Plasmids bearing hfq and the hns-like gene stpA complement hns mutants in modulating arginine decarboxylase gene expression in Escherichia coli.

Biodegradative arginine decarboxylase is inducible by acid and is derepressed in an hns mutant. Several plasmids from an Escherichia coli library that could complement the hns phenotype were characterized and placed into groups. One group includes plasmids that contain the hns gene and are considered true complements. Another group was found to carry the hfq gene, which encodes the host factor HF-1 for bacteriophage Q beta replication. Plasmids of the third group contain inserts that mapped at 60.2 min on the E. coli chromosome. We identified an open reading frame (stpA) with a deduced amino acid sequence showing more than 60% identity with the sequences of H-NS proteins from several species as being responsible for the hns complementing phenotype of the third group.     

Roles of the DNA binding proteins H-NS and StpA in homologous recombination and repair of bleomycin-induced damage in Escherichia coli

The DNA binding protein H-NS promotes homologous recombination in Escherichia coli, but the role of its paralog StpA in this process remains unclear. Here we show that an hns mutant, but not an stpA mutant, are marginally defective in conjugational recombination and is sensitive to the double-strand-break-inducing agent bleomycin. Interestingly, the hns stpA double mutant is severely defective in homologous recombination and more bleomycin-sensitive than is the hns or stpA single mutant, indicating that the stpA mutation synergistically enhances the defects of homologous recombination and the increased bleomycin-sensitivity in the hns mutant. In addition, the transduction analysis in the hns stpA double mutant indicated that the stpA mutation also enhances the defect of recombination in the hns mutant. These results suggest that H-NS plays an important role in both homologous recombination and repair of bleomycin-induced damage, while StpA can substitute the H-NS function. The recombination analysis of hns single, stpA single, and hns stpA double mutants in the recBC sbcA and recBC sbcBC backgrounds suggested that the reduction of the hns single or hns stpA double mutants may not be due to the defect in a particular recombination pathway, but may be due to the defect in a common process of the pathways. The model for the functions of H-NS and StpA in homologous recombination and double-strand break repair is discussed.     

rpoS Function Is Essential for bgl Silencing Caused by C-Terminally Truncated H-NS in Escherichia coli

From evolutionary and physiological viewpoints, the Escherichia coli bgl operon is intriguing because its expression is silent (Bgl(-) phenotype), at least under several laboratory conditions. H-NS, a nucleoid protein, is known as a DNA-binding protein involved in bgl silencing. However, we previously found that bgl expression is still silent in a certain subset of hns mutations, each of which results in a defect in its DNA-binding ability. Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the cis-acting element of the bgl promoter and the mutated H-NS. To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl(+) phenotype in the background of hns60, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn10cam transposon. These isolated mutations were mapped to five loci on the chromosome. Among these loci, three appeared to be leuO, hns, and bglJ, which were previously characterized, while the other two were novel. Genetic analysis revealed that the two insertions are within the rpoS gene and in front of the lrhA gene, respectively. The former encodes the stationary-phase-specific sigma factor, sigma(S), and the latter encodes a LysR-like DNA-binding protein. It was found that sigma(S) is defective in both types of mutant cells. These results showed that the rpoS function is involved in the mechanism underlying bgl silencing, at least in the hns60 background used in this study. We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed. Our results did not support the idea that StpA has an adapter function in the genetic background used.     

Effect of p-Fluorophenylalanine on Chromosome Replication in Escherichia coli

The effect of p-fluorophenylalanine (FPA) on deoxyribonucleic acid (DNA) synthesis and chromosome replication was studied in a thymine-requiring mutant of Escherichia coli. The rate and extent of chromosome replication were followed by labeling the DNA with isotopic thymine and a density marker, bromouracil. The DNA was extracted and analyzed by CsCl gradient centrifugation. The block in chromosome replication caused by high concentrations of FPA occurred at the same point on the chromosome as that caused by amino acid starvation. In a random culture, DNA in cells treated with FPA replicated only slightly slower than the DNA from cells that were not exposed to the analogue. In cultures which had been previously starved for thymine, however, the DNA from the cells treated with FPA showed a marked decrease in the rate and extent of replication. It was concluded that the E. coli cell is most sensitive to FPA when a new cycle of chromosome replication is being initiated at the beginning of the chromosome.     

