Genetic map of the opp (Oligopeptide permease) locus of Salmonella typhimurium.

The uptake of peptides by Salmonella typhimurium is mediated by three apparently independent transport systems. One of these systems, the oligopeptide permease, is encoded by a genetic locus (opp) which has been mapped at 34 min on the S. typhimurium chromosomal map. We accurately mapped the location of opp by cotransduction frequencies and by deletion analysis and show that the gene order for this region of the chromosome is cysB-trp-tonB-opp-galU-tdk. All opp mutants, independently isolated by a variety of means, mapped at this one locus, between tonB and galU. Spontaneous and transposon Tn10-generated deletions were used to construct a fine-structure genetic map of opp. Evidence is presented which indicates that opp covers a 5- to 6-kb segment of DNA and is therefore likely to consist of more than one gene.     

Genetic analysis of Escherichia coli oligopeptide transport mutants.

The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.     

Molecular characterization of the oligopeptide permease of Salmonella typhimurium

The oligopeptide permease (Opp) of Salmonella typhimurium is a periplasmic binding protein-dependent transport system and handles any peptides containing from two to five amino acid residues. Opp plays an important nutritional role and is also required for the recycling of cell wall peptides. We have determined the nucleotide sequence of the opp operon. In addition to the four opp genes identified previously by genetic means (oppABCD) a fifth gene, oppF, is shown to be cotranscribed as part of the opp operon. Using reverse genetics, we show that oppF also encodes an essential component of the Opp transport system. The five proteins, OppABCDF, are shown to be the only proteins required for Opp function. Regulation of opp expression and of the differential expression of genes within the operon is investigated. We have devised a simple means of constructing lacZ gene fusions to any S. typhimurium chromosomal gene in vivo, using derivatives of bacteriophage Mu. Using this procedure, opp-lacZ gene fusions were selected. The resultant Opp-LacZ hybrid proteins were used to show that OppB, OppC and OppD are membrane-associated proteins. A detailed comparison of the Opp components with those of other binding protein-dependent transport systems provides insight into the mechanisms and evolution of these transport systems.     

Phaseolotoxin transport in Escherichia coli and Salmonella typhimurium via the oligopeptide permease.

Phaseolotoxin [(N delta-phosphosulfamyl)ornithylalanylhomoarginine], a phytotoxic tripeptide produced by Pseudomonas syringae pv. phaseolicola that inhibits ornithine carbamoyltransferase, is transported into Escherichia coli and Salmonella typhimurium via the oligopeptide transport system (Opp). Mutants defective in oligopeptide permease (Opp-) were resistant to phaseolotoxin. Spontaneous phaseolotoxin-resistant mutants (Toxr) lacked the Opp function as evidenced by their cross-resistance to triornithine and failure to utilize glycylhistidylglycine as a source of histidine. Growth inhibition by phaseolotoxin was prevented by peptides known to be transported via the Opp system and by treatment of the toxin with L-aminopeptidase. In both E. coli and S. typhimurium, Toxr mutations were cotransducible with trp, suggesting that the opp locus occupies similar positions in genetic maps of these bacteria.     

The ami locus of the Gram-positive bacterium Streptococcus pneumoniae is similar to binding protein-dependent transport operons of Gram-negative bacteria

The complete nucleotide sequence of the ami locus of Streptococcus pneumoniae revealed the presence of six open reading frames, amiABCDEF. The predicted Ami proteins are probably involved in a transport system. The AmiA, C, D, E, and F proteins exhibit homology with components of the oligopeptide permeases (opp) of Salmonella typhimurium and Escherichia coli. Intriguingly, the AmiB protein is homologous to ArsC, a cytosolic modifier subunit of the anion pump encoded by the arsenical resistance operon of the R-factor R773 from E. coli. Data are presented which indicate that Ami is indeed a transport system.     

Cloning, sequencing, expression and characterization of DNA photolyase from Salmonella typhimurium.

We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5.     

Repair of cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium and Escherichia coli.

The role of nucleotide excision repair and 3-methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3-methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed.     

Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes.

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.     

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.     

Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, D-alanine:D-alanine ligase (ADP forming)

A gene coding for D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) (EC 6.3.2.4) activity has been isolated from a lambda library of Salmonella typhimurium DNA. Insertion mutations in the gene indicate that the gene is not essential for growth of the bacterium. The encoded enzyme was purified from an overproducing strain of S. typhimurium. D-Ala-D-Ala ligase is a protein of 39,271 molecular weight and has a kcat of 644 min-1 at pH 7.2. A 2.4-kilobase SalI-SphI fragment containing the gene was sequenced, and the ddlA gene consists of 1092 nucleotides. The gene sequence was compared to the sequence of the ddl gene of Escherichia coli [Robinson, A. C., Kenan, D. J., Sweeney, J., & Donachie, W. D. (1986) J. Bacteriol. 167, 809-817]. Because of differences between the S. typhimurium gene and the E. coli ddl gene, the S. typhimurium gene has been named ddlA.     

Identification of the enzymatic reactions encoded by the purG and purI genes of Escherichia coli

The chromosomal locations of the genes purG and purI on the Escherichia coli linkage map are the opposites of those of Salmonella typhimurium. By methods which permit the identification of lesions in any of the five early enzymes of the purine de novo pathway, the gene-enzyme relationships of the purG and purI loci have been reevaluated in these two organisms. The results demonstrate that the relative locations of the genes encoding the two enzymes (phosphoribosylformylglycinamidine synthetase and phosphoribosylaminoimidazole synthetase) are similar in the two organisms. The gene products have been correctly determined in S. typhimurium. The gene products currently listed for the loci in E. coli are incorrect. The E. coli purG locus is equivalent to the S. typhimurium purI locus, and the E. coli purI locus is equivalent to the S. typhimurium purG locus.     

