his operons of Escherichia coli and Salmonella typhimurium are regulated by DNA supercoiling.

The hisW mutations of Salmonella typhimurium are highly pleiotropic mutations that elevate his operon expression, reduce ilv gene expression, alter stable RNA metabolism, and confer defective growth properties. The hisW mutations are highly linked to a naladixic acid-resistant gyrA mutation of S. typhimurium. Multicopy recombinant plasmids containing the Escherichia coli gyrA gene are able to complement both the growth defects and the elevated his operon expression associated with the hisW mutations. We conclude that hisW mutations are alleles of the gyrA gene. The hisU1820 mutant of S. typhimurium exhibits many of the same phenotypes as hisW mutants. Several lines of evidence, including high transduction linkage to recF, suggest that hisU1820 is an allele of gyrB. Finally, well-characterized gyrA and gyrB alleles of E. coli are also his regulatory mutations. We propose that a wild-type degree of chromosomal superhelicity is required for maximal production of histidyl-tRNA and normal his operon regulation.     

Gene for the RNA polymerase sigma subunit mapped in Salmonella typhimurium and Escherichia coli by cloning and deletion.

The genes for the RNA polymerase sigma subunit (rpoD) and DNA primase (dnaG) of Salmonella typhimurium have been cloned into lambda vectors. Combined restriction, deletion and functional analysis of the cloned fragment allows us to map the genes precisely on the fragment, establishes the direction in which rpoD is transcribed, and reveals the existence of at least one new gene in the vicinity. A closely homologous, smaller fragment of Escherichia coli DNA, also cloned into lambda, contains rpoD and at least part of dnaG.     

Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes.

S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system. The S-30 extracts prepared from cells that have been shifted to 45 degrees C express HS genes in vitro approximately 8 times better than extracts from cells at 33 degrees C. In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% of that seen in extracts from cells at 33 degrees C. These results correlate well with the levels of HS sigma factor and normal sigma factor bound to RNA polymerase. Thus, there was an 8-fold increase in the HS sigma factor and a 60% decrease in the normal sigma factor associated with RNA polymerase at the higher temperature. Part of the increase in the level of the HS sigma factor could be accounted for by a 3-fold increase in the level of HS sigma factor mRNA during heat induction.     

Nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli: an evolutionary comparison.

The complete nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli were determined. The nucleotide sequences are 24.8% divergent, compared with amino acid sequence divergence of 14.9%. Over half of the codons of each gene contain synonymous nucleotide changes. The pattern of synonymous nucleotide changes is consistent with the interpretation that such changes result from random mutational events. We do not find any evidence indicating that codon selection or RNA structure is of major selective value. We conclude that polypeptide function is the primary basis of selection in trpA and that most synonymous codon changes are selectively neutral.     

Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli.

The rpoS gene of Escherichia coli encodes a putative RNA polymerase sigma factor that is considered to be the central regulator of gene expression in stationary phase. The gene product (sigma 38) was overproduced using the cloned rpoS gene and purified to homogeneity. Reconstituted RNA polymerase holoenzyme (E sigma 38) was found to recognize in vitro a number of typical sigma 70-type promoters, including the lacUV5 and trp promoters. Some, however, were recognized exclusively or preferentially by E sigma 70, whereas at least one, fic, was favored by E sigma 38. Thus E. coli promoters can be classified into three groups: the first group is recognized by E sigma 70 and E sigma 38, but the second and third groups are recognized substantially by either E sigma 70 or E sigma 38 alone. In contrast to other minor sigma factors, sigma 38 shares a set of amino acid sequences common among the principal sigma factors of eubacteria and is therefore a member of the RpoD-related protein family. The intracellular level of sigma 38 was demonstrated to increase in vivo upon entry into stationary phase. These results together indicate that sigma 38 is a second principal sigma factor in stationary-phase E. coli.     

Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli

A triazole mimic of a DNA phosphodiester linkage has been produced by templated chemical ligation of oligonucleotides functionalized with 5'-azide and 3'-alkyne. The individual azide and alkyne oligonucleotides were synthesized by standard phosphoramidite methods and assembled using a straightforward ligation procedure. This highly efficient chemical equivalent of enzymatic DNA ligation has been used to assemble a 300-mer from three 100-mer oligonucleotides, demonstrating the total chemical synthesis of very long oligonucleotides. The base sequences of the DNA strands containing this artificial linkage were copied during PCR with high fidelity and a gene containing the triazole linker was functional in Escherichia coli.     

Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific.

Specialized type I topoisomerases catalyze DNA strand transfer during site-specific recombination in prokaryotes and fungi. As a rule, the site specificity of these systems is determined by the DNA binding and cleavage preference of the topoisomerase per se. The Mr 32,000 topoisomerase I encoded by vaccinia virus (a member of the eukaryotic family of "general" type I enzymes) is also selective in its interaction with DNA; binding and cleavage occur in vitro at a pentameric motif 5'-(C or T)CCTT in duplex DNA. Expression of vaccinia virus DNA topoisomerase I in a lambda lysogen of Escherichia coli promotes int-independent excisive recombination of the prophage. To address whether the topoisomerase directly catalyzes DNA strand transfer in vivo, the recombination junctions of plaque-purified progeny phage were cloned and sequenced. In five of six distinct excision events examined, a topoisomerase cleavage sequence is present in one strand of the DNA duplex of both recombining partners. Recombination entails no duplication, insertion, or deletion of nucleotides at the crossover points, consistent with excision via conservative strand exchange at sites of topoisomerase cleavage. Three of these five recombination events are distinguished by the presence of direct repeats at the parental half-sites that extend beyond the pentameric cleavage motif, suggesting that sequence homology may facilitate excision. The data are consistent with a model in which vaccinia topoisomerase catalyzes reciprocal strand transfer, leading to the formation of a nonmigrating Holliday junction, the resolution of which can lead to excisive recombination.     

Integration host factor is required for the DNA inversion that controls phase variation in Escherichia coli.

The on-and-off expression (phase variation) of type 1 fimbriae, encoded by fimA, in Escherichia coli is controlled by the inversion of a promoter-containing 314-base-pair DNA element. This element is flanked on each side by a 9-base-pair inverted, repeat sequence and requires closely linked genes for inversion. Homology analysis of the products of these genes, fimB and fimE, reveals a strong similarity with the proposed DNA binding domain of lambda integrase, which mediates site-specific recombination in the presence of integration host factor. Integration host factor, encoded by himA and hip/himD, binds to the sequence 5' TNYAANNNRTTGAT 3', where Y = pyrimidine and R = purine, in mediating integration-excision. In analyzing the DNA flanking the fim 314-base-pair inversion sequence, we found the adjacent sequence 5' TTTAACTTATTGAT 3', which corresponds perfectly with the consensus integration host factor binding site. To characterize the role of himA in phase variation, we transduced either a deletion of himA or an insertionally inactivated hip/himD gene into an E. coli strain with a fimA-lacZ operon fusion. We found the rate of phase variation decreases sharply from 10(-3) to less than 10(-5) per cell per generation. Southern hybridization analysis demonstrates that the himA mutation results in a failure of the switch-generated genetic rearrangement. When the transductant was transformed with a himA+ plasmid, normal switching returned. Thus integration host factor is required for normal type 1 fimbriae phase variation in E. coli.     

Mutants of Salmonella typhimurium and Escherichia coli Pleiotropically Defective in Active Transport

Two classes of mutants isolated from E. coli and Salmonella typhimurium are altered in respiration-coupled active transport, as studied in whole cells and/or isolated membrane vesicles. Mutant cells defective in D-lactate dehydrogenase (dld) transport amino acids and lactose normally. Membrane vesicles prepared from these mutants do not exhibit D-lactate-dependent transport, D-lactate oxidation, or D-lactate: dichlorophenolindophenol reductase activity. However, succinate-dependent transport is markedly enhanced in these mutants, without a corresponding increase in succinic dehydrogenase activity.     