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.     

Dielectrophoresis as a tool to characterize and differentiate isogenic mutants of Escherichia coli

In this study we report on an experimental method based on dielectrophoretic analysis to identify changes in four Escherichia coli isogenic strains that differed exclusively in one mutant allele. The dielectrophoretic properties of wild-type cells were compared to those of hns, hha, and hha hns mutant derivatives. The hns and hha genes code respectively for the global regulators Hha and H-NS. The Hha and H-NS proteins modulate gene expression in Escherichia coli and other Gram negative bacteria. Mutations in either hha or hns genes result in a pleiotropic phenotype. A two-shell prolate ellipsoidal model has been used to fit the experimental data, obtained from dielectrophoresis measurements, and to study the differences in the dielectric properties of the bacterial strains. The experimental results show that the mutant genotype can be predicted from the dielectrophoretic analysis of the corresponding cultures, opening the way to the development of microdevices for specific identification. Therefore, this study shows that dielectrophoresis can be a valuable tool to study bacterial populations which, although apparently homogeneous, may present phenotypic variability.     

Delta dnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures.

Major heat shock proteins, such as the Escherichia coli DnaK protein, not only are required for cell growth after heat shock but seem to possess important functions in cellular metabolism at normal growth temperatures as well. E. coli delta dnaK52 mutants have severe cellular defects at 30 degrees C, one of which is in cell division (B. Bukau and G. C. Walker, J. Bacteriol, 171:2337-2346, 1989). Here we show that at 30 degrees C, delta dnaK52 mutants have defects in chromosome segregation and in maintenance of low-copy-number plasmids. Fluorescence microscopic analysis revealed that chromosomes were frequently lacking at peripheries of cell filaments of delta dnaK52 mutants and clustered at other locations. In other parts of the cell filaments, chromosomes were apparently normally distributed and they were also present in most of the small cells found in populations of delta dnaK52 cells. These defects might be at the level of DNA replication, since delta dnaK52 mutants have a threshold lower rate of DNA synthesis than wild-type cells. Chromosome segregation defects of delta dnaK52 mutants were also observed in an rnh dnaA mutant background, in which initiation of DNA replication is DnaA-oriC independent. We also found that low-copy-number P1 miniplasmids could not be stably maintained in delta dnaK52 mutants at 30 degrees C. delta par P1 miniplasmids that carry the P1-encoded rep functions required for their replication but lack the P1-encoded par functions required for faithful partitioning of the plasmids during cell division were also unstable in delta dnaK52 mutants. Taken together, our results indicate important, although not absolutely essential, functions for DnaK at 30 degrees C in one or more processes necessary for correct replication and/or partitioning of chromosomes and P1 miniplasmids. Furthermore, we found that P1 miniplasmids were also highly unstable in dnaJ259 mutants, indicating a role for the DnaJ heat shock protein in maintenance of these plasmids.     

Methylation of GATC sites is required for precise timing between rounds of DNA replication in Escherichia coli.

We have used the Koppes and Nordstr?m (Cell 44:117-124, 1986) CsCl density transfer approach for analysis of DNA from exponentially growing, isogenic Escherichia coli dam+ and dam mutant cells to show that timing between DNA replication initiation events is precise in the dam+ cells but is essentially random in the dam cells. Thus, methylation of one or more GATC sites, such as those found in unusual abundance within the origin, oriC, is required for precise timing between rounds of DNA replication, and precise timing between initiation events is not required for cell viability. Both the dam-3 point mutant and the delta(dam)100 complete deletion mutant were examined. The results were independent of the mismatch repair system; E. coli mutH cells showed precise timing, whereas timing in the isogenic E. coli mutH delta(dam)100 double mutant was random. The mechanism is thus different from the role of Dam methylation in mismatch repair and probably involves conversion of hemimethylated GATC sites present in daughter origins just after initiation to a fully methylated state.     

Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium

Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns.     

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.     

Bacillus subtilis SMC is required for proper arrangement of the chromosome and for efficient segregation of replication termini but not for bipolar movement of newly duplicated origin regions

SMC protein is required for chromosome condensation and for the faithful segregation of daughter chromosomes in Bacillus subtilis. The visualization of specific sites on the chromosome showed that newly duplicated origin regions in growing cells of an smc mutant were able to segregate from each other but that the location of origin regions was frequently aberrant. In contrast, the segregation of replication termini was impaired in smc mutant cells. This analysis was extended to germinating spores of an smc mutant. The results showed that during germination, newly duplicated origins, but not termini, were able to separate from each other in the absence of SMC. Also, DAPI (4',6'-diamidino-2-phenylindole) staining revealed that chromosomes in germinating spores were able to undergo partial or complete replication but that the daughter chromosomes were blocked at a late stage in the segregation process. These findings were confirmed by time-lapse microscopy, which showed that after duplication in growing cells the origin regions underwent rapid movement toward opposite poles of the cell in the absence of SMC. This indicates that SMC is not a required component of the mitotic motor that initially drives origins apart after their duplication. It is also concluded that SMC is needed to maintain the proper layout of the chromosome in the cell and that it functions in the cell cycle after origin separation but prior to complete segregation or replication of daughter chromosomes. It is proposed here that chromosome segregation takes place in at least two steps: an SMC-independent step in which origins move apart and a subsequent SMC-dependent step in which newly duplicated chromosomes condense and are thereby drawn apart.     

RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12.

Escherichia coli rnh mutants lacking RNase H activity are capable of recA+-dependent DNA replication in the absence of concomitant protein synthesis (stable DNA replication). In rnh dnaA::Tn10 and rnh delta oriC double mutants in which the dnaA+-dependent initiation of DNA replication at oriC is completely blocked, the recA200 mutation encoding a thermolabile RecA protein renders both colony formation and DNA synthesis of these mutants temperature sensitive. To determine which stage of DNA replication (initiation, elongation, or termination) was blocked, we analyzed populations of these mutant cells incubated at 30 or 42 degrees C in the presence or absence of chloramphenicol (CM) by dual-parameter (DNA-light scatter) flow cytometry. Incubation at 30 degrees C in the presence of CM resulted in cells with a continuum of DNA content up to seven or more chromosome equivalents per cell. The cultures which had been incubated at 42 degrees C in the absence or presence of CM consisted of cells with integral numbers of chromosomes per cell. It is concluded that active RecA protein is required specifically for the initiation of stable DNA replication.     

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.     

SLG1 plays a role during G1 in the decision to enter or exit the cell cycle

Saccharomyces cerevisiae cells decide to divide during G1. If nutrients are abundant, cells pass through START and coordinately undergo DNA replication, bud emergence, and spindle pole body duplication. Phenotypic analysis of the slg1delta mutant revealed that this mutation uncouples post-START events. At the nonpermissive temperature, slg1delta cells that have undergone bud emergence but not DNA replication or SPB duplication accumulate. Furthermore, while wild-type cells arrest in GO when starved, the slg1delta mutant fails to arrest at this point; instead, cells with small buds accumulate. The slg1delta mutation displayed genetic interactions with cdc34, which encodes a regulator of exit from G1. This is consistent with a role of SLG1 in G1 regulation. Epitope-tagged Slg1p cofractionated with the plasma membrane, suggesting that Slglp may function by integrating external cues and relaying them to the interior of the cell. We propose that SLG1 plays a regulatory role in bud emergence or stationary phase.