A Cross-System Comparison of Bacterial and Fungal Biomass in Detritus Pools of Headwater Streams

The absolute amount of microbial biomass and relative contribution of fungi and bacteria are expected to vary among types of organic matter (OM) within a stream and will vary among streams because of differences in organic matter quality and quantity. Common types of benthic detritus [leaves, small wood, and fine benthic organic matter (FBOM)] were sampled in 9 small (1st-3rd order) streams selected to represent a range of important controlling factors such as surrounding vegetation, detritus standing stocks, and water chemistry. Direct counts of bacteria and measurements of ergosterol (a fungal sterol) were used to describe variation in bacterial and fungal biomass. There were significant differences in bacterial abundance among types of organic matter with higher densities per unit mass of organic matter on fine particles relative to either leaves or wood surfaces. In contrast, ergosterol concentrations were significantly greater on leaves and wood, confirming the predominance of fungal biomass in these larger size classes. In general, bacterial abundance per unit organic matter was less variable than fungal biomass, suggesting bacteria will be a more predictable component of stream microbial communities. For 7 of the 9 streams, the standing stock of fine benthic organic matter was large enough that habitat-weighted reach-scale bacterial biomass was equal to or greater than fungal biomass. The quantities of leaves and small wood varied among streams such that the relative contribution of reach-scale fungal biomass ranged from 10% to as much as 90% of microbial biomass. Ergosterol concentrations were positively associated with substrate C:N ratio while bacterial abundance was negatively correlated with C:N. Both these relationships are confounded by particle size, i.e., leaves and wood had higher C:N than fine benthic organic matter. There was a weak positive relationship between bacterial abundance and streamwater soluble reactive phosphorus concentration, but no apparent pattern between either bacteria or fungi and streamwater dissolved inorganic nitrogen. The variation in microbial biomass per unit organic matter and the relative abundance of different types of organic matter contributed equally to driving differences in total microbial biomass at the reach scale.     

Substrate recognition properties of oligopeptidase B from Salmonella enterica serovar Typhimurium

Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P(1). While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu(576) and Glu(578), that define P(1) specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp(460) and Asp(462), that may be involved in defining P(2) specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.     

cDNA nucleotide sequence encoding the ZPC protein of Australian hydromyine rodents: a novel sequence of the putative sperm-combining site within the family Muridae

This comparative study of the cDNA sequence of the zona pellucida C (ZPC) glycoprotein in murid rodents focuses on the nucleotide and amino acid sequence of the putative sperm-combining site. We ask the question: Has divergence evolved in the nucleotide sequence of ZPC in the murid rodents of Australia? Using RT-PCR and (RACE) PCR, the complete cDNA coding region of ZPC in the Australian hydromyine rodents Notomys alexis and Pseudomys australis, and a partial cDNA sequence from a third hydromyine rodent, Hydromys chrysogaster, has been determined. Comparison between the cDNA sequences of the hydromyine rodents reveals that the level of amino acid sequence identity between N. alexis and P. australis is 96%, whereas that between the two species of hydromyine rodents and M. musculus and R. norvegicus is 88% and 87% respectively. Despite being reproductively isolated from each other, the three species of hydromyine rodents have a 100% level of amino acid sequence identity at the putative sperm-combining site. This finding does not support the view that this site is under positive selective pressure. The sequence data obtained in this study may have important conservation implications for the dissemination of immunocontraception directed against M. musculus using ZPC antibodies.     

Efficacy of different administration routes for vaccination against Vibrio anguillarum in Atlantic halibut (Hippoglossus hippoglossus L.)

Atlantic halibut (Hippoglossus hippoglossus L.) is a potentially important new species to cold-water aquaculture. Development of a viable industrial farming technique has been hampered by continued pathogen problems within the rearing cycle and there are several reports that indicated how susceptible juvenile halibut are to bacterial and viral diseases. Interest has been expressed, within the industry, over the possibility of vaccinating suitably sized animals to protect against the more common aquaculture pathogens. Vibrio spp. are of particular concern due to their ubiquitous nature and the relatively frequent occurrence of these pathogens within marine aquaculture. We have previously investigated the susceptibility of Atlantic halibut to infection by Vibrio anguillarum and the efficacy of intraperitoneal injected delivery of a commercial vaccine in protecting against the disease. Given the very high rate of protection offered by immunisation we wanted to investigate the effect of alternate routes of administration on the efficacy of the vaccine.     

Nucleotide variation,haplotype structure,and association with end-stage renal disease of the human interleukin-1 gene cluster

A dense gene-based SNP map was constructed across a 360-kb region containing the interleukin-1 gene cluster (IL1A, IL1B, and IL1RN), focusing on IL1RN. In total, 95 polymorphisms were confirmed or identified primarily by direct sequencing. Polymorphisms were precisely mapped to completed BAC and genomic sequences spanning this region. The polymorphisms were typed in 443 case-control subjects from Caucasian and African American groups. Consecutive pair-wise marker linkage disequilibrium was not strictly correlated with distance and ranged from D'=0.0079 to 1.000 and D'=0.0521 to 1.0000 in Caucasians and African Americans, respectively. Single markers and haplotypes in IL1 cluster genes were evaluated for association with end-stage renal disease (ESRD). Eleven SNPs show some evidence of association with ESRD, with the strongest associations in two IL1A variants, one SNP, rs1516792-3, in intron 5 (p=0.0015) and a 4-bp insertion/deletion within the 3'UTR, rs16347-2 (p=0.0024), among African Americans with non-T2DM-associated ESRD.     

