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.     

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.     

4-phenyldiazenyl 2-(phenylimino methyl) phenols; synthesis and in-vitro biological evaluation as potential antibacterial agents

A new series of 4-phenyldiazenyl 2-(phenylimino methyl) phenols were synthesized by the condensation of 5-[(2-chloro phenyl) diazenyl] 2-hydroxybenzaldehyde with different substituted aromatic amines and sulphonamides. All the synthesized compounds were screened in-vitro for their antibacterial activity against different human pathogens viz: B. anthracis, E.coli, S. aureus, S. typhimurium, and P. aeruginosa using disk diffusion assay. All the compounds exhibited considerable inhibition against the bacteria tested.     

Azide-induced mutagenesis in gram-negative bacteria is recA-and lexA-independent.

Azide-induced mutagenesis was investigated in Salmonella typhimurium and Escherichia coli. Azide was highly effective in inducing mutation in uvrB, uvrB recA and uvrB recB mutants of S. typhimurium. The mutagenic effect of azide was also observed in uvrA lexA mutants of E. coli K12 and E. coli B/r. These results suggest that azide-induced mutagenesis is due to mis-replication of DNA.     

Genes affecting coliphage BF23 and E colicin sensitivity in Salmonella typhimurium.

Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants resistant to BF23 (bfe) were isolated, and the trait was mapped using phage P1. The bfe gene in S. typhimurium was located between argF (66% co-transducible) and rif (61% co-transducible). The BF23-sensitive S. typhimurium strains were not sensitive to the E colicins. Cells of these rough strains absorbed colicin, as measured by loss of E2 or E3 killing units from colicin solutions and by specific adsorption of 125I-colicin E2 to bfe+ cells. Sensitivity to colicins E1, E2, and E3 was observed in a S. typhimurium strain carrying the F'8 gal+ episome. This episome complemented the tolB mutation of Escherichia coli. We conclude that the bfe+ protein satisfies requirements for adsorption of both phage BF23 and the E colicins. In addition, expression of a gene from E. coli, possibly tolB, is necessary for efficient E colicin killing of S. typhimurium.     

Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium.

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.     

An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp

The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonella typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-1 and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.     

4-phenyldiazenyl 2-(phenylimino methyl) phenols; synthesis and in-vitro biological evaluation as potential antibacterial agents

A new series of 4-phenyldiazenyl 2-(phenylimino methyl) phenols were synthesized by the condensation of 5-[(2-chloro phenyl) diazenyl] 2-hydroxybenzaldehyde with different substituted aromatic amines and sulphonamides. All the synthesized compounds were screened in-vitro for their antibacterial activity against different human pathogens viz: B. anthracis, E.coli, S. aureus, S. typhimurium, and P. aeruginosa using disk diffusion assay. All the compounds exhibited considerable inhibition against the bacteria tested.     

Suitability of recombinant Escherichia coli and Pseudomonas putida strains for selective biotransformation of m-nitrotoluene by xylene monooxygenase

Escherichia coli JM101(pSPZ3), containing xylene monooxygenase (XMO) from Pseudomonas putida mt-2, catalyzes specific oxidations and reductions of m-nitrotoluene and derivatives thereof. In addition to reactions catalyzed by XMO, we focused on biotransformations by native enzymes of the E. coli host and their effect on overall biocatalyst performance. While m-nitrotoluene was consecutively oxygenated to m-nitrobenzyl alcohol, m-nitrobenzaldehyde, and m-nitrobenzoic acid by XMO, the oxidation was counteracted by an alcohol dehydrogenase(s) from the E. coli host, which reduced m-nitrobenzaldehyde to m-nitrobenzyl alcohol. Furthermore, the enzymatic background of the host reduced the nitro groups of the reactants resulting in the formation of aromatic amines, which were shown to effectively inhibit XMO in a reversible fashion. Host-intrinsic oxidoreductases and their reaction products had a major effect on the activity of XMO during biocatalysis of m-nitrotoluene. P. putida DOT-T1E and P. putida PpS81 were compared to E. coli JM101 as alternative hosts for XMO. These promising strains contained an additional dehydrogenase that oxidized m-nitrobenzaldehyde to the corresponding acid but catalyzed the formation of XMO-inhibiting aromatic amines at a significantly lower level than E. coli JM101.     

Demonstration of cel operon expression of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa at elevated temperatures refractory to their growth.

