The Kinome of Pacific Oyster Crassostrea gigas, Its Expression during Development and in Response to Environmental Factors

Oysters play an important role in estuarine and coastal marine habitats, where the majority of humans live. In these ecosystems, environmental degradation is substantial, and oysters must cope with highly dynamic and stressful environmental constraints during their lives in the intertidal zone. The availability of the genome sequence of the Pacific oyster Crassostrea gigas represents a unique opportunity for a comprehensive assessment of the signal transduction pathways that the species has developed to deal with this unique habitat. We performed an in silico analysis to identify, annotate and classify protein kinases in C. gigas, according to their kinase domain taxonomy classification, and compared with kinome already described in other animal species. The C. gigas kinome consists of 371 protein kinases, making it closely related to the sea urchin kinome, which has 353 protein kinases. The absence of gene redundancy in some groups of the C. gigas kinome may simplify functional studies of protein kinases. Through data mining of transcriptomes in C. gigas, we identified part of the kinome which may be central during development and may play a role in response to various environmental factors. Overall, this work contributes to a better understanding of key sensing pathways that may be central for adaptation to a highly dynamic marine environment

Deciphering the molecular adaptation of the king scallop (Pecten maximus) to heat stress using transcriptomics and proteomics

Background The capacity of marine species to survive chronic heat stress underpins their ability to survive warming oceans as a result of climate change. In this study RNA-Seq and 2-DE proteomics were employed to decipher the molecular response of the sub-tidal bivalve Pecten maximus, to elevated temperatures. Results Individuals were maintained at three different temperatures (15, 21 and 25 °C) for 56 days, representing control conditions, maximum environmental temperature and extreme warming, with individuals sampled at seven time points. The scallops thrived at 21 °C, but suffered a reduction in condition at 25 °C. RNA-Seq analyses produced 26,064 assembled contigs, of which 531 were differentially expressed, with putative annotation assigned to 177 transcripts. The proteomic approach identified 24 differentially expressed proteins, with nine identified by mass spectrometry. Network analysis of these results indicated a pivotal role for GAPDH and AP-1 signalling pathways. Data also suggested a remodelling of the cell structure, as revealed by the differential expression of genes involved in the cytoskeleton and cell membrane and a reduction in DNA repair. They also indicated the diversion of energetic metabolism towards the mobilization of lipid energy reserves to fuel the increased metabolic rate at the higher temperature. Conclusions This work provides preliminary insights into the response of P. maximus to chronic heat stress and provides a basis for future studies examining the tipping points and energetic trade-offs of scallop culture in warming oceans

Rôle de la (p)ppGpp synthétase RelA dans l adaptation aux contraintes environnementales et la virulence chez Enterococcus faecalis ([thèse soutenue sur un ensemble de travaux ])

Dans leur environnement naturel, les bactéries sont continuellement soumises à des fluctuations des conditions physico-chimiques du milieu, et ont développé des mécanismes d adaptation extrêmement performants. Parmi ces mécanismes, la synthèse des (p)ppGpp [guanosine (penta-) tetra-phosphate] a d abord été décrite chez les bactéries pour leur rôle dans la réponse à une carence en acides aminés (réponse stringente). Chez les bactéries à Gram positif, la synthèse et la dégradation des (p)ppGpp sont sous la dépendance d une enzyme bifonctionnelle, RelA, mais peut aussi impliquer des synthétases alternatives (eg. RelQ). Nous avons construit et analysé chez Enterococcus faecalis les mutants de délétion relA, relQ, relA- relQ, ainsi qu un mutant DrelAsp produisant une protéine RelA tronquée de ses 241 acides aminés C-terminaux. RelA est l enzyme majoritairement responsable de l accumulation des (p)ppGpp. Par rapport à la souche sauvage V583, les deux mutants relA sont plus résistants à différents stress (sels biliaires, éthanol, acidité), mais ont des phénotypes contrastés vis-à-vis de certaines contraintes. En particulier, le mutant relAsp est résistant vis-à-vis d un stress oxydatif létal. La virulence des mutants a été testée dans le modèle animal Galleria mellonella, montrant une hyper virulence du mutant relAsp, et l atténuation du double mutant relA- relQ. Dans l optique de mieux comprendre le rôle de relA dans l établissement de ces phénotypes, nous avons analysé l expression des gènes et des protéines dans les mutants relA par des démarches transcriptomique et protéomique globales, et mené une analyse transcriptomique par puces à ADN de la souche V583 soumise à une contrainte oxydative.Bacteria have developed very powerful adaptive mechanisms to cope with the fluctuations of the physicochemical conditions of the natural environment. The alarmones (p)ppGpp [guanosine (penta-) tetra-phosphate] were initially described in bacteria for their role in the response to amino acids starvation (ie, stringent response). In Gram positive bacteria, the synthesis and degradation of (p)ppGpp is dependent on a bifunctional enzyme, RelA, but may also imply alternative synthetases (eg. RelQ). We created and analyzed in Enterococcus faecalis V583 the deletion mutants relA, relQ, relA- relQ, as well as a mutant producing a RelA protein truncated from its 241 C-term amino acids (named relAsp). We showed that RelA is the main enzyme responsible for the accumulation of (p)ppGpp in E. faecalis. Compared to the wild type strain V583, the two relA mutants revealed more resistant to various stresses (bile salts, ethanol, acidity), but displayed contrasting phenotypes to other stresses. In particular, the relAsp revealed highly resistant towards a lethal oxidative stress. The virulence of the mutants was assayed towards the animal virulence model Galleria mellonella, showing an increased virulence of the relAsp mutant, and an attenuated phenotype for the double relA- relQ mutant. Aiming at better understanding the role of relA in the development of these phenotypes, we analyzed the genes and proteins expressions in the relA mutants by using transcriptomic and proteomic global approaches, and performed a transcriptomic analysis in the V583 strain subjected to an oxidative stress.CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Starvation and osmotic stress induced multiresistances. Influence of extracellular compounds.

