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International audience; Cell separation is dependent on cell wall hydrolases that cleave the peptidoglycan shared between daughter cells. In Streptococcus thermophilus, this step is performed by the Cse protein whose depletion resulted in the formation of extremely long chains of cells. Cse, a natural chimeric enzyme created by domain shuffling, carries at least two important domains for its activity: the LysM expected to be responsible for the cell wall-binding and the CHAP domain predicted to contain the active centre. Accordingly, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against the N-terminal end of this protein. Immunolocalization shows the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus sub-tilis and S. thermophilus. Additionally, RP-HPLC analysis of muropeptides released from B. subtilis and S. thermophilus cell wall after digestion with the CHAP domain shows that Cse is an endopeptidase. Altogether, these results suggest that Cse is a cell wall hydrolase involved in daughter cell separation of S. thermophilus.

Thirty-five putative integrative conjugative elements and related elements were identified at 15 locations in the eight sequenced genomes of Streptococcus agalactiae . Twelve are composite, likely resulting from site-specific accretions. Circular forms were detected for five elements. Macroarray analysis confirmed their high plasticity and wide distribution in S. agalactiae .

Cell division is a dynamic process ending by separation of the daughter cells. This final step requires the cleavage of the murein septum synthetized during cell division. In Streptococcus thermophilus, cse plays an important role in cell separation. Cse protein contains, at its N-terminal end, a signal peptide and a putative LysM motif suggesting that it is secreted and able to bind to the cell wall. Furthermore, the C-terminus of Cse carries a putative cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain conferring to the protein a potential catalytic activity. To gain insight into the role of Cse in the cell division process, in silico analysis of the Firmicutes proteins displaying CHAP-related domain was undertaken. This work allowed us to distinguish and characterize within the Firmicutes the 2 families of proteins (CHAP and NlpC/p60) belonging to the CHAP superfamily. These 2 families regroup mainly peptidoglycan hydrolases. Data from the literature indicate that NlpC/p60 and CHAP proteins cleave distinct peptidoglycan bonds. Among the enzymes characterized within the Firmicutes, NlpC/p60 proteins are γ-D-glutamate-meso-diaminopimelate muropeptidase. Instead, CHAP enzymes involved in cell separation are N-acetylmuramoyl-L-alanine amidase and CHAP lysins have endopeptidase activity.

International audience; Comparative analysis of the Streptomyces chromosome sequences, between Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces ambofaciens ATCC23877 (whose partial sequence is released in this study), revealed a highly compartmentalized genetic organization of their genome. Indeed, despite the presence of specific genomic islands, the central part of the chromosome appears highly syntenic. In contrast, the chromosome of each species exhibits large species-specific terminal regions (from 753 to 1,393 kb), even when considering closely related species (S. ambofaciens and S. coelicolor). Interestingly, the size of the central conserved region between species decreases as the phylogenetic distance between them increases, whereas the specific terminal fraction reciprocally increases in size. Between highly syntenic central regions and species-specific chromosomal parts, there is a notable degeneration of synteny due to frequent insertions/deletions. This reveals a massive and constant genomic flux (from lateral gene transfer and DNA rearrangements) affecting the terminal contingency regions. We speculate that a gradient of recombination rate (i.e., insertion/deletion events) toward the extremities is the force driving the exclusion of essential genes from the terminal regions (i.e., chromosome compartmentalization) and generating a fast gene turnover for strong adaptation capabilities

Insertional mutagenesis was used to isolate clones from Streptococcus thermophilus CNRZ368 that were modified in their abilities to tolerate oxidative stress. During this process, two menadione-sensitive clones (6G4 and 18C3) were found to display abnormal cell morphologies and distorted chain topologies and were further studied. Molecular characterization of both 6G4 and 18C3 mutants indicated that they were disrupted in open reading frames homologous to rodA and pbp2b , respectively. Both genes encoded proteins in Escherichia coli that were described as being implicated in peptidoglycan synthesis during the process of cell elongation and to function in determining the rod shape of the cell. This work reports a possible connection between peptidoglycan biosynthesis and oxidative stress defense in S. thermophilus CNRZ368.

Component PP3 is a phosphoglycoprotein isolated from bovine milk with unknown biological function, which displays in its C-terminal region a basic amphipathic α-helix, a feature often involved in membrane association. According to that, the behaviour of PP3 and of a synthetic peptide from the C-terminal domain (residues 113–135) was investigated in lipid environment. Conductance measurements indicated that the peptide was able to associate and form channels in planar lipid bilayers composed of neutral or charged phospholipids. Electrostatic interactions seemed to promote voltage-dependant channel formation but this was not absolutely required since the pore-forming ability of the 113–135 C-terminal peptide was also detected with the zwitterionic lipid bilayer. Additionally, a spectroscopic study using circular dichroism argues that the peptide adopts an α-helical conformation in interaction with neutral or charged micelles. Thus, the conducting aggregates in bilayers might be composed of a bundle of peptides in helical conformation. Besides, similar conductance measurements performed with the whole PP3 protein did not induce any channel fluctuations. However, with the latter, an early breakdown of the bilayers occurred, a finding that can be tentatively explained by a massive incorporation of PP3. In the light of the present results, it could be inferred that PP3 membrane attachment may be achieved by oligomerization of the C-terminal amphipathic helical region.

International audience; – Streptococcus thermophilus CNRZ368 is an anaerobic aerotolerant bacteria and its ability to survive under aerobic growth conditions raises the question of the existence of a putative defence system against oxidative stress. Thus, survival of CNRZ368 in the presence of increasing concentrations of hydrogen peroxide was studied. Moreover, the influence of the physiologic state of the cells, as well as that of a preexposition with sublethal doses of hydrogen peroxide, upon S. thermophilus CNRZ368 survival were determined. The results suggest that S. thermophilus displays a defence system against oxidative stress and that this system is inducible.  2001 Éditions scientifiques et médicales Elsevier SAS hydrogen peroxide / oxidative stress / S. thermophilus CNRZ368

International audience; The suitability of random amplified polymorphic DNA PCR for the detection of differences between Streptomyces species and strains was evaluated. For this purpose, a protocol of RAPD specific for Streptomyces DNA, i.e. suitable for DNA presenting a high G+C content, was developed using S. ambofaciens ATCC23877. Among the 30 primers tested, all containing 80% G+C, 17 gave a pattern with this strain. Six oligonucleotides were chosen to compare 12 strains belonging to six species of Streptomyces. These oligonucleotides were then used to determine whether these strains could be differentiated at the DNA level with this method. All fingerprints obtained with six primers differed from one species to another. We showed that the RAPD method could be used to reveal intraspecific and intraclonal polymorphisms. Thus, RAPD allows for the rapid, sensitive and specific detection of genetic diversity among species and strains of Streptomyces.

International audience; In Streptomyces ambofaciens, colony pigmentation is an unstable character. Very unstable mutants selected from twelve wild type (WT) subclones of S. ambofaciens ATCC23877 were investigated. This research showed that the polymorphism in colony pigmentation had distinct features. The first aspect is the coexistence of four types of colonies: pigmented colonies (Pig+), pigment-defective colonies (Pigcol-), pigmented colonies harboring pigment-defective sectors (Pigsec+) or pigment-defective papillae (Pigpap+). The second feature was revealed by the study on Pigpap+ colonies. We showed that WT progeny after 14 days of growth consisted almost totally of Pigpap+ colonies. Pigpap+ colonies were also found to be genetically different from each other. Characterization of twelve colonies presenting more than 20 papillae (Hyperpap colonies) led to the isolation of twelve mutator strains which produced at high frequency Pigcol- and Hyperpap colonies. Each exhibited a specific mutator phenotype and were distinct from each other. Such strains constituted a part of the polymorphism observed in each of the WT progeny and also generated a high variability. Finally, we showed that pigment-defective papillae were mutants and constituted a new form of genetic instability.

