Tatiana Rochat

Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis

Outside In Acquisition and analysis of large data sets promises to move us toward a greater understanding of the mechanisms by which biological systems are dynamically regulated to respond to external cues. Now, two papers explore the responses of a bacterium to changing nutritional conditions (see the Perspective by Chalancon et al.). Nicolas et al. (p. 1103) measured transcriptional regulation for more than 100 different conditions. Greater amounts of antisense RNA were generated than expected and appeared to be produced by alternative RNA polymerase targeting subunits called sigma factors. One transition, from malate to glucose as the primary nutrient, was studied in more detail by Buescher et al. (p. 1099) who monitored RNA abundance, promoter activity in live cells, protein abundance, and absolute concentrations of intracellular and extracellular metabolites. In this case, the bacteria responded rapidly and largely without transcriptional changes to life on malate, but only slowly adapted to use glucose, a shift that required changes in nearly half the transcription network. These data offer an initial understanding of why certain regulatory strategies may be favored during evolution of dynamic control systems. A horizontal analysis reveals the breadth of genes turned on and off as nutrients change. Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Anti-inflammatory effects of Lactobacillus casei BL23 producing or not a manganese-dependant catalase on DSS-induced colitis in mice.

BackgroundHuman immune cells generate large amounts of reactive oxygen species (ROS) throughout the respiratory burst that occurs during inflammation. In inflammatory bowel diseases, a sustained and abnormal activation of the immune system results in oxidative stress in the digestive tract and in a loss of intestinal homeostasis. We previously showed that the heterologous production of the Lactobacillus plantarum ATCC14431 manganese-dependant catalase (MnKat) in Lb. casei BL23 successfully enhances its survival when exposed to oxidative stress. In this study, we evaluated the preventive effects of this antioxidative Lb. casei strain in a murine model of dextran sodium sulfate (DSS)-induced moderate colitis.ResultsEither Lb. casei BL23 MnKat- or MnKat+ was administered daily to mice treated with DSS for 10 days. In contrast to control mice treated with PBS for which DSS induced bleeding diarrhea and mucosal lesions, mice treated with both Lb. casei strains presented a significant (p < 0.05) reduction of caecal and colonic inflammatory scores.ConclusionNo contribution of MnKat to the protective effect from epithelial damage has been observed in the tested conditions. In contrast, these results confirm the high interest of Lb. casei as an anti-inflammatory probiotic strain.

Production of a Heterologous Nonheme Catalase by Lactobacillus casei: an Efficient Tool for Removal of H2O2 and Protection of Lactobacillus bulgaricus from Oxidative Stress in Milk

ABSTRACT Lactic acid bacteria (LAB) are generally sensitive to H2O2, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H2O2 through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H2O2, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H2O2 from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.

Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice

Human immune cells release large amounts of reactive oxygen species (ROS) such as superoxide radical and hydrogen peroxide via respiratory burst. In inflammatory bowel diseases, a sustained and abnormal activation of the immune response results in oxidative stress of the digestive tract and in a loss of intestinal homeostasis. We previously reported that heterologous production of the Lactobacillus plantarum manganese catalase (MnKat) enhances the survival of Lb. casei BL23 when exposed to oxidative stress. Anti-inflammatory effects were observed after Lb. casei BL23 oral administrations in moderate murine dextran sodium sulfate (DSS)-induced colitis, without added effects of the MnKat production. Here, we evaluated the protective effects obtained by an improved antioxidative strategy. The Lactococcus lactis sodA gene was expressed in Lb. casei BL23 which acquired an efficient manganese superoxide dismutase (MnSOD) activity. The effects of Lb. casei MnSOD alone or in combination with Lb. casei MnKat were compared first in eukaryotic cell PMA-induced oxidative stress model and then in severe murine DSS-induced colitis. Based on ROS production assays as well as colonic histological scores, a significant reduction of both oxidative stress and inflammation was observed with Lb. casei MnSOD either alone or in combination with Lb. casei MnKat. No added effect of the presence of Lb. casei MnKat was observed. These results suggest that Lb. casei BL23 MnSOD could have anti-inflammatory effects on gut inflammation.

Lactococcus lactis SpOx spontaneous mutants: a family of oxidative-stress-resistant dairy strains.

ABSTRACT Numerous industrial bacteria generate hydrogen peroxide (H2O2), which may inhibit the growth of other bacteria in mixed ecosystems. We isolated spontaneous oxidative-stress-resistant (SpOx) Lactococcus lactis mutants by using a natural selection method with milk-adapted strains on dairy culture medium containing H2O2. Three SpOx mutants displayed greater H2O2 resistance. One of them, SpOx3, demonstrated better behavior in different oxidative-stress situations: (i) higher long-term survival upon aeration in LM17 and milk and (ii) the ability to grow with H2O2-producing Lactobacillus delbrueckii subsp. delbrueckii strains. Furthermore, the transit kinetics of the SpOx3 mutant in the digestive tract of a human flora-associated mouse model was not affected.

Gene expression control by selective RNA processing and stabilization in bacteria

RNA maturation is a key event regulating genes at post-transcriptional level. In bacteria, it is employed to adjust the amounts of proteins and functional RNAs, often in response to environmental constraints. During the process of RNA maturation, enzymes and factors that would otherwise promote RNA degradation convert a labile RNA into a stable and biologically functional molecule.

Carrageenan catabolism is encoded by a complex regulon in marine heterotrophic bacteria

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.Carrageenans, major cell wall polysaccharides of red macroalgae, are metabolised by marine heterotrophic bacteria through unclear mechanisms. Here, the authors identify an unusual polysaccharide-utilization locus encoding carrageenan catabolism in a marine bacterium, and characterise the complete pathway.

Proteomic analysis of spontaneous mutants of Lactococcus lactis: Involvement of GAPDH and arginine deiminase pathway in H2O2 resistance

Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H2O2‐resistant (SpOx) strains. In the parental strain, glyceraldehyde‐3‐phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H2O2 resistance of exponential growing cells. Upon H2O2 exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be upregulated and disruption of arcA or arcB genes abolished H2O2 resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.

Genomic Characterization of Flavobacterium psychrophilum Serotypes and Development of a Multiplex PCR-Based Serotyping Scheme

Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

Complete Genome Sequence of Flavobacterium psychrophilum Strain OSU THCO2-90, Used for Functional Genetic Analysis

ABSTRACT We report here the complete annotated genome sequence of Flavobacterium psychrophilum OSU THCO2-90, isolated from Coho salmon (Oncorhynchus kisutch) in Oregon. The genome consists of a circular chromosome with 2,343 predicted open reading frames. This strain has proved to be a valuable tool for functional genomics.