Zooplankton composition and distribution off the coast of Galicia, Spain

During June and September 1984, zooplankton samples were collectedwith other hydrographic and biological data along the Galiciancoast (NW of Spain). In June copepods contributed {small tilde}60%to the total zooplankton community, with larvaceans, siphonophoresand cladocerans also abundant. In September >90% of the zooplanktonsampled were copepods. The dominant species of copepods in bothJune and September were Acartia clausi, Paracalanus parvus andTemora longicornis. The meroplankton was dominated by echinoderms,bryozoans, barnacle larvae and bivalve larvae. In June the averagezooplankton biomass was 31.08 mg C m^–3; the Septemberaverage was 41.69 mg C m^–3. The relationship between theslopes of the regression equations (biomass versus abundance)suggests that the zooplankton assemblage in June was composedby larger animals than in September. The major concentrationof zooplankton was between 0 and 50 m, with both June and Septemberdaytime surface samples having 6–7 times the amount oforganisms than the lower water column (50–100 m). Therewere no distinct differences in total zooplankton abundancesat the inshore and offshore stations; however, the inshore stationsoften had a higher percentage of meroplankton than the offshorestations. In June zooplankton abundance at the northern transectsand the western transects was similar. In September there weregreater concentrations of zooplankton in the western Galicianshelf as compared with the northern shelf. These differencesin the horizontal distribution of the zooplankton were relatedto upwelling events.     

Impact of Postovulatory Food Deprivation on the Ova Transport, Hormonal Profiles and Metabolic Changes in Sows

The effect of food deprivation on ova transport, hormonal profiles and metabolic changes was studied in 20 crossbred multiparous sows during their second oestrus after weaning. To determine the time of ovulation, transrectal ultrasonographic examination was performed. The sows were divided into 2 groups, one control group (C-group), which was fed according to Swedish standards, and one experimental group (E-group). The E-group sows were deprived of food from the first morning meal after ovulation until slaughter. Blood samples were collected every second hour from about 12 h before expected ovulation in the second oestrus after weaning until slaughter and were analysed for progesterone, prostaglandin F_2α-metabolite, insulin, glucose, free fatty acids and triglycerides. All sows were slaughtered approximately 48 h after ovulation and the genital tract was recovered. The isthmic part of the oviduct was divided into 3 equally long segments and flushed separately with phosphate buffered saline (PBS). Uterine horns were also flushed with PBS. A significantly greater number of ova were found in the first and second part of the isthmus in the E-group (p = 0.05) while in the C-group most of the ova were found in the third part of the isthmus or the uterus (p = 0.01). The level of prostaglandin F_2α-metabolite was significantly higher in the E-group compared with the C-group. The concentration of progesterone increased in both groups after ovulation but there were no significant differences between the groups. The other blood parameters showed that the food-deprived sows were in a catabolic state. The 48 h period of fasting results, directly or indirectly in an delayed ova transport, which may be due to a delayed relaxation in the smooth circular muscle layer of the isthmus.     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

Genome diversification within a clonal population of pandemic Vibrio parahaemolyticus seems to depend on the life circumstances of each individual bacteria

Background

New strains of Vibrio parahaemolyticus that cause diarrhea in humans by seafood ingestion periodically emerge through continuous evolution in the ocean. Influx and expansion in the Southern Chilean ocean of a highly clonal V. parahaemolyticus (serotype O3:K6) population from South East Asia caused one of the largest seafood-related diarrhea outbreaks in the world. Here, genomics analyses of isolates from this rapidly expanding clonal population offered an opportunity to observe the molecular evolutionary changes often obscured in more diverse populations.

Results

Whole genome sequence comparison of eight independent isolates of this population from mussels or clinical cases (from different years) was performed. Differences of 1366 to 217,729 bp genome length and 13 to 164 bp single nucleotide variants (SNVs) were found. Most genomic differences corresponded to the presence of regions unique to only one or two isolates, and were probably acquired by horizontal gene transfer (HGT). Some DNA gain was chromosomal but most was in plasmids. One isolate had a large region (8,644 bp) missing, which was probably caused by excision of a prophage. Genome innovation by the presence of unique DNA, attributable to HGT from related bacteria, varied greatly among the isolates, with values of 1,366 (ten times the number of highest number of SNVs) to 217,729 (a thousand times more than the number of highest number of SNVs).

