Completion of genome of Aeromonas salmonicida subsp. salmonicida 01-B526 reveals how sequencing technologies can influence sequence quality and result interpretations.

Aeromonas salmonicida subsp. salmonicida is a pathogen that primarily infects salmonids. A strain of this bacterium, 01-B526, has been used in several studies as a reference. The genomic sequence of this strain is available, but comes from pyrosequencing and is the second most fragmented assembly for this bacterium. We generated its closed genome sequence and found a pitfall in result interpretations associated with low-quality genomic sequences.

Genomic and phenotypic characterization of an atypical Aeromonas salmonicida strain isolated from a lumpfish and producing unusual granular structures.

Aeromonas salmonicida strains are roughly classified into two categories, typical and atypical strains. The latter mainly regroup isolates that present unusual phenotypes or hosts, comparatively to the typical strains that belong to the salmonicida subspecies. This study focuses on an uncharacterized atypical strain, M18076-11, isolated from lumpfish (Cyclopterus lumpus) and not part of the four recognized Aeromonas salmonicida subspecies. This isolate presents an unreported phenotype in the A. salmonicida species: the formation of large granular aggregates. Granules are formed of a heterogeneous mix of live and dead cells, with live cells composing the majority of the population. Even if no mechanism was determined to cause cellular aggregation, small globular structures at the cell surface were observed, which might affect granular formation. Pan-genome phylogenetic analysis indicated that this strain groups alongside the masoucida subspecies. However, phenotypic tests showed that these strains have diverging phenotypes, suggesting that M18076-11 might belong to a new subspecies. Also, a pAsal1-like plasmid, which was only reported in strains of the subspecies salmonicida, was discovered in M18076-11. This study sheds light on unsuspected diversity in A. salmonicida subspecies and stresses the need of thorough identification when a new strain is encountered, as unique traits might be discovered.

Identification of dichloroacetic acid degrading Cupriavidus bacteria in a drinking water distribution network model.

AIMS: Bacterial community structure and composition of a drinking water network were assessed to better understand this ecosystem in relation to haloacetic acid (HAA) degradation and to identify new bacterial species having HAA degradation capacities. METHODS AND RESULTS: Biofilm samples were collected from a model system, simulating the end of the drinking water distribution network and supplied with different concentrations of dichloroacetic and trichloroacetic acids at different periods over the course of a year. The samples were analysed by culturing, denaturing gradient gel electrophoresis (DGGE) and sequencing. Pipe diameter and HAA ratios did not impact the bacterial community profiles, but the season had a clear influence. Based on DGGE profiles, it appeared that a particular biomass has developed during the summer compared with the other seasons. Among the bacteria isolated in this study, those from genus Cupriavidus were able to degrade dichloroacetic acid. Moreover, these bacteria degrade dichloroacetic acid at 18°C but not at 10°C. CONCLUSIONS: The microbial diversity evolved throughout the experiment, but the bacterial community was distinct during the summer. Results obtained on the capacity of Cupriavidus to degrade DCAA only at 18°C but not at 10°C indicate that water temperature is a major element affecting DCAA degradation and confirming observations made regarding season influence on HAA degradation in the drinking water distribution network. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first demonstration of the HAA biodegradation capacity of the genus Cupriavidus.

A kinase-independent function of Ask1 in caspase-independent cell death.

Ask1 (apoptosis signal-regulating kinase 1) is activated as a consequence of cell exposure to a variety of stresses and can then initiate apoptosis. A known pathway of apoptosis downstream of Ask1 involves the activation of the stress-activated protein kinases, the release of cytochrome c from mitochondria, the activation of caspases, and the fragmentation of nuclei. Here, we characterized a novel mechanism of Ask1-mediated cell killing that is triggered by the interaction with Daxx. Co-transfection of Ask1 and Daxx induced a caspase-independent cell-death process characterized at the morphological level by distinctive crumpled nuclei easily distinguishable from the condensed and fragmented nuclei seen during classical caspase-dependent apoptosis. The kinase activity of Ask1 was not involved in this process, because mutants lacking kinase activity were as efficient as wild type Ask1 in mediating Daxx-induced cell death. Ask1N, a deletant that lacks the C-terminal half including the kinase domain of Ask1, was constitutively active in producing crumpled nuclei. In contrast, Ask1DeltaN, the reciprocal deletant that possesses constitutive kinase activity, produced fragmented nuclei typical of caspase-dependent death processes. We conclude that in addition to a caspase-dependent pro-apoptotic function that depends on its kinase activity, Ask1 possesses a caspase-independent killing function that is independent on its kinase activity and is activable by interaction with Daxx. In the physiological situation, such an activity is induced as a consequence of the translocation of Daxx from the nucleus to the cytoplasm, a condition that occurs following activation of the death receptor Fas.

