Degradation of Escherichia coli beta-galactosidase fragments in protease-deficient mutants of Salmonella typhimurium.

The degradation rates of several mutationally generated fragments of Escherichia coli beta-galactosidase were determined in wild-type strains of Salmonella typhimurium and in mutant Salmonella strains lacking several proteases and peptidases. Three termination fragments (produced by lacZ545, lacZ521, and lacZX90) and one internal reinitiation (restart) fragment [lacZpi(1)] are degraded in wild-type Salmonella strains at the same rates observed in wild-type Escherichia coli strains. Mutations that lead to loss of peptidases N, A, B, P, and Q or to loss of protease I or II do not affect the decay rates of any of these fragments. In addition, all of these peptidases and proteases are present in E coli mutants carrying deg mutations (deg mutations are known to stabilize beta-galactosidase fragments). Apparently, none of the proteases and peptidases that are currently accessible to direct genetic analysis plays a role in the early steps of the degradation of protein fragments.     

Isolation of glutamate-inserting ochre suppressor mutants of Salmonella typhimurium and Escherichia coli.

Glutamate-inserting ochre suppressors have been identified among late-arising, spontaneous revertants of a hisG428 mutant of Salmonella typhimurium and an argE3 mutant of Escherichia coli. The S. typhimurium suppressors mapped in the tRNA2(Glu) gene gltU at 82 min; those in E. coli were found to be in tRNA2(Glu) genes gltW at 56 min, gltU at 85 min, and gltT at 90 min.     

Galactose metabolism in gal mutants of Salmonella typhimurium and Escherichia coli

Abstract We report a new pathway for galactose metabolism in Escherichia coli and Salmonella typhimurium . Growth of gal mutants on galactose is restored by the addition of pyrrolo-quinoline quinone (PQQ) to the medium. In such strains galactose is oxidized to galactonate by a PQQ-dependent, membrane-bound dehydrogenase. A pathway for galactonate metabolism in these organisms has already been described.     

his-Linked hydrogen sulfide locus of Salmonella typhimurium and its expression in Escherichia coli.

A his-linked H2S locus of Salmonella typhimurium has been further defined by direct isolation of H2S mutants. Expression of this locus in Escherichia coli has been demonstrated.     

A Col E1 hybrid plasmid containing Escherichia coli genes complementing d-xylose negative mutants of Escherichia coli and Salmonella typhimurium

The Clarke and Carbon bank of Col El - Escherichia coli DNa hybrid plasmids was screened for complementation of d-xylose negative mutants of E. coli. Of several obtained, the smallest, pRM10, was chosen for detailed study. Its size was 16 kilobases (kb) and that of the insert was 9.7 kg. By transformation or F'-mediated conjugation this plasmid complemented mutants of E. coli defective in either D-xylose isomerase or D-xylulose kinase activity, or both. The activity of D-xylulose kinase in E. coli transformants which bear an intact chromosomal gene for this enzyme was greater than that for the host, due to a gene dosage effect. The plasmid also complemented D-xylose negative mutants of Salmonella typhimurium by F'-mediated conjugation between E. coli and S. typhimurium. Salmonella typhimurium mutants complemented were those for D-xylose isomerase and for D-xylulose kinase in addition to pleiotropic D-xylose mutants which were defective in a regulatory gene of the D-xylose operon. In addition, the plasmid complemented the glyS mutation in E. coli and S. typhimurium. The glyS mutant of E. coli was temperature sensitive, indicating that the plasmid carried the structural gene for glycine synthetase. The glyS mutation in E. coli maps at 79 min, as do the xyl genes. The behaviour of the plasmid is consistent with the existence of a d-xylose operon in E. coli. The data also suggest that the plasmid carries three of the genes of this operon, specifically those for D-xylose isomerase, D-xylulose kinase, and a regulatory gene.     

CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli

The putative multi-copper oxidase CueO had previously been implicated in intrinsic copper resistance in Escherichia coli. In this report we showed that the presence of CueO in the periplasm protected alkaline phosphatase from copper-induced damage. CueO contained four copper atoms per molecule and displayed spectroscopic properties typical of blue copper oxidases. CueO catalyzed the oxidation of p-phenylenediamine (pPD), 2,6-dimethoxyphenol (DMP) and exhibited ferroxidase activity in vitro.     

envM genes of Salmonella typhimurium and Escherichia coli.