International audience; Pulsed-field gel electrophoresis (PFGE) proved to be a powerful approach to study bacterial genomics. The genome structure and genetic polymorphism of Gram-positive bacteria from the high G+C (Streptomyces) and low G+C (Streptococcus) groups have been studied. PFGE allowed the estimation of the size of their genome at about 8 Mbp and 1.8 Mbp, respectively, and to get an insight into their chromosome geometry. Thus, physical mapping of the genome of wild-type Streptomyces ambofaciens strains revealed the linearity of the 8 Mbp chromosomal DNA and its typical invertron structure, while the 1.8 Mbp chromosome of Streptococcus thermophilus was shown to be circular. These findings disproved the long-standing idea of universality of bacterial chromosome circularity. In addition, strains belonging to the species S. ambofaciens and S. thermophilus allowed us to characterize the genetic polymorphism at the intraspecific level. Within the S. thermophilus species, comparison of the physical maps showed a relative conservation of gene order as well as restriction sites along the chromosome. In contrast, variable loci were characterized that revealed localized genome rearrangements. The most spectacular of these corresponded to horizontal gene transfer events of sequences. In S. ambofaciens, the physical maps of three isolates pointed to the conservation of the genetic organization. However, a strong polymorphism was observed in the terminal regions of the linear chromosomal DNA. Previous PFGE studies in S. ambofaciens gave proof of a high structural instability of a limited region of the chromosome called unstable region (i.e., DNA rearrangements such as deletions and amplifications). These intraclonal rearrangements create an impressive intraspecific polymorphism of genome size and shape (linear or circular). In both organisms, the DNA rearrangements are restricted to particular regions of the chromosome.

The wild Rhizopus strain spT3 extracted from tempeh of Bali, Indonesia (Institute of Microbial Resources, Taichung, Taiwan) had first been identified as Rhizopus microspores var. oligosporus spT3. This strain is used to ferment soybeans, to detoxify them and improve their nutritional value. Analysis of fermented seeds showed that this strain strongly degrades numerous noxious antinutritional factors, which is unusual for R. microsporus var. oligosporus. The activity of strain spT3 on carbohydrates, compared with that of R. microsporus var. oligosporus NRRL 2710 (= CBS 338.62 = ATCC 22959 = IMI 174457 = IFO 8631 = Scholer's M140), presented differences suggesting that strain spT3 was not a R. microsporus var. oligosporus. As a matter of fact, strain spT3 hydrolyzed sucrose and raffinose, whereas R. microsporus var. oligosporus NRRL 2710 did not. A taxonomic study including morphology, growth temperature, and mating showed that the strain spT3 was similar to R. microsporus var. chinensis CBS 261.28 (= Scholer's M213).Key words: Rhizopus microsporus var. oligosporus, Rhizopus microsporus var. chinensis, Rhizopus microsporus var. rhizopodiformis, mating, carbohydrate utilization, taxonomy, scanning electron microscopy.

Mammary epithelial cells, obtained from lactating cows, were cultured onto inserts coated with several components of extracellular matrix. The influence of these components upon the maintenance of differentiation has been determinated. Every day, alpha S1-casein secretion was measured by radioimmunoassay (RIA) in apical and basal compartments. Reorganization of functional tight junctions was evaluated by measurement of transepithelial electrical resistance (TER). On EHS matrix, cells underwent alveolar structures and never established TER. alpha S1-casein secretion strongly fluctuated with the day of culture. When plated onto fibronectin, cells reorganized a typical pavement and established TER. Nevertheless, TER and casein secretion highly fluctuated. On laminin-coated inserts, a few cells bound to the substratum, dedifferentiated, and proliferated to confluency within 9 days. TER progressively increased to a stable level after 15 days. Casein was not recovered after 6 days. Cells on type I collagen-coated inserts reorganized an epithelial pavement within 2 days and quickly established a stable TER. They secreted apically high levels of casein during 2 weeks. As cells maintained their biochemical differentiation, the culture on type I collagen-coated inserts seems an efficient model for primary culture of bovine mammary epithelial cells and allows studies of polarized alpha S1-casein secretion.

SummaryA microparticle-enhanced nephelometric immunoassay has been developed for the determination of caseinomacropeptide (CMP) in bovine milk. It is based on the nephelometric quantification of the competitive immunoagglutination of a microparticle–CMP conjugate with an anti-κ-casein (κ-CN) antiserum. This one step immunoassay was sensitive (detection limit in reaction mixture, 16μg/l), accurate (linear recovery of CMP in dilution overloading) and reproducible (CV 7–14% for within and between run precision). Because of the specificity of the polyclonal antiserum used, it was necessary to separate CMP from κ-CN by ultrafiltration before the quantification of bovine milk CMP. Under the conditions of milk ultrafiltration used, κ-CN was entirely retained (> 99·5%) but the concentration of CMP measured in milk ultrafiltrates was underestimated (by ∼25%) compared with its concentration in whole milk. Microparticle-enhanced nephelometric immunoassay of CMP, with a calibration range from 0·32 to 20 mg/1 for 20- fold diluted milk ultrafiltrate, allowed contamination of bovine milk by rennet whey as low as 5 ml/1 to be detected. Applied to ultrafiltrates from milk stored at 4 °C, this immunoassay also detected proteolysis of κ-CN not revealed by measurement of κ-CN concentration in milk. A statistical lower limit of 3·21 mg/1 was determined as the increase in CMP concentration in milk ultrafiltrates that indicated probable κ-CN proteolysis in the milk sample. Previously demonstrated to be an easy to perform method for assaying the main proteins of bovine milk, microparticle-enhanced nephelometric immunoassay thus also appeared to be appropriate to quantify CMP so as to detect slight contamination of milk by whey and to indicate the proteolysis of κ-CN during milk storage at low temperature.

Ribosomal DNA polymorphism was studied in order to demonstrate intra- and interspecies differentiation of 42 Bifidobacterium strains. DNA from these strains was digested with the endonucleases BamHI, EcoRV, HindIII and PvuII and then analysed by Southern blotting. Ribosomal patterns using a part of an rRNA 23S gene as a probe clearly differentiated the majority of species from each other. Only B. indicum ATCC 25912T and B. infantis ATCC 15697T displayed identical ribosomal patterns, even though they are classified into two different species. Moreover, ribotypes were able to distinguish between strains belonging to the same species. Furthermore, these strains generally showed common bands, except for B. infantis strains and two strains of B. animalis., L'ADN ribosomique de 42 souches du genre Bifidobacterium a été étudié afin de démontrer leur variabilité intra- et interspécifique. L'ADN de ces souches a été digéré par les endonucleases BamHI, EcoRV, HindIII and PvuII puis analysé par Southern blot. Les profils ribosomiques obtenus par l'utilisation d'une sonde correspondant à une fraction du gène ribosomique 23S différentie clairement la majorité des espèces. Seules les souches B. indicum ATCC 25912T et B. infantis ATCC 15697T présentent le même profil abien qu'elles aient été classées dans des espèces différentes. Les ribotypes obtenus permettent également de distinguer des souches de la même espèce. Les profils de ces dernières présentent le plus généralement des bandes communes, exceptées celles de l'espèce B. infantis et les deux souches de B. animalis.

