DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli

Nucleotide sequences of the cysB region of Salmonella typhimurium and Escherichia coli have been determined and compared. A total of 1759 nucleotides were sequenced in S. typhimurium and 1840 in E. coli. Both contain a 972-nucleotide open reading frame identified as the coding region for the cysB regulatory protein on the basis of sequence homology and by comparison of the deduced amino acid sequences with known physicochemical properties of this protein. The DNA sequence identity for the cysB coding region in the two species is 80.5%. The deduced amino acid sequences are 95% identical. The predicted cysB polypeptide molecular weights are 36,013 for S. typhimurium and 36,150 for E. coli. For both proteins a helix-turn-helix region similar to that found in other DNA-binding proteins is predicted from the deduced amino acid sequence. Sequences upstream to cysB contain open reading frames which represent the carboxyl-terminal end of the topA gene product, DNA topoisomerase I. A pattern of highly conserved nucleotide sequences in the 151 nucleotides immediately preceding the cysB initiator codon in both species suggests that this region may contain multiple signals for the regulation of cysB expression.     

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.     

araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora.

The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.     

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.     

Gene-protein relationships in the flagellar hook-basal body complex of Bacillus subtilis: sequences of the flgB, flgC, flgG, fliE and fliF genes

The nucleotide sequence of five genes from the major Bacillus subtilis chemotaxis locus has been determined. Four of these genes encode proteins that are homologous to the Salmonella typhimurium FlgB, FlgC, FlgG and FliF proteins. One gene encodes a protein that is homologous to the Escherichia coli FliE protein. The data from S. typhimurium and E. coli suggest that all of these proteins form part of the hook-basal body (HBB) complex of the bacterial flagella. The FlgB, FlgC and FlgG proteins are components of the proximal and distal rods. The FliF protein forms the M-ring that anchors the rod assembly to the membrane. The role of the FliE protein within the HBB complex has not yet been determined. The similarity between the B. subtilis and S. typhimurium proteins suggests that the structure of the M-ring and the rod may be similar in the two species. However, we observed some differences in size and amino acid composition between some of the corresponding homologues that suggest the basal body proteins may be organized slightly differently within B. subtilis.     

Genes on the 90-kilobase plasmid of Salmonella typhimurium confer low-affinity cobalamin transport: relationship to fimbria biosynthesis genes.

A cloned fragment of Salmonella typhimurium DNA complemented the defect in cobalamin uptake of Escherichia coli or S. typhimurium btuB mutants, which lack the outer membrane high-affinity transport protein. This DNA fragment did not carry btuB and was derived from the 90-kb plasmid resident in S. typhimurium strains. The cobalamin transport activity engendered by this plasmid had substantially lower affinity and activity than that conferred by btuB. Complementation behavior and maxicell analyses of transposon insertions showed that the cloned fragment encoded five polypeptides, at least two of which were required for complementation activity. The nucleotide sequence of the coding region for one of these polypeptides, an outer membrane protein of about 84,000 Da, was determined. The deduced polypeptide had properties typical of outer membrane proteins, with an N-terminal signal sequence and a predicted preponderance of beta structure. This outer membrane protein had extensive amino acid sequence homology with PapC and FaeD, two E. coli outer membrane proteins involved in the export and assembly of pilus and fimbria subunits on the cell surface. This homology raises the likelihood that the observed cobalamin transport did not result from the production of an authentic transport system but that overexpression of one or more outer membrane proteins allowed leakage of cobalamins through the perturbed outer membrane. These results also suggest that the 90-kb plasmid carries genes encoding an adherence mechanism.     

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.     

Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium.

The gene determining the structure of a major outer membrane protein of Escherichia coli, protein Ia, has been located between serC and pyrD, at the min 21 region of the linkage map. This is based on the isolation and characterization of E. coli-Salmonella typhimurium intergeneric hybrids as well as analyses of a mutation (ompF2) affecting the formation of protein Ia. When the serC region of the S. typhimurium chromosome was transduced by phage P1 into E. coli, two classes of transductants were obtained; one produced protein Ia like the parental strain of E. coli, whereas the other produced not protein Ia but a pair of outer membrane proteins structurally related to 35K protein, one of the major outer membrane proteins of S. typhimurium. Furthermore, a strain of S. typhimurium harboring an F' plasmid which carries the ompF region of the E. coli chromosome was found to produce a protein indistinguishable from protein Ia, beside the outer membrane proteins characteristic to the parental Salmonella strain. These results suggest that the structural genes for protein Ia (E. coli) and for 35K protein (S. typhimurium) are homologous to each other and are located at the ompF region of the respective chromosome. The bearing of these findings on the genetic control of protein Ia formation is discussed.     

