Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences.

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.

Cloning and DNA sequence analysis of the wild-type and mutant cyclic AMP receptor protein genes from Salmonella typhimurium.

The crp gene from Salmonella typhimurium, as well as two mutant adenylate cyclase regulation genes designated crpacr-3 and crpacr-4, were cloned into the EcoRI site of plasmid pUC8. Initially cloned on 5.6-kilobase fragments isolated from EcoRI digests of chromosomal DNA, these genes were further subcloned into the BamHI-EcoRI site of plasmid pBR322. When tested, Escherichia coli crp deletion strains harboring the clones regained their ability to pleiotropically ferment catabolite-repressible sugars. Also, the crpacr-containing strains displayed sensitivity to exogenous cyclic AMP (cAMP) when grown on eosin-methylene blue medium with xylose as the carbon source. The proteins encoded by the S. typhimurium wild-type and mutant crp genes were found to have similar molecular weights when compared with the wild-type cAMP receptor protein (CRP) from E. coli. DNA sequence analysis of the wild-type crp gene showed only a three-nucleotide difference from the E. coli sequence, suggesting little divergence of the crp gene between these organisms. The crpacr sequences, however, each contained single nucleotide changes resulting in amino acid substitutions at position 130 of the CRP. Based on the site at which these substitutions occur, the crpacr mutations are believed to affect CRP-cAMP interactions.

Transduction analysis of transposon Tn551 insertions in the trp-thy region of the Staphylococcus aureus chromosome.

Previous studies have shown that Tn551, a 5.2-kilobase-pair transposon that determines constitutive resistance to erythromycin, can occupy a variety of chromosomal sites between thy-101 and trp-103 in Staphylococcus aureus 8325. Although many of these insertions were "silent," many others, including lys, thr, met, tyr, and trp, resulted in auxotrophic mutations. The close proximity and erythromycin-resistant phenotypes of the insertions in this region have made their mapping by transformation difficult. Analysis of these sites and similar chemically induced mutations by generalized transduction with phage 80 alpha have defined the order and relationship of these insertion sites and provided a detailed map of this region of the chromosome, including the orientation of the trp operon. The results of this study and a limited phenotypic characterization of the mutants have shown that the divergent pathway from aspartate to lysine, threonine, and methionine, several reactions in tyrosine biosynthesis, and the entire tryptophan operon are determined by this region of the chromosome. The linkage results obtained by transduction have been compared with similar data obtained previously by transformation; this comparison suggests the existence, between thy and lys, of a preferred headful cutting site for transducing phage DNA morphogenesis from the host chromosome.