A "dock, lock, and latch" structural model for a staphylococcal adhesin binding to fibrinogen

Gram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bbeta chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic "dock, lock, and latch" mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria.     

Expression of three <Emphasis Type="Italic">spalt </Emphasis>(<Emphasis Type="Italic">sal</Emphasis>) gene homologues in zebrafish embryos

Three homologues of the Drosophilaregion-specific homeotic gene spalt (sal) have been isolated in zebrafish, sall1a, sall1b and sall3. Phylogenetic analysis of these genes against known salDNA sequences showed zebrafish sall1aand sall1b to be orthologous to other vertebrate sal-1 genes and zebrafish sall3to be orthologous to other vertebrate sal-3 genes, except Xenopus sall3. Phylogenetic reconstruction suggests that zebrafish sall1a and sall1bresulted from a gene duplication event occurring prior to the divergence of the ray-finned and lobe-finned fish lineages. Analysis of the expression pattern of the zebrafish sal genes shows that sall1a and sall3 share expression domains with both orthologous and non-orthologous vertebrate sal genes. Both are expressed in various regions of the CNS, including in primary motor neurons. Outside of the CNS, sall1a expression is observed in the otic vesicle (ear), heart and in a discrete region of the pronephric ducts. These analyses indicate that orthologies between zebrafish sal genes and other vertebrate sal genes do not imply equivalence of expression pattern and, therefore, that biological functions are not entirely conserved. However we suggest that, like other vertebrate sal genes, zebrafish sal genes have a role in neural development. Also, expression of zebrafish sall1a in the otic vesicle, heart sac and the pronephric ducts of zebrafish embryos is possibly consistent with some of the abnormalities seen in Sall1-deficient mice and in Townes-Brocks Syndrome, a human disorder which is caused by mutations in the human spalt gene SALL1.     

Igf2 imprinting in development and disease

Igf2 is one of the first imprinted genes discovered and occupies a centre stage in the study of imprinting. This is because it has dramatic effects on the control of fetal growth, it is involved in growth disorders and in cancer, it interacts with products of other imprinted genes, and its imprinting status is under complex regulation in a cluster of tightly linked imprinted genes. Here we review briefly the key features of Igf2 imprinting in normal development and in disease, and hope to show what a fascinating subject of study this gene and its biology provides.     

Screening marine natural products for selective inhibitors of key kynurenine pathway enzymes

Kynurenine, a metabolite of tryptophan along the 'kynurenine pathway', is at a branch point of the pathway which can lead to the synthesis of both quinolinic acid (QUIN) and kynurenic acid (KYNA). KYNA is an antagonist of glutamate receptors; however, QUIN is a selective agonist of NMDA receptors, and has been shown to act as an excitotoxic agent. A high QUIN/KYNA ratio has been implicated in a variety of neurological diseases in which excitotoxic neuronal cell death is found, e.g. AIDS-related dementia, stroke, etc. Inhibiting the key enzymes of this pathway (i.e. kynureninase and kynurenine 3-hydroxylase) would lower the QUIN/KYNA ratio, which may potentially have neuroprotective effects. We have developed high through-put assays for kynurenine pathway enzymes which allow us to screen extracts from marine organisms for selective enzyme inhibitors. Active metabolites are purified, isolated and identified by HPLC, high-field NMR and mass spectral techniques. Extracts from a sponge of the Aka species were found to contain a selective inhibitor of kynureninase. We have recently purified and identified the active principal as being serotonin sulfate. Related indoleamines, serotonin and 5-hydroxyindoleacetic acids are inactive. This finding may be suggestive of a novel interaction between the serotoninergic and excitatory amino acid pathways.     

The Myxococcus xanthus lipopolysaccharide O-antigen is required for social motility and multicellular development

The gliding bacterium Myxococcus xanthus aggregates to form spore-filled fruiting bodies when nutrients are limiting. Defective fruiting-body formation and sporulation result from mutations in the sasA locus, which encodes the wzm wzt wbgA (formerly rfbABC ) lipopolysaccharide (LPS) O-antigen biosynthesis genes. Mutants carrying these same sasA mutations are defective in social motility and form small glossy colonies. We report here that the developmental and motility phenotypes of four mutants each containing different Tn 5 insertions in LPS O-antigen biosynthesis genes are similar to those of the original sasA locus mutants. All of the LPS O-antigen mutants tested exhibited defective developmental aggregation and sporulated at only 0.02–15% of the wild-type level. In addition, all of the LPS O-antigen mutants were determined by genetic analyses to be wild type for adventurous motility and defective in social motility, indicating that the LPS O-antigen is necessary for normal development and social motility. The two previously identified cell-surface components required for social motility, type IV pili and the protein-associated polysaccharide material termed fibrils, were detected on the surfaces of all of the LPS O-antigen mutants. This indicates that LPS O-antigen is a third cell-surface component required for social motility.