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A chromosomal repeated sequence from Streptococcus thermophilus was identified as a new insertion sequence (IS), IS1191. This is the first IS element characterized in this species. This 1313 bp element has 28 bp imperfect terminal inverted repeats and is flanked by short direct repeats of 8 bp. The single large open reading frame of IS1191 encodes a 391-amino-acid protein which displays homologies with transposases encodes by IS1201 from Lactobacillus helveticus (44.5% amino-acid sequence identity) and by the other ISs of the IS256 family. One of the copies of IS1191 is inserted into a truncated iso-IS981 element. The nucleotide sequences of two truncated iso-IS981s from S. thermophilus and the sequence of IS981 element from Lactococcus lactis share more than 99% identity. The distribution of these insertion sequences in L. lactis and S. thermophilus strains suggests that intergeneric transfers occur during cocultures used in the manufacture of cheese.

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The three restriction endonucleases Sfil, BssHl, and SmaI were found to generate fragments with suitable size distributions for mapping the genome of Streptococcus thermophilus A054. A total of 5, 8, and 24 fragments were produced with SfiI, BssHI, and SmaI, respectively. An average genome size of 1,824 kb was determined by summing the total fragment sizes obtained by digestions with these three enzymes. Partial and multiple digestions of genomic DNA in conjunction with Southern hybridization were used to map SfI, BssHII, and SmaI fragments. All restriction fragments were arranged in a unique circular chromosome. Southern hybridization analysis with specific probes allowed 23 genetic markers to be located on the restriction map. Among them, six rrn loci were precisely located. The area of the chromosome containing the ribosomal operons was further detailed by mapping some of the ApaI and SgrAI sites. Comparison of macrorestriction patterns from three clones derived from strain A054 revealed two variable regions in the chromosome. One was associated with the tandem rinD and rnnE loci, and the other was mapped in the region of the lactose operon.Streptococcus thermophilus is an economically important lactic acid bacterium used in the manufacture of yoghurt and some hard cheeses. It is a gram-positive, facultative anaerobic microorganism that is exclusively isolated from milk and fermented dairy products. Despite its importance in the dairy industry, genetic studies of S. thermophilus have been limited until recently. To date, basic methods of gene transfer by transformation, transduction, or conjugative mobilization have been reported (34). Several genes have been cloned and analyzed, including genes involved in glucide metabolism (lacZ, lacS, galE, galM, and ldh [5,25,43,44,51]) and DNA recombination (recA [15]), ribosomal sequences (4, 23, 38), and the aspartate racemase gene (62). A marked restriction polymorphism has been detected between different strains of this species by analysis of hybridization patterns obtained with a species-specific probe (13) and with probes by genes coding for rRNA (38). Despite the availability of gene transfer systems and cloned genes, the structure and organization of the S. thermophilus genome are not well defined.Pulsed-field gel electrophoresis (PFGE) (9, 52) has proved to be an efficient method for genome size estimation and construction of chromosomal maps, as well as being a useful tool for the characterization of bacterial species. Preliminary results from PFGE have indicated a genome size of 1.75 Mb for strain STi of S. thermophilus (31) and allowed restriction polymorphism within S. thermophilus to be observed (48).In this study, we used PFGE to estimate the size of the chromosome of S. thermophilus A054 and to construct a physical map by using the restriction enzymes SMaI, Sfil, BssHII, ApaI, and SgrAI. Hybridization probes were used to locate 23 genetic markers on the chromosomal map. Genetic polymorphism within strain A054 was analyzed by comparing the macrorestric...

The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3 end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1 could be or could be derived from an integrative conjugative element.Cocultures of various lactic acid bacteria are used during the manufacture of dairy products. Sequence comparisons and hybridizations reveal that horizontal transfers between a large array of species of lactic acid bacteria have occurred, most likely during dairy cocultures (13,32). The most convincing evidence indicates that insertion sequences IS1191, IS981, ISS1, and IS1194 (4,5,14) and some open reading frames (ORFs) involved in exopolysaccharide synthesis (6) or in restriction-modification (24) were transferred between the lactic acid bacteria Streptococcus thermophilus and Lactococcus lactis in cocultures used during cheese manufacture. However, the mechanism of genetic exchange between these two species remains unknown, and no conjugative element has been previously characterized in S. thermophilus.Cloning of var1C and localization of its limits. The Sm4 fragment of the S. thermophilus CNRZ368 chromosome was previously found to contain the 35-kb variable region var1C, which was absent from the corresponding chromosomal fragments of strains A054 and NST2280 (28). A region containing an IS1191 copy inserted in a truncated IS981 element (14) was cloned and found to be included in var1C (28). Chromosome walking using a GEM11 genomic library of CNRZ368 (25) was performed to isolate recombinant bacteriophages overlapping the var1C region. Their inserts were subcloned in pBC KSϩ and used as hybridization probes on A054 and NST2280 DNAs. S35, ES27, I132.3, ES13, and SC02 fragments hybridized to A054 and NST2280 DNAs. On the contrary, all of the probes covering the 35.5-kb region (except IS1191 and IS981) and located between the HindIII sites H L and H R (Fig. 1) did not hybridize to A054 and NST2280 DNAs (data not shown). Furthermore, CNRZ368, A054, and NST2280 showed identical restriction maps in regions located to the left of the HindIII site H L and to the right of the HindIII site H R (Fig. 1). These data indicated that var1C limits are located near these HindIII sites. When ES27 including the left end and ES13 including the right end were hybridized to DNAs of the three strains digested by ClaI, EcoO109, EcoRI, PstI, or XbaI, they revealed the same fragment from A054 and NST2280, but two different fragments from CNRZ368. Thus, the flanking regions of var1C in CNRZ368 are adjacent to each other in strains A054 and NST2280 (Fig. 1).Because A054 and CNRZ368 are very closely related to each other, but distantly rel...

In this study, a DNA construct encoding the urease B subunit was assessed for its immune profile and its ability to reduce bacterial colonization in the murine stomach. Our studies suggest that local innate immune responses may play a greater role than previously supposed in limiting H. pylori colonization in the gastric mucosa.