Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.     

Alteration of copper physiology in mice overexpressing the human Menkes protein ATP7A

The Menkes protein (ATP7A) is defective in the Cu deficiency disorder Menkes disease and is an important contributor to the maintenance of physiological Cu homeostasis. To investigate more fully the role of ATP7A, transgenic mice expressing the human Menkes gene ATP7A from chicken beta-actin composite promoter (CAG) were produced. The transgenic mice expressed ATP7A in lung, heart, liver, kidney, small intestine, and brain but displayed no overt phenotype resulting from expression of the human protein. Immunohistochemical analysis revealed that ATP7A was found primarily in the cardiac muscle, smooth muscle of the lung, distal tubules of the kidney, intestinal enterocytes, and patches of hepatocytes, as well as in the hippocampus, cerebellum, and choroid plexus of the brain. In 60-day- and 300-day-old mice, Cu concentrations were reduced in most tissues, consistent with ATP7A playing a role in Cu efflux. The reduction in Cu was most pronounced in the hearts of older T22#2 females (24%), T22#2 males (18%), and T25#5 females (23%), as well as in the brains of 60-day-old T22#2 females and males (23% and 30%, respectively).     

Mitochondrial dihydrolipoyl succinyltransferase deficiency accelerates amyloid pathology and memory deficit in a transgenic mouse model of amyloid deposition

Mitochondrial dysfunction and oxidative stress are involved in Alzheimer disease (AD) pathogenesis. In human AD brains, the activity of the α-ketoglutarate dehydrogenase enzyme complex (α-KGDHC) is reduced. KGDHC is mostly involved in NADH production. It can also participate in oxidative stress and reactive oxygen species (ROS) production. The mitochondrial dihydrolipoyl succinyltransferase enzyme (DLST) is a key subunit specific to the α-KGDHC. In cultured cells, reduction of DLST increased H₂O₂-induced ROS generation and cell death. Thus, we asked whether partial genetic deletion of DLST could accelerate the onset of AD pathogenesis, using a transgenic mouse model of amyloid deposition crossed with DLST^+/− mice. Tg19959 mice, which carry the human amyloid precursor protein with two mutations, develop amyloid deposits and progressive behavioral abnormalities. We compared Tg19959 mice to Tg19959-DLST^+/− littermates at 2–3 months of age and studied the effects of DLST deficiency on amyloid deposition, spatial learning and memory, and oxidative stress. We found that α-KGDHC activity was reduced in DLST^+/− mice. We also found that DLST deficiency increased amyloid plaque burden, Aβ oligomers, and nitrotyrosine levels and accelerated the occurrence of spatial learning and memory deficits in female Tg19959 mice. Our data suggest that α-KGDHC may be involved in AD pathogenesis through increased mitochondrial oxidative stress.     

Molecular characterization of bacteria associated with the trophosome and the tube of Lamellibrachia sp., a siboglinid annelid from cold seeps in the eastern Mediterranean

Specimens of Lamellibrachia ( Annelida: Siboglinidae ) were recently discovered at cold seeps in the eastern Mediterranean. In this study, we have investigated the phylogeny and function of intracellular bacterial symbionts inhabiting the trophosome of specimens of Lamellibrachia sp. from the Amon mud volcano, as well as the bacterial assemblages associated with their tube. The dominant intracellular symbiont of Lamellibrachia sp. is a gammaproteobacterium closely related to other sulfide-oxidizing tubeworm symbionts. In vivo uptake experiments show that the tubeworm relies on sulfide for its metabolism, and does not utilize methane. Bacterial communities associated with the tube form biofilms and occur from the anterior to the posterior end of the tube. The diversity of 16S rRNA gene phylotypes includes representatives from the same divisions previously identified from the tube of the vent species Riftia pachyptila , and others commonly found at seeps and vents.     

Proteinase-3 induces procaspase-3 activation in the absence of apoptosis: potential role of this compartmentalized activation of membrane-associated procaspase-3 in neutrophils

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.     

Dopaminergic transmission in STOP null mice

Neuroleptics are thought to exert their anti-psychotic effects by counteracting a hyper-dopaminergic transmission. Here, we have examined the dopaminergic status of STOP (stable tubule only polypeptide) null mice, which lack a microtubule-stabilizing protein and which display neuroleptic-sensitive behavioural disorders. Dopamine transmission was investigated using both behavioural analysis and measurements of dopamine efflux in different conditions. Compared to wild-type mice in basal conditions or following mild stress, STOP null mice showed a hyper-locomotor activity, which was erased by neuroleptic treatment, and an increased locomotor reactivity to amphetamine. Such a behavioural profile is indicative of an increased dopaminergic transmission. In STOP null mice, the basal dopamine concentrations, measured by quantitative microdialysis, were normal in both the nucleus accumbens and the striatum. When measured by electrochemical techniques, the dopamine efflux evoked by electrical stimulations mimicking physiological stimuli was dramatically increased in the nucleus accumbens of STOP null mice, apparently due to an increased dopamine release, whereas dopaminergic uptake and auto-inhibition mechanisms were normal. In contrast, dopamine effluxes were slightly diminished in the striatum. Together with previous results, the present study indicates the association in STOP null mice of hippocampal hypo-glutamatergy and of limbic hyper-dopaminergy. Such neurotransmission defects are thought to be central to mental diseases such as schizophrenia.     

