Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by MNNG both in E. coli and in S. typhimurium. The AdaST protein encoded by the plasmid increased expression of the ada'-lacZ chromosome fusion about 5-fold when an E. coli strain carrying both the fusion operon and the plasmid was exposed to a low concentration of MNNG, whereas the E. coli Ada protein encoded by a low-copy-number plasmid increased it about 40-fold under the same conditions. The low ability of AdaST to function as a positive regulator could account for the apparent lack of an adaptive response to alkylation damage in S. typhimurium.     

Genetic characterization of the pnuC gene, which encodes a component of the nicotinamide mononucleotide transport system in Salmonella typhimurium.

The pnuC gene, which encodes a component of the nicotinamide mononucleotide transport system, has been mapped and oriented. The gene order of the pnuC region, which is at min 17 of the Salmonella chromosome, is nadA-pnuC-aroG-gal. Polarity tests, with pnuC::Mu d-lac operon fusions, reveal that the pnuC gene is the promoter distal gene in an operon with the nadA gene, which encodes the second enzyme of the pyridine biosynthetic pathway. The nadA pnuC operon is regulated by the NadI repressor. The pnuC gene also has its own promoter, since strains with a nadA::Tn10d(Tc) insertion still express the pnuC gene at a low, unregulated level.     

Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase.

The phoN gene of Salmonella typhimurium encodes nonspecific acid phosphatase (EC 3.1.3.2), which is regulated by a two-component regulatory system consisting of the phoP and phoQ genes. We cloned the phoN region into a plasmid vector by complementation of a phoN mutant strain and determined the nucleotide sequence of the phoN gene and its flanking regions. The phoN gene could encode a 26-kDa protein, which was identified by the maxicell method as the product of phoN. Results of the enzyme assay and Southern hybridization with chromosomal DNA of Escherichia coli K-12 suggests that there is no phoN gene in E. coli. The regulatory pattern of phoN in E. coli and Southern hybridization analysis of the E. coli chromosome with the S. typhimurium phoP gene suggest that E. coli K-12 also harbors the phoP and phoQ genes.     

Molecular cloning, characterization, and nucleotide sequence of the rfc gene, which encodes an O-antigen polymerase of Salmonella typhimurium.

The rfc gene of Salmonella typhimurium was located in a 1.75-kb HindIII fragment and restored wild-type lipopolysaccharide synthesis ability to both an older rfc point mutant and new rfc::IS10 mutants. DNA sequencing of the HindIII fragment revealed an open reading frame which could encode a protein of 407 amino acids with an Mr of 47,472 and also revealed potential translation signals. Modulator codons accounted for 12.5% of the total codon content, providing a possible explanation for the nondetectability of the protein in subcellular systems. Secondary structure analysis suggested the presence of transmembrane beta-sheet structures, implying a possible role for the protein in translocation of hydrophilic O-antigen-containing materials. Salmonella strains of groups A, B, and D1 contained rfc-homologous DNA, but strains of groups C1, C2, C3, D2, and E2 did not.     

The Escherichia coli orthologue of the Salmonella ushB gene (ushB(c)) produces neither UDP-sugar hydrolase activity nor detectable protein, but has an identical sequence to that of Escherichia coli cdh

Salmonella ushB, which encodes a membrane-bound UDP-sugar hydrolase, has an Escherichia coli orthologue (ushB(c)) which does not detectably produce this activity. In this report, we show that ushB(c) does not produce any detectable protein either, despite being transcribed normally. Remarkably, ushB(c) is shown to have 100% sequence identity with E. coli cdh, previously characterised as encoding an active CDP-diglyceride hydrolase, an apparent contradiction with implications regarding enzyme evolution. We suggest that a useful gene designation is cdh (ushB(c)) rather than either ushB(c) or cdh, alone.     

Cloning and molecular characterization of the ompA gene from Salmonella typhimurium

The ompA gene from Salmonella typhimurium, encoding a major heat-modifiable protein of the outer membrane, has been cloned and extensively characterized. When expressed in Escherichia coli the gene directs the synthesis of an OmpA protein which is functionally and topologically indistinguishable from that made in S. typhimurium, thus indicating that export and membrane incorporation are very similar in the two organisms. The S. typhimurium protein effectively substitutes for the E. coli polypeptide in F-dependent conjugation and in the uptake of certain colicins, although it cannot serve as the receptor for the OmpA-specific phages K3 and TuII. On examination of the primary sequence of the protein, predicted from the nucleotide sequence of its gene, it was found that those domains likely to be exposed on the cell surface were significantly different to the corresponding regions of the E. coli polypeptide. These differences in the structure of the two proteins have been used to interpret differences in their biological activities.     

Studies on the UDP-sugar hydrolases from Escherichia coli and Salmonella typhimurium

A simple procedure for purification of the UDP-sugar hydrolase from Escherichia coli is described. The enzyme has an apparent molecular weight of 61,000. The UDP-sugar hydrolase from Salmonella typhimurium has been solubilized and partially purified from total cell membranes. According to several criteria, antibodies raised against the purified E. coli enzyme do not seem to react with partially purified Salmonella enzyme.     

Isolation and characterization of a substitution mutation in the ompR gene of Salmonella typhimurium LT2.