Accumulation of trospectomycin by strains of Salmonella typhimurium, Escherichia coli and Haemophilus influenzae

Trospectomycin, unlike aminoglycosidic aminocyclitols, is accumulated by a nonsaturable, energy-independent, diffusional process in Salmonella typhimurium, Escherichia coli and Haemophilus influenzae. A deep rough mutant of S typhimurium was more susceptible and accumulated the drug faster, and F porin deficient mutants of E coli were more resistant than parental strains. Trospectomycin likely uses both porin and nonporin pathways to cross the outer membrane. An E coli strain effectively accumulated the drug anaerobically, explaining its anaerobic activity. An H influenzae strain accumulated trospectomycin at concentrations below those for which detectable uptake could be observed with E coli or S typhimurium strains, consistent with greater activity in Haemophilus species.     

OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins.

The oxyR gene is required for the induction of a regulon of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium. The E. coli oxyR gene has been cloned and sequenced, revealing an open reading frame (305 amino acids) that encodes a 34.4-kDa protein, which is produced in maxicells carrying the oxyR clone. The OxyR protein shows homology to a family of positive regulatory proteins including LysR in E. coli and NodD in Rhizobium. Like them, oxyR appears to be negatively autoregulated: an oxyR::lacZ gene fusion produced 5-fold higher levels of beta-galactosidase activity in oxyR null mutants compared to oxyR+ controls, and extracts from an OxyR-overproducing strain were able to protect regions (-27 to +21) of the oxyR promoter from DNase I digestion. DNA sequence analysis of the oxyR2 mutation, which causes overexpression of oxyR-regulated proteins in the absence of oxidative stress, showed that the oxyR2 phenotype is due to a missense mutation (C.G to T.A transition) that changes alanine to valine at amino acid position 234 of OxyR.     

Similarity of genes horizontally acquired by Escherichia coli and Salmonella enterica is evidence of a supraspecies pangenome

Most bacterial and archaeal genomes contain many genes with little or no similarity to other genes, a property that impedes identification of gene origins. By comparing the codon usage of genes shared among strains (primarily vertically inherited genes) and genes unique to one strain (primarily recently horizontally acquired genes), we found that the plurality of unique genes in Escherichia coli and Salmonella enterica are much more similar to each other than are their vertically inherited genes. We conclude that E. coli and S. enterica derive these unique genes from a common source, a supraspecies phylogenetic group that includes the organisms themselves. The phylogenetic range of the sharing appears to include other (but not all) members of the Enterobacteriaceae. We found evidence of similar gene sharing in other bacterial and archaeal taxa. Thus, we conclude that frequent gene exchange, particularly that of genetic novelties, extends well beyond accepted species boundaries.     

Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes.

The ada-alkB operon protects Escherichia coli against the effects of many alkylating agents. We have subcloned it into the pSV2 mammalian expression vector to yield pSV2ada-alkB, and this plasmid has been introduced into Mer- HeLa S3 cells, which are extremely sensitive to killing and induction of sister chromatid exchange by alkylating agents. One transformant (the S3-9 cell line) has several integrated copies of pSV2ada-alkB and was found to express a very high level of the ada gene product, the 39-kDa O6-methylguanine-DNA methyltransferase. S3-9 cells were found to have become resistant to killing and induction of sister chromatid exchange by two alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine and N,N'-bis(2-chloroethyl)-N-nitro-sourea. This shows that bacterial DNA alkylation-repair genes are able to suppress the alkylation-repair defects in human Mer- cells.     

Dimorphic transition in Escherichia coli and Salmonella typhimurium: surface-induced differentiation into hyperflagellate swarmer cells.

We describe a new behavioral response in Escherichia coli and Salmonella typhimurium in which the bacteria differentiate into filamentous, multinucleate, hyperflagellate cells that navigate the surface of solid media by means of coordinated swarming motility. The cue for differentiation into swarmer cells is provided by the concentration and composition of the agar. Examination of the behavior of various mutants shows that the flagellar apparatus used for swimming motility and the chemotaxis system are indispensable for swarming motility.