protein that is preferentially associated with transcriptionally active chromatin. To assess the effect of HMG-14 on transcription by RNA polymerase II, in vivo-assembled chromatin with elevated amounts of HMG-14 was obtained. Here it is shown that HMG-14 enhanced transcription on chromatin templates but not on DNA templates. This protein stimulated the rate of elongation by RNA polymerase II but not the level of initiation of transcription. These findings suggest that the association of HMG-14 with nucleosomes is part of the cellular process involved in the generation of transcriptionally active chromatin.

Modulated expression of promoters containing upstream curved DNA sequences by the Escherichia coli nucleoid protein H-NS.

Replacement of the CRP-binding site of the gal control region by curved sequences can lead to the restoration of promoter strength in vivo. One curved sequence called 5A6A, however, failed to do so. The gene hns exerts a strong negative control on the resulting 5A6A gal promoter as well as on the distant bla promoter, specifically in a 5A6A gal context. The product of this gene, H-NS, displays a better affinity for this particular insert compared to other curved sequences. Mechanisms by which H-NS may repress promoters both at short and long distances from a favoured binding site are discussed.

Association of nucleosome-free regions and basal transcription factors with in vivo-assembled chromatin templates active in vitro.

Using SV40 minichromosomes assembled in vivo, we have studied the relationship between a nucleosome-free promoter-region and initiation of transcription by RNA polymerase II on chromatin templates in vitro. Our data suggest that accessibility of DNA to transcription factors, programmed into the structure of the chromatin, is crucial for initiation of transcription. First, minichromosomes competent to be transcribed in vitro contained nucleosome-free promoter regions. Second, tsC219 minichromosomes, most of which contain the nucleosome-free promoter region, supported transcription more efficiently both in vivo and in vitro than wild-type minichromosomes, in which only a subset contain the nucleosome-free region. We have also identified basal transcription factors associated with the in vivo-assembled chromatin templates. A striking correlation was observed between minichromosomes associated with in vivo initiated RNA polymerases and those associated with the basal transcription factors TFIID and TFIIE/F, and to a lesser extent, TFIIB. Of these associated factors, only TFIID was poised for ready assembly into preinitiation complexes and therefore for subsequent initiation of transcription. However, an active chromatin template could also be maintained in the absence of the binding of TFIID. Finally, our data are consistent with the presence of TFIIF in elongating ternary complexes on the chromatin templates.

Sequence determinants for H1 binding on Escherichia coli lac and gal promoters.

The H1 protein is a likely candidate for structuring DNA in the bacterial nucleoid. We have studied determinants leading to its binding to DNA (and in particular to Escherichia coli lac and gal promoters) in vitro through the pattern of attack of both DNaseI and the copper-o-phenanthroline complex [(OP)2Cu+]. The binding of H1 depends on the primary sequence of DNA. H1 also associates with recognition sites for specific proteins, in particular with the Pribnow box and the CRP binding site. Binding of H1 to the Pribnow box of the wild-type lac promoter does not change the pattern of nucleolytic digestion with (OP)2Cu+. In contrast, binding of H1 to the strong lac promoter mutants Ps and UV5 appears to change the conformational state of this DNA. Similar changes in accessibility of the minor groove surrounding the respective binding sites were observed for both H1-DNA and CRP-DNA complexes.

Correlation between the conformation of Escherichia coli -10 hexamer sequences and promoter strength: use of orthophenanthroline cuprous complex as a structural index.

The lac and gal control regions contain two functional overlapping promoters P1 and P2. Point mutations can shift transcription from P1 to P2 and vice versa. We show that the reactivity of DNA fragments towards nucleolytic attack with orthophenanthroline cuprous complex can be used to predict which promoter competes more efficiently for RNA polymerase binding. Furthermore, similar changes in reactivity are observed as closed complexes isomerize to form the final open complexes, provided that the functional start is taken as a reference. We found a correlation between the reactivity pattern of -10 regions in uncomplexed DNA and the rate of open complex formation.

H1a, an E. coli DNA-binding protein which accumulates in stationary phase, strongly compacts DNA in vitro.

We characterize a component of the E. coli bacterial nucleoid H1a, which accumulates in stationary phase. This protein, identical with the major component of a plasmid-protein complex previously isolated in our laboratory, has a pI close to 7.5. Acrylamide gel electrophoresis and sedimentation in sucrose gradient have shown that the protein H1a induces significant compaction into DNA. This compaction is equivalent to that observed in nucleosome core although it introduces only a slight change in linking number. In addition, the structural change induced in the lactose L8UV5 promoter by H1a results in the decrease in the kinetic of formation of the open complex with RNA polymerase.

A DNA-binding protein from E. coli isolation, characterization and its relationship with proteins H1 and B1.

A protein of Mr 16,000 (protein 16 K) which tightly binds to DNA has been isolated from E. coli. The amino acid composition and amino-terminal amino acid sequence of this protein have been determined. On the basis of its physico-chemical characteristics the protein 16 K was shown to be identical to the protein H1 [Cukier-Kahn et al. (1972) Proc. Natl. Acad. Sci. USA 69, 3643-3647; Spassky and Buc (1977) Eur. J. Biochem. 81, 79-90] which corresponds itself to the protein B1 [Bakaev (1981) Molekulyarnaya Biologya 15, 1350-1362].