Conjugation and bacteriophage P1 transduction experiments in Escherichia coli showed that resistance to the antibacterial compound diazaborine is caused by an allelic form of the envM gene. The envM gene from Salmonella typhimurium was cloned and sequenced. It codes for a 27,765-dalton protein. The plasmids carrying this DNA complemented a conditionally lethal envM mutant of E. coli. Recombinant plasmids containing gene envM from a diazaborine-resistant S. typhimurium strain conferred the drug resistance phenotype to susceptible E. coli cells. A guanine-to-adenine exchange in the envM gene changing a Gly codon to a Ser codon was shown to be responsible for the resistance character. Upstream of envM a small gene coding for a 10,445-dalton protein was identified. Incubating a temperature-sensitive E. coli envM mutant at the nonpermissive temperature caused effects on the cells similar to those caused by treatment with diazaborine, i.e., inhibition of fatty acid, phospholipid, and lipopolysaccharide biosynthesis, induction of a 28,000-dalton inner membrane protein, and change in the ratio of the porins OmpC and OmpF.     

Isolation and Characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium.

Mutations in the ack (acetate kinase) and pta (phosphotransacetylase) genes in Salmonella typhimurium were characterized and determined to be analogous to those of previously described Escherichia coli mutants. We established that in both bacterial species these genes were cotransducible with the neighboring histidine transport operon and were distally located relative to purF. pta mutants were sensitive to the dye alizarin yellow and were unable to grow on medium containing inositol as a carbon source. We selected mutants of both species with deletions covering both the ack and the pta genes; some deletions extended into the histidine transport operon.     

Periplasmic space in Salmonella typhimurium and Escherichia coli.

The volume of the periplasmic space in Escherichia coli and Salmonella typhimurium cells was measured. This space, in cells grown and collected under conditions routinely used in work with these bacteria, was shown to comprise from 20 to 40% of the total cell volume. Further studies were conducted to determine the osmotic relationships between the periplasm, the external milieu, and the cytoplasm. Results showed that there is a Donnan equilibrium between the periplasm and the extracellular fluid, and that the periplasm and cytoplasm are isoosmotic. In minimal salts medium, the osmotic strength of the cell interior was estimated to be approximately 300 mosM, with a net pressure of approximately 3.5 atm being applied to the cell wall. A corollary of these findings was that an electrical potential exists across the outer membrane. This potential was measured by determining the distributions of Na+ and Cl- between the periplasm and the cell exterior. The potential varied with the ionic strength of the medium; for cells in minimal salts medium it was approximately 30 mV, negative inside.     

Fine-structure genetic map of the cysB locus in Salmonella typhimurium.

A genetic map of the cysB region of the Salmonella typhimurium chromosome was constructed using bacteriophage P22-mediated transduction. Strains bearing delta (supX cysB) mutations were employed to divide this regulatory locus into 12 segments containing a total of 39 single-site mutations. Twenty-five of these single-site mutations were further ordered by reciprocal three-point crosses. The results do not support the concept of multiple cistrons at cysB and suggest that the abortive transductants previously observed in crosses between certain cysB mutants were due to intracistronic complementation. The prototrophic cys-1352 mutation, which causes the constitutive expression of the cysteine biosynthetic enzymes, was found to lie within the cysB region itself. It is bracketed by mutations, which lead to an inability to derepress for these enzymes and result in auxotrophy for cysteine.     

Expression of Escherichia coli fol alleles in Salmonella typhimurium.

Studies of the expression of Escherichia coli fol alleles in Salmonella typhimurium indicated that fol regulatory functions are highly conserved between these bacterial species.     

Oligopeptidase-deficient mutants of Salmonella typhimurium.

An oligopeptidase that hydrolyzes N-acetyl-L-alanyl-L-alanyl-L-alanyl-L-alanine (AcAla4) has been identified in extracts of Salmonella typhimurium. Mutants lacking this activity have been isolated in dcp mutant strains by screening extracts of mutagenized clones for failure to hydrolyze AcAla4 or by screening colonies for inability to use AcAla4 as a nitrogen source. Double mutants (dcp optA) lacking both oligopeptidase A and dipeptidyl carboxypeptidase cannot use AcAla4 as a nitrogen source, although dcp+ optA and dcp optA+ strains grow on this peptide. The mutations responsible for the loss of activity map at a locus (optA) between asd (75 map units) and xylA (78 map units). Oligopeptidase A hydrolyzes certain N-blocked tetrapeptides, unblocked pentapeptides, and unblocked hexapeptides, usually but not always liberating the C-terminal tripeptide. These two activities seem to be responsible for the production of a large fraction of the dipeptides that accumulate during protein breakdown in a pepN pepA pepB pepD strain.     

