A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli.

Three distinct clones from a Salmonella typhimurium genomic library were identified which suppressed the copper-sensitive (Cu(s)) phenotype of cutF mutants of Escherichia coli. One of these clones, pCUTFS2, also increased the copper tolerance of cutA, -C, and -E mutants, as well as that of a lipoprotein diacylglyceryl transferase (lgt) mutant of E. coli. Characterization of pCUTFS2 revealed that the genes responsible for suppression of copper sensitivity (scs) reside on a 4.36-kb DNA fragment located near 25.4 min on the S. typhimurium genome. Sequence analysis of this fragment revealed four open reading frames (ORF120, ORF627, ORF207, and ORF168) that were organized into two operons. One operon consisted of a single gene, scsA (ORF120), whereas the other operon contained the genes scsB (ORF627), scsC (ORF207), and scsD (ORF168). Comparison of the deduced amino acid sequences of the predicted gene products showed that ScsB, ScsC, and ScsD have significant homology to thiol-disulfide interchange proteins (CutA2, DipZ, CycZ, and DsbD) from E. coli and Haemophilus influenzae, to an outer membrane protein (Com1) from Coxiella burnetii, and to thioredoxin and thioredoxin-like proteins, respectively. The two operons were subcloned on compatible plasmids, and complementation analyses indicated that all four proteins are required for the increased copper tolerance of E. coli mutants. In addition, the scs locus also restored lipoprotein modification in lgt mutants of E. coli. Sequence analyses of the S. typhimurium scs genes and adjacent DNAs revealed that the scs locus is flanked by genes with high homology to the cbpA (predicted curved DNA-binding protein) and agp (acid glucose phosphatase) genes of E. coli located at 22.90 min (1,062.07 kb) and 22.95 min (1,064.8 kb) of the E. coli chromosome, respectively. However, examination of the E. coli chromosome revealed that these genes are absent at this locus and no evidence has thus been obtained for the occurrence of the scs locus elsewhere on the genome.     

Structural characterization of the Salmonella typhimurium LT2 umu operon.

The umuDC operon of Escherichia coli encodes functions required for mutagenesis induced by radiation and a wide variety of chemicals. The closely related organism Salmonella typhimurium is markedly less mutable than E. coli, but a umu homolog has recently been identified and cloned from the LT2 subline. In this study the nucleotide sequence and structure of the S. typhimurium LT2 umu operon have been determined and its gene products have been identified so that the molecular basis of umu activity might be understood more fully. S. typhimurium LT2 umu consists of a smaller 417-base-pair (bp) umuD gene ending 2 bp upstream of a larger 1,266-bp umuC gene. The only apparent structural difference between the two operons is the lack of gene overlap. An SOS box identical to that found in E. coli is present in the promoter region upstream of umuD. The calculated molecular masses of the umuD and umuC gene products were 15.3 and 47.8 kilodaltons, respectively, which agree with figures determined by transpositional disruption and maxicell analysis. The S. typhimurium and E. coli umuD sequences were 68% homologous and encoded products with 71% amino acid identity; the umuC sequences were 71% homologous and encoded products with 83% amino acid identity. Furthermore, the potential UmuD cleavage site and associated catalytic sites could be identified. Thus the very different mutagenic responses of S. typhimurium LT2 and E. coli cannot be accounted for by gross differences in operon structure or gene products. Rather, the ability of the cloned S. typhimurium umuD gene to give stronger complementation of E. coli umuD77 mutants in the absence of a functional umuC gene suggests that Salmonella UmuC protein normally constrains UmuD protein activity.     

Nucleotide sequence analysis of purH and purD genes from Salmonella typhimurium

The purH and purD genes coding for the 5'-phosphoribosyl 5-amino-imidazole-4-carboxamide (AICAR) transformylase and 5'-phosphoribosyl-glycinamide (GAR) synthetase, respectively, were identified on a 4.8 kb Eco RI fragment of chromosomal DNA from Salmonella typhimurium. Nucleotide sequence analysis of the cloned fragment revealed the presence of two large open reading frames (O.R.F.), which were separated by 11 base pairs (bp). Substantial DNA and amino acid sequence homology was noted between the purH and purD genes of S. typhimurium and Escherichia coli. Expression of the Salmonella purD gene in a T7 polymerase/promoter system revealed the presence of a 49 kDa protein band by SDS-PAGE and subsequent autoradiography. The purH gene of Salmonella was not expressed since the 5' end of this gene was not cloned.     