Quantitative and qualitative impact of hospital effluent on dissemination of the integron pool

There is increasing evidence that human activity, and especially the resulting effluent, has a major role in the dissemination of bacterial antibiotic-resistance determinants in the environment. Hospitals are the major antibiotic consumers and thus facilitate the spread of antibiotic resistance. Questions are increasingly being raised about the management of hospital effluents, but their involvement in antibiotic-resistance dissemination has never been assessed. Integrons are a paradigm of genetic transfer between the environmental resistome and both commensal and pathogenic bacteria. In order to assess the impact of hospital activities on antibiotic-resistance dissemination in the environment, we monitored integrons and their gene cassettes in hospital effluents, and their release in the environment. We found that bacterial communities present in a hospital effluent contained a high proportion of integrons. In terms of both their gene cassette diversity and gene cassette arrays, the urban effluent and municipal wastewater treatment plant (WWTP) influent were most similar, whereas the hospital effluent and recirculation sludge exhibited very specific patterns. We found that anthropogenic activities led to the release of abundant integrons and antibiotic-resistance gene cassettes, but we observed no specific impact of hospital activities on the receiving environment. Furthermore, although the WWTP did not reduce the normalized integron copy number, it reduced the diversity of gene cassette arrays contained in the raw wastewater, underlining the effect of the biological treatment on the anthropogenic integron pool arriving at the WWTP.     

Crossing Borders for Science

Exchanging ideas with like-minded, enthusiastic people interested in the same topic is crucial for the advancement of a scientist''s career. Several Regional Student Groups (RSGs) of the International Society for Computational Biology (ISCB) Student Council have cooperated in the last six years to organize scientific workshops and conferences. With motivated students, it is possible to create a memorable event for fellow scientists; in doing so, the organizers gain valuable experiences. While collaborating across borders and time zones can be difficult, feedback from event organizers was always positive. When limited resources are juxtaposed with great ideas and a network of contacts, the outcome is always an amazing experience, despite organizers being separated geographically across different countries.     

Evidence of diversity and recombination in <Emphasis Type=Italic>Arsenophonus</Emphasis> symbionts of the <Emphasis Type=Italic>Bemisia tabaci</Emphasis>species complex

BACKGROUND: Maternally inherited bacterial symbionts infecting arthropods have major implications on host ecology and evolution. Among them, the genus Arsenophonus is particularly characterized by a large host spectrum and a wide range of symbiotic relationships (from mutualism to parasitism), making it a good model to study the evolution of host-symbiont associations. However, few data are available on the diversity and distribution of Arsenophonus within host lineages. Here, we propose a survey on Arsenophonus diversity in whitefly species (Hemiptera), in particular the Bemisia tabaci species complex. This polyphagous insect pest is composed of genetic groups that differ in many ecological aspects. They harbor specific bacterial communities, among them several lineages of Arsenophonus, enabling a study of the evolutionary history of these bacteria at a fine host taxonomic level, in association to host geographical range and ecology. RESULTS: Among 152 individuals, our analysis identified 19 allelic profiles and 6 phylogenetic groups, demonstrating this bacterium's high diversity. These groups, based on Arsenophonus phylogeny, correlated with B. tabaci genetic groups with two exceptions reflecting horizontal transfers. None of three genes analyzed provided evidence of intragenic recombination, but intergenic recombination events were detected. A mutation inducing a STOP codon on one gene in a strain infecting one B. tabaci genetic group was also found. Phylogenetic analyses of the three concatenated loci revealed the existence of two clades of Arsenophonus. One, composed of strains found in other Hemiptera, could be the ancestral clade in whiteflies. The other, which regroups strains found in Hymenoptera and Diptera, may have been acquired more recently by whiteflies through lateral transfers. CONCLUSIONS: This analysis of the genus Arsenophonus revealed a diversity within the B. tabaci species complex which resembles that reported on the larger scale of insect taxonomy. We also provide evidence for recombination events within the Arsenophonus genome and horizontal transmission of strains among insect taxa. This work provides further insight into the evolution of the Arsenophonus genome, the infection dynamics of this bacterium and its influence on its insect host's ecology.     

Molecular and ultrastructural characterization of two ascomycetes found on sunken wood off Vanuatu Islands in the deep Pacific Ocean

A new genus of a deep-sea ascomycete with one new species, Alisea longicolla, is described based on analyses of 18S and 28S rDNA sequences and morphological characters. A. longicolla was found together with Oceanitis scuticella, on small twigs and sugar cane debris trawled from the bottom of the Pacific Ocean off Vanuatu Islands. Molecular and morphological characters indicate that both fungi are members of Halosphaeriaceae. Within this family, O. scuticella is phylogenetically related to Ascosalsum and shares similar ascospore morphology and appendage ontogeny. The genus Ascosalsum is considered congeneric with Oceanitis and Ascosalsum cincinnatulum, Ascosalsum unicaudatum and Ascosalsum viscidulum are transferred to Oceanitis, an earlier generic name.