UV-mutable hybrids of Salmonella incorporating escherichia coli Region adjacent to Tryptophan operon

Summary Salmonella typhimurium recombinants have been constructed which have acquired UV-induced mutability. Such recombinants were obtained after transfer of plasmid F126 from E. coli. UV-mutability was acquired by UV-stable Salmonella as a nonselective marker after selection of trp ⁺ marker in 2.5% of the cases. Possible deficiency in UV-stable Salmonella of the umuC gene is discussed.     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome

Many Salmonella typhimurium genes are required for bacterial entry into host cells. P22 transduction analysis has localized several invasion loci near minute 59 on the S. typhimurium chromosome. To further characterize the 59–60 min chromosomal region, we determined the physical and genetic map of 85 kb of S. typhimurium DNA between srl and cysC. It was previously shown that some of the invasion genes from this region are not present in Escherichia coli K-12. We examined whether other S. typhimurium genes on the 85 kb of DNA were similarly absent from E. coli We found that a contiguous 40 kb fragment of the S. typhimurium chromosome which encodes invasion genes is absent from the corresponding region of the E. coli K-12 chromosome and may represent a pathogenicity island. We speculate that acquisition of the 40 kb region must have significantly advanced the evolution of Salmonella as a pathogen.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

envB mutations confer UV-sensitivity to Salmonella typhimurium and UV-resistance to Escherichia coli

Summary An envB mutation isolated in Salmonella typhimurium LT2 was transferred by conjugation to Escherichia coli K-12. The mutation produced the same alterations in E. coli as in S. typhimurium concerning cell shape, sensitivity to drugs, autolysis, and fermentation of carbohydrates. However, although the mutation conferred sensitivity to UV irradiation in Salmonella, in E. coli it behaved as a genuine envB mutation producing resistance to UV inactivation. The fact that the mutation produced opposite effects in the survival of UV-irradiated S. typhimurium and E. coli discloses an intriguing difference between these closely related species.Career Investigator of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the over-expressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.     

Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutropbus pbbB and to Rbizobium meliloti nodG

Summary A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. mehloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the pbbB gene and involved in poly--hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli ⁷⁰ consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.     

OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium

Summary The tppB locus of Salmonella typhimurium encodes the anaerobically-induced tripeptide permease. We have demonstrated that expression of tppB requires the function of the ompR and envZ gene products, originally identified as positive regulatory proteins required for the osmotic regulation of porin expression. Significantly, tppB expression is not osmotically regulated. We have also identified three additional genes whose expression depends on OmpR. Thus OmpR and EnvZ serve a more general regulatory role than has previously been supposed. This study provides the first detailed genetic analysis of the ompB locus of S. typhimurium.     

Cloning and characterization of the alkaline phosphatase positive regulator gene (phoB) of Escherichia coli

Summary The positive regulator gene (phoB) for alkaline phosphatase of Escherichia coli was cloned into the EcoRI site of pBR322 from the E. coli chromosome by a shotgun method. phoB was then constructed in vitro by replacing the C fragment of gtC by the phoB chromosomal fragment obtained from the hybrid plasmid. When the phoB mutant was lysogenized by phoB, the lysogen became PhoB⁺. The integration site of the phage was identified by P1 phage transduction to be around phoB site on the chromosome. From these results, we conclude that the cloned gene is phoB and not a gene which suppresses phenotypically phoB mutation when it is in a multi-copy state. The restriction map was constructed. Based on this information, several PhoB deletion plasmids and smaller PhoB⁺ plasmids were constructed in vitro. By examining PhoB phenotype when these plasmids were introduced into phoB mutant, we could define the phoB gene locus in 2 kb on the restriction map of the cloned chromosomal fragment. Cells carrying the multi-copy phoB gene produced alkaline phosphatase qualitatively under normal phosphate regulation. The phoB gene product was identified by the maxicell method as a protein with a molecular weight of approximately 31,000 daltons.     

Cloning of bacterial DNA replication genes in bacteriophage lambda

Summary Recombinant lambda phages containing the genes for dnaZ protein (the subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichi coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.Abbreviations DTT dithiothreitol - Ec Escherichia coli - EDTA ethylene diamine tetra acetic acid - kb kilobase 1,000 bases or base-pairs - moi multiplicity of infection - pol I E. coli DNA polymerase I - pol III holoenzyme E. coli DNA polymerase III holoenzyme - pri dnaG, primase-coding gene - SSB single-strand binding protein - St Salmonella typhimurium - sup gene coding for suppressor - ts temperature-sensitive     

A DNA sequence from Dictyostelium discoideum complements ura3 and ura5 mutations of Saccharomyces cerevisiae

