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Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomie islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in Streptococcus thermophilus, ICESt1 and ICESt3. Mitomycin C, a DNA-damaging agent, derepresses ICESt3 conjugative transfer almost 25-fold. The ICESt3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICESt3 transconjugants of Streptococcus pyogenes and Enterococcus faecalis were recovered, only one transconjugant of Lactococcus lactis was obtained. The newly incoming ICEs, except the one from L. lactis, are site-specifically integrated into the 3' end of the fda gene and are still able to excise in these transconjugants. Furthermore, ICESt3 was retransferred from E. faecalis to S. thermophilus. Recombinant plasmids carrying different parts of the ICESt1 recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.

A DNA-damaging agent, mitomycin C, derepresses the site-specific excision of two integrative and potentially conjugative elements from Streptococcus thermophilus, ICESt1 and ICESt3. The regulation pathway involves a repressor related to phage lambda cI repressor. It could also involve a putative regulator related to another type of phage repressors, the 'cI-like' repressors.

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.

The isolation of a Streptococcus thermophilus CNRZ368 mutant displaying a long-chain phenotype allowed us to identify the cse gene (for cellular segregation). The N terminus of Cse exhibits high similarity to Streptococcus agalactiae surface immunogenic protein (SIP), while its C terminus exhibits high similarity to S. thermophilus PcsB. In CNRZ368, deletion of the entire cse open reading frame leads to drastic lengthening of cell chains and altered colony morphology. Complementation of the [DELTA]cse mutation with a wild-type allele restored both wild-type phenotypes. The central part of Cse is a repeat-rich region with low sequence complexity. Comparison of cse from CNRZ368 and LMG18311 strains reveals high variability of this repeat-rich region. To assess the impact of this central region variability, the central region of LMG18311 cse was exchanged with that of CNRZ368 cse. This replacement did not affect chain length, showing that divergence of the central part does not modify cell segregation activity of Cse. The structure of the cse locus suggests that the chimeric organization of cse results from insertion of a duplicated sequence deriving from the pcsB 3' end into an ancestral sip gene. Thus, the cse locus illustrates the module-shuffling mechanism of bacterial gene evolution.

A type II polyketide synthase gene cluster located in the terminal inverted repeats of Streptomyces ambofaciens ATCC 23877 was shown to be responsible for the production of an orange pigment and alpomycin, a new antibiotic probably belonging to the angucycline/angucyclinone class. Remarkably, this alp cluster contains five potential regulatory genes, three of which (alpT, alpU, and alpV) encode proteins with high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family. Deletion of the two copies of alpV (one in each alp cluster located at the two termini) abolished pigment and antibiotic production, suggesting that AlpV acts as a transcriptional activator of the biosynthetic genes. Consistent with this idea, the transcription of alpA, which encodes a ketosynthase essential for orange pigment and antibiotic production, was impaired in the alpV mutant, while the expression of alpT, alpU, and alpZ, another regulatory gene encoding a [gamma]-butyrolactone receptor, was not significantly affected. Real-time PCR experiments showed that transcription of alpV in the wild-type strain increases dramatically after entering the transition phase. This induction precedes that of alpA, suggesting that AlpV needs to reach a threshold level to activate the expression of the structural genes. When introduced into an S. coelicolor mutant with deletions of actII-ORF4 and redD, the SARP-encoding genes regulating the biosynthesis of actinorhodin and undecylprodigiosin, respectively, alpV was able to restore actinorhodin production only. However, actII-ORF4 did not complement the alpV mutant, suggesting that AlpV and ActII-ORF4 may act in a different way.

The duplicated hasR and hasL genes of Streptomyces ambofaciens encode alternative sigma factors (named [[sigma].sup.B.sub.k] and [[sigma].sup.B.sub.L] belonging to the [[sigma].sup.B] general stress response family in Bacillus subtilis. The duplication appears to be the result of a recent event that occurred specifically in S. ambofaciens. The two genes are 98% identical, and their deduced protein products exhibit 97% identity at the amino acid level. In contrast with the coding sequences, their genetic environments and their transcriptional control are strongly divergent. While hasL is monocistronic, hasR is arranged in a polycistronic unit with two upstream open reading frames, arsR and prsR, that encode putative anti-anti-[sigma] and anti-[sigma] factors, respectively. Transcription of each has gene is initiated from two promoters. In each case, one promoter was shown to be developmentally controlled and to be similar to those recognized by the B. subtilis general stress response sigma factor [[sigma].sup.B]. Expression from this type of promoter for each of the has genes dramatically increases during the course of growth in liquid or on solid media and following oxidative and osmotic stresses. Reverse transcription-PCR measurements indicate that hasR is 100 times more strongly expressed than hasL from the [[sigma].sup.B]-like promoter. Transcription from the second promoter of each gene (located upstream of arsR in the case of the hasR locus) appears to be constitutive and weak. Quantitative transcriptional analysis in single and double has mutant strains revealed that [[sigma].sup.B.sub.R] and [[sigma].sup.B.sub.L] direct their own transcription as well as that of their duplicates. Only a slight sensitivity in response to oxidative conditions could be assigned to either single or double mutants, revealing the probable redundancy of the [sigma] factors implied in stress response in Streptomyces.

