Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1Tyr)

virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.     

Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli

The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.     

Characterization of hns genes from Erwinia amylovora

The small basic histone-like protein H-NS is known for bacteria to attenuate virulence of several animal pathogens. An hns homologue from E. amylovora was identified by complementing an E. coli hns-mutant strain with a cosmid library from E. amylovora. A 1.6 kb EcoRI-fragment complemented the mucoid phenotype and repressed the ss-glucosidase activity of E. coli PD32. The open reading frame encoding an H-NS-like protein of 134 amino acid was later shown to be located on plasmid pEA29 (McGhee and Jones 2000). A chromosomal hns gene was amplified with PCR consensus primers and localized near galU of E. amylovora. E. amylovora mutants were created by insertion of a resistance cassette, and the intact gene was inserted into a high copy number plasmid for constitutive expression. Purified chromosomal H-NS protein preferentially bound to a DNA fragment from the lsc region and bending was predicted for an adjacent fragment with the rlsB-promoter. Levan production was significantly increased by hns mutations. Synthesis of the capsular exopolysaccharide amylovoran and of levan were reduced, when hns from the E. amylovora plasmid was overexpressed. A mutation in chromosomal hns of E. amylovora increased amylovoran synthesis, and both mutations retarded symptom formation on immature pears.     

Role of the nucleoid-associated protein H-NS in the synthesis of virulence factors in the phytopathogenic bacterium Erwinia chrysanthemi

The ability of the enterobacterium Erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). Additional factors, including flagellar proteins and exopolysaccharides (EPS), also are required for the efficient colonization of plants. Production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental conditions. The possible involvement of the protein H-NS in this process was investigated. The E. chrysanthemi hns gene was cloned by complementation of an Escherichia coli hns mutation. Its nucleotide sequence contains a 405-bp open reading frame that codes for a protein with 85% identity to the E. coli H-NS protein. An E. chrysanthemi hns mutant was constructed by reverse genetics. This mutant displays a reduced growth rate and motility but an increased EPS synthesis and sensitivity toward high osmolarity. Furthermore, pectate lyase production is dramatically reduced in this mutant. The hns mutation acts on at least two conditions affecting pectate lyase synthesis: induction of pectate lyase synthesis at low temperatures (25 degrees C) is no longer observed in the hns mutant and induction of pectate lyase production occurs in the late stationary growth phase in the hns background, instead of in the late exponential growth phase as it does in the parental strain. Moreover, the E. chrysanthemi hns mutant displays reduced virulence on plants. Taken together, these data suggest that H-NS plays a crucial role in the expression of the virulence genes and in the pathogenicity of E. chrysanthemi.     

Effects of multicopy LeuO on the expression of the acid-inducible lysine decarboxylase gene in Escherichia coli.

We previously reported that mutations in hns, the structural gene for the histone-like protein H-NS, cause derepressed expression of cadA, which encodes the acid-inducible lysine decarboxylase at noninducing pH (pH 8.0). This study reports the characterization of a plasmid isolated from an Escherichia coli library that suppresses the effect of an hns mutation on cadA expression. A previously sequenced open reading frame, leuO, proves to be the gene that causes the hns-complementing phenotype. The mechanism for this phenotype appears to be overexpression of leuO from a multicopy plasmid, which drastically reduces production of CadC, the essential activator for cadA induction. These results show an in vivo regulatory phenotype for leuO, consistent with its proposed protein sequence.     

The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants

The StpA protein is closely related to H-NS, the well-characterised global regulator of gene expression which is a major component of eubacterial chromatin. Despite sharing a very high degree of sequence identify and having biochemical properties in common with H-NS, the physiological function of StpA remains unknown. We show that StpA exhibits similar DNA-binding activities to H-NS. Although both display a strong preference for binding to curved DNA, StpA binds DNA with a four-fold higher affinity than H-NS, with K(d)s of 0.7 microM and 2.8 microM, respectively. It has previously been reported that expression of stpA is derepressed in an hns mutant. We have quantified the amount of StpA protein produced under this condition and find it to be only one-tenth the level of H-NS protein in wild-type cells. Our findings explain why the presence of StpA does not compensate for the lack of H-NS in an hns mutant, and why the characteristic pleiotropic hns mutant phenotype is observed.     

Expression and mutational analysis of the nucleoid-associated protein H-NS of Salmonella typhimurium

The H-NS (H1) protein is a major component of bacterial chromatin. Mutations in the hns (osmZ) gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes in an allele-specific manner. H-NS expression was found not to vary with growth phase or growth medium osmolarity. Additionally, 10 independent hns mutations were isolated and characterized. Five of these mutations were the result of an IS10 insertion, each generating a truncated polypeptide. The other five mutations were the same specific deletion of one amino acid, delta Ala46. The various hns mutations exhibited different phenotypes and influenced DNA topology to variable extents. Implications for the mechanism by which H-NS influences gene expression are discussed.