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.     

Utilization of orotate as a pyrimidine source by Salmonella typhimurium and Escherichia coli requires the dicarboxylate transport protein encoded by dctA.

Mutants deficient in orotate utilization (initially termed out mutants) were isolated by selection for resistance to 5-fluoroorotate (FOA), and the mutations of 12 independently obtained isolates were found to map at 79 to 80 min on the Salmonella typhimurium chromosome. A gene complementing the mutations was cloned and sequenced and found to possess extensive sequence identity to characterized genes for C4-dicarboxylate transport (dctA) in Rhizobium species and to the sequence inferred to be the dctA gene of Escherichia coli. The mutants were unable to utilize succinate, malate, or fumarate as sole carbon source, an expected phenotype of dctA mutants, and introduction of the cloned DNA resulted in restoration of both C4-dicarboxylate and orotate utilization. Further, succinate was found to compete with orotate for entry into the cell. The S. typhimurium dctA gene encodes a highly hydrophobic polypeptide of 45.4 kDa, and the polypeptide was found to be enriched in the membrane fraction of minicells harboring a dctA+ plasmid. The DNA immediately upstream of the deduced -35 region contains a putative cyclic AMP-cyclic AMP receptor protein complex binding site, thus affording an explanation for the more effective utilization of orotate with glycerol than with glucose as carbon source. The E. coli dctA gene was cloned from a lambda vector and shown to complement C4-dicarboxylate and orotate utilization in FOA-resistant mutants of both E. coli and S. typhimurium. The accumulated results demonstrate that the dctA gene product, in addition to transporting C4-dicarboxylates, mediates the transport of orotate, a cyclic monocarboxylate.     

The porin OmpC of Salmonella typhimurium mediates adherence to macrophages.

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequence of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.     

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.     

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.     

Molecular characterization of the gene coding for major outer membrane protein OmpA from Enterobacter aerogenes

The ompA gene from Enterobacter aerogenes was subcloned into a low-copy-number plasmid vector and the resultant plasmid, pTU7En, used to study its expression in Escherichia coli K12. Although the gene was strongly expressed and large amounts of OmpA protein were present in the outer membrane its product was not functionally identical to the E. coli polypeptide. In particular, the E. aerogenes OmpA protein was unable to confer sensitivity to OmpA-specific phages of E. coli. When the primary structure of the protein was deduced from the nucleotide sequence of its gene it was found that three domains differed extensively from the corresponding regions of the E. coli protein. As two of these are known to be exposed on the cell surface we inferred that these alterations are responsible for differences in the biological activity of the two proteins.     

The ompH gene of Yersinia enterocolitica: cloning, sequencing, expression, and comparison with known enterobacterial ompH sequences

We have recently described a previously uncharacterized outer membrane protein of Salmonella typhimurium and Escherichia coli and cloned and sequenced the corresponding gene, the ompH gene, of S. typhimurium (P. Koski, M. Rhen, J. Kantele, and M. Vaara, J. Biol. Chem. 264:18973-18980, 1989). We report here the cloning, sequencing, and expression of the corresponding gene of Yersinia enterocolitica. It is significantly homologous to the ompH genes of E. coli and S. typhimurium (homology percentages, 65 and 64%, respectively), has a promoter region strongly homologous to the E. coli 17-bp class consensus promoter, and encodes a protein consisting of 165 amino acids (22 of which form the signal sequence). The plasmid-borne Y. enterocolitica ompH was found to be expressed both in the E. coli host and in minicells. The isolated outer membrane of Y. enterocolitica was shown to contain OmpH. The homology of the Y. enterocolitica OmpH protein is 66% with E. coli OmpH and 64% with S. typhimurium OmpH. All OmpH proteins have almost identical hydrophobic profiles, charge distributions, and predicted secondary structures. Because yersiniae are considered rather distant relatives of E. coli and S. typhimurium in the Enterobacteriaceae family, these results might indicate that most or all strains of the family Enterobacteriaceae have OmpH proteins remarkably homologous to those now sequenced.     