The 34 734-bp integrative and potentially conjugative element (putative ICE) ICESt1 has been previously found to be site-specifically integrated in the 3' end of the fda locus of Streptococcus thermophilus CNRZ368. Four types of genomic islands related to ICESt1 are integrated in the same position in seven other strains of S. thermophilus. One of these elements, ICESt3, harbours conjugation and recombination modules closely related to those of ICESt1 and excises by site-specific recombination. Two other types of elements, CIME19258 and CIME302, are flanked by site-specific attachment sites closely related to attL and attR of ICESt1 and ICESt3, whereas [DELTA]CIME308 only possesses a putative attR site; none of these three elements carry complete conjugation and recombination modules. ICESt1 contains a functional internal recombination site, attL', that is almost identical to attL of CIME19258. The recombination between attL' and attR of ICESt1 leads to the excision of the expected circular molecule (putative ICE); a cis-mobilizable element (CIME) flanked by an attL site and an attB' site remains integrated into the 3' end of fda. Furthermore, sequences that could be truncated att sites were found within ICESt1, ICESt3 and CIME302. All together, these data suggest that these genomic islands evolved by deletion and tandem accretion of ICEs and CIMEs resulting from site-specific recombination. A model for this evolution is proposed and its application to other genomic islands is discussed.

Molecular analysis of ten menadione-sensitive mutants that were obtained from insertional mutagenesis was undertaken for identifying the genes of Streptococcus thermophilus CNRZ368 involved in oxidative stress defense. Higher sensitivity to superoxide stress could be caused by the inactivation of sufD and iscU genes and a critical role could be played by [Fe-S] cluster repair in the oxidative stress defense of anaerobic bacteria.

Research has been conducted on genes implicated in Streptococcus thermophilus CNRZ368 oxidative stress defense. The identification of these genes has been carried out via insertional mutagenesis and the results indicate that the resulting clones are sensitive to menadione and have altered morphologies and reduced growth rates.

Research has been conducted on the rDNA operons of Streptomyces ambofaciens strains. The nucleotide composition of the operons' internally transcribed sequences has been investigated and the results indicate that recombination occurs between the rDNA loci and leads to the nucleotide block exchange.

Researchers describe a plasmid gene in Streptococcus thermophilus that codes a type II restriction-modification system similar to LlaKR21 of Lactococcus lactis and Sau3AI of Staphylococcus aureus. All of these type II R-M systems recognize GATC sequences.

The ICESt1 element of Streptococcus thermophilus CNRZ368 codes a site-specific integrase, excisionase enzymes, and putative conjugate transfer proteins related to the Tn916 transposon. It is 35.5 kb and integrated into the 3' end of a gene encoding fructose-1,6-biphosphate aldolase.

The integration of a polyketide synthase (PKS) gene within the amplified unit DNA (AUD205) of Streptomyces ambofaciens has been found to alter the lipid metabolism rate of the strain. Experimental results revealed that all samples introduced with a mutant strain NSA205 strain manifested reduced ability to produce spiramycin. Majority of the strain further exhibited partial deletion of the AUD205 DNA. Environmental conditions and the physiological condition of bacteria have also been shown to influence the lipid composition of S. ambofaciens.

Pulsed-field gel electrophoresis and sequence probing were used to determine the chromosomal characteristics of mutant strains of Streptomyces ambofaciens that came about as result of genetic instability. Three types of structures were differentiated according to the manner of deletion of sequences. Such deletion were either internal to one chromosomal arm or affected all mutant strains.