IlvHI locus of Salmonella typhimurium.

In Escherichia coli K-12, the ilvHI locus codes for one of two acetohydroxy acid synthase isoenzymes. A region of the Salmonella typhimurium genome adjacent to the leucine operon was cloned on plasmid pBR322, yielding plasmids pCV47 and pCV49 (a shortened version of pCV47). This region contains DNA homologous to the E. coli ilvHI locus, as judged by hybridization experiments. Plasmid pCV47 did not confer isoleucine-valine prototrophy upon either E. coli or S. typhimurium strains lacking acetohydroxy acid synthase activity, suggesting that S. typhimurium lacks a functional ilvHI locus. However, isoleucine-valine prototrophs were readily isolated from such strains after mutagenesis with nitrosoguanidine. In one case we found that the Ilv+ phenotype resulted from an alteration in bacterial DNA on the plasmid (new plasmid designated pCV50). Furthermore, a new acetohydroxy acid synthase activity was observed in Ilv+ revertants; this enzyme was similar to E. coli acetohydroxy acid synthase III in its lack of activity at low pH. This new activity was correlated with the appearance in minicells of a new polypeptide having an approximate molecular weight of 61,000. Strains carrying either pCV49 or pCV50 produced a substantial amount of ilvHI-specific mRNA. These results, together with results from other laboratories, suggest that S. typhimurium has functional ilvB and ilvG genes and a cryptic ilvHI locus. E. coli K-12, on the other hand, has functional ilvB and ilvHI genes and a cryptic ilvG locus.     

Fertility of Salmonella typhimurium Crosses with Escherichia coli

At least one factor that causes low fertility of Salmonella typhimurium LT2 strains in crosses with Escherichia coli K-12 Hfr's can be inhibited by growing the female strains in supplemented minimal salts medium rather than in nutrient broth and by incubating the female strains at 50 C immediately before mating with the Hfr. These pretreatments can enhance the recovery of prototrophic recombinants for markers injected early by the Hfr by a factor of as much as 10(4). The heat treatment is effective only on the female in intergeneric crosses and gradually loses (within 50 min) its effectiveness after return of heat-treated cells to 37 C. It is concluded that the restriction system of the female is heat-sensitive. Since markers injected late by the male enter females in which the heat-impaired restriction system has recovered, few recombinants for late markers are found. The presence of the leading end of an E. coli Hfr in an S. typhimurium-E. coli hybrid enhances by up to sevenfold the frequency of lac(+) recombinants in subsequent crosses with an E. coli Hfr if the E. coli segment is integrated into the chromosome of the hybrid; the effect is less marked if the E. coli segment is not integrated.     

Oxidative stress responses in Escherichia coli and Salmonella typhimurium.

Oxidative stress is strongly implicated in a number of diseases, such as rheumatoid arthritis, inflammatory bowel disorders, and atherosclerosis, and its emerging as one of the most important causative agents of mutagenesis, tumorigenesis, and aging. Recent progress on the genetics and molecular biology of the cellular responses to oxidative stress, primarily in Escherichia coli and Salmonella typhimurium, is summarized. Bacteria respond to oxidative stress by invoking two distinct stress responses, the peroxide stimulon and the superoxide stimulon, depending on whether the stress is mediated by peroxides or the superoxide anion. The two stimulons each contain a set of more than 30 genes. The expression of a subset of genes in each stimulon is under the control of a positive regulatory element; these genes constitute the OxyR and SoxRS regulons. The schemes of regulation of the two regulons by their respective regulators are reviewed in detail, and the overlaps of these regulons with other stress responses such as the heat shock and SOS responses are discussed. The products of Oxy-R- and SoxRS-regulated genes, such as catalases and superoxide dismutases, are involved in the prevention of oxidative damage, whereas others, such as endonuclease IV, play a role in the repair of oxidative damage. The potential roles of these and other gene products in the defense against oxidative damage in DNA, proteins, and membranes are discussed in detail. A brief discussion of the similarities and differences between oxidative stress responses in bacteria and eukaryotic organisms concludes this review.     

