Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus

Abstract

A promoter probe vector, pTG244, was constructed with the aim of isolating transcription initiation signals from Streptococcus thermnophilus (Streptococcus salivarius subsp. thermophilus). pTG244 is based on the Escherichia coli-streptococcus shuttle vector pTG222, into which the promoterless chloramphenicol acetyltransferase gene of Bacillus pumilus (cat-86) was cloned. Random Sau3A fragments from the S. thermophilus A054 chromosomal DNA were cloned upstream of the cat-86 gene by using E. coli as the host. The pool of recombinant plasmids were introduced into S. thermnophilus and Lactococcus lactis subsp. lactis in order to search for promoter activity in these hosts. For S. thermophilus, it was necessary to first select erythromycinresistant transformants and then to screen for chloramphenicol resistance among these. Direct selection of chloramphenicol-resistant clones was, however, possible in L. lactis subsp. lactis. Six fragments exhibiting promoter activity were characterized in S. thermophilus by measuring the levels of cat-86 transcription and/or chloramphenicol acetyltransferase specific activity. Three of the promoter-carrying fragments were sequenced. The 5' ends of their corresponding mRNAs were determined by S1 mapping and shown to correspond to a purine residue in all cases. Upstream from these potential transcription start points, sequences homologous to the E. coli o70 and the Bacillus subtilis vegetative o43 (or cA) consensus promoters were identified.

New insights in the molecular biology and physiology of revealed by comparative genomics

“…The genome contains three pseudogenes (w dexT, w pulA, w sacB) encoding remnants of a dextranase, a pullulanase and a levansucrase. However, a complete amylase gene was identified (amyL), which is in agreement with the finding that this species displays low amylolytic activity [85].…”

Identification of four loci isolated from two Streptococcus thermophilus phage genomes responsible for mediating bacteriophage resistance

“…Transformation of E. coli XLI-Blue (Stratagene) and positive selection of recombinant plasmids was performed as previously described [3]. S. thermophilus cells were made competent for transformation using the method described by Slos et al [8]. The DNA inserts of the recombinant plasmids were sequenced to con¢rm the ¢delity of the PCR fragment.…”

Isolation and characterisation of promoter regions fromStreptococcus thermophilus

“…Erythromycin was added at 5 /xg ml-1 for S. thermophilus and 150 /xg ml -I for E. coli, and chloramphenicol was used at 5 /~g ml-~ and 20 /zg ml-~ respectively. Plasmid DNA was introduced into S. thermophilus via electroporation by published procedures [1,2]…”

“…However, one of these thermophilic LAB, Streptococcus thermophilus, used in yoghurt fermentations, is now amenable to genetic analysis through virtue of transformation procedures * Corresponding author. [1,2]. Because of its status as a GRAS (generally regarded as safe) organism, S. thermophilus is an attractive host system for the production of homologous and heterologous proteins.…”