The heat-stable acid-soluble phosphoglycoprotein component PP3 was isolated from the bovine milk proteose peptone fraction by concanavalin A affinity chromatography. Glycopeptides from the ConA-bound fraction corresponding to the component PP3 were obtained by Pronase digestion and were separated by gel filtration into high and low-molecular-mass glycopeptides. In a previous work, we have investigated the structure of the N-glycans from the high-molecular-mass glycopeptides [Girardet et al. (1995) Eur J Biochem 234: 939–46]. Here, we describe the structure of the O-glycans from the low-molecular-mass glycopeptides. By combining methylation analysis, mass spectrometry, 400 MHz 1H-NMR spectroscopy and peptide sequence analysis, we show that the low-molecular-mass fraction contains several neutral glycopeptides. A mixture of the following three glycan structures linked to the Thr86 has been identified: GalNacα1-O-Thr, Gal(β1-3)GalNAcα1-O-Thr and Gal(β1-4)GlcNAc(β1-6)[Galβ1-3)]GalNAcα1-O-Thr. © 1998 Rapid Science Ltd

Bioinformatics analyses have identified gene clusters encoding cryptic polyketide biosynthetic pathways, not associated with the production of known metabolites, in several actinomycete genome sequences. Discovery of the metabolic products of such cryptic gene clusters promises to unearth a hitherto untapped wealth of novel bioactive compounds. However, a major obstacle to the discovery of novel natural products by genome mining is that many cryptic pathways are expressed poorly or not at all under normal laboratory conditions. The discovery of the stambomycins, a new family of 51-membered macrolides with promising anti-cancer activity, as the products of a novel type I modular polyketide synthase (PKS) system identified in the partial genome sequence of Streptomyces ambofaciens will be described. Activation of expression of the pathway by genetic manipulation of a putative pathway-specific regulatory gene was key to this discovery. The structures of these novel macrolides suggests that their biosynthesis involves novel features, including in trans hydroxylation during polyketide chain assembly to provide the hydroxyl group required for offloading of the fully-assembled polyketide chain from the PKS via macrocyclisation. Experiments aimed at probing this unusual transformation will be described.

Genetic exchanges between Streptococci occur frequently and contribute to their genome diversification. Most of sequenced streptococcal genomes carry multiple mobile genetic elements including Integrative and Conjugative Elements (ICEs) that play a major role in these horizontal gene transfers. In addition to genes involved in their mobility and regulation, ICEs also carry genes that can confer selective advantages to bacteria. Numerous elements have been described in S. agalactiae especially those integrated at the 3' end of a tRNA(Lys) encoding gene. In strain 515 of S. agalactiae, an invasive neonate human pathogen, the ICE (called 515_tRNA(Lys)) is functional and carries different putative virulence genes including one encoding a putative new CAMP factor in addition to the one previously described. This work demonstrated the functionality of this CAMP factor (CAMP factor II) in Lactococcus lactis but also in pathogenic strains of veterinary origin. The search for co-hemolytic factors in a collection of field strains revealed their presence in S. uberis, S. dysgalactiae, but also for the first time in S. equisimilis and S. bovis. Sequencing of these genes revealed the prevalence of a species-specific factor in S. uberis strains (Uberis factor) and the presence of a CAMP factor II encoding gene in S. bovis and S. equisimilis. Furthermore, most of the CAMP factor II positive strains also carried an element integrated in the tRNA(Lys) gene. This work thus describes a CAMP factor that is carried by a mobile genetic element and has spread to different streptococcal species. Citation: Chuzeville S, Puymege A, Madec J-Y, Haenni M, Payot S (2012) Characterization of a New CAMP Factor Carried by an Integrative and Conjugative Element in Streptococcus agalactiae and Spreading in Streptococci. PLoS ONE 7(11): e48918. doi:10.1371/journal.pone.0048918

International audience; The high level of genetic instability in Streptomyces ambofaciens is related to large scale DNA rearrangements (deletions and DNA amplifications) which occur within a 2 Mb chromosomal region. The genome of several Streptomyces species is linear and the unstable region is present at the chromosomal extremities. This has raised the questions of the role of the unstable region (which is dispensable under laboratory conditions), the functions of the genes present in this area, and the relationships between instability and chromosomal linearity. The unstable region of Streptomyces and the replication termini of several other microorganisms, including Escherichia coli, share numerous common traits. This suggests that the unstable region of Streptomyces includes the replication terminus, and that chromosomal instability is related to the termination process.

International audience; In Streptomyces ambofaciens NSA2002, pigmented wild-type colonies spontaneously give rise to pigment-negative (Pig-) mutants at a frequency of about 0.5%. This genetic instability is related to large deletions which can be associated with amplifications of DNA sequences. The influence of three fluoroquinolones (ciprofloxa-cin, enoxacin, and norfloxacin) on this property was investigated. At a survival rate higher than 60%o, most colonies showed a patchwork phenotype consisting of phenotypically heterogeneous colonies harboring numerous mutant sectors. Moreover, the frequency of Pig-mutants rose to more than 90%o at survival rates equal to or higher than 10%0. Induced Pig-mutants showed the same phenotypical features as did spontaneous mutants. Most of them also harbored deletions, associated in some cases with DNA amplifications, in two loci of the large unstable region, AUD6 and AUD90 (derived from amplifiable unit of DNA). The size of deletions in induced mutants could rise to 1.5 Mb. These results show that ciprofloxacin, enoxacin, and norfloxacin greatly stimulate genetic instability and the occurrence of DNA rearrangements in S. ambofaciens. Moreover, these three fluoroquinolones had the same rank order for both toxic (i.e., antibacterial) and genotoxic activities. If the antibacterial effect of fluoroquinolones in S. ambofaciens is due to their interference with DNA gyrase, as shown for some other organisms, the genotoxic effect observed could be due to their interaction with this type II topoisomerase. This suggests that DNA gyrase is involved in the process of genetic instability in S. ambofaciens.

Component 3 corresponds to the hydrophobic fraction of bovine milk proteose-peptone and is composed of three principal glycoproteins with molecular masses of 11, 19, and 29 kDa. To understand better its interesting emulsifying properties and its capability of inhibiting spontaneous lipolysis in milk, isolation and characterization of the three glycoproteins are needed. The newly introduced technique of high performance electrophoresis chromatography is an efficient tool to prepare these glycoproteins in high purity. This preparative technique offers the resolving power of gel electrophoresis with the automatization associated with HPLC. Parameters that influence protein separation are discussed. The isolated 19- and 29-kDa glycoproteins, which bind to immobilized concanavalin A, are then characterized by analysis of their glycan and amino acid compositions. In particular, the glycan molar ratio of the 19-kDa glycoprotein is in good agreement with a biantennary N-acetyllactosamine-type glycan structure.