International audienceGrowth restriction due to stasis and/or hyperosmolarity is a common situation encountered by microorganisms in nature. Therefore, they have developed defence systems allowing them to withstand these periods. Bacteria respond to these conditions by a metabolic reprogramming which leads to a cellular state of enhanced resistance. This communication reviews recent advances in knowledge of the molecular basis of this phenomenon in different bacteria

New lnu(C) Gene Conferring Resistance to Lincomycin by Nucleotidylation in Streptococcus agalactiae UCN36

Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 μg/ml) but susceptible to clindamycin (MIC = 0.12 μg/ml) and erythromycin (MIC = 0.06 μg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuA(N2), LnuA, and LnuA′ lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl(2). Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin

Changes in Protein Synthesis and Morphology during Acid Adaptation of Propionibacterium freudenreichii

Survival of bacteria in changing environments depends on their ability to adapt to abiotic stresses. Microorganisms used in food technology face acid stress during fermentation processes. Similarly, probiotic bacteria have to survive acid stress imposed within the stomach in order to reach the intestine and play a beneficial role. Propionibacteria are used both as cheese starters and as probiotics in human alimentation. Adaptation to low pH thus constitutes a limit to their efficacy. Acid stress adaptation in the probiotic SI41 strain of Propionibacterium freudenreichii was therefore investigated. The acid tolerance response (ATR) was evidenced in a chemically defined medium. Transient exposure to pH 5 afforded protection toward acid challenge at pH 2. Protein neosynthesis was shown to be required for optimal ATR, since chloramphenicol reduced the acquired acid tolerance. Important changes in genetic expression were observed with two-dimensional electrophoresis during adaptation. Among the up-regulated polypeptides, a biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair were identified during the early stress response, while the universal chaperonins GroEL and GroES corresponded to a later response. The beneficial effect of ATR was evident at both the physiological and morphological levels. This study constitutes a first step toward understanding the very efficient ATR described in P. freudenreichii

Rapid and efficient protocol to introduce exogenous DNA in Vibrio harveyi and Pseudoalteromonas sp.

Vibrio campbellii BAA-1116 is renowned for its bioluminescence properties, and genetic tools are available to genetically track this strain. However, many other ecologically important V. harveyi strains exist, for which only few genetic tools are available. In this study, a rapid electroporation protocol was developed to transform replicative plasmids in various environmental V. harveyi and Pseudoalteromonas strains. Moreover, a mini-Tn7 delivery system was modified to site-specifically integrate mini-transposons in the genome of V. harveyi ORM4. As a proof-of-principle, replicative plasmids carrying bioreporters were introduced by electroporation in V. harveyi ORM4 cells, and gene expression was followed at the single cell level. We could demonstrate that a flagellar gene is subjected to bimodal gene expression in V. harveyi ORM4, being highly expressed in 10% of the population in stationary phase. This study extends the possibilities to study environmental Vibrio strains and uncovers the occurrence of phenotypic heterogeneity in flagellar expression in Vibrio

Studying host – pathogen interactions by a mutagenesis approach the case of Vibrio tapetis type IV secretion system in the brown ring disease (BRD) affecting the manila clam Ruditapes philippinarum

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