SOS Response Activation and Competence Development Are Antagonistic Mechanisms in Streptococcus thermophilus

Streptococcus includes species that either contain or lack the LexA-like repressor (HdiR) of the classical SOS response. In Streptococcus pneumoniae, a species which belongs to the latter group, SOS response inducers (e.g., mitomycin C [Mc] and fluoroquinolones) were shown to induce natural transformation, leading to the hypothesis that DNA damage-induced competence could contribute to genomic plasticity and stress resistance. Using reporter strains and microarray experiments, we investigated the impact of the SOS response inducers mitomycin C and norfloxacin and the role of HdiR on competence development in Streptococcus thermophilus. We show that both the addition of SOS response inducers and HdiR inactivation have a dual effect, i.e., induction of the expression of SOS genes and reduction of transformability. Reduction of transformability results from two different mechanisms, since HdiR inactivation has no major effect on the expression of competence (com) genes, while mitomycin C downregulates the expression of early and late com genes in a dose-dependent manner. The downregulation of com genes by mitomycin C was shown to take place at the level of the activation of the ComRS signaling system by an unknown mechanism. Conversely, we show that a ComX-deficient strain is more resistant to mitomycin C and norfloxacin in a viability plate assay, which indicates that competence development negatively affects the resistance of S. thermophilus to DNA-damaging agents. Altogether, our results strongly suggest that SOS response activation and competence development are antagonistic processes in 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. Data from these experiments allowed us to distinguish genes whose action might be specific to oxidative stress defense (tgt, ossF, and ossG) from those whose action may be generalized to other stressful conditions (mreD, rodA, pbp2b, cpsX, and iscU). Among the mutants, two harbored an independently inserted copy of pGh9:ISS1 in two loci close to each other. More precisely, these two loci are homologous to the sufD and iscU genes, which are involved in the biosynthesis of iron-sulfur clusters. This region, called the suf region, was further characterized in S. thermophilus CNRZ368 by sequencing and by construction of DeltasufD and iscU(97) nonpolar mutants. The streptonigrin sensitivity levels of both mutants suggest that these two genes are involved in iron metabolism.     

Galactokinase activity in Streptococcus thermophilus

ATP-dependent phosphorylation of [14C]galactose by 11 strains of Streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK). Activities were 10 times higher in fully induced, galactose-fermenting (Gal+) strains than in galactose-nonfermenting (Gal-) strains. Lactose-grown, Gal- cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels. Gal+ S. thermophilus 19258 also released galactose into the medium, but when lactose was depleted growth on galactose commenced, and galK increased from 0.025 to 0.22 micromol of galactose phosphorylated per min per mg of protein. When lactose was added to galactose-grown cells of S. thermophilus 19258, galK activity rapidly decreased. These results suggest that galK in Gal+ S. thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal- strains.     

Galactose transport in Streptococcus thermophilus

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Molecular genetics of Streptococcus thermophilus

Abstract The metabolism and genetics of Streptococcus thermophilus (presently Streptococcus salivarius ssp. thermophilus ) have only been investigated recently despite its widespread use in milk fermentation processes. The development of recombinant DNA technology has allowed impressive progress to be made in the knowledge of thermophilic dairy streptococci. In particular, it has permitted a careful analysis of phenotypically altered variants which were derived from a mother strain by plasmid or chromosomal DNA reorganization. While natural phage defense mechanisms of S. thermophilus remain poorly documented, information on the bacteriophages responsible for fermentation failures has accumulated. The lysogenic state of two S. thermophilus strains has also been demonstrated for the first time. Gene transfer techniques for this species have been established and improved to the point that targeted manipulation of their chromosomal determinants is now feasible. Cloning and expression vectors have been constructed, and a few heterologous genes were successfully expressed in S. thermophilus . The first homologous genes, involved in carbohydrate utilization, have been cloned and sequenced, shedding some light on the molecular organization of key metabolic steps.     

Galactokinase activity in Streptococcus thermophilus.

ATP-dependent phosphorylation of [14C]galactose by 11 strains of Streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK). Activities were 10 times higher in fully induced, galactose-fermenting (Gal+) strains than in galactose-nonfermenting (Gal-) strains. Lactose-grown, Gal- cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels. Gal+ S. thermophilus 19258 also released galactose into the medium, but when lactose was depleted growth on galactose commenced, and galK increased from 0.025 to 0.22 micromol of galactose phosphorylated per min per mg of protein. When lactose was added to galactose-grown cells of S. thermophilus 19258, galK activity rapidly decreased. These results suggest that galK in Gal+ S. thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal- strains.     