When Escherichia coli was incubated at the growth-refractory temperatures of 48 and 54 degrees C, expression of the cel operon was demonstrated by phospho-beta-glucosidase activity. This enzyme activity was also detected at the growth-refractory temperatures in Salmonella typhimurium and Pseudomonas aeruginosa. Thermotolerant and mesothermophilic mutants of E. coli, S. typhimurium, and P. aeruginosa, able to grow with generation times of 30 to 40 min at 48 and 54 degrees C, exhibited phospho-beta-glucosidase activity at their growth temperatures of 48 and 54 degrees C. Thus, the cel operon previously described as a cryptic operon in E. coli and S. typhimurium was found to be expressed at growth-refractory temperatures of the mesophilic parent and growth-permissive temperatures (48 and 54 degrees C) of the thermotolerant and mesothermophilic mutants.     

Demonstration of cel operon expression of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa at elevated temperatures refractory to their growth.

When Escherichia coli was incubated at the growth-refractory temperatures of 48 and 54 degrees C, expression of the cel operon was demonstrated by phospho-beta-glucosidase activity. This enzyme activity was also detected at the growth-refractory temperatures in Salmonella typhimurium and Pseudomonas aeruginosa. Thermotolerant and mesothermophilic mutants of E. coli, S. typhimurium, and P. aeruginosa, able to grow with generation times of 30 to 40 min at 48 and 54 degrees C, exhibited phospho-beta-glucosidase activity at their growth temperatures of 48 and 54 degrees C. Thus, the cel operon previously described as a cryptic operon in E. coli and S. typhimurium was found to be expressed at growth-refractory temperatures of the mesophilic parent and growth-permissive temperatures (48 and 54 degrees C) of the thermotolerant and mesothermophilic mutants.     

Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair.

Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli.     

A single point mutation in 3-deoxy-D-manno-octulosonate-8-phosphate synthase is responsible for temperature sensitivity in a mutant strain of Salmonella typhimurium

Salmonella typhimurium mutants conditionally deficient in 3-deoxy-d-manno-octulosonate-8-phosphate (KDO8P) synthase activity play a central role in our understanding of lipopolysaccharide function in enteric bacteria. The detailed characterization of KDO8P synthase from such a mutant, however, has not been previously reported. To address this issue KDO8P synthase from S. typhimurium AG701 and from a related temperature-sensitive strain (S. typhimurium AG701i50) have been overexpressed in Escherichia coli and purified to homogeneity. The enzyme from the temperature-sensitive strain has a single proline to serine substitution at position 145, leading to an increase in K(m) for both substrates, d-arabinose 5-phosphate and phosphoenolpyruvate. Analytical gel filtration and native polyacrylamide gel electrophoresis indicate that this enzyme also has an altered oligomeric state. These observations are rationalized through an examination of the structure of E. coli KDO8P synthase, which has 93% sequence identity to the enzyme from S. typhimurium.     

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.     

Mapping and characterization of the nad genes in Salmonella typhimurium LT-2.

An ampicillin enrichment technique was used to isolate 39 nicotinic acid-requiring mutants of Salmonella typhimurium LT-2. Using interrupted-mating and transductional mapping procedures, three loci, designated nadA, nadB, and nadC, were identified. These loci mapped at 33, 82, and 6 min, respectively, on the S. typhimurium linkage map. The arrangement of the loci on the Salmonella linkage map corresponded closely to the nadA, nadB, and nadC loci on the Escherichia coli K-12 linkage map, indicating that the de novo pathway to nicotinamide adenine dinucleotide and the genes governing the enzymes involved in this pathway in S. typhimurium are very similar to those in E. coli. Evidence is also presented which indicates that the product of the nadC locus in S. typhimurium LT-2 is the enzyme quinolinic acid phosphoribosyltransferase. All nadC mutants of S. typhimurium secreted between 2 and 8 mumol of quinolinic acid per 100 ml of secretion medium. In addition, none of the nadC mutants isolated were able to grow in 10(-3) M quinolinic acid, whereas all nadA and nadB mutants of S. typhimurium grew well in the presence of quinolinic acid. Transductional crosses between nadB mutants provided evidence suggestive of more than one locus in the nadB region.     

Salmonella typhimurium newD and Escherichia coli leuC genes code for a functional isopropylmalate isomerase in Salmonella typhimurium-Escherichia coli hybrids.