The expression of the genes ompC and ompF encoding major outer membrane proteins is dependent on the ompR-envZ operon. Here we describe the isolation and characterization of an ompR mutation, a single-base-pair change, that results in an Arg-to-Cys substitution. When present in multiple copies, the mutant allele conferred a dominant OmpC- OmpF+ phenotype. Furthermore, the mutant allele exhibited allele-specific negative complementation with other ompR mutations. This ability, together with its dominant character, suggested that the OmpR protein is capable of multimerization.     

Isolation and characterization of oxaloacetate decarboxylase of Salmonella typhimurium,a sodium ion pump

Anaerobic growth of Salmonella typhimurium on citrate is Na⁺-dependent and requires induction of the necessary enzymes during a 20–40 h lag phase. The citrate fermentation pathway involves citrate lyase and oxaloacetate decarboxylase. The decarboxylase is a membrane-bound. Na⁺-activated, biotin-containing enzyme that functions as a Na⁺ pump. Oxaloacetate decarboxylase was isolated by affinity chromatography of a Triton X-100 extract of the bacterial membranes on avidin-Sepharose. The enzyme consists of three subunits , , , with apparent molecular weights of 63800, 34500 and 10600. The -chain contains a covalently attached biotin group and binds to antibodies raised against the -subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae. The Na⁺ transport function was reconstituted by incorporation of the puriried enzyme into proteoliposomes.     

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.     

Cloning and characterization of the UDP-sugar hydrolase gene (ushA) of Enterobacter aerogenes IFO 12010

A bacterial alkaline phosphatase (BAP, the phoA gene product) is primarily responsible for the hydrolysis of the substrates 5-bromo-4-chloro-3-indolylphosphate-p-toluidine (XP) and p-nitrophenyl phosphate (pNPP). Using these substrates and an E. coli phoA mutant, we have cloned Enterobacter aerogenes genes conferring an XP(+) phenotype. Two types of clones were identified based on phenotypic tests and DNA sequences. One of them is a E. aerogenes phoA gene (XP(+), pNPP(+)) as expected; surprisingly the other one was found to be a ushA gene (XP(+), pNPP(-)), which encodes an UDP (uridine 5'-diphosphate)-sugar hydrolase. The E. aerogenes ushA gene shares high sequence identity with ushA of E. coli and the mutationally silent ushA0 gene of Salmonella typhimurium at both the nucleotide (over 79%) and amino acid (over 93%) levels. Expression of the E. aerogenes ushA gene in E. coli produced high level of UDP-sugar hydrolase, as confirmed by TLC (thin layer chromatography) analysis together with a presence of a strong band due to a XP hydrolysis on a polyacrylamide gel.     

Isolation and characterization of proline peptidase mutants of Salmonella typhimurium

The proline requirement of Salmonella typhimurium strain proB25 can be satisfied by either of the peptides Leu-Pro or Gly-Pro-Ala. A mutant derivative of strain proB25 isolated by penicillin selection in medium containing Leu-Pro as proline source fails to use either Leu-Pro or Gly-Pro-Ala as a source of proline. This strain is a double mutant that lacks two aminoacyl-proline-specific peptidases. One of these enzymes (peptidase Q) catalyzes the rapid hydrolysis of Leu-Pro but does not hydrolyze Gly-Pro-Ala or poly-l-proline. Mutations at a site (pepQ) near metE lead to loss of this activity. The other peptidase (peptidase P) catalyzes the hydrolysis of Gly-Pro-Ala and poly-l-proline but is only weakly active with Leu-Pro as substrate. This enzyme is similar to aminopeptidase P previously described in Escherichia coli (16). Mutations at a locus (pepP) near serA lead to loss of this enzyme.     

Isolation and characterization of lon mutants in Salmonella typhimurium.

In this paper we report the isolation and characterization of lon mutants in Salmonella typhimurium. The mutants were isolated by using positive selection by chlorpromazine resistance. The physiological and biochemical properties of the lon mutants in S. typhimurium are very similar to those of Escherichia coli lon mutants. Mutants altered at this locus contain little or no activity of the ATP-dependent protease La and show a number of pleiotropic phenotypes, including increased production of capsular polysaccharides, increased sensitivity to UV light and other DNA-damaging agents, and a decreased ability to degrade abnormal proteins.     

Cloning,expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5 end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg²⁺. In the presence of Mg²⁺ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.     

Molecular characterization of the Salmonella typhimurium parE gene.

The DNA sequence of the wild type S. typhimurium parE gene was determined. The predicted protein has 96.7% amino acid identity with the ParE protein of E.coli, but is 29 amino acids longer, due to an additional basepair in the 3' end of the S. typhimurium gene. Subclones of the S. typhimurium parE gene localized the sites of four heat sensitive mutations within parE. The parE206 and parE374 mutations are identical (Val67-Met) and lie in a highly conserved region corresponding to the ATP binding pocket of GyrB. Two additional heat sensitive mutations were sequenced and predict the following amino acid substitutions: parE377 (Gly399-Ser) and parE493 (Thr583-Pro). All of the heat sensitive mutations lie in regions with strong amino acid homology to GyrB.