Summary A 3.7 kilobase fragment of Dictyostelium discoideum genomic DNA has been cloned by its ability to complement a yeast ura5 mutation affecting the activity of orotidine-5-phosphate carboxy-lyase (EC 4.1.1.23). This fragment also complements a yeast ura5 mutation that leads to a defect in orotate phosphoribosyl transferase (EC 2.4.2.10). The orotidine-5-phosphate carboxy-lyase and the orotate phosphoribosyl transferase activities that result from Dictyostelium gene expression in yeast have been detected. The size of the DNA required for both complementations has been localised to a segment of less than 2 kb. A unique Dictyostelium RNA species of 1,600 base pairs hybridises to this fragment. In vitro deletions in this fragment lead to the simultaneous loss of the two activities. The two enzymatic activities coelute as a protein of 120.000 daltons during gel filtration of a Dictyostelium extract. These results favour the existence, on the cloned Dictyostelium DNA fragment, of a unique structural gene which codes for a bifunctional enzyme carrying the two activities, orotidine-5-phosphate carboxy-lyase and orotate phosphoribosyl transferase.Abbreviations bp basepair - kb kilobasepair - MOPS Morpholino propane sulfonic acid     

Functional homology of fla genes between Salmonella typhimurium and Escherichia coli

Summary Genetic studies have shown the presence of more than 20 fla genes indispensable for the formation of flagella in Salmonella typhimurium and Escherichia coli. Functional homology of the fla genes in these two bacterial species was examined through intergeneric complementation tests by bacteriophage Pl-mediated transduction from E. coli donors to S. typhimurium recipients. It was found that most of the fla gene products in these two bacterial species were interchangeable and the following correspondence was established (S. typhimurium genes vs. E. coli genes): flaFIV to flaV; flaFV to flaK; flaFVII to flaL; flaFIX to flaM; flaC to flaH; flaM to flaG; flaE to flaI; flaAI to flaN; flaAII·1 to flaB; flaAIII to flaC; flaS to flaO; flaR to flaE; flaQ to flaA; and flaB to flaR. These results suggest that the chromosomal alignment of the functionally homologous genes is very similar in these two bacterial species. Furthermore, five additional fla genes were inferred to exist in E. coli in addition to the fla genes already identified. They were termed flaU, flaX, flaY, flaZ, and flbB (flb is equivalent to fla), which corresponded to flaFI, flaFVI, flaFVIII, flaFX, and flaK of Salmonella in this order. The flaK mutants of E. coli showed no complementation with any of the flaFV, flaFVI, flaFVII, flaFVIII, or flaFIX mutants of Salmonella.     

Nucleotide sequence of the Vibrio alginolyticus glnA region

The nucleotide sequence of a 4 kb fragment containing the Vibrio alginolyticus glnA, ntrB and ntrC genes was determined. The upstream region of the glnA gene contained tandem promoters. The upstream promoter resembled the consensus sequence for Escherichia coli ⁷⁰ promoters whereas the presumptive downstream promoter showed homology with nitrogen regulated promoters. Four putative NR_I binding sites were located between the tandem promoters. The ntrB gene was preceded by a single presumptive NR_I binding site. The ntrC gene was located 45 base pairs downstream from the ntrB gene. The V. alginolyticus ntrB and ntrC genes were able to complement ntrB, ntrC deletions in E. coli.Abbreviations bp base pair(s) - CAP catabolite-activating protein - GS glutamine synthetase - kb kilobase(s) - ORF open reading frame - SD Shine-Dalgarno     

Identification of an indigenous plasmid carrying a gene for trimethoprim resistance in Pseudomonas syringae pv. glycinea

Summary Pseudomonas syringae pv. glycinea Race 8 strain PgB3 is naturally resistant to trimethoprim (Tp) at concentrations up to 500 g/ml. A genomic library of total PgB3 DNA was constructed by ligating EcoRI-restricted DNA into the EcoRI site of the cosmid vector, pLAFR1, packaging the DNA in vitro into bacteriophage lambda, and transducing E. coli DH1 cells. Of 960 cosmid clones selected for resistance to tetracycline, six were resistant to trimethoprim at 500 g/ml. An insert into pLAFR1 of about 9.4 kb was shown to be consistently present in the tirmethoprim-resistant clones. Southern blot analysis using radioactively labeled insert DNA as probe indicated that the 9.4 kb fragment hybridized only with a 40 kb indigenous plasmid from PgB3 designated pPg2.     

