Morphometric and cladistic analyses of the phylogeny of Macropodinium (Ciliophora : Litostomatea : Macropodiniidae)

Phylogenetic studies of the genus Macropodinium were conducted using two methods; phenetics and cladistics. The phenetic study of morphometrics suggested that the genus could be divided into 3 groups attributable mostly to cell size and shape. The cladistic study also split the genus into 3 groups related to cell size but groups were further distinguished by patterns of ornamentation. Reconciliation of both approaches revealed considerable congruence, however, it also suggested the existence of convergences in the phenetic study and a lack of resolution in the cladistic study. The morphological diversity of Macropodinium is probably due to evolutionary trends such as increasing body size, allometry and polymerisation of structures. None of these trends, however, was uniformly directional and differential effects were observed in different regions of the phylogenetic tree. Comparison of the phylogeny of Macropodinium to a consensus phylogeny of the macropodids revealed limited incongruence between the 2 trees. The ciliate groups could be related to 2 host groups; the wallaby genera and the kangaroo and wallaroo subgenera. The association with these host groups may be the result of phyletic codescent, ecological resource tracking or a combination of both. Further studies of both host and ciliate phylogeny are necessary to resolve these effects.

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha- D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (K-d = 0.15 muM) than mannose (K-d = 2.3 muM). Exploration of the binding affinities of alpha-D-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.

Pseudomonas aeruginosa fimL regulates multiple virulence functions by intersecting with Vfr-modulated pathways

Virulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene (fimL) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa. The predicted product of fimL is homologous to the N-terminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL. vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa.

Do different surrogate methods detect lateral genetic transfer events of different relative ages?

Non-tree-based ('surrogate') methods have been used to identify instances of lateral genetic transfer in microbial genomes but agreement among predictions of different methods can be poor. It has been proposed that this disagreement arises because different surrogate methods are biased towards the detection of certain types of transfer events. This conjecture is supported by a rigorous phylogenetic analysis of 3776 proteins in Escherichia coli K12 MG1655 to map the ages of transfer events relative to one another.

Pseudotrypanosoma elphinstonae sp n., a trichomonad symbiotic in Schedorhinotermes (Isoptera : Rhinotermitidae)

A new species of Pseudotrypanosoma, P. elphinstonae sp. n., is described which is symbiotic within the hindguts of the rhinotermitid termites Schedorhinotermes secundus and S. intermedius. P. elphinstonae possesses most of the features of the genus: 4 anterior flagella, prominent costa and recurrent flagellum forming an undulating membrane and simple bean-shaped parabasal body. The mastigont complex is of similar composition and arrangement to other trichomonads but the pelto-axostylar complex is greatly simplified being composed of a single layer of microtubules which do not over lap and cannot be distinguished into separate structures. The undulating membrane is much smaller than in related species; the costa is smaller and simpler and there are no microtubular bundles connecting it to the recurrent flagellum. Comparison of the ultrastructure of P. elphinstonae sp. n. with that of P. giganteum demonstrated that P. elphinstonae sp. n. in addition to being much smaller in size had a correspondingly simper ultrastructural organisation lacking several organelles which characterise the latter species.

Trichodinids (Ciliophora : Trichodinidae) from native and exotic Australian freshwater fishes

There are very few data on trichodinids of freshwater fishes in Australia. 2003 fishes were surveyed across Eastern Australia to investigate the diversity of trichodinids present, to determine which species have been introduced with exotic fishes and to determine the extent to which these species have crossed into native fish Populations. Twenty-one putative trichodinid species were recovered from the 33 fish species examined. Trichodina heterodentata, T. mutabilis and T. reticulata were the exotic species recovered regularly; a single specimen matched a fourth exotic species, T acuta. All four exotic species are redescribed from Australian material. Trichodina heterodentata was recorded from 17 species of fishes, 15 of which were new host records; this species is identified as one of emerging importance in fish parasitology and a list of its known hosts is presented. Two new native species are also described based on silver stained specimens: T cribbi sp. n. from Hypseleotris galii, H. klunzingeri, and Hypseleotris sp. 5; and T. bassonae sp. n. from Selenotoca multifasciata. Trichodina cribbi is characterised by a large circular central inclusion and approximately 28 denticles, which have a blade length slightly greater than the ray length. Trichodina bassonae is characterised by a small, round, central inclusion and approximately 25 denticles, which have straight, non tapering rays that are in line with the leading edge of the denticle blade. It is estimated that the Australian trichodinid fauna may include up to 150 as yet undescribed species and represents a major source of unexplored biodiversity.