Conclusions

The evolutionary forces (SNVs, HGT) acting on each isolate of the same population were found to differ to an extent that probably depended on the ecological scenario and life circumstances of each bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1385-8) contains supplementary material, which is available to authorized users.     

Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA,EsxB, and EspA

Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately α-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.The WXG100 (WXG; pfam06013) proteins are a class of effector molecules found in gram-positive bacteria (26). WXG proteins are characterized by their small size (∼ 100 amino acids [aa]) and the presence of a WXG motif, or its structural equivalent, near the midpoint of their primary sequence (26). Bioinformatic analyses have shown that one WXG gene is frequently positioned near, or directly adjacent to, a second, related, WXG gene (14). The gene pairs characterized thus far encode proteins that associate to form 1:1 complexes (20, 31). The WXG proteins were once thought to be restricted to the mycobacteria, but homologues have now been detected in species of Bacillus, Listeria, Streptomyces, and Corynebacterium, among others, and the Pfam server lists >89 distinct WXG-encoding species and strains (10).The identification of WXG proteins encoded by the pathogens Mycobacterium tuberculosis (15, 17, 19, 36), Mycobacterium marinum (13), and Staphylococcus aureus (5) has created significant interest in the proteins'' biological activity. Nevertheless, these proteins are not a priori virulence factors (39), since organisms expressing WXG proteins are not necessarily capable of causing disease. In addition to pathogenesis, the WXG proteins are associated with processes as disparate as zinc homeostasis (24) and conjugal gene transfer (9, 11). A model for the mechanism(s) of action of these proteins that includes an explanation for their apparent functional versatility is at present lacking. One reason for this ambiguity may be the near-absence of studies comparing virulence-associated and non-virulence-associated WXG proteins, which is a goal of this study.The M. tuberculosis secreted virulence factors EsxA (also called ESAT-6, or Rv3875) and EsxB (CFP-10; Rv3874) are the founding members of the WXG family, and M. tuberculosis derivatives defective in EsxA and EsxB are attenuated (17, 19, 36). The results of biochemical and structural studies indicate that EsxA and EsxB form a tightly associated heterodimer, EsxAB (25, 30, 31). The M. tuberculosis genome contains 23 WXG genes, named esxA to esxW, and the majority of these are expressed as tandem pairs (26). Of the pairs, five, including esxA and esxB, are contained within larger, highly conserved genetic loci, called esx-1 to esx-5 (Fig. ​(Fig.1).1). These loci have been the focus of much research, since mutants of esx-1 are attenuated, and esx-3 and esx-5 are necessary for in vitro growth of M. tuberculosis and M. marinum (1, 2, 32-34). The esx loci are proposed to encode secretory apparatuses dedicated to the secretion of their cognate WXG proteins (1).Open in a separate windowFIG. 1.Genetic map of the esx-1 loci of M. tuberculosis and M. smegmatis. The M. tuberculosis esx-1 genes discussed in the text are indicated by white arrows, as are their M. smegmatis homologues. The M. tuberculosis map also shows the Rv3884 and Rv3885 genes, which are part of the adjacent esx-2 locus. pRD1-2F9 is the cosmid that was used to create an esx-1-specific prey library. pRD1-2F9 includes the Rv3860 to Rv3885 genes, thus encompassing the entire esx-1 locus and part of esx-2. The four genes below the M. smegmatis map include defective insertion sequences (ISs) inserted into MSMEG_0075.Although the majority of genes required for the secretion of the EsxAB heterodimer are encoded from within esx-1, additional non-esx-1 genes are necessary for secretion. In particular, one M. tuberculosis locus, esp, encodes three proteins essential for EsxAB secretion (12, 23). The first gene of the operon encodes a protein, EspA, that is cosecreted with EsxAB via the ESX-1 apparatus (12). Although no direct physical evidence has been presented, the inference from the interdependent cosecretion of the three proteins is that they likely form a complex, which is secreted by the ESX-1 apparatus. In this paper we provide the first genetic evidence