ALBERT

Notes: 1. Large amounts of coarse particulate organic matter (CPOM) are buried in the sand and gravel beds of many rivers during spates. The effects of these patchily distributed resources on hyporheic invertebrates and water chemistry are poorly understood. Buried CPOM may provide local ‘hot-spots’ of food for hyporheic detritivores and their predators, alter nutrient supply to nearby sediment biofilms, and generate habitat for some invertebrates.2. To examine potential short-term effects on hyporheic water chemistry, nutrient concentrations and invertebrate assemblage composition, leaf packs were buried in downwelling (surface water infiltrating the hyporheic zone) and upwelling (hyporheic water emerging to the surface) zones at two sites along a gravel-bed river in northern New South Wales. At one site, pits were excavated to simulate leaf burial (procedural control) and plastic ‘leaves’ were buried to test whether invertebrates might respond to leaves as refuges rather than food. Hyporheic CPOM, sediment size fractions, and interstitial silt content were also quantified at these sites.3. Dry weights of naturally buried CPOM (leaf litter and wood fragments) varied substantially (0.6–71.7 g L–1 sediment). Amounts of CPOM did not differ between up- vs. downwelling zones or between sites. Hyporheic dissolved oxygen saturation was generally high (〉 75%), and was lower in upwelling zones. The hyporheos was dominated taxonomically by water mites (≈ 20 species), whereas small oligochaetes were most abundant (40% of total abundance). Tiny instars of elmid beetle larvae and leptophlebiid mayfly nymphs were also common. Before experimental manipulation, faunal composition differed between up- and downwelling zones. In upwelling zones, bathynellaceans and blind peracarids were found, whereas small individuals of the surface benthos were common in samples from downwelling zones. This validated stratification of the experiment across zones of hydrologic exchange.4. Twenty days after leaf burial, there was no effect of the treatments at either site on changes in most variables, including mean numbers of taxa and individuals per sample. Similarly, changes in faunal composition of the hyporheos in the treatments paralleled those in the controls except for a weak response in the buried leaves treatment in the upwelling zone at site 1. Artificially buried leaf litter does not seem to influence hyporheic water chemistry or fauna at these two sites. It is probable that naturally buried leaf litter is swiftly processed soon after entrainment and that repeating this experiment immediately after a flood may yield different results.

Notes: The ability of Staphylococcus aureus cells to induce platelet aggregation has long been recognized. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial receptors required remains poorly understood. Using genetic manipulation, this study for the first time provides clear evidence that several S. aureus surface proteins participate in the inter-action with platelets. Mutants of S. aureus strain Newman lacking one or more surface proteins were tested for their ability to stimulate platelet aggre-gation. This approach was complemented by the expression of a number of candidate proteins in the non-aggregating Gram-positive bacterium Lacto-coccus lactis. S. aureus-induced aggregation was monophasic and was dependent on the platelet receptor GPIIb/IIIa. The fibrinogen-binding proteins, clumping factors A and B and the serine-aspartate repeat protein SdrE could each induce aggregation when expressed in L. lactis. Although protein A expressed in L. lactis was not capable of inducing aggregation independently, it enhanced the aggregation response when expressed on the surface of S. aureus. Thus, S. aureus has multiple mechanisms for stimulating platelet aggregation. Such functional redundancy suggests that this phenomenon may be important in the pathogenesis of invasive diseases such as infective endocarditis.

Notes: Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle-forming pili (BFP) are positively regulated by the plasmid-encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild-type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.

Notes: Clumping factor of Staphylococcus aureus is a fibrinogen-binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen-binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild-type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative N-terminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild-type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti-ClfA serum and in binding to fibrinogen-coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti-ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.

Notes: Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σs-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli. The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.

Notes: Salmonella typhimurium responds to a variety of environmental stresses by accumulating the alternative sigma factor σS. The repertoire of σS -dependent genes that are subsequently expressed confers tolerance to a variety of potentially lethal conditions including low pH and stationary phase. The mechanism(s) responsible for triggering σS accumulation are of considerable interest, because they help to ensure survival of the organism during encounters with suboptimal environments. Two genes associated with regulating σS levels in S. typhimurium have been identified. The first is clpP, encoding the protease known to be responsible for degrading σS in Escherichia coli. The second is dksA, encoding a protein of unknown function not previously associated with regulating σS levels. As predicted, clpP mutants accumulated large amounts of σS even in log phase. However, dksA mutants failed to accumulate σS in stationary phase and exhibited lower accumulation during acid shock in log phase. DksA appears to be required for the optimal translation of rpoS based upon dksA mutant effects on rpoS transcriptional and translational lacZ fusions. The region of rpoS mRNA between codons 8 and 73 is required to see the effects of dksA mutations. This distinguishes the role of DksA from that of HF-I (hfq ) in rpoS translation, as the HF-I target area occurs well upstream of the rpoS start codon. DksA appears to be involved in the expression of several genes in addition to rpoS based on two-dimensional SDS–PAGE analysis of whole-cell proteins. As a result of their effects on gene expression, mutations in clpP and dksA decreased the virulence of S. typhimurium in mice, consistent with a role for σS in pathogenesis.