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 rgg(0182) 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 Rgg(0182) is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Delta rgg(0182) 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 Rgg(0182) transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

International audience; Genomic islands, flanked by attachment sites, devoid of conjugation and recombination modules and related to the integrative and conjugative element (ICE) ICESt3, were previously found in Streptococcus thermophilus. Here, we show that ICESt3 transfers to a recipient harbouring a similar engineered genomic island, CIMEL3catR3, and integrates by site-specific recombination into its attachment sites, leading to their accretion. The resulting composite island can excise, showing that ICESt3 mobilizes CIMEL3catR3 in cis. ICESt3, CIMEL3catR3 and the whole composite element can transfer from the strain harbouring the composite structure. The ICESt3 transfer to a recipient bearing CIMEL3catR3 can also lead to retromobilization, i.e. its capture by the donor. This is the first demonstration of specific conjugative mobilization of a genomic island in cis and the first report of ICE-mediated retromobilization. CIMEL3catR3 would be the prototype of a novel class of non-autonomous mobile elements (CIMEs: CIs mobilizable elements), which hijack the recombination and conjugation machinery of related ICEs to excise, transfer and integrate. Few genome analyses have shown that CIMEs could be widespread and have revealed internal repeats that could result from accretions in numerous genomic islands, suggesting that accretion and cis mobilization have a key role in evolution of genomic islands.

International audience

International audience; A combination of gene loss and acquisition through horizontal gene transfer (HGT) is thought to drive Streptococcus thermophilus adaptation to its niche, i.e. milk. In this study, we describe an in silico analysis com- bining a stochastic data mining method, analysis of homologous gene distribution and the identification of features frequently associated with horizontally transferred genes to assess the proportion of the S. thermophilus gen- ome that could originate from HGT. Our mining approach pointed out that about 17.7% of S. thermophilus genes (362 CDSs of 1,915) showed a composition bias; these genes were called 'atypical'. For 22% of them, their functional annotation strongly support their acquisition through HGT.; Une combinaison de perte / acquisition de gènes dans un processus de transfert horizontal (HGT) a conduit le Streptococcus thermophilus à s'adapter dans sa niche écologique, c-a-d le lait. Dans ce travail, nous décrivons une méthode de fouille de donnéeS fondée sur la modélisation stochastique, l'analyse de la distribution des gènes homologues et l'identification de traits fréquemment associés à des gènes transférés horizontalement afin d'évaluer quelle proportion du génome a été acquise par HGT. Nous approche montre que 17,7% approximativement des gènes de S. thermophilus (362 CDSs des 1915) ont un biais de composition; ces gènes sont dits "atypiques" . Pour 22% d'entre eux, leur annotation fonctionnelle laisse supposer fortement une acquisition par HGT.

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Markov models represent a powerful way to approach the problem of mining time and spatial signals whose variability is not yet fully understood. In this chapter, we will present a general methodology to mine different kinds of temporal and spatial signals having contrasting properties: continuous or discrete with few or many modalities. This methodology is based on a high order Markov modelling as implemented in a free software: carottAge (Gnu GPL), Les modèles de Markov sont des modèles puissants pour analyser des signaux temporels et spatiaux dont la variabilité n'est pas entièrement comprise. Dans ce chapitre, nous présentons notre méthodologie pour fouiller différentes sortes de signaux ayant des propriétés différentes: signaux continus ou discrets, simples ou composites. Cette méthodologie s'appuie sur des modèles de Markov cachés du second-ordre tels qu'implantés dans la boîte à outils CarottAge (licence Gnu-GPL).

International audience; Members of the Streptomyces genus are among the most prolific microorganisms producing secondary metabolites with wide uses in medicine and in agriculture. Sequencing of the genome of the model Streptomyces, Streptomyces coelicolor, has highlighted an unexpected feature, i.e. that the potential of these organisms to synthesise secondary metabolites has been largely underestimated. They indeed possess many more gene clusters encoding natural product-like biosynthetic pathways than there are known natural products. Similar observations have since been made for other bacterial or fungal genomes. Thus, it became clear that microbial secondary metabolism had been seriously underestimated and that genome-based approaches were very promising for the search of new bioactive compounds. Here, we present an overview of the secondary metabolite biosynthetic potential of Streptomyces ambofaciens, a species known for decades as producer of the macrolide spiramycin and the pyrrolamide congocidine. Interestingly, genome analysis has revealed that despite of the close phylogenetic relatedness between S. coelicolor and S. ambofaciens, most of its secondary metabolite gene clusters are species-specific.

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.

Streptococcus thermophilus, bacterium used in dairy industry, is subjected to many environmental changes (oxygen concentration modification, temperature shift, pH variation...) during manufacturing of fermented products. To adapt itself to these modifications, bacterium has defense systems which are controlled by regulation networks involving, in particular, transcriptional regulators. Among them, they are transcriptional regulators of the Rgg family. rgg genes code transcriptional regulators which are very polymorph. Several studies showed their involvement in stress response. The originality of S. thermophilus is the presence of many different rgg copies on genomes of this bacterial species (between 5 and 7 copy according to strain). Objective of this study is the characterization of the Rgg family and study of the S. thermophilus rgg gene function, and, in particular, involvement of these genes in S. thermophilus adaptation to environmental changes. In silico analyses has allowed to show that rgg genes are specific to the Streptocococcaceae family of and the Lactobacillus and the Listeria genus. Proteins belonging to this family are polymorphic and don?t form a monophyletic group. Analyses nevertheless showed that these proteins were characterized by two domains: a "HTH domain" in N-terminal position, very probably involved in the DNA interaction, and a "median domain" in central position which the role remains to determine. Analyses realized during this work show that S. thermophilus LMG18311 rgg0182 gene codes a transcriptional regulator which participates, in particular, in the adaptive response of S. thermophilus exposed to an increase of its growth temperature. Furthermore, the deletion of the rgg0182 gene confers on cells the capacity of a hydrophobic surface adhesion and leads to a reduction of cells chains size. All these phenotypes suggest that Rgg0182 protein is a global regulator of S. thermophilus LMG18311 genic expression. Target genes of this regulator were looked for among genes of thermal stress response and indicate that Rgg0182 protein is involved in the chaperons and proteases homeostasis., Streptococcus thermophilus, bactérie utilisée en industrie agro-alimentaire, est soumise à de nombreuses modifications environnementales (modification de la concentration en oxygène, de la température, du pH, ...) lors de la fabrication de produits fermentés. Pour s'adapter à ces modifications, la bactérie dispose de systèmes de défense qui sont contrôlés par des réseaux de régulation impliquant notamment des régulateurs transcriptionnels. Parmi eux, se trouvent les régulateurs de la famille Rgg. Les gènes Rgg codent des régulateurs transcriptionnels très polymorphes. Plusieurs études ont montré leur implication dans la réponse aux stress. L'originalité de S. thermophilus est que de nombreuses copies Rgg différentes sont présentes sur les génomes de cette espèce bactérienne (entre 5 et 7 copie selon les souches). Cette étude a pour objectif la caractérisation de la famille Rgg et l'étude du rôle des gènes Rgg de S. thermophilus, et, notamment, l'implication de ces gènes dans l'adaptation de S. thermophilus à son environnement. Les analyses réalisées in silico ont permis de montrer que les gènes Rgg sont spécifiques de la famille des Streptocococcaceae et des genres Lactobacillus et Listeria. Les protéines appartenant à cette famille sont polymorphes et ne forment pas un groupe monophylétique. Les analyses ont néanmoins montré que ces protéines se caractérisaient par deux domaines : un « domaine HTH » en position N-terminale, très probablement impliqué dans la liaison à l'ADN et un « domaine médian » en position centrale, dont le rôle reste à déterminer. Les analyses réalisées au cours de ce travail montrent que le gène rgg0182 de S. thermophilus LMG18311 code un régulateur transcriptionnel qui participe, notamment, à la réponse adaptative de S. thermophilus exposée à une augmentation de sa température de croissance. De plus, la délétion du gène Rgg0182 confère aux cellules une capacité d'adhésion à une surface hydrophobe et induit une réduction de la taille des chaînes. L'ensemble de ces phénotypes suggère que la protéine Rgg0182 est un régulateur global de l'expression génique de S. thermophilus LMG18311. Des gènes cibles de ce régulateur ont été recherchés parmi les gènes de réponses au choc thermique et indique que la protéine Rgg0182 est impliquée dans l'homéostasie des chaperons et des protéases.

