Differential regulation of two closely related integrative and conjugative elements from Streptococcus thermophilus.

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).

The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation.

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.

Site-specific accretion of an integrative conjugative element together with a related genomic island leads to cis mobilization and gene capture.

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.

Conjugative transfer of the integrative conjugative elements ICESt1 and ICESt3 from Streptococcus thermophilus.

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, ICESt1 and ICESt3. Mitomycin C, a DNA-damaging agent, derepresses ICESt3 conjugative transfer almost 25-fold.

The CHAP domain of Cse functions as an endopeptidase that acts at mature septa to promote Streptococcus thermophilus cell separation.

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.

Diversity of Firmicutes peptidoglycan hydrolases and specificities of those involved in daughter cell separation.

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.

Characterization of proteins belonging to the CHAP-related superfamily within the Firmicutes.

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.

Regulation of excision of integrative and potentially conjugative elements from Streptococcus thermophilus: role of the arp1 repressor.

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'.

Derepression of excision of integrative and potentially conjugative elements from Streptococcus thermophilus by DNA damage response: implication of a cI-related repressor.

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 constant gene orf14.9, which belongs to the variable eps (exopolysaccharide) cluster, is involved in the cell growth of Streptococcus thermophilus.

In Streptococcus thermophilus, the eps clusters involved in exopolysaccharide (EPS) biosynthesis are very polymorphic, nevertheless they all contain a highly conserved sequence corresponding to that of orf14.9. This open reading frame (ORF) is transcribed in a reverse direction with respect to eps genes.

Evolution of the terminal regions of the Streptomyces linear chromosome.

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.

Intraspecific variability of the terminal inverted repeats of the linear chromosome of Streptomyces ambofaciens.

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.

High genetic variability of the Streptococcus thermophilus cse central part, a repeat rich region required for full cell segregation activity.

The cse gene of Streptococcus thermophilus encodes an extracytoplasmic protein involved in cell segregation. The Cse protein consists of two putative domains: a cell wall attachment LysM domain and a catalytic CHAP domain. These two domains are spaced by an interdomain linker, known as Var-Cse, previously reported to be highly divergent between two S.

The rggC locus, with a frameshift mutation, is involved in oxidative stress response by Streptococcus thermophilus.

In Streptococcus thermophilus, the locus rggC contains a frameshift mutation and thus consists of two open reading frames (ORFs), rggC (1) and rggC (2), which encode proteins exhibiting similarity with the Rgg transcriptional regulator family. In this work, mutants showing a partial deletion of rggC (1) and rggC (2 )were constructed and their response to menadione, a superoxide-generating compound, was analysed. These mutants exhibited different behaviour to this oxidative stress compared with the wild-type strain.

Genetic instability of whiG gene during the aerial mycelium development of Streptomyces ambofaciens ATCC23877 under different conditions of nitrogen limitations.

In Streptomyces ambofaciens, white papillae that genetic instability events generate during aerial mycelium growth, give rise to Pig-pap mutants which are unable to sporulate and devoid of large genome rearrangement. Knowing that genetic and environmental factors can influence the number of papillae per colony, we investigated the effect of nutrient limitated conditions of growth on the formation of white papillae. We observed that under nitrogen limitation and, most particularly, under amino acid limitation, the number of papillae per colony dramatically increased.

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

Streptococcus thermophilus is a major dairy starter used for the manufacture of yoghurt and cheese. The access to three genome sequences, comparative genomics and multilocus sequencing analyses suggests that this species recently emerged and is still undergoing a process of regressive evolution towards a specialised bacterium for growth in milk. Notably, S.

Sigma factor WhiG and its regulation constitute a target of a mutational phenomenon occurring during aerial mycelium growth in Streptomyces ambofaciens ATCC23877.

The genetic instability of Streptomyces ambofaciens affects the pigmentation of colonies and generates a variety of mutants the majority of which display large genome rearrangements. Among them, the Pig-pap mutants, which probably result from a mutational event occurring during aerial mycelium growth, display specific features, since they are unable to sporulate and do not harbor any large detectable genome rearrangements. To identify the mutational event causing their phenotype, three Pig-pap mutants originating from three independent mutational events were characterized.

cse, a Chimeric and variable gene, encodes an extracellular protein involved in cellular segregation in Streptococcus thermophilus.

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.

Involvement of AlpV, a new member of the Streptomyces antibiotic regulatory protein family, in regulation of the duplicated type II polyketide synthase alp gene cluster in Streptomyces ambofaciens.

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.

Characterization of oxidative stress-resistant mutants of Streptococcus thermophilus CNRZ368.

During industrial processes, the dairy organism Streptococcus thermophilus is exposed to stress conditions. Its ability to survive and grow in an aerobic environment indicates that it must possess defensive mechanisms against reactive oxygen species. To identify the genes involved in oxidative stress defence, a collection of mutants was generated by random insertional mutagenesis and screened for menadione sensitivity and resistance.

Differential and cross-transcriptional control of duplicated genes encoding alternative sigma factors in Streptomyces ambofaciens.

The duplicated hasR and hasL genes of Streptomyces ambofaciens encode alternative sigma factors (named sigma(B(R)) and sigma(B(L))) belonging to the sigma(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.

Evolution of genomic islands by deletion and tandem accretion by site-specific recombination: ICESt1-related elements from Streptococcus thermophilus.

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.

Identification of Streptococcus thermophilus CNRZ368 genes involved in defense against superoxide stress.

To better understand the defense mechanism of Streptococcus thermophilus against superoxide stress, molecular analysis of 10 menadione-sensitive mutants, obtained by insertional mutagenesis, was undertaken. This analysis allowed the identification of 10 genes that, with respect to their putative functions, were classified into five categories: (i) those involved in cell wall metabolism, (ii) those involved in exopolysaccharide translocation, (iii) those involved in RNA modification, (iv) those involved in iron homeostasis, and (v) those whose functions are still unknown. The behavior of the 10 menadione-sensitive mutants exposed to heat shock was investigated.

Functional angucycline-like antibiotic gene cluster in the terminal inverted repeats of the Streptomyces ambofaciens linear chromosome.

Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the beta-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS.

End-to-end fusion of linear deleted chromosomes initiates a cycle of genome instability in Streptomyces ambofaciens.

Two mutant strains harbouring a linear chromosome whose size reached 13 Mb (versus approximately 8 Mb for the wild type) were characterized. This chromosomal structure resulted from the fusion in inverted orientation of two chromosomes partially deleted on the same arm. The fusion occurred by illegitimate recombination between 6 bp repeats.