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.     

Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium.

We report the analysis of three open reading frames of Salmonella typhimurium LT2 which we identified as rfaF, the structural gene for ADP-heptose:LPS heptosyltransferase II; rfaD, the structural gene for ADP-L-glycero-D-manno-heptose-6-epimerase; and part of kbl, the structural gene for 2-amino-3-ketobutyrate CoA ligase. A plasmid carrying rfaF complements an rfaF mutant of S. typhimurium; rfaD and kbl are homologous to and in the same location as the equivalent genes in Escherichia coli K-12. The RfaF (heptosyl transferase II) protein shares regions of amino acid homology with RfaC (heptosyltransferase I), RfaQ (postulated to be heptosyltransferase III), and KdtA (ketodeoxyoctonate transferase), suggesting that these regions function in heptose binding. E. coli contains a block of DNA of about 1,200 bp between kbl and rfaD which is missing from S. typhimurium. This DNA includes yibB, which is an open reading frame of unknown function, and two promoters upstream of rfaD (P3, a heat-shock promoter, and P2). Both S. typhimurium and E. coli rfaD genes share a normal consensus promoter (P1). We postulate that the yibB segment is an insertion into the line leading to E. coli from the common ancestor of the two genera, though it could be a deletion from the line leading to S. typhimurium. The G+C content of the rfaLKZYJI genes of both S. typhimurium LT2 and E. coli K-12 is about 35%, much lower than the average of enteric bacteria; if this low G+C content is due to lateral transfer from a source of low G+C content, it must have occurred prior to evolutionary divergence of the two genera.     

Salmonella typhimurium has two homologous but different umuDC operons: cloning of a new umuDC-like operon (samAB) present in a 60-megadalton cryptic plasmid of S. typhimurium.

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The DNA which can restore UV mutability to a umuD44 strain and to a umuC122::Tn5 strain of E. coli has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the cloned DNA potentially encoded proteins with calculated molecular weights of 15,523 and 47,726 and was an analog of the E. coli umuDC operon. We have termed this cloned DNA the samAB (for Salmonella mutagenesis) operon and tentatively referred to the umuDC operon of S. typhimurium LT2 (C. M. Smith, W. H. Koch, S. B. Franklin, P. L. Foster, T. A. Cebula, and E. Eisenstadt, J. Bacteriol. 172:4964-4978, 1990; S. M. Thomas, H. M. Crowne, S. C. Pidsley, and S. G. Sedgwick, J. Bacteriol. 172:4979-4987, 1990) as the umuDCST operon. The samAB operon is 40% diverged from the umuDCST operon at the nucleotide level. Among five umuDC-like operons so far sequenced, i.e., the samAB, umuDCST, mucAB, impAB, and E. coli umuDC operons, the samAB operon shows the highest similarity to the impAB operon of TP110 plasmid while the umuDCST operon shows the highest similarity to the E. coli umuDC operon. Southern hybridization experiments indicated that (i) S. typhimurium LT2 and TA1538 had both the samAB and the umuDCST operons and (ii) the samAB operon was located in a 60-MDa cryptic plasmid. The umuDCST operon is present in the chromosome. The presence of the two homologous but different umuDC operons may be involved in the poor mutability of S. typhimurium by UV and chemical mutagens.     

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.     

Molecular cloning and characterization of supQ/newD, a gene substitution system for the leuD gene of Salmonella typhimurium.

