Effects of rodA and pbp2b Disruption on Cell Morphology and Oxidative Stress Response of Streptococcus thermophilus CNRZ368

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

Characterisation of Streptococcus thermophilus CNRZ368 oxidative stress-resistant mutants: involvement of a potential Rgg-like transcriptional regulator

International audienceEight mutants of Streptococcus thermophilus CNRZ368 presenting a menadione-resistant phenotype were selected and the locus mutated in each mutant was identified. Among these clones, 5 were disrupted in a gene of unknown function, 2 were impaired in genes involved in cellular metabolism and the last one (the mutant 15H7) was disrupted in rgg encoding a putative transcriptional regulator. To determine if rgg could be a key regulator of the superoxide defence of S. thermophilus, the 15H7 mutant was further characterised. The results from our work indicate that the rgg gene, mutated in the 15H7 genome, is a pseudogene composed of 2 ORFs (rggA and rggB) that are potentially translated in two different frames. Moreover, transcription analysis demonstrated the existence of a transcript containing rggA, rggB and orf2. Construction of $\Delta$rggA, $\Delta$rggB and orf2 mutants and their phenotypic analysis confirmed the involvement of rggA and rggB in the oxidative stress response. The question of the role of the pseudogene rgg is still open.Caractérisation de mutants résistants au stress oxydant chez Streptococcus thermophilus CNZR368 : implication d'un régulateur transcriptionnel putatif de type Rgg. Huit mutants de Streptococcus thermophilus CNRZ368, présentant un phénotype de résistance à la ménadione ont été sélectionnés et le locus muté a été identifié pour chacun d'eux. Parmi ces clones, 5 sont interrompus dans un gène de fonction inconnue, 2 sont mutés dans des gènes impliqués dans le métabolisme cellulaire et un autre (15H7) est interrompu dans le gène rgg codant potentiellement un régulateur transcriptionnel. Afin de déterminer si rgg est un régulateur majeur de la défense contre le stress superoxyde le mutant 15H7 a été plus amplement étudié. Les résultats de notre travail indiquent que le gène rgg, muté dans le génome de 15H7, est un pseudogène composé de deux ORF (rggA and rggB) potentiellement traduites dans deux phases de lecture différentes. De plus, l'analyse transcriptionelle démontre l'existence d'un transcrit englobant rggA, rggB et orf2. La construction des mutants $\Delta$rggA, $\Delta$rggB et orf2 ainsi que l'analyse de leur phénotype confirme l'implication de rggA et rggB dans la réponse contre le stress oxydant. La question concernant le rôle du pseudogène rgg est toujours ouverte

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 ΔsufD and iscU(97) nonpolar mutants. The streptonigrin sensitivity levels of both mutants suggest that these two genes are involved in iron metabolism

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. In CNRZ368, deletion of the entire cse open reading frame leads to drastic lengthening of cell chains and altered colony morphology. Complementation of the Δcse mutation with a wild-type allele restored both wild-type phenotypes. The central part of Cse is a repeat-rich region with low sequence complexity. Comparison of cse from CNRZ368 and LMG18311 strains reveals high variability of this repeat-rich region. To assess the impact of this central region variability, the central region of LMG18311 cse was exchanged with that of CNRZ368 cse. This replacement did not affect chain length, showing that divergence of the central part does not modify cell segregation activity of Cse. The structure of the cse locus suggests that the chimeric organization of cse results from insertion of a duplicated sequence deriving from the pcsB 3′ end into an ancestral sip gene. Thus, the cse locus illustrates the module-shuffling mechanism of bacterial gene evolution