The mglB sequence of Salmonella typhimurium LT2; promoter analysis by gene fusions and evidence for a divergently oriented gene coding for the mgl repressor

Summary The mglB gene of Salmonella typhimurium LT2 coding for the galactose-binding protein (GBP) was sequenced. We compared the deduced amino acid sequence with the GBP sequence of Escherichia coli K12. The mature proteins differ in only 19 of 309 amino acid residues, corresponding to 94% homology. Analysis of the mglB control region by promoter-probe vectors revealed that two promoters, P1 and P2, constitute the mgl control region (P _mgl ). P1 and P2 function in a synergistic way. P1 is the main promoter of the operon; its activity is 20 times the activity of P2. Both promoters are activated by the cyclic adenosine monophosphate catabolite activator protein (cAMP/CAP) complex. While P1 is inactive in the absence of the cAMP/CAP complex, there is residual activity of P2 under these conditions. Studies on the inducibility of the mglBAEC operon using multicopy plasmid promoter-probe vectors were hampered by the titration of the mgl repressor resulting in a partially constitutive expression of the mgl operon. The results indicate that only P1 is responding to induction by D-fucose. A weak promoter, P _D , within the P1 region but divergent to it was found. P _D is neither stimulated by the cAMP/CAP complex nor by D-fucose. We cloned the gene located downstream to P _D and found it to strongly repress the expression of the mgl operon. We termed this gene mglD. The presence of D-fucose abolished the repression caused by the plasmid-encoded mglD gene product.Abbreviations IPTG isopropyl-1-thic--D-galatopyranoside - ONPG 2-nitrophenyl--D-galatopyranoside - XG 5-bromo-4-chloro-3-indolyl--D-galatopyranoside - Kan^r Kanamycin resistance     

The nucleotide sequence of the araC regulatory gene in Salmonella typhimurium LT2

The nucleotide sequence of the araC regulatory gene of Salmonella typhimurium LT2 has been determined. This sequence and the predicted araC translational product are compared to their counterparts in Escherichia coli. The two genes code for similar products although the S. typhimurium protein is eleven amino acids shorter than the E. coli protein. The predicted amino acid sequences are 92% conserved and the DNA sequences are 82% conserved for the common regions of the two genes.     

Use of lambda vehicles to isolate ompC-lacZ gene fusions in Salmonella typhimurium LT2

Summary A novel plasmid vector, pAMH70 carrying both the lamB and nusA genes of Escherichia coli K12 was constructed. Introduction of this plasmid into Salmonella typhimurium LT2 renders this bacterium both sensitive to adsorption and able to sustain growth and lysogenization by . Using this strain as a recipient, stable gene fusions to the gene encoding a major outer membrane porin protein OmpC, were constructed with a vehicle placMu. To confirm the actual site of fusions they were genetically mapped and transducing phages carrying the ompC-lacZ fusion were isolated and relysogenized. The fusions were also shown to be to ompC by their regulatory properties.     

The Salmonella typhimurium LT2 uvrD gene is regulated by the lexA gene product.

The uvrD gene product apparently plays a role in the repair of UV damage, in mismatch repair, and in genetic recombination. A lower level of expression of the Salmonella typhimurium LT2 uvrD gene was observed in maxicells prepared from an Escherichia coli strain that contained a lexA+ plasmid than in maxicells prepared from an E. coli strain that lacked functional LexA protein. These results suggest that the uvrD+ gene is repressed by the LexA protein and is thus a member of the set of genes whose expression is increased by "SOS"-inducing treatments.     

The plasmid of Salmonella typhimurium LT2

Summary Methods of clonal analysis were applied to the study of heterogeneity of the progeny after crosses of 4 donor strains (Hfr H, Hfr C, KL 16 and KL 99) with 3 recipient strains (PC 0212, AB 712 and ECK 022). Three markers were used in each cross. The distal one was the selective marker. The inheritance of two additional proximal markers characterized the heterogeneity of clones originating from particular zygotes. In most crosses the percentage of heterogeneity exceeded 30. One of the recipient strains, obtained by conjugation of the conventional strain PC 0212 with the donor Hfr H revealed unusual properties in respect to heterogeneity. Exconjugants derived from this recipient (ECK 022) and donor Hfr H and Hfr C had a heterogeneity index of about 5%. It is shown that this unusual behavior reflects a very fast process of segregation of recombinants.In crosses with the donors KL 16 and KL 99 the same recipient revealed normal indices of heterogeneity. All these data are explained assuming that there exists a specific genetic marker which determines the process of decay of merozygotes. Tentatively it is called het. Its approximate localization was deduced from specifically designed experiments, in which the heterogeneity of the progeny was found very different, when the donor KL 16 transmitted different parts of its chromosome to the recipient ECK 022.     

Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene.

Regulation of the Salmonella typhimurium metJ gene was examined by measuring beta-galactosidase activity in Escherichia coli strains lysogenic for a phage carrying a metJ-lacZ gene fusion. The results indicated that the metJ gene is regulated by its own gene product and by methionine supplementation to the growth medium. This autoregulatory mechanism involved two tandem promoters, pJ1 and pJ2, separated by approximately 65 base pairs. Deletion analysis permitted the assessment of the activity of promoters pJ1 and pJ2 individually. Promoter Pj1 was negatively regulated by the metJ gene product and by methionine. Although Pj2 regulation remained unclear, evidence is presented which suggests that it is not negatively regulated like pJ1.     

Cloning of mglB, the structural gene for the galactose-binding protein of Salmonella typhimurium and Escherichia coli

Summary From libraries of EcoRI fragments of Salmonella thyphimurium and Escherichia coli DNA in gt7, phages could be isolated that carry mglB, the structural gene of the galactose-binding protein as well as other mgl genes. Lysogenization of an E. coli mutant carrying a defective galactose-binding protein with gt7 mglB (Salmonella) restores full galactose transport and galactose chemotaxis. Both the E. coli mutant protein as well as the wild-type Salmonella galactose-binding protein are synthesized in this strain. The EcoR1 fragments of both organisms carrying the mgl genes were 6 Kb long. They were subcloned into the multicopy plasmid pACUC184. The hybrid plasmid containing the Salmonella mgl DNA gives rise to the synthesis of large amounts of galactose-binding protein in the periplasm of E. coli. The protein can be precipitated by antibodies against the E. coli binding protein and is identical to the fully processed protein isolated from Salmonella typhimurium LT2. In vitro protein synthesis (Zubay-system) with either gt7 mgl phages as well as the hybrid plasmid as DNA matrix produces the galactose-binding protein mainly in precursor form that is precipitable by specific antibodies.