Culturable bacterial symbionts isolated from two distinct sponge species (Pseudoceratina clavata and Rhabdastrella globostellata) from the Great Barrier Reef display similar phylogenetic diversity

The diversity of the culturable microbial communities was examined in two sponge species-Pseudoceratina clavata and Rhabdastrella globostellata. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis. The bacterial community structures represented in both sponges were found to be similar at the phylum level by the same four phyla in this study and also at a finer scale at the species level in both Firmicutes and Alphaproteobacteria. The majority of the Alphaproteobacteria isolates were most closely related to isolates from other sponge species including alpha proteobacterium NW001 sp. and alpha proteobacterium MBIC3368. Members of the low %G + C gram-positive (phylum Firmicutes), high %G + C gram-positive (phylum Actinobacteria), and Cytophaga-Flavobacterium-Bacteroides (phylum Bacteroidetes) phyla of domain Bacteria were also represented in both sponges. In terms of culturable organisms, taxonomic diversity of the microbial community in the two sponge species displays similar structure at phylum level. Within phyla, isolates often belonged to the same genus-level monophyletic group. Community structure and taxonomic composition in the two sponge species P. clavata and Rha. globostellata share significant features with those of other sponge species including those from widely separated geographical and climatic regions of the sea.

Real-time PCR in the microbiology laboratory

Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

Enzymology and molecular biology of prokaryotic sulfite oxidation

Despite its toxicity, sulfite plays a key role in oxidative sulfur metabolism and there are even some microorganisms which can use it as sole electron source. Sulfite is the main intermediate in the oxidation of sulfur compounds to sulfate, the major product of most dissimilatory sulfur-oxidizing prokaryotes. Two pathways of sulfite oxidation are known: (1) direct oxidation to sulfate catalyzed by a sulfite: acceptor oxidoreductase, which is thought to be a molybdenum-containing enzyme; (2) indirect oxidation under the involvement of the enzymes adenylylsulfate (APS) reductase and ATP sulfurylase and/or adenylylsulfate phosphate adenylyltransferase with APS as an intermediate. The latter pathway allows substrate phosphorylation and occurs in the bacterial cytoplasm. Direct oxidation appears to have a wider distribution; however, a redundancy of pathways has been described for diverse photo- or chemotrophic, sulfite-oxidizing prokaryotes. In many pro- and also eukaryotes sulfite is formed as a degradative product from molecules containing sulfur as a heteroatom. In these organisms detoxification of sulfite is generally achieved by direct oxidation to sulfate. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Wolbachia: Invasion biology in South Pacific butterflies

Wolbachia ensdosymbionts are well known for their ability to manipulate the population biology and development of their hosts. One of the less studied outcomes of Wolbachia infection with this symbiont is the selective killing of male embryos. Recent work on butterflies living on different South Pacific islands is beginning to help us understand the complexity of the co-evolutionary interactions between these partners.

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

The cell biology of atherosclerosis - new developments

In the development of atherosclerotic lesions, three basic processes occur: 1) invasion of the artery wall by leucocytes, particularly monocytes and T-lymphocytes; 2) smooth muscle phenotypic modulation, proliferation, and synthesis of extracellular matrix; and 3) intracellular (macrophage and smooth muscle) lipoprotein uptake and lipid accumulation. Invasion of the vessel wall by leucocytes is mediated through the expression of adhesion molecules on both leucocytes and the endothelium making them 'sticky'. The adhesion molecules are induced by high serum cholesterol levels or complement fragments. Leucocytes which have adhered to the endothelium are chemo-attracted into the vessel wall by cytokines produced by early arriving leucocytes or by low density lipoprotein which has passively passed into the wall, in the process being trapped and oxidised. The oxidised low density lipoprotein is taken up by scavenger receptors (which are not subject to down-regulation) on both macrophages and smooth muscle cells. The overaccumulation of lipid is toxic to the cells and they die contributing to the central necrotic core. The macrophages and T-lymphocytes produce substances which induce smooth muscle cells of the artery wall to change from a 'contractile' (high volume fraction of myofilaments [V(v)myo]) to a 'synthetic' (low V(v)myo) phenotype. In this altered state they respond to growth factors released from macrophages, platelets, regenerating endothelial cells and smooth muscle cells; produce large amounts of matrix; express lipoprotein scavenger receptors; express adhesion molecules for leucocytes; and express HLA-DR following exposure to the T-lymphocyte product, IFN-delta, suggesting that they can become involved in a generalised immune reaction.