Integrative and Conjugative Elements (ICEs) are genomic islands, which excise from the chromosome, self-transfer by conjugation and integrate. They harbor a modular organization: genes and sequences involved in the same biological process are grouped in the same region. This work concerns the modality of transfer and maintenance of ICESt1 and ICESt3, two ICEs of Streptococcus thermophilus that share closely related core region. ICESt1 excises much less frequently than ICESt3. Nevertheless, excision of the two elements is activated by the same stimuli (DNA damage, stationary phase and/or cell density) and depends of the host strain. Bioinformatical and transcriptional analyses highlight several differences in their organization. However, each of these two ICEs would encode two different regulators, cI and ImmR, suggesting that a complex and original pathway govern to ICESt1' and ICESt3' regulation. This regulation would be shared with numerous ICEs that we identified in the genome of various commensal or pathogenic streptococci. According to the original definition, ICE's maintenance would be exclusively due to their integration in the host chromosome, and ICEs would not be able of extracellular replication. However, in addition to the induction of ICESt3' excision and transfer, DNA damage cause replication of its extrachromosomal form. This unexpected property is encoded by the core region and would be implicated in the maintenance of the element. Comparision with data recently published on other ICEs suggest that intracellular replication could be involved in the maintenance of numerous ICEs, besides their integration., Les éléments intégratifs conjugatifs (ICE) sont des îlots génomiques qui codent leur excision du chromosome, leur transfert par conjugaison et leur intégration. Ils présentent une organisation modulaire, chaque module incluant tous les gènes nécessaires pour conférer une fonction biologique. Ce travail a porté sur l'étude de la régulation ainsi que les modalités de transfert et de maintien d'ICESt1 et ICESt3, deux ICE de Streptococcus thermophilus présentant une région core étroitement apparentée et une région variable non apparentée. Les résultats obtenus ont montré que, bien qu'ICESt3 s'excise et se transfère à beaucoup plus haute fréquence qu'ICESt1, l'excision des deux éléments est activée par des stimuli identiques et est dépendante de la souche hôte. Chacun de ces ICE code des homologues de deux types de régulateurs différents, cI et ImmR, ce qui implique un mécanisme de régulation complexe et original qui pourrait être conservée chez de nombreux ICE apparentés identifiés lors de ce travail. Selon la définition initiale, les ICE se maintiendraient uniquement sous forme intégrée et ne se répliqueraient pas de façon intracellulaire. Cependant, les dommages à l'ADN induisent non seulement l'excision et le transfert d'ICESt3, mais aussi sa présence en copies multiples extrachromosomiques. Les résultats obtenus impliquent une réplication sous forme extrachromosomique, réplication codée par la région core et qui serait impliquée dans le maintien de l'élément. Une telle réplication pourrait être impliquée dans le maintien de nombreux ICE en plus de leur intégration.

We identified a genetic context encoding a transcriptional regulator of the Rgg family and a small hydrophobic peptide (SHP) in nearly all streptococci and suggested that it may be involved in a new quorumsensing mechanism, with SHP playing the role of a pheromone. Here, we provide further support for this hypothesis by constructing a phylogenetic tree of the Rgg and Rgg-like proteins from Gram-positive bacteria and by studying the shp/rgg1358 locus of Streptococcus thermophilus LMD-9. We identified the shp1358 gene as a target of Rgg1358, and used it to confirm the existence of the steps of a quorumsensing mechanism including secretion, maturation and reimportation of the pheromone into the cell. We used surface plasmon resonance to demonstrate interaction between the pheromone and the regulatory protein and performed electrophoretic mobility shift assays to assess binding of the transcriptional regulator to the promoter regions of its target genes. The active form of the pheromone was identified by mass spectrometry. Our findings demonstrate that the shp/rgg1358 locus encodes two components of a novel quorum-sensing mechanism involving a transcriptional regulator of the Rgg family and a SHP pheromone that is detected and reimported into the cell by the Ami oligopeptide transporter.

The present invention relates to Stambomycin compounds, their derivatives and their pharmaceutically acceptable salt thereof.

Second-order Hidden Markov Models (HMM2) are stochastic processes with a high efficiency in exploring bacterial genome sequences. Different types of HMM2 (M1M2, M2M2, M2M0) combined to combinatorial methods were developed in a new approach to discriminate genomic regions without a priori knowledge on their genetic content. This approach was applied on two bacterial models in order to validate its achievements: Streptomyces coelicolor and Streptococcus thermophilus. These bacterial species exhibit distinct genomic traits (base composition, global genome size) in relation with their ecological niche: soil for S. coelicolor and dairy products for S. thermophilus. In S. coelicolor, a first HMM2 architecture allowed the detection of short discrete DNA heterogeneities (5-16 nucleotides in size), mostly localized in intergenic regions. The application of the method on a biologically known gene set, the SigR regulon (involved in oxidative stress response), proved the efficiency in identifying bacterial promoters. S. coelicolor shows a complex regulatory network (up to 12% of the genes may be involved in gene regulation) with more than 60 sigma factors, involved in initiation of transcription. A classification method coupled to a searching algorithm (i.e. R?MES) was developed to automatically extract the box1-spacer-box2 composite DNA motifs, structure corresponding to the typical bacterial promoter -35/-10 boxes. Among the 814 DNA motifs described for the whole S. coelicolor genome, those of sigma factors (?B, ?WhiG) could be retrieved from the crude data. We could show that this method could be generalized by applying it successfully in a preliminary attempt to the genome of Bacillus subtilis, Les modèles de Markov d'ordre 2 (HMM2) sont des modèles stochastiques qui ont démontré leur efficacité dans l'exploration de séquences génomiques. Cette thèse explore l'intérêt de modèles de différents types (M1M2, M2M2, M2M0) ainsi que leur couplage à des méthodes combinatoires pour segmenter les génomes bactériens sans connaissances a priori du contenu génétique. Ces approches ont été appliquées à deux modèles bactériens afin d'en valider la robustesse : Streptomyces coelicolor et Streptococcus thermophilus. Ces espèces bactériennes présentent des caractéristiques génomiques très distinctes (composition, taille du génome) en lien avec leur écosystème spécifique : le sol pour les S. coelicolor et le milieu lait pour S. thermophilus

•An extensive survey of nodulation in the legume genus Mimosa was undertaken in two major biomes in Brazil, the Cerrado and the Caatinga, in both of which there are high degrees of endemicity of the genus.•Nodules were collected from 67 of the 70 Mimosa spp. found. Thirteen of the species were newly reported as nodulating. Nodules were examined by light and electron microscopy, and all except for M. gatesiae had a structure typical of effective Mimosa nodules. The endosymbiotic bacteria in nodules from all of the Mimosa spp. were identified as Burkholderia via immunolabelling with an antibody against Burkholderia phymatum STM815.•Twenty of the 23 Mimosa nodules tested were shown to contain nitrogenase by immunolabelling with an antibody to the nitrogenase Fe- (nifH) protein, and using the δ15N (15N natural abundance) technique, contributions by biological N2 fixation of up to 60% of total plant N were calculated for Caatinga Mimosa spp.•It is concluded that nodulation in Mimosa is a generic character, and that the preferred symbionts of Brazilian species are Burkholderia. This is the first study to demonstrate N2 fixation by beta-rhizobial symbioses in the field., This work was funded by the Natural Environment Research Council, UK (NE/B505038/1). The collection and transfer of nodules were authorized by permit no. 007/2005 from the Ministério do Meio Ambiente, Brazil (IBAMA).

The constant and urgent need of novel bioactive compounds is the result of the emergence in the last decades of new and old infectious diseases, a sore for humankind. Actinomycetes and especially the genus Streptomyces are the principal producers of microbial drugs producing nearly 60-70% of the natural products. The majority of secondary metabolites belong to the class of polyketides that are synthesised by multienzymatic complexes named polyketides synthases (PKS). PKSs condensate simple small carboxylic acids to generate a wide range of complex polyketide structures. In the search for new drugs, the genome mining approach proved to be a powerful tool in the identification of cryptic secondary metabolite pathways. The sequencing and the analysis of Streptomyces ambofaciens ATCC23877 genome has revealed a large type I PKS cluster, on the right arm of the chromosome. The cluster contains 9 PKS, composed of 25 functional modules. In silico analysis of the PKS genes and of the tailoring genes enabled to predict the structure of the expected metabolite, a macrolide. In the laboratory standard conditions, the cluster showed to be silent. Therefore, to promote the expression of the cluster, the regulatory gene samR0484, encoding a LAL regulator (Large ATP binding protein of the LuxR family) was overexpressed in the wt strain. Transcriptional analyses showed that the PKS genes were expressed. Subsequently, by comparative metabolic profiling between the mutant strain and the wt, we were able to detect the novel metabolite produced by S. ambofaciens. Structural elucidation revealed four form of a 50-membered macrolide, named sambomycin. The compounds endow antibacterial and antitumoral activities. The structure unveiled unique and interesting characteristics of sambomycin, i.e. the cyclization reaction and the presence of an atypical extender unit. The mechanisms of biosynthesis have been analysed more in details in this work. We also investigated in the regulation of the cluster. The LAL regulator was shown to be an essential transcriptional activator, binding to the promoter regions of the PKS genes and probably to other genes in the cluster.; La recherche de nouveaux métabolites d'intérêt médical est toujours d'actualité, surtout si l'on considère que d'anciennes, mais aussi des nouvelles infections bactériennes ou virales apparaissent régulièrement. Les actinomycètes, et plus particulièrement les Streptomyces, sont les principaux producteurs de molécules anti-microbiennes. En effet, ils produisent presque 60-70% des produits naturels d'origine microbienne. La plupart de ces métabolites appartient à la classe des polycétides, qui sont synthétisés par des complexes multienzymatiques, les polycétide-synthétases (PKS). Les PKS utilisent des acides gras simples pour assembler des structures polycétidiques très diversifiées. Une approche très prometteuse pour identifier des nouvelles voies de biosynthèse de métabolites secondaires est basée sur une approche génomique ou « genome mining ». Le séquençage du chromosome linéaire de Streptomyces ambofaciens ATCC23877 a, entre autre, révélé sur le bras chromosomique droit, un cluster cryptique de gènes de PKS de type I de grande taille. Ce cluster contient 25 gènes, dont 9 gènes de biosynthèse, pour un total de 25 modules, tous fonctionnels. Les analyses in silico des gènes de PKS ont permis de prédire que le cluster serait responsable de la synthèse d'une molécule appartenant à la famille des macrolides. Dans des conditions standard de laboratoire, le cluster était silencieux. Afin d'activer l'expression du cluster, un gène de régulation, samR0484, présent dans le cluster et codant un régulateur de la famille LAL (Large ATP binding protein of the LuxR family), a été surexprimé dans la souche sauvage. Les analyses transcriptionelles ont montré que cela se traduisait par l'induction de l'expression des gènes de biosynthèse. Par conséquence, une stratégie de « comparative metabolic profiling » a été menée entre la souche sauvage et la souche mutante afin d'identifier le nouveau métabolite. Quatre formes différentes d'un macrolide avec un cycle lactonique de 50 carbones, ont été isolées et caractérisées. Ces composants, nommés sambomycine A, B, C et D, ont montré une activité antibactérienne et une activité antiproliférative intéressante. La détermination de la structure de la sambomycine a révélé des caractéristiques uniques et intéressantes, concernant la réaction de cyclisation et la synthèse d'un précurseur atypique. Ces mécanismes de biosynthèse ont fait l'objet d'une étude plus approfondie. Nous nous sommes également intéressés à la régulation de ce cluster. Le régulateur LAL agit comme un activateur transcriptionnel essentiel. Des analyses préliminaires indiquent que ce régulateur se fixe aux régions promotrices de certains gènes, notamment celles des gènes de biosynthèse ainsi que celles d'autres gènes de post-modification, activant ainsi leur transcription.

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.

International audience; We present a new data mining method based on stochastic analysis (HMM for Hidden Markov Model) and combinatorial methods for discovering new transcriptional factors in bacterial genome sequences. Sigma factor binding sites (SFBSs) were described as patterns of box1 - spacer - box2 corresponding to the -35 and -10 DNA motifs of bacterial promoters. We used a high-order Hidden Markov Model in which the hidden process is a second-order Markov chain. Applied on the genome of the model bacterium Streptomyces coelicolor (2), the a posteriori state probabilities revealed local maxima or peaks whose distribution was enriched in the intergenic sequences (``iPeaks'' for intergenic peaks). Short DNA sequences underlying the iPeaks were extracted and clustered by a hierarchical classification algorithm based on the SmithWaterman local similarity. Some selected motif consensuses were used as box1 (-35 motif) in the search of a potential neighbouring box2 (-10 motif) using a word enumeration algorithm. This new SFBS mining methodology applied on Streptomyces coelicolor was successful to retrieve already known SFBSs and to suggest new potential transcriptional factor binding sites (TFBSs). The well defined SigR regulon (oxidative stress response) was also used as a test quorum to compare first and second-order HMM. Our approach also allowed the preliminary detection of known SFBSs in Bacillus subtilis.

International audience

International audience; Congocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the S. ambofaciens chromosome, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletions of 8 of the 22 genes confirm the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned specific functions in regulation, resistance, or congocidine assembly. In contrast, the biosynthetic origin of the precursors cannot be easily inferred from in silico analyses. Congocidine is assembled by a nonribosomal peptide synthetase (NRPS) constituted of a free-standing module and several single-domain proteins encoded by four genes. The iterative use of its unique adenylation domain, the utilization of guanidinoacetyl-CoA as a substrate by a condensation domain, and the control of 4-aminopyrrole-2-carboxylate polymerization constitute the most original features of this NRPS.

Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic 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 , ICE St1 and ICE St3 . Mitomycin C, a DNA-damaging agent, derepresses ICE St3 conjugative transfer almost 25-fold. The ICE St3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICE St3 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, ICE St3 was retransferred from E. faecalis to S. thermophilus . Recombinant plasmids carrying different parts of the ICE St1 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.