Notes: The pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post-translationally modified by the addition of an O-linked trisaccharide, Gal (β1–4) Gal (α1–3) 2,4-diacetimido-2,4,6-trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with well-defined lipopolysaccharide (LPS) or pilin-linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin-linked trisaccharide structure. The pglA mutation, like the galE mutation reported previously, had no effect on pilus-mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.

Notes: As an important facet of host–pathogen interaction, Staphylococcus aureus has the ability to adhere to human extracellular matrix (ECM) components via a range of surface proteins. Here we have shown that IsdA has broad-spectrum ligand-binding activity, including fibrinogen and fibronectin. Mapping studies revealed a distinct domain responsible for ligand binding. This domain is present in a number of iron-regulated proteins of S. aureus and in other Gram-positive organisms. The isdA gene is only expressed in iron-limited conditions under the control of Fur and not in standard laboratory media. Such conditions occur in serum in vitro and during infection. Whole cell binding and clumping assays revealed that when the bacteria are grown under iron-limited conditions, IsdA constitutes a physiologically relevant adhesin to both fibrinogen and fibronectin. Thus for S. aureus, iron is an important marker for the host environment, to which the bacterium responds by differential regulation of at least one element of its adhesive strategy.

Notes: We report the complete sequence of Staphylococcal pathogenicity island bovine 2 (SaPIbov2), encoding the biofilm-associated protein Bap. SaPIbov2 contains 24 open reading frames, including sip, which encodes a functional staphylococcal integrase protein. SaPIbov2 is bordered by 18 bp direct repeats. The integration site into the chromosome lies at the 3′ end of a gene encoding GMP synthase. SaPIbov2 has extensive similarity to previously described pathogenicity islands of Staphylococcus aureus. The principal difference is that toxin genes present in the other pathogenicity islands are exchanged for a transposon-like element that carries the bap gene and genes encoding an ABC transporter and a transposase. Also, SaPIbov2 can be excised to form a circular element and can integrate site-specifically and RecA-independently at a chromosomal att site in a Sip-dependent manner. This was demonstrated both in S. aureus and with plasmid substrates ectopically in Escherichia coli. Thus, SaPIbov2 encodes a functional recombinase of the integrase family that promotes element excision and insertion/integration. In addition, we demonstrated that the presence of SaPIbov2 facilitated the persistence of S. aureus in an intramammary gland infection model. Finally, different bovine isolates of S. aureus were found to carry islands related to SaPIbov2, suggesting the existence of a family of related pathogenicity islands.

Notes: We present the first synthesis of the life history and ecology of seahorses, compare relationships for seahorses with other marine teleosts and identify research needs. Seahorses occurred primarily amidst temperate seagrasses and tropical coral reefs. Population densities were generally low, ranging from 0 to 0·51 individuals m−2, but reached 10 m−2 in some patches. Inferred life spans ranged from 1 to 5 years. Seahorses consumed live prey and possibly changed diet as they grew. Growth rates are poorly investigated to date. Reproduction and mating systems are the best-studied aspects of seahorse ecology. The relationship between size at first maturity and maximum size in seahorses conformed to that for other marine teleosts. All seahorse species were monogamous within a cycle, but some were polygamous across cycles. Direct transfer of clutches to the brood pouch of the male fish made it difficult to measure clutch size in live seahorses. After brooding, males released from c. 5 to 2000 young, depending on species and adult size. Newborn young measured from 2 to 20 mm in length, which was a narrower size range than the 17-fold difference that occurred in adult size. Newborn body size had no relationship to adult size. Both eggs and young were larger than expected among marine teleosts, even when considering only those with parental care, but brood size at release was lower than expected, perhaps because the young were more developed. The size of adults, eggs and young increased with increasing latitude, although brood size did not. Considerable research is needed to advance seahorse conservation and management, including (a) fisheries-dependent and fisheries-independent abundance estimates, (b) age- or stage-based natural and fishing mortalities, (c) growth rates and age at first maturity, and (d) intrinsic rates of increase and age- or size-specific reproductive output. Current data confirm that seahorses are likely to be vulnerable to high levels of exploitation.