Nucleotide sequence of the Salmonella typhimurium mutS gene required for mismatch repair: homology of MutS and HexA of Streptococcus pneumoniae.

The mutS gene product of Escherichia coli and Salmonella typhimurium is one of at least four proteins required for methyl-directed mismatch repair in these organisms. A functionally similar repair system in Streptococcus pneumoniae requires the hex genes. We have sequenced the S. typhimurium mutS gene, showing that it encodes a 96-kilodalton protein. Amino-terminal amino acid sequencing of purified S. typhimurium MutS protein confirmed the initial portion of the deduced amino acid sequence. The S. typhimurium MutS protein is homologous to the S. pneumoniae HexA protein, suggesting that they arose from a common ancestor before the gram-negative and gram-positive bacteria diverged. Overall, approximately 36% of the amino acids of the two proteins are identical when the sequences are optimally aligned, including regions of stronger homology which are of particular interest. One such region is close to the amino terminus. Another, located closer to the carboxy terminus, includes homology to a consensus sequence thought to be diagnostic of nucleotide-binding sites. A third one, adjacent to the second, is homologous to the consensus sequence for the helix-turn-helix motif found in many DNA-binding proteins. We found that the S. typhimurium MutS protein can substitute for the E. coli MutS protein in vitro as it can in vivo, but we have not yet been able to demonstrate a similar in vitro complementation by the S. pneumoniae HexA protein.     

The nucleotide sequence coding for major outer membrane protein OmpA of Shigella dysenteriae.

The nucleotide sequence of the ompA gene from Shigella dysenteriae has been determined and the amino acid sequence of the pro-OmpA protein predicted. Sequence comparison between the ompA genes of S.dysenteriae and Escherichia coli showed that features such as mRNA secondary structure and codon usage, as well as polypeptide function, have been conserved during evolution. The pro-OmpA protein of S.dysenteriae consists of 351 residues, as opposed to the 346 of the E.coli protein and also shows several amino acid changes. These changes have been used to interpret differences in the biological activity of the two proteins.     

Glutamyl-tRNA reductase from Escherichia coli and Synechocystis 6803. Gene structure and expression.

In the cyanobacterium Synechocystis sp. PCC 6803 and in the enterobacterium Escherichia coli delta-amino-levulinic acid (ALA) is formed from glutamyl-tRNA by the sequential action of two enzymes, glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase. E. coli has two GluTR proteins with sizes of 45 kDa (GluTR45) and 85 kDa (GluTR85) (Jahn, D., Michelsen, U., and S?ll, D. (1991) J. Biol. Chem. 266, 2542-2548). The hemA gene, isolated from E. coli and several other eubacteria, has been proposed to encode a structural component of GluTR. Because of the inability to overexpress this gene in E. coli, we demonstrate directly GluTR function for the E. coli hemA gene product by its expression and functional analysis in yeast, which does not form ALA from Glu-tRNA. Gel filtration experiments demonstrated definitively that the yeast-expressed HemA protein corresponded to GluTR45. Furthermore, analysis of GluTR activity in an E. coli strain with a disrupted hemA gene displayed GluTR85, but not GluTR45 activity. The hemA gene from Synechocystis 6803 was cloned by functional complementation in E. coli. DNA sequence analysis revealed an open reading frame capable of encoding a 427-amino acid polypeptide (molecular mass of 47,525 Da). The Synechocystis 6803 amino acid sequence shows significant similarity upon alignment with HemA sequences from E. coli, Bacillus subtilis, Salmonella typhimurium, and Chlorobium vibrioforme but does not contain the amino acid sequence derived from the N terminus of the previously purified GluTR protein (Rieble, S., and Beale, S. I. (1991) J. Biol. Chem. 266, 9740-9745). These experiments are the first direct demonstration of GluTR activity of the HemA protein and provide further evidence for two pathways of ALA formation in prokaryotes.