International audience

Streptomycessp. US 24 andStreptomycessp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed usingE. coliET12567 (pUZ8002), as a conjugal donor, carrying the integrative plasmid pSET152. For theStreptomycessp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas forStreptomycessp. TN 58, the integration was in single copy at theattBsite. Plasmid pSET152 was inherited every time for all analysedStreptomycessp. US 24 andStreptomycessp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA) fromStreptomycessp. SK was expressed in strainStreptomycessp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of theStreptomycessp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch.

Analyses of genomes had suggested that numerous bacterial genomic islands would be integrative conjugative elements (ICEs) or elements deriving from them. ICEs excise under a circular form by site-specific recombination, transfer by conjugation, and integrate in a recipient cell. The type of elements is very widespread in genomes of bacteria and archaea. Various related genomic islands are integrated at the 3' of the fda ORF in different Streptococcus thermophilus strains. This family includes 2 integrative and potentially conjugative elements, of which ICESt3, and 4 elements deriving from ICEs by deletion and named CIMEs (cis mobilizable elements). This work has demonstrated that ICESt3 transfers between S. thermophilus and related species. This element is the first conjugative element identified in this streptococcus. The ICESt3 transfer to a cell already carrying an ICE or a CIME leads to the characterization of site-specific accretions of ICESt3 and a related genomic island. Using donor cell harboring a CIME-ICE tandem, the co-transfer of the CIME and the ICE, the transfer of the ICE and the transfer of the only CIME were obtained, demonstrating conjugative mobilization of a CIME by ICESt3. Thus, the genomic islands from S. thermophilus evolve by site-specific accretion and conjugative mobilization. Moreover, an analysis of sequences from databases and an analysis of literature strongly suggest that CIMEs are widespread and that site-specific accretions between genomic islands play a key role in bacterial evolution., Des analyses de génomes avaient suggéré que de nombreux îlots génomiques bactériens seraient des éléments intégratifs conjugatifs (ICE) ou des éléments en dérivant. Les ICE s'excisent sous forme circulaire par la recombinaison site-spécifique, se transfèrent par conjugaison et s'intègrent chez une cellule réceptrice. Les éléments de ce type sont très abondants aux seins des génomes de bactéries et d'archées. Divers îlots génomiques apparentés sont intégrés dans l'extrémité 3' de l'ORF fda chez plusieurs souches de Streptococcus thermophilus. Cette famille inclut 2 éléments intégratifs potentiellement conjugatifs, dont ICESt3, et 4 éléments qui dérivent d'ICE par délétion, les CIME (cis mobilizable elements). Ce travail a montré qu'ICESt3 se transfère entre souches de S. thermophilus et entre espèces proches. Cet élément est le premier élément conjugatif identifié chez ce streptocoque. Le transfert d'ICESt3 vers une cellule portant un ICE ou un CIME intégré a conduit à l'accrétion site-spécifique d'ICESt3 et d'un îlot génomique apparenté. À partir de cellules portant un tandem CIME-ICE, le co-transfert du CIME et de l'ICE ainsi que le transfert du CIME seul ont été obtenus, démontrant la mobilisation conjugative d'un CIME par ICESt3. Ainsi, les îlots génomiques de S. thermophilus évoluent par accrétion site-spécifique et mobilisation conjugative. Par ailleurs, une analyse de séquences disponibles dans les bases de données et une analyse bibliographique suggèrent très fortement que les CIME sont très répandus et que les événements d'accrétion site-spécifique entre îlots génomiques jouent un rôle important dans l'évolution bactérienne.

International audience; Genetic instability in Strepfomyces ambofaciens DSM 40697 involves genomic rearrangements such as amplifications and deletions of particular DNA sequences. Most amplifications were located in two amplifiable regions, one of which, called AUD6 (amplifiable unit of DNA no. 6) was revealed to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied had amplifications, deletions or both at the AUD6 locus. This locus contains several reiterations which are specific to this AUD. Moreover, one of the endpoints of the AUD6 shows homology with an internal sequence. Deletions occurred exclusively at one side of the amplified DNA sequence (ADS) and removed part of the proximal copy of this ADS, leading to the conclusion that multiple rearrangements can occur at this AUD locus.

Streptomyces ambofaciens produces an orange pigment and the antibiotic alpomycin, both of which are products of a type II polyketide synthase gene cluster identified in each of the terminal inverted repeats of the linear chromosome. Five regulatory genes encoding Streptomyces antibiotic regulatory proteins ( alpV , previously shown to be an essential activator gene; alpT ; and alpU ) and TetR family receptors ( alpZ and alpW ) were detected in this cluster. Here, we demonstrate that AlpZ, which shows high similarity to γ-butyrolactone receptors, is at the top of a pathway-specific regulatory hierarchy that prevents synthesis of the alp polyketide products. Deletion of the two copies of alpZ resulted in the precocious production of both alpomycin and the orange pigment, suggesting a repressor role for AlpZ. Consistent with this, expression of the five alp -located regulatory genes and of two representative biosynthetic structural genes ( alpA and alpR ) was induced earlier in the alpZ deletion strain. Furthermore, recombinant AlpZ was shown to bind to specific DNA sequences within the promoter regions of alpZ , alpV , and alpXW , suggesting direct transcriptional control of these genes by AlpZ. Analysis of solvent extracts of S. ambofaciens cultures identified the existence of a factor which induces precocious production of alpomycin and pigment in the wild-type strain and which can disrupt the binding of AlpZ to its DNA targets. This activity is reminiscent of γ-butyrolactone-type molecules. However, the AlpZ-interacting molecule(s) was shown to be resistant to an alkali treatment capable of inactivating γ-butyrolactones, suggesting that the AlpZ ligand(s) does not possess a lactone functional group.

Within Streptococcus thermophilus, Cse was identified as the major cell disconnecting peptidoglycan hydrolase (PGH) and was demonstrated to be species-specific. To identify cell disconnecting PGHs encoded by other Streptococcus genomes, we explored the diversity of domains carried by Firmicutes PGHs, and especially that of enzymes involved in daughter cell separation. This work brings to light the diversity of PGHs and reveals that each species recruits its own cell-separating enzyme distinct from that of the others. This specificity is probably correlated with the diversity of substrates found in the bacterial cell wall

Background Many programs have been developed to identify transcription factor binding sites. However, most of them are not able to infer two-word motifs with variable spacer lengths. This case is encountered for RNA polymerase Sigma (σ) Factor Binding Sites (SFBSs) usually composed of two boxes, called -35 and -10 in reference to the transcription initiation point. Our goal is to design an algorithm detecting SFBS by using combinational and statistical constraints deduced from biological observations. Results We describe a new approach to identify SFBSs by comparing two related bacterial genomes. The method, named SIGffRid (SIGma Factor binding sites Finder using R'MES to select Input Data), performs a simultaneous analysis of pairs of promoter regions of orthologous genes. SIGffRid uses a prior identification of over-represented patterns in whole genomes as selection criteria for potential -35 and -10 boxes. These patterns are then grouped using pairs of short seeds (of which one is possibly gapped), allowing a variable-length spacer between them. Next, the motifs are extended guided by statistical considerations, a feature that ensures a selection of motifs with statistically relevant properties. We applied our method to the pair of related bacterial genomes of Streptomyces coelicolor and Streptomyces avermitilis. Cross-check with the well-defined SFBSs of the SigR regulon in S. coelicolor is detailed, validating the algorithm. SFBSs for HrdB and BldN were also found; and the results suggested some new targets for these σ factors. In addition, consensus motifs for BldD and new SFBSs binding sites were defined, overlapping previously proposed consensuses. Relevant tests were carried out also on bacteria with moderate GC content (i.e. Escherichia coli/Salmonella typhimurium and Bacillus subtilis/Bacillus licheniformis pairs). Motifs of house-keeping σ factors were found as well as other SFBSs such as that of SigW in Bacillus strains. Conclusion We demonstrate that our approach combining statistical and biological criteria was successful to predict SFBSs. The method versatility autorizes the recognition of other kinds of two-box regulatory sites.