The isopropylmalate isomerase of Salmonella typhimurium and Escherichia coli is a complex of the leuC and leuD gene products. The supQ/new D gene substitution system in S. typhimurium restores leucine prototrophy to leuD mutants of S. typhimurium. Previous genetic evidence supports a model that indicates the replacement of the missing LeuD polypeptide by the newD gene product. This model proposed that this gene substitution is possible when a mutation at the supQ locus (near newD) liberates unaltered newD polypeptide from its normal complex with the supQ protein product. In this study, recombinant plasmids carrying newD, supQ, or both were transformed into E. coli and S. typhimurium strains deleted for the leuD and supQ genes to test the supQ/newD gene substitution model for suppression of leucine auxotrophy. It was determined that the newD gene encodes a 22-kilodalton polypeptide which can restore leucine prototrophy to leuD deletion strains and that a functional supQ gene prevents this suppression. It was also determined that the supQ and newD genes are separated by a gene encoding a 50-kilodalton protein, pB. While there is extensive DNA sequence homology between the leucine operons of S. typhimurium and E. coli, DNA hybridization experiments did not indicate substantial homology between the newD and leuD genes. These data, taken together with previously obtained genetic data, eliminate the possibility that supQ and newD are recently translocated segments of the leucine operon.     

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.     

Metabolic regulation of the histidine operon in Escherichia coli and Salmonella typhimurium

Expression of the histidine operon in Escherichia coli cells in contrast to the one in Salmonella typhimurium is changed proportionally to cells growth rate on the different carbon sources. The specific activity of histidinol-dehydrogenase is repressed by addition of 19 amino acids both in Escherichia coli and Salmonella typhimurium independent of the growth medium used. Using of Escherichia coli and Salmonella typhimurium strains containing the heterologous histidine operons made possible to demonstrate the dependence of the histidine operon metabolic regulation to be determined by the operon itself but not by the specificity of the recipient cells. ppGpp was shown to be a positive regulator of the histidine operon expression in Escherichia coli.     

Cloning and sequence analysis of hydrogenase regulatory genes (hydHG) from Salmonella typhimurium.

The nucleotide sequence of the hydHG operon, comprised of chromosomal genes that regulate labile hydrogenase activity in Salmonella typhimurium, was compared with the reported hydHG sequence of Escherichia coli. Nucleotide sequence analysis of a 4.8 kb EcoRI fragment of Salmonella chromosomal DNA revealed that one of the open reading frames (ORF) encoded a protein of 441 amino acid residues. This large ORF was identified on a 2.7 kb Eco RI/HindIII fragment and coded for the complete hydG gene. The carboxy-terminus (626 bp) of the hydH gene also was present immediately upstream of hydG. Expression of the Salmonella hydG gene in a T7 promoter/polymerase system revealed the presence of a unique 45 kDa protein band. The incomplete hydH gene was not expressed. It is proposed that the labile hydrogenase activity in S. typhimurium may be regulated by the multiple component system.     

Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli

Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E. coli strains deleted for the gal operon. Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322. Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype. Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site. The system was used efficiently for cloning lambda, yeast, and human DNA. The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance. Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites. Construction of several related vectors is described.     

Mapping and spacer identification of rRNA operons of Salmonella typhimurium.

The rRNA operons of Salmonella typhimurium have been characterized with respect to their map position, orientation, and type of tRNA spacer. One of the seven rrn operons was found to be linked to pheA and another was found to be linked to aroE. This information, together with published information about the other five rrn operons, shows that S. typhimurium and Escherichia coli are essentially identical in terms of the number, the map position, and the orientation of all seven operons. S. typhimurium and E. coli were also similar in that four of the rrn spacer regions code for tRNAGlu2 and three code for tRNAAla1B. However, the two species differed in that rrnD coded for tRNAGlu2 and rrnB coded for tRNAAla1B in S. typhimurium. This is the opposite of the arrangement in E. coli. We have tabulated the coordinates of the BamHI and PstI sites flanking six of the S. typhimurium rrn genes and present revisions for the coordinates of some of the E. coli sites.     

Identification of a umuDC locus in Salmonella typhimurium LT2.

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV light and many other DNA-damaging agents. The existence of a umuDC analog in Salmonella typhimurium has been questioned. With DNA probes to the E. coli umuD and umuC genes, we detected, by Southern blot hybridization, sequences similar to both of these genes in S. typhimurium LT2. We also confirmed that the presence of cloned E. coli umuD enhances the UV mutability and resistance of S. typhimurium. Our data strongly suggest that S. typhimurium contains a functional umuDC operon.     

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.