Phenotypic Analysis of Random hns Mutations Differentiate DNA-Binding Activity from Properties of fimA Promoter Inversion Modulation and Bacterial Motility

H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of the proU and fimB genes, increased fimA promoter inversion rates, and repression of the flhCD master operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proU expression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimB occurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hns mutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.     

Expression inEscherichia coliof Y5-Mutant and N-terminal Domain-Deleted DNA Gyrase B Proteins Affects Strongly Plasmid Maintenance

Escherichia coliDNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43-kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, competein vivowith the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoilingin vivo.This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activityin vivo.Thus, ourin vivoapproach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or the GyrB/GyrB or GyrB/GyrA protein interactions.     

55.2, a Phage T4 ORFan Gene,Encodes an Inhibitor of Escherichia coli Topoisomerase I and Increases Phage Fitness

Topoisomerases are enzymes that alter the topological properties of DNA. Phage T4 encodes its own topoisomerase but it can also utilize host-encoded topoisomerases. Here we characterized 55.2, a phage T4 predicted ORF of unknown function. High levels of expression of the cloned 55.2 gene are toxic in E. coli. This toxicity is suppressed either by increased topoisomerase I expression or by partial inactivation of the ATPase subunit of the DNA gyrase. Interestingly, very low-level expression of 55.2, which is non-lethal to wild type E. coli, prevents the growth of a deletion mutant of the topoisomerase I (topA) gene. In vitro, gp55.2 binds DNA and blocks specifically the relaxation of negatively supercoiled DNA by topoisomerase I. In vivo, expression of gp55.2 at low non-toxic levels alters the steady state DNA supercoiling of a reporter plasmid. Although 55.2 is not an essential gene, competition experiments indicate that it is required for optimal phage growth. We propose that the role of gp55.2 is to subtly modulate host topoisomerase I activity during infection to insure optimal T4 phage yield.     

nifH promoter activity is regulated by DNA supercoiling in Sinorhizobium meliloti

In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown ceils, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S.meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoiling-dependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.     

Identification of DNA topoisomerases involved in immediate and transient DNA relaxation induced by heat shock inEscherichia coli

The linking number of plasmid DNA in exponentially growingEscherichia coli increases immediately and transiently after heat shock. The purpose of this study was to search for DNA topoisomerases that catalyze this relaxation of DNA. Neither introduction of atopA deletion mutation nor treatment of cells with DNA gyrase inhibitors affected the DNA relaxation induced by heat shock. Thus, DNA topoisomerase I and DNA gyrase are apparently not involved in the process. However, the reaction was inhibited by nalidixic acid or by oxolinic acid in thetopA mutant and the reaction was resistant to nalidixic acid in atopA mutant carrying, in addition, thenalA26 mutation. These results are interpreted as indicating that both DNA topoisomerase I and DNA gyrase are involved in the DNA relaxation induced by heat shock.     

Heterogeneity in the level of ampicillin resistance conferred by pBR322 derivatives with different DNA supercoiling

Summary Cloning of an EcoRI restriction fragment, containing the 900 bp -terminal sequence of transposon Tn1000, into pBR322, resulted in two plasmids, pICV63 and pICV64, which differed in the orientation of the cloned fragment within the replicon and in the level of ampicillin resistance conferred on the host cell. The DNAs of these plasmids differ in superhelicity and we suggest that a change in supercoiling of pICV63 DNA leads to this plasmid conferring resistance to only low levels of ampicillin, probably by reducing the expression of the bla gene. This hypothesis is supported by the fact that topA or supX mutations, which abolish topoisomerase I, reduce still further the level of resistance to ampicillin of pICV63-containing cells, whereas the gyrB226 compensatory mutation renders these cells more ampicillin resistant. Plasmid pICV63, therefore, enables mutant alleles of genes governing DNA topology to be recognized.     

Topoisomerase mutations affect the relative abundance of many Escherichia coli proteins

The relative abundance of 88 proteins was measured in extracts from three strains of Escherichia coli K-12 that are isogenic except for the topA and gyrB genes. Mutations in these genes slightly raise or lower, respectively, steady-state DNA supercoiling levels but have little effect on growth rate. Altered protein abundances were observed in the mutant strains relative to wild type. Many proteins exhibited minimum abundance at wild-type supercoiling levels, and other proteins exhibited maximal abundance at relaxed levels. A smaller number showed maximal abundance at elevated levels of supercoiling. These data suggest that small, non-lethal changes in DNA supercoiling can have widespread effects on patterns of gene expression.