The supQ newD gene substitution system in Salmonella typhimurium restores leucine prototrophy to leuD mutants by providing the newD gene product which is capable of replacing the missing leuD polypeptide in the isopropylmalate isomerase, a complex of the leuC and leuD gene product. Mutations in the supQ gene are required to make the newD protein available. An Escherichia coli F' factor was constructed which carried supQ- newD+ from S. typhimurium on a P22-specialized transducing genome. This F' pro lac (P22dsupQ394newD) episome was transferred into S. typhimurium strains containing th leuD798-ara deletion; the resulting merodiploid strains had a Leu+ phenotype, indicating that supQ- newD+ is dominant over supQ+ newD+, and eliminating the possibility that the supQ gene codes for a repressor of the newD gene. Furthermore, transfer of the F' pro lac (P22dsupQ39newD) into E. coli leuD deletion strains restored leucine prototrophy, showing that the S. typhimurium newD gene can complment the E. coli leuC gene. Growth rates of the S. typhimurium-E coli hybrid strains indicated that the mutant isopropylmalate isomerase in these strains does not induce a leucine limitation, as it does in S. typhimurium leuD supQ mutants. In vitro activity of the mutant isopropylmalate isomerase was demonstrated; the Km values for alpha-isopropylmalate of both the S. typhimurium leuC-newD isomerase and the S. typhimurium-E. coli hybrid isomerase were as much as 100 times higher than the Km values for alpha-isopropylmalate of the wild-type enzyme, which was 3 x 10(-4) M. Mutagenesis of E. coli leuD deletion strains failed to restore leucine prototrophy, indicating that E. coli does not have genes analogous to the S. typhimurium supQ newD genes, of that, if present, activation of a newD is a rare event or is lethal to the cell.     

Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background.     

Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium, mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity.

A transposon Tn10 insertion into the Salmonella typhimurium poxA gene was identified among a set of mutations conferring sulfometuron methyl (SM) hypersensitivity. This Tn10 insertion mapped to 95 min on the S. typhimurium chromosome, a location analogous to that of poxA in the Escherichia coli genome. Like the E. coli poxA mutant, this mutant had reduced pyruvate oxidase activity, reduced cross-reacting material to antiserum to purified E. coli pyruvate oxidase, and reduced growth rates. In addition, the following phenotypes were identified for the E. coli and S. typhimurium poxA mutants: hypersensitivity to SM and alpha-ketobutyrate (AKB), deficiency in AKB metabolism, reduced activity of acetolactate synthase, and hypersensitivity to a wide range of bacterial growth inhibitors, including antibiotics, amino acid analogs, and dyes. An E. coli mutant defective in poxB, the structural gene encoding pyruvate oxidase, did not have these phenotypes; therefore, they are not solely a consequence of a pyruvate oxidase deficiency. Comparisons were made with mutant alleles of two other genes that are located near poxA and confer related phenotypes. The S. typhimurium poxA mutant differed both genetically and phenotypically from an miaA mutant. E. coli abs mutants had somewhat reduced pyruvate oxidase activity but had normal AKB metabolism. The relationship of the pleiotropic phenotypes of the poxA mutants to their SM hypersensitivity is discussed.     

Cloning, sequencing, and expression of the genes encoding the adenosylcobalamin-dependent ethanolamine ammonia-lyase of Salmonella typhimurium

Ethanolamine ammonia-lyase is a bacterial enzyme that catalyzes the adenosylcobalamin-dependent conversion of certain vicinal amino alcohols to oxo compounds and ammonia. Studies of ethanolamine ammonia-lyase from Clostridium sp. and Escherichia coli have suggested that the enzyme is a heterodimer composed of subunits of Mr approximately 55,000 and 35,000. Using a partial Sau3A Salmonella typhimurium library ligated into pBR328 and selecting by complementation of a mutant lacking ethanolamine ammonia-lyase activity, we have cloned the genes for the 2 subunits of the S. typhimurium enzyme. The genes were localized to a 6.5-kilobase fragment of S. typhimurium DNA, from which they could be expressed in E. coli under noninducing conditions. Sequencing of a 2526-base pair portion of this 6.5-kilobase DNA fragment revealed two open reading frames separated by 21 base pairs. The open reading frames encoded proteins of 452 and 286 residues whose derived N-terminal sequences were identical to the N-terminal sequences of the 2 subunits of the E. coli ethanolamine ammonia-lyase, except that residue 16 of the large subunit was asparagine in the E. coli sequence and aspartic acid in the S. typhimurium sequence.