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.

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...

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The physical maps of Streptococcus thermophilus CNRZ368 and NST2280 strains were constructed by analysing PFGE patterns obtained with the low-frequencycutting enzymes Smal, BssHll and Sfil. Their chromosomes are 1864 and 1840 kb circular molecules, respectively. Comparison of their physical maps with that of the reference A054 strain revealed a relatively conserved organization of the restriction sites. Three variable regions were detected with the map of CNRZ368 whereas 15 were found with the map of NST2280. To construct the genetic maps, probes corresponding to 10 single-copy genes, the rm genes and the insertion sequences lS1191,1S981 and IS51 were hybridized to Southern blots of chromosomal DNA digested with the different mapping enzymes. Comparison of the genetic maps of the three strains showed a conserved location of the mapped single-copy genes. However, six rrn loci were present in the chromosome of A054 and CNRZ368 whereas five were present in the NST2280 chromosome. A polymorphism was also found in the copy number of the insertion sequences between the three strains.

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Three ribosomal probes from Streptococcus salivarius subsp. thermophilus were cloned. Sequence data demonstrate that their juxtaposition corresponds to an entire operon. They were used in order to study ribosomal operon number and organization. rRNA genes were shown to be clustered in the order 5'-16S-23S-5S-3' and the number of rrn loci to vary within the subspecies. The smallest of the 3 probes was used for strain characterization. Substantial variability in hybridization patterns was observed among strains, resulting not only from, restriction fragment length polymorphism (RFLP) but also from the variability of ribosomal operon number.

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Summary -The thermophilic and homofermentative lactic acid bacterium Streptococcus thermo-phi/us has a relatively small genome ('= 1.8 Mb). Nevertheless, seve rai loci which were unstable or exhibiting sequence polymorphism were found. The rRNA gene number varies according to the strain. Whereas the CNRZ368 strain contains 6 rm loci, 5% of its offspring only have 5. This alteration was shown to result from deletionsoccurring within the 2 close rRNA loci, rmO and rmE. Deletions gave rise to hybrid rmOIE loci and probably originated from homologous recombination events. Intraspecific genetic polymorphism also affects the locus varA. The cloned 121 fragment belongs to the varA locus and reveals the various structures of this locus. Whereas sorne strains carry the whole 121 fragment, sorne others lack either its right side or its left side. Both deletions and insertions of various length could result in such a situation. Moreover, evidence was provided that part of the 121 right side is repeated and dispersed in the genome. Another system of genetic instability affects colony morphology. Four variants of Streptococcus thermophi/us CNRZ368, differing in slze, shape and opacity of colonies have been isolated. Switches between phenotypes occur at different frequencies (Hl--3 to 10-1). The 4 genotypes are different, as demonstrated by the 4 characteristic Xhol patterns of the 4 variants. In the case of the NST1403 strain, a CNRZ368 subclone exhibiting the ring phenotype, the mutation was shown to be either an insertion or a duplication. Thus S thermophi/us genomic instability results from various molecular mechanisms, ail of them leading to intraspecific polymorphism. Streptococcus thermophilus 1genetic instability