Galactose transport in Streptococcus thermophilus.

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Efficient insertional mutagenesis in Streptococcus thermophilus

Bacteria have always been considered ideal organisms for genetic analysis. While this is true for some model organisms, like Escherichia coli, Bacillus subtilis and, more recently, Lactococcus lactis, genetic analysis of other organisms is often prevented by lack of valuable tools, like vectors, transposons and methods for transformation, gene inactivation and random insertional mutagenesis. This is the case of the moderately thermophilic bacterium Streptococcus thermophilus, an organism that, in spite of its widespread use for food fermentations, is only poorly characterized. We report here an insertional mutagenesis system that allows efficient random mutagenesis, easy characterization of the interrupted genes and construction of stable null mutations. This may become a powerful S. thermophilus tool for both genetic analysis and construction of 'food-grade' mutants of this biotechnologically relevant microorganism.     

BlpC-regulated bacteriocin production in Streptococcus thermophilus

Streptococcus thermophilus B59671 produces a bacteriocin with anti-pediococcal activity, but genes required for its production are not characterized. Genome sequencing of S. thermophilus has identified a genetic locus encoding a quorum sensing (QS) system that regulates production of class II bacteriocins. However, in strains possessing this gene cluster, production of bacteriocin like peptides (Blp) was only observed when excess pheromone was provided. PCR analysis revealed this strain possessed blpC, which encodes the 30-mer QS pheromone. To investigate if BlpC regulates bacteriocin production in S. thermophilus B59671, an integrative vector was used to replace blpC with a gene encoding for kanamycin resistance and the resulting mutant did not inhibit the growth of Pediococcus acidilactici. Constitutive expression of blpC from a shuttle vector restored the bacteriocin production, confirming the blp gene cluster is essential for bacteriocin activity in S. thermophilus B59671.     

Costs of CRISPR-Cas-mediated resistance in Streptococcus thermophilus

CRISPR-Cas is a form of adaptive sequence-specific immunity in microbes. This system offers unique opportunities for the study of coevolution between bacteria and their viral pathogens, bacteriophages. A full understanding of the coevolutionary dynamics of CRISPR-Cas requires knowing the magnitude of the cost of resisting infection. Here, using the gram-positive bacterium Streptococcus thermophilus and its associated virulent phage 2972, a well-established model system harbouring at least two type II functional CRISPR-Cas systems, we obtained different fitness measures based on growth assays in isolation or in pairwise competition. We measured the fitness cost associated with different components of this adaptive immune system: the cost of Cas protein expression, the constitutive cost of increasing immune memory through additional spacers, and the conditional costs of immunity during phage exposure. We found that Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins. Increasing immune memory by acquiring up to four phage-derived spacers was not associated with fitness costs. In addition, the activation of the CRISPR-Cas system during phage exposure induces significant but small fitness costs. Together these results suggest that the costs of the CRISPR-Cas system arise mainly due to the maintenance of the defence system. We discuss the implications of these results for the evolution of CRISPR-Cas-mediated immunity.     

Functional characterization of roles of GalR and GalS as regulators of the gal regulon.

An isorepressor of the gal regulon in Escherichia coli, GalS, has been purified to homogeneity. In vitro DNase I protection experiments indicated that among operators of the gal regulon, GalS binds most strongly to the external operator of the mgl operon, which encodes the high-affinity beta-methylgalactoside galactose transport system, and with less affinity to the operators controlling expression of the gal operon, which codes for enzymes of galactose metabolism. GalS has even less affinity for the external operator of galP, which codes for galactose permease, the major low-affinity galactose transporter in the cell. This order of affinities is the reverse of that of GalR, which binds most strongly to the operator of galP and most weakly to that of mgl. Our results also show that GalS, like its homolog, GalR, is a dimeric protein which in binding to the bipartite operators of the gal operon selectively represses its P1 promoter. Consistent with the fact that GalR is the exclusive regulator of the low-affinity galactose transporter, galactose permease, and that the major role of GalS is in regulating expression of the high-affinity galactose transporter encoded by the mgl operon, we found that the DNA binding of GalS is 15-fold more sensitive than that of GalR to galactose.     

Characterization of superoxide dismutase in Streptococcus thermophilus.

Streptococcus thermophilus AO54 possesses a single manganese-containing superoxide dismutase (MnSOD). The enzyme was found to be insensitive to cyanide or to a modified H2O2 treatment. The enzyme is expressed in a growth-phase-dependent fashion, increasing three- to fourfold upon entry into stationary phase. The specific activity for MnSOD was the same under anaerobic or aerobic conditions and was not induced by the presence of paraquat under aerobic conditions.