Stabilization of glucose-starved Escherichia coli K12 and Salmonella typhimurium LT2 by peptidase-deficient mutants

Escherichia coli K12 and Salmonella typhimurium LT2 cells were stabilized during carbon starvation in the presence of peptidase-deficient mutant strains. The rate of loss of viability of the wild-type S. typhimurium strain was decreased an average of 2-fold, and the rate for the wild-type E. coli strain was decreased about 2.3-fold, when either was starved in the presence of the multiply peptidase-deficient S. typhimurium strain TN852; other peptidase-deficient strains exhibited similar stabilizing effects. Starving wild-type S. typhimurium LT2 cells utilized peptides excreted by the starving peptidase-deficient cells for protein synthesis, and, to a lesser extent, as respiratory substrates. Provision of free amino acids in steady-state levels to starving E. coli K12 cells in a cell recycle apparatus had a stabilizing effect similar to that of mixing with peptidase-deficient cells.     

Transition of deletion mutants of the composite resistance plasmid NR1 in Escherichia coli and Salmonella typhimurium.

Derivatives of the composite R plasmid NR1 from which a portion of the resistance determinants (r-determinants) component had been deleted were found to undergo amplification of the remaining r-determinants region in Escherichia coli and Salmonella typhimurium. The wild-type NR1 plasmid does not amplify in these genera, although all of these plasmids undergo amplification in Proteus mirabilis. The deletion mutants retained the mercuric ion resistance operon (mer) but conferred a much lower level of sulfonamide resistance than NR1. The remaining r-determinants region, which is bounded by direct repeats of the insertion element IS1, formed multiple tandem duplications in E. coli, S. typhimurium, and P. mirabilis after subculturing the host cells in medium containing high concentrations of sulfonamide. Gene amplification was characterized by restriction endonuclease analysis, analytical buoyant density centrifugation, DNA-DNA hybridization, and sedimentation in sucrose gradients. The tandem repeats remained attached to the resistance transfer factor component of the plasmid in at least part of the plasmid population; autonomous tandem repeats of r-determinants were probably also present. Amplification did not occur in host recA mutants. Amplified strains subcultured in drug-free medium lost the amplified r-determinants. By using a strain temperature sensitive for the recA gene, it was possible to obtain gene amplification at the permissive temperature. Loss of r-determinants took place at the permissive temperature, but not at the nonpermissive temperature. The termini of the deletions of several independent mutants which conferred low sulfonamide resistance were found to be located within the adjacent streptomycin-spectinomycin resistance gene.     

Altered growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level in hisT mutants of Salmonella typhimurium and Escherichia coli.

In Escherichia coli, the level of 6-phosphogluconate dehydrogenase is directly proportional to the cellular growth rate during growth in minimal media. This contrasts with the report by Winkler et al. (M. E. Winkler, J. R. Roth, and P. E. Hartman, J. Bacteriol. 133:830-843, 1978) that the level of the enzyme in Salmonella typhimurium LT-2 strain SB3436 is invariant. The basis for the difference in the growth-rate-dependent regulation between the two genera was investigated. Expression of gnd, which encodes 6-phosphogluconate dehydrogenase, was growth rate uninducible in strain SB3436, as reported previously, but it was 1.4-fold growth rate inducible in other S. typhimurium LT-2 strains, e.g., SA535. Both the SB3436 and SA535 gnd genes were growth rate inducible in E. coli K-12. Moreover, the nucleotide sequences of the regulatory regions of the two S. typhimurium genes were identical. We concluded that a mutation unlinked to gnd is responsible for the altered growth rate inducibility of 6-phosphogluconate dehydrogenase in strain SB3436. Transductional analysis showed that the altered regulation is due to the presence of a mutation in hisT, the gene for the tRNA modification enzyme pseudouridine synthetase I. A complementation test showed that the regulatory defect conferred by the hisT mutation was recessive. In E. coli, hisT mutations reduced the extent of growth rate induction by the same factor as in S. typhimurium. The altered regulation conferred by hisT mutations was not simply due to their general effect of reducing the polypeptide chain elongation rate, because miaA mutants, which lack another tRNA modification and have a similarity reduced chain growth rate, had higher rather than lower 6-phosphogluconate dehydrogenase levels. Studies with genetic fusions suggested that hisT mutations lower the gnd mRNA level. The data also indicated that hisT is involved in translational control of gnd expression, but not the aspect mediated by the internal complementary sequence.