Acquisition of maltose chemotaxis in Salmonella typhimurium by the introduction of the Escherichia coli chemosensory transducer gene.

Escherichia coli and Salmonella typhimurium are closely related species. However, E. coli cells show maltose chemotaxis but S. typhimurium cells do not. When an E. coli chemotransducer gene (tarE), the product of which is required for both aspartate and maltose chemotaxis, was introduced by using a plasmid vector into S. typhimurium cells with a defect in the corresponding gene (tarS), the transformant cells acquired the ability for both aspartate and maltose chemotaxis. In contrast, when the tars gene was introduced into tarE-deficient E. coli cells, the transformant cells acquired aspartate chemotaxis but not maltose chemotaxis. These results indicate that the absense of maltose chemotaxis in S. typhimurium is a consequence of the properties of the tars gene product.     

Expression of the cloned Escherichia coli O9 rfb gene in various mutant strains of Salmonella typhimurium.

To investigate the effect of chromosomal mutation on the synthesis of rfe-dependent Escherichia coli O9 lipopolysaccharide (LPS), the cloned E. coli O9 rfb gene was introduced into Salmonella typhimurium strains defective in various genes involved in the synthesis of LPS. When E. coli O9 rfb was introduced into S. typhimurium strains possessing defects in rfb or rfc, they synthesized E. coli O9 LPS on their cell surfaces. The rfe-defective mutant of S. typhimurium synthesized only very small amounts of E. coli O9 LPS after the introduction of E. coli O9 rfb. These results confirmed the widely accepted idea that the biosynthesis of E. coli O9-specific polysaccharide does not require rfc but requires rfe. By using an rfbT mutant of the E. coli O9 rfb gene, the mechanism of transfer of the synthesized E. coli O9-specific polysaccharide from antigen carrier lipid to the R-core of S. typhimurium was investigated. The rfbT mutant of the E. coli O9 rfb gene failed to direct the synthesis of E. coli O9 LPS in the rfc mutant strain of S. typhimurium, in which rfaL and rfbT functions are intact, but directed the synthesis of the precursor. Because the intact E. coli O9 rfb gene directed the synthesis of E. coli O9 LPS in the same strain, it was suggested that the rfaL product of S. typhimurium and rfbT product of E. coli O9 cooperate to synthesize E. coli O9 LPS in S. typhimurium.     

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2.

Two pathways exist for cleavage of the carbon-phosphorus (C-P) bond of phosphonates, the C-P lyase and the phosphonatase pathways. It was previously demonstrated that Escherichia coli carries genes (named phn) only for the C-P lyase pathway and that Enterobacter aerogenes carries genes for both pathways (K.-S. Lee, W. W. Metcalf, and B. L. Wanner, J. Bacteriol. 174:2501-2510, 1992). In contrast, here it is shown that Salmonella typhimurium LT2 carries genes only for the phosphonatase pathway. Genes for the S. typhimurium phosphonatase pathway were cloned by complementation of E. coli delta phn mutants. Genes for these pathways were proven not to be homologous and to lie in different chromosomal regions. The S. typhimurium phn locus lies near 10 min; the E. coli phn locus lies near 93 min. The S. typhimurium phn gene cluster is about 7.2 kb in length and, on the basis of gene fusion analysis, appears to consist of two (or more) genes or operons that are divergently transcribed. Like that of the E. coli phn locus, the expression of the S. typhimurium phn locus is activated under conditions of Pi limitation and is subject to Pho regulon control. This was shown both by complementation of the appropriate E. coli mutants and by the construction of S. typhimurium mutants with lesions in the phoB and pst loci, which are required for activation and inhibition of Pho regulon gene expression, respectively. Complementation studies indicate that the S. typhimurium phn locus probably includes genes both for phosphonate transport and for catalysis of C-P bond cleavage.     

Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs

Mutation of a single gene, referred to as selA1 in Salmonella typhimurium and as selD in Escherichia coli, results in the inability of these organisms to insert selenium specifically into the selenopolypeptides of formate dehydrogenase and into the 2-selenouridine residues of tRNAs. The mutation does not involve transport of selenite into the cell or reduction of selenite to selenide since both mutant strains synthesize selenocysteine and selenomethionine from added selenite and incorporate these selenoamino acids non-specifically into numerous proteins of the bacterial cells. Complementation of the mutation in S. typhimurium with the selD gene from E. coli indicates functional identity of the selA1 and selD genes. Although the selA1 gene maps at approximately 21 min on the S. typhimurium chromosome and the selD gene at approximately 38 min on the E. coli chromosome, only a single gene in wild-type S. typhimurium hybridized to the E. coli selD gene probe. Transformation of the mutant Salmonella strain with a plasmid bearing the E. coli selD gene restored formate dehydrogenase activity, 75Se incorporation into formate dehydrogenase seleno-polypeptides and [75Se]seleno-tRNA synthesis. Transformation with an additional plasmid carrying an E. coli formate dehydrogenase selenopolypeptide-lacZ gene fusion showed that the selD gene allowed readthrough of the UGA codon and synthesis of beta-galactosidase in the Salmonella mutant.     

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.     

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.