Molecular cloning of ompRS, a regulatory locus controlling production of outer membrane proteins in Erwinia carotovora subsp. carotovora

Summary A locus, ompRS, controlling synthesis of outer membrane proteins was cloned from Erwinia carotovora subsp. carotovora (Ecc) by complementation of an Escherichia coli ompR—envZ mutant. The Ecc ompRS locus was both structurally and functionally similar to the ompR—envZ operon controlling porin gene expression in E. coli as shown by DNA hybridization and complementation of E. coli ompR and envZ mutants. Furthermore, introduction of ompRS into E. coli (ompR—envZ) strains restored the osmoregulation of the major outer membrane protein genes ompC and ompF Maxicell analysis of ompRS-carrying plasmids suggested that proteins similar in size to the E. coli ompR and envZ gene products were encoded by the Ecc ompR and ompS genes, respectively. Introduction of an ompRS transposon mutant onto the Ecc chromosome by marker exchange mutagenesis showed that ompRS is essential for production of a major outer membrane porin in Ecc. This mutational defect could be complemented by clones carrying Ecc ompRS or E. coli ompR envZ. The lack of the porin did not, however, compromise the virulence of these Ecc mutants.     

Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome

Summary Mutations at the cpxA locus of Escherichia coli K-12 affect cellular processes that are not otherwise related. We have now determined the physical and genetic structure of the E. coli chromosome in the region of cpxA (87.5 min). Our results indicate that cpxA is a single gene. Previous studies showed cpxA to be linked to tpiA. We therefore isolated two tpiA ⁺ recombinant plasmids, pRA200 and pRA300, from EcoRI and BamHI digests of F133, respectively. By genetic complementation or enzyme overproduction, the 9.5 kb EcoRI fragment in pRA200 was shown to include glpK, tpiA and cdh. The 13.6 kb BamHI fragment of pRA300 lacks glpK, but includes tpiA, pfkA and cpxA. Neither fragment complemented a deletion of the rha operon. These data indicate the chromosomal gene order: 87 min-rha-cpxA-pfkA-cdh-tpiA-glpK-88 min. The EcoRI and BamHI fragments overlap in an interval corresponding to about 8.2 kb of DNA. The total region of the E. coli K12 chromosome covered by the two fragments is about 15 kb. A terminal 2 kb EcoRI-BamHI fragment from pRA300 complemented the chromosomal cpxA2[Ts] allele with respect to isoleucine and valine synthesis, RNA bacteriophage sensitivity and surface exclusion in Hfr strains, and envelope protein composition. Complementation occurred when the fragment was subcloned in pBR325 but not when it was subcloned in pBR322, suggesting that the 2 kb fragment lacks expression sequences that are supplied by cat (chloramphenicol acetyltransferase gene) expression sequences of pBR325. The cpxA locus on the E. coli chromosome was established with respect to two chromosomal Tn10 insertions by a combination of genetic and physical analyses. The locus established by those analyses was consistent with the location of the 2 kb EcoRI-BamHI fragment in the physical map of the region. Physical analyses of (rha-pfkA) and (rha-tpiA) deletion strains showed that they lack cpxA and surrounding genes. Since these strains were viable, cpxA is not essential under all growth conditions.     

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome

Summary A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E. coli and B. subtilis. Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment.The cloned plasmid pMS102-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B. subtilis chromosome. (1) E. coli cells harboring this plasmid stuck to each other and to glass. This property was more apparent when cells were grown in poor media. (2) E. coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid. (3) The frequency of transformation of B. subtilis by pMS102-B7 was less than 1/1,000 of that by the vector plasmid pMS102. The number of copies of pMS102-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B. subtilis. This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts.Both in E. coli and B. subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system. The implication of the function of the B7 fragment in the initiation of the B. subtilis chromosome will be discussed.     

Cloning and expression inEscherichia coli of the sucrase gene fromBacillus subtilis

Summary A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank ofE. coli harboring recombinant cosmids representative of theB. subtilis genome. It was shown that thesacA gene is located in a 2 kbEcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2 kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for inB. subtilis andE. coli. Complementation of asacA mutation was observed in Rec⁺ and Rec^- strains ofB. subtilis. Expression of sucrase was also demonstrated inE.coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic inE. coli since the presence of the recombinant plasmids did not allow the transport of [U¹⁴C] sucrose and the growth of the cells.It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport ofB. subtilis.     

Discontinuity of homology of Escherichia coli and Salmonella typhimurium DNA in the lac region

Summary Partial homology of Salmonella typhimurium DNA to Escherichia coli DNA was demonstrated by Southern hybridization blots to exist on either side of the lac operon of E. coli but no homology was detected between S. typhimurium DNA and about 12 kb of E. coli DNA including the lac genes as well as about 5 kb of E. coli DNA between lac and proC. Thus portions of DNA seem to have been either added to the E. coli genome or deleted from the S. typhimurium genome since their divergence from a common ancestor. Although an IS1 element was located near the lac operon of E. coli, the insertional element was shown not to be near any of the junctures of discontinuity of E. coli - S. typhimurium homology near lac.