The genetic polymorphism existing between three Streptococcus thermophilus strains by pulsed-field gel electrophoresis was analyzed. The chromosomes of CNRZ368 and NST2280 strains were 1864 and 1840 kb circular molecules, respectively. With respect to A054 strain, their physical maps showed a relatively conserved organization of restriction sites. Genetic maps of the three strains revealed a conserved location in the chromosome of A054 and CNRZ368. Polymorphism was found in the copy number of insertions between the strains.

Molecular techniques revealed that production of the macrolide antibiotic spiramycin by Streptomyces ambofaciens RP181110 is governed by an ORF (orfPS) in the AUD205 locus. Genomic rearrangements resulted in the loss of spiromycin production but amplification of the AUD205 locus restored spiromycin biosynthesis. Sequencing of the orfPS gene revealed four sequences including a reiterated sequence corresponding to the acyltransferase domain of the protein. Since amplification caused overtranscription of the gene, it was suggested that orfPS encodes a polyketide synthase that affects spiramycin biosynthesis.

Pulse-field gel electrophoresis and three restriction endonucleases SfiI, BssHII and SmaI aid the construction of a physical map and the estimation of the chromosome size of Streptococcus thermophilus A054. The average size of each genome is 1,824 kb. Hybridization experiments indicate the presence of 23 genetic markers on the chromosomal map. The presence of two variable regions in the chromosome is shown by comparisons of macrorestriction patterns of the three restriction enzymes.

Bifidobacterium strains are identified and classified by employing restriction patterns of rRNA genes. Industrial Bifidobacterium strains and 121 Bifidobacterium strains isolated from the colonic flora of human volunteers are compared. Colonization by exogenous microorganism is resisted by anaerobic component of the endogenous intestinal microflora.

Genetic instability in Streptomyces ambofaciens DSM 40697 is linked to genomic instability induced by several alterations occurring in a large unstable site where two amplifiable DNA loci are expressed. The nucleotide sequence of one AUD6 fragment of 1.9kb shows that two open reading frames, which are termed ORF1 and ORF2, are present. The unstable site of S. ambofaciens is not silent due to the presence of a few loci.

DNA gyrase is directly involved in genetic instability, as proved by pig mutants arising at a frequency of 0.5%. Oxolinic acid and novobiocin are responsible for inducing several pig mutant sectors in more than 80% of the colonies. This leads to the heterogeneous patchwork phenotype. Subinhibitory concentrations also result in genetic instability. Induced pig mutants resemble those that arise spontaneously at a frequency of 0.5% in wild-type streptomyces ambofaciens.

Abstract Background Two closely related ICEs, ICESt1 and ICESt3, have been identified in the lactic acid bacterium Streptococcus thermophilus. While their conjugation and recombination modules are almost identical (95% nucleotide identity) and their regulation modules related, previous work has demonstrated that transconjugants carrying ICESt3 were generated at rate exceeding by a 1000 factor that of ICESt1. Results The functional regulation of ICESt1 and ICESt3 transcription, excision and replication were investigated under different conditions (exponential growth or stationary phase, DNA damage by exposition to mitomycin C). Analysis revealed an identical transcriptional organization of their recombination and conjugation modules (long unique transcript) whereas the transcriptional organization of their regulation modules were found to be different (two operons in ICESt1 but only one in ICESt3) and to depend on the conditions (promoter specific of stationary phase in ICESt3). For both elements, stationary phase and DNA damage lead to the rise of transcript levels of the conjugation-recombination and regulation modules. Whatever the growth culture conditions, excision of ICESt1 was found to be lower than that of ICESt3, which is consistent with weaker transfer frequencies. Furthermore, for both elements, excision increases in stationary phase (8.9-fold for ICESt1 and 1.31-fold for ICESt3) and is strongly enhanced by DNA damage (38-fold for ICESt1 and 18-fold for ICESt3). Although ICEs are generally not described as replicative elements, the copy number of ICESt3 exhibited a sharp increase (9.6-fold) after mitomycin C exposure of its harboring strain CNRZ385. This result was not observed when ICESt3 was introduced in a strain deriving ICESt1 host strain CNRZ368, deleted for this element. This finding suggests an impact of the host cell on ICE behavior. Conclusions All together, these results suggest a novel mechanism of regulation shared by ICESt1, ICESt3 and closely related ICEs, which we identified by analysis of recently sequenced genomes of firmicutes. This is the first report of a partial shutdown of the activity of an ICE executed by a strain belonging to its primary host species. The sharp increase of ICESt3 copy number suggests an induction of replication; such conditional intracellular replication may be common among ICEs.

Abstract Background Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. Results In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. Conclusions These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.