Inhibition of Daxx-mediated apoptosis by heat shock protein 27.

Heat shock protein 27 (HSP27) confers cellular protection against a variety of cytotoxic stresses and also against physiological stresses associated with growth arrest or receptor-mediated apoptosis. Phosphorylation modulates the activity of HSP27 by causing a major change in the supramolecular organization of the protein, which shifts from oligomers to dimers. Here we show that phosphorylated dimers of HSP27 interact with Daxx, a mediator of Fas-induced apoptosis, preventing the interaction of Daxx with both Ask1 and Fas and blocking Daxx-mediated apoptosis. No such inhibition was observed with an HSP27 phosphorylation mutant that is only expressed as oligomers or when apoptosis was induced by transfection of a Daxx mutant lacking its HSP27 binding domain. HSP27 expression had no effect on Fas-induced FADD- and caspase-dependent apoptosis. However, HSP27 blocked Fas-induced translocation of Daxx from the nucleus to the cytoplasm and Fas-induced Daxx- and Ask1-dependent apoptosis. The observations revealed a new level of regulation of the Fas pathway and suggest a mechanism for the phosphorylation-dependent protective function of HSP27 during stress and differentiation.

The interaction of HSP27 with Daxx identifies a potential regulatory role of HSP27 in Fas-induced apoptosis.

The heat shock protein HSP27 protects cells against a wide variety of toxic treatments and blocks apoptosis induced by exposures to anticancer drugs and activation of the death receptor Fas. The molecular mechanisms of protection are unknown but appear to be regulated by phosphorylation of HSP27. Two apoptotic pathways can be activated downstream of Fas. The Fas-adaptor FADD mediates a caspase-dependent pathway. Fas also activates a caspase-independent pathway which correlates with Fas-induced translocation of Daxx from the nucleus to the cytoplasm and involves the interaction of Daxx with Fas and Ask1. We found that phosphorylated dimers of HSP27 interact with Daxx, preventing its interaction with Ask1 and Fas and blocking Daxx-mediated apoptosis. Expression of HSP27 also prevents the translocation of Daxx from the nucleus to the cytoplasm which is induced upon expression of Ask1 or stimulation of Fas. The observations reveal a new level of regulation of the Fas pathway. Whereas the FADD axis can be modulated by expression of FLIP, a natural inhibitor of FADD, our results show that HSP27 can accomplish a similar function for the Daxx axis.

Cloning and characterization of hGMEB1, a novel glucocorticoid modulatory element binding protein.

A 21-bp element called glucocorticoid modulatory element (GME) modulates the glucocorticoid receptor-mediated responses via the binding of an as yet poorly characterized transacting complex of proteins containing the 88-kDa GMEB1 and the 67-kDa GMEB2. Using heat shock protein 27 (HSP27) as bait in the yeast two-hybrid assay, we cloned a 1.83-kb cDNA encoding a novel 573-amino acid protein called human GMEB1 (hGMEB1). hGMEB1 possesses a KDWK domain, contains sequences almost identical (36/38) to three tryptic peptides of rat GMEB1 and shares 38% identity with rat GMEB2. hGMEB1 is ubiquitously expressed as a 85-kDa protein in all cell lines and tissues examined. In vitro translated hGMEB1 bound specifically to GME oligonucleotides yielding a complex of similar size to the complex obtained using rat liver nuclear extracts. Both complexes were supershifted with an antibody specific to hGMEB1. Co-immunoprecipitation experiments confirmed the in vivo interaction of HSP27 with hGMEB1.

HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus.

Distinct biochemical activities have been reported for small and large molecular complexes of heat shock protein 27 (HSP27), respectively. Using glycerol gradient ultracentrifugation and chemical cross-linking, we show here that Chinese hamster HSP27 is expressed in cells as homotypic multimers ranging from dimers up to 700-kDa oligomers. Treatments with arsenite, which induces phosphorylation on Ser15 and Ser90, provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers. Ser90 phosphorylation was sufficient and necessary for causing this change in structure. Dimer formation was severely inhibited by replacing Ser90 with Ala90 but not by replacing Ser15 with Ala15. Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions. One domain was insensitive to phosphorylation and corresponded to the C-terminal alpha-crystallin domain. The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein. Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation. A deletion within this domain of residues Arg5-Tyr23, which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers. We propose that HSP27 forms stable dimers through the alpha-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain.