Mutagenesis of RpoE-like sigma factor genes in Bdellovibrio reveals differential control of groEL and two groES genes

Background

In recent years, Staphylococcus epidermidis ( Se) has become a major nosocomial pathogen and the most common cause of infections of implanted prostheses and other indwelling devices. This is due in part to avid biofilm formation by Se on device surfaces. However, it still remains unknown that how the process of Se biofilm development is associated with relapsed infection in such patients.

Results

We have identified clinical Se isolates displaying enhanced biofilm dispersal and self-renewal relative to reference strain. These isolates also exhibit enhanced initial cell attachment, extracellular DNA release, cell autolysis and thicker microcolonies during biofilm development relative to reference strain. Our genetic analyses suggest that these clinical isolates exhibit significant downregulation of RNAIII, the effector molecule of the agr quorum sensing system, and upregulation of the autolysin gene atlE. Isogenic deletion of the agr system in Se 1457 confirmed that agr negatively regulating atlE resulted in enhanced initial cell attachment, extracellular DNA release, cell autolysis and biofilm formation abilities. In contrast, double deletion of agr and atlE significantly abolished these features.

Conclusions

Collectively, these data reveal the role of agr system in long-term biofilm development and pathogenesis during Se caused indwelling devices-related relapsed infection.     

Schilbetrematoides manizani n. sp. (Dactylogyridea) monogenean parasite of Schilbe mandibularis (Günther, 1867) (Schilbeidae) in the Ivory Coast

The authors describe a new species in the genus Schilbetrematoides Kritsky & Kulo, 1992 from the gills of Schilbe mandibularis (Günther, 1867) (Schilbeidae) caught in the rivers Bia and Agneby in Ivory Coast. S. manizani n. sp. differs from S. pseudodactylogyrus Kritsky & Kulo, 1992, a parasite of Schilbe intermedius Rüppell, 1832 by having morphologically distinct gripi.     

Adenylate cyclase stimulation by VIP in rat and human parotid membranes

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 microM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 microM). The relative potency of peptides of the VIP family was: VIP greater than peptide histidine isoleucinamide (PHI) greater than secretin. The beta-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 microM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP greater than PHI greater than secretin). In both rat and human parotid membranes, two proteins (Mr 44 kDa and 53 kDa) of the alpha-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.     

Overexpression of human apolipoprotein A-II in mice induces hypertriglyceridemia due to defective very low density lipoprotein hydrolysis

Two lines of transgenic mice, hAIItg-delta and hAIItg-lambda, expressing human apolipoprotein (apo)A-II at 2 and 4 times the normal concentration, respectively, displayed on standard chow postprandial chylomicronemia, large quantities of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) but greatly reduced high density lipoprotein (HDL). Hypertriglyceridemia may result from increased VLDL production, decreased VLDL catabolism, or both. Post-Triton VLDL production was comparable in transgenic and control mice. Postheparin lipoprotein lipase (LPL) and hepatic lipase activities decreased at most by 30% in transgenic mice, whereas adipose tissue and muscle LPL activities were unaffected, indicating normal LPL synthesis. However, VLDL-triglyceride hydrolysis by exogenous LPL was considerably slower in transgenic compared with control mice, with the apparent Vmax of the reaction decreasing proportionately to human apoA-II expression. Human apoA-II was present in appreciable amounts in the VLDL of transgenic mice, which also carried apoC-II. The addition of purified apoA-II in postheparin plasma from control mice induced a dose-dependent decrease in LPL and hepatic lipase activities. In conclusion, overexpression of human apoA-II in transgenic mice induced the proatherogenic lipoprotein profile of low plasma HDL and postprandial hypertriglyceridemia because of decreased VLDL catabolism by LPL.     

Characterisation of physiological and immunological differences between Pacific oysters (Crassostrea gigas) genetically selected for high or low survival to summer mortalities and fed different rations under controlled conditions

Within the framework of a national scientific program named “MORtalités ESTivales de l'huître creuse Crassostrea gigas” (MOREST), a family-based experiment was developed to study the genetic basis of resistance to summer mortality in the Pacific oyster, Crassostrea gigas. As part of the MOREST project, the second generation of three resistant families and two susceptible families were chosen and pooled into two respective groups: “R” and “S”. These two groups of oysters were conditioned for 6 months on two food levels (4% and 12% of oyster soft-tissue dry weight in algal dry weight per day) with a temperature gradient that mimicked the Marennes-Oléron natural cycle during the oyster reproductive period. Oyster mortality remained low for the first two months, but then rapidly increased in July when seawater temperature reached 19 °C and above. Mortality was higher in “S” oysters than in “R” oysters, and also higher in oysters fed the 12% diet than those fed 4%, resulting in a decreasing, relative order in cumulative mortality as follows; 12% “S” > 12% “R” > 4% “S” > 4% “R”. Although the observed mortality rates were lower than those previously observed in the field, the mortality differential between “R” and “S” oysters was similar. Gonadal development, estimated by tissue lipid content, followed a relative order yielding a direct, positive relationship between reproductive effort and mortality as we reported precedently by quantitative histology. Regarding hemocyte parameters, one of the most striking observations was that reactive oxygen species (ROS) production was significantly higher in “S” oysters than in “R” oysters in May and June, regardless of food level. The absence of known environmental stress under these experimental conditions suggests that the ROS increase in “S” oyster could be related to their higher reproductive activity. Finally, a higher increase in hyalinocyte counts was observed for”S” oysters, compared to “R” oysters, in July, just before mortality. Taken together, our results suggest an association of genetically based resistance to summer mortality, reproductive strategy and hemocyte parameters.     

Extracting kinetic rate constants from surface plasmon resonance array systems

Surface plasmon resonance imaging systems, such as Flexchip from Biacore, are capable of monitoring hundreds of reaction spots simultaneously within a single flow cell. Interpreting the binding kinetics in a large-format flow cell presents a number of potential challenges, including accounting for mass transport effects and spot-to-spot sample depletion. We employed a combination of computer simulations and experimentation to characterize these effects across the spotted array and established that a simple two-compartment model may be used to accurately extract intrinsic rate constants from the array under mass transport-limited conditions. Using antibody systems, we demonstrate that the spot-to-spot variability in the binding kinetics was <9%. We also illustrate the advantage of globally fitting binding data from multiple spots within an array for a system that is mass transport limited.     

Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase.

1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these 'surface' amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.     

Complete genome sequence of Rhizobium leguminosarum bv trifolii strain WSM2304, an effective microsymbiont of the South American clover Trifolium polymorphum

Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.