Streptococcus thermophilus is a lactic bacteria used a stater of fermentation in dairy factories for the production of yogurt and many cheeses. S. thermophilus grows as chains of ovoid cells. However, the genetic basis of S. thermophilus cell separation is still unknown. A S. thermophilus mutant displaying extremely long chains of cells was characterized and demonstrated to be impaired a gene that we named cse for cell separation. The originality of this gene is that cse creation resulted from domain shuffling. Indeed, the analysis has revealed that its 5?extremity has homology with that of sip from S. salivarius, while its 3?extremity shares homology with pcsB from S. thermophilus.The cse gene specific from S. thermophilus, encodes a modular protein. The N-terminal end of Cse contains a secretion signal and cell-wall binding LysM domain. Its C-terminal end includes a CHAP domain found in bacterial cell-wall hydrolases. In this study, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against this protein. Immunolocalization shows that the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a lytic activity on the cell wall of S. thermophilus that has been demonstrated by zymogram. Additionally, RP-HPLC analysis of muropeptides released from S. thermophilus after digestion with the CHAP domain shows that Cse is a cell wall hydrolase that can function as an endopeptidase. Alltogether, these results suggest that Cse is a major cell wall hydrolase involved in daughter cell separation of S. thermophilus., Streptococcus thermophilus est une bactérie lactique utilisée dans l'industrie laitière pour la fabrication de yaourts et de divers fromages. S. thermophilus se développe en chaîne de cellules. Le mécanisme de la séparation des cellules n'est pas connu chez S. thermophilus. Un mutant de S. thermophilus présentant des chaînes de cellules extrêmement longues a été caractérisé. Le gène identifié est nommé cse pour cell separation. La particularité de cse est qu'il résulte d'un réassortiment de modules. En effet, l'analyse a montré que son extrémité 5' est homologue à celle de sip de S. salivarius, tandis que son extrémité 3' est homologue à celle de pcsB de S. thermophilus. Le gène cse spécifique de S. thermophilus code une protéine modulaire. A son extrémité N-terminale, Cse possède un peptide signal et un domaine de liaison à la paroi, LysM. Et à son extrémité C-terminale, Cse possède un domaine CHAP, présent dans les hydrolases du peptidoglycane. Dans cette étude, la localisation de Cse à la surface des cellules de S. thermophilus a été réalisée par microscopie électronique à transmission et immunofluorescence à l'aide d'anticorps dirigés contre cette protéine. Cse est localisée spécifiquement aux septa matures de S. thermophilus. De plus, l'activité de Cse a été démontré par zymogramme et présente une activité lytique qui est conférée par son domaine CHAP. L'analyse par RP-HPLC des muropeptides de la paroi de S. thermophilus après digestion avec le domaine CHAP a révélé que Cse est une hydrolase du peptidoglycane et plus précisément une endopeptidase. L'ensemble de ces résultats montre que Cse est l'enzyme majeure de la séparation cellulaire de S. thermophilus.

The Streptomyces are soil bacteria with a large linear chromosome, typically 8 to 10 Mb, high part of G-C bases (72.1% for S. coelicolor) and are subject to a high degree of genetic instability, correlated with the formation of large rearrangement occurring in the terminal chromosomal regions. The analysis of the terminal regions sequences and partial sequence central region of S. ambofaciens ATCC23877 shows that the size of the terminal species-specific regions increases as the phylogenetic distance between compared species increases. The synteny observed between central regions degenerates progressively over several hundreds of kilobases before reaching the terminal species-specific regions. This synteny appears as gradually parceled out by multiple insertions/deletions (indels) of genes. The comparison of the sequences of the Terminal Inverted Repeat (TIR) of two S. ambofaciens strains shows the two strains share the same ancestral boundaries of TIRs. On the other hand, the terminals regions are strain-specific suggesting that exchanges of replicon extremities, potentially plasmidic, have occurred, contributing to the terminal variability observed at the intraspecific level. The mechanisms of double breaks repair or the frequency of these double breaks could be the reasons of this variability. Sometimes, the chromosome becomes dicentric and enters in a break-fusion-bridge (BFB) cycle. This cycle is the consequence of a end-to-end fusion of two deleted chromosomes. The analysis of S. ambofaciens DSM40697 strains with a second locus involved in the partitioning narrows the implication of this one in the chromosomic instability and the Streptomyces' evolution., Les Streptomyces sont des bactéries du sol possédant un chromosome linéaire de grande taille (8-11 Mb), un fort taux en bases G-C (72,1% chez S. coelicolor) ainsi que l'apparition à haute fréquence de mutants, présentant de grands réarrangements touchant les régions terminales chromosomiques. L'analyse des séquences des régions terminales et des séquences partielles de la région centrale de la souche ATCC23877 de S. ambofaciens a permis de montrer que la taille de la région spécifique d'espèce augmente avec l'éloignement phylogénétique. Cette perte progressive de synténie est le résultat d'évènements d'insertions/délétions (indels) de petits fragments d'ADN. La comparaison des séquences des Répétitions Terminales Inversées de deux souches de S. ambofaciens a montré la présence de frontières ancestrales communes et les régions spécifiques situées aux extrémités. Cette variabilité serait la conséquence de remplacements d'extrémités de réplicons linéaires potentiellement d'origine plasmidique. L'intervention différentielle de mécanismes de réparation de cassures double brin ou une fréquence plus importante des cassures le long du chromosome pourraient être à la base de cette variabilité. Une conséquence de la formation de cassures double brin est l'entrée dans le cycle de cassure-fusion-pont (CFP) dont un intermédiaire est un chromosome dicentrique. L'entrée dans ce cycle serait la conséquence de la fusion de deux chromosomes délétés d'un bras chromosomique. L'analyse de souches de S. ambofaciens DSM40697 contenant un second centre de partition précisera l'implication des systèmes de partition dans l'instabilité et l'évolution du chromosome des Streptomyces.

International audience; The integrative and conjugative elements (ICEs) excise by site-specific recombination between attL and attR flanking sites, self-transfer the resulting circular form and integrate into the genome of the recipient cell. Two putative ICEs, ICESt1 and ICESt3, are integrated in the same locus in 2 strains of Streptococcusthermophilus. ICESt1 is a composite element harbouring an internal recombination site, attL’. The recombination between attL’ and attR leads to the excision of a shorter putative ICE, ICESt2. ICESt1/ICESt2 and ICESt3 carry related regulation modules sharing the open reading frame arp1 that encodes a protein related to the cI repressor of the phage λ. The repressors belonging to this family autoproteolyse in the presence of damaged DNA. Treatments with mitomycin C induce an increase in the excision of ICESt1, ICESt2 and ICESt3. Furthermore, the arp1 deletion leads to a 1,000-fold increase in the excision of ICESt1 and ICESt2 and to a decrease in the excision induction by mitomycin C. Thus, all together, these results suggest that the autocleavage of the arp1 repressor is involved in derepression of the S. thermophilus putative ICE excision by mitomycin C.