that these three proteins interact.The lack of a genetic assay for the study of ESX-1 activity in M. tuberculosis has hindered the identification of all of the protein components of the apparatus and all of the substrates that it secretes. However, the fast-growing, nonpathogenic organism Mycobacterium smegmatis has a conserved esx-1 locus that is essential for DNA transfer, and we have exploited this requirement for genetic studies (9). These analyses have shown that the M. smegmatis ESX-1 apparatus is functionally related to that of M. tuberculosis (11) and that M. smegmatis encodes non-esx-1 genes necessary for the secretion of the EsxAB heterodimer, including orthologues of EspA (9).Here we have examined whether the secondary and quaternary structures of M. tuberculosis EsxA and EsxB are prototypical for other, functionally distinct and evolutionarily distant members of the WXG family (Fig. ​(Fig.2A).2A). Comparisons were made to homologues encoded by M. smegmatis (esxA and esxB), Corynebacterium diphtheriae (esxA and esxB), and an additional non-virulence-related pair from M. tuberculosis (esxG and esxH, encoded from the esx-3 locus). Structural characterization of these proteins establishes that their secondary and quaternary structures are conserved, with each pair folding into a predominately α-helical structure and associating to form a heterodimer. We next devised and tested the utility of a novel strategy to identify proteins that interact specifically with these WXG heterodimers. This involved fusing EsxB and EsxA to create a biomimetic heterodimer for use in mycobacterial two-hybrid experiments. We reasoned that the use of this unique bait would allow the detection of proteins that interact with both components of the native heterodimer and that these proteins would normally go undetected in the conventional, single-protein two-hybrid screens. Indeed, using this approach, we identified novel protein partners of M. tuberculosis EsxBA (MtbEsxBA). We show for the first time that EspA proteins from M. tuberculosis and M. smegmatis interact with the EsxBA heterodimer (from both species) but not with EsxA or EsxB alone. We also provide evidence for promiscuity between the different M. tuberculosis ESX apparatuses by showing that EsxBA, encoded by esx-1, can interact with Esx proteins encoded by esx-2. Taken together, our studies suggest that the WXG proteins possess similar structures and properties, regardless of the host species and the apparent biological function.Open in a separate windowFIG. 2.Sequence alignment of WXG proteins characterized in this study and the strategy used to facilitate their expression. (A) Amino acid sequence alignment of four pairs of WXG proteins. Conserved sequences are in boldface, and the signature WXG motif is indicated with asterisks. Three residues in Rv3874 (EsxB) and a single residue in Rv3875 (EsxA) are underlined; they are the sites of amino acid substitutions discussed in the text that abrogate Rv3871 interactions. (B) (Bottom) Scheme for coexpression of tandemly arranged WXG genes. (Top) The ribbon cartoon (30) shows how the two monomers are freed from the expressed fusion protein by thrombin cleavage (scissors) at the peptide tether (balls and sticks).     

Recombinant antibodies: a new reagent for biological agent detection

Antibodies are critical reagents used in several biodetection platforms for the identification of biological agents. Recent advances in phage display technology allow isolation of high affinity recombinant antibody fragments (Fabs) that may bind unique epitopes of biological threat agents. The versatility of the selection process lends itself to efficient screening methodologies and can increase the number of antigen binding clones that can be isolated. Pilot scale biomanufacturing can then be used for the economical production of these immunoglobulin reagents in bacterial fermentation systems, and expression vectors with hexahistidine tags can be used to simplify downstream purification. One such Fab reagent directed against botulinum neurotoxin A/B has been shown to be sensitive in a variety of assay formats including surface plasmon resonance (SPR), flow cytometry, enzyme linked immunosorbent assay (ELISA), and hand-held immunochromatographic assay. Recombinant antibodies can provide another source of high quality detection reagents in our arsenal to identify or detect pathogens in environmental samples.