The Na ؉ -translocating NADH:quinone oxidoreductase is the entry site for electrons into the respiratory chain and the main sodium pump in Vibrio cholerae and many other pathogenic bacteria. In this work, we have employed steady-state and transient kinetics, together with equilibrium binding measurements to define the number of cation-binding sites and characterize their roles in the enzyme. Our results show that sodium and lithium ions stimulate enzyme activity, and that Na ؉ -NQR enables pumping of Li ؉ , as well as Na ؉ across the membrane.We also confirm that the enzyme is not able to translocate other monovalent cations, such as potassium or rubidium. Although potassium is not used as a substrate, Na ؉ -NQR contains a regulatory site for this ion, which acts as a nonessential activator, increasing the activity and affinity for sodium. Rubidium can bind to the same site as potassium, but instead of being activated, enzyme turnover is inhibited. Activity measurements in the presence of both sodium and lithium indicate that the enzyme contains at least two functional sodium-binding sites. We also show that the binding sites are not exclusively responsible for ion selectivity, and other steps downstream in the mechanism also play a role. Finally, equilibrium-binding measurements with 22 Na ؉ show that, in both its oxidized and reduced states, Na ؉ -NQR binds three sodium ions, and that the affinity for sodium is the same for both of these states.The sodium-pumping NADH:quinone oxidoreductase (Na ϩ -NQR) 2 is the entry site for electrons into the respiratory chain of many marine and pathogenic bacteria, including Vibrio cholerae (1-9). Na ϩ -NQR catalyzes the electron transfer from NADH to ubiquinone, and uses the energy released by the redox reaction to pump sodium ions, creating an electrochemical gradient (10 -13). In different bacteria, the sodium gradient supplies energy for a variety of fundamental processes, including amino acid and sugar transport, ATP synthesis, pH regulation, drug efflux, and toxin extrusion (12). Na ϩ -NQR is composed of five subunits with transmembrane spanning segments (NqrB-F) and one hydrophilic subunit (NqrA) (14). The enzyme contains five redox cofactors: two covalently bound FMNs, in the hydrophilic domains of subunits NqrB (FMN B ) and NqrC (FMN C ) (15-19), one FAD and one 2Fe-2S center, in the cytosolic domain of NqrF (20, 21), and one riboflavin in . With the exception of riboflavin, all of the cofactor-binding sites have been identified in the sequence. Flavin molecules are flexible electron carriers that can participate in one-or two-electron reactions, although the two-electron reactions are thermodynamically favored and produce relatively more stable products. Interestingly, in Na ϩ -NQR, the two FMNs and the riboflavin cofactor undergo only one-electron transitions in the reaction cycle of the enzyme. Furthermore, riboflavin is found as a stable neutral radical (RibH ⅐ ) in the air-oxidized form of the enzyme (15).Our group has previously shown that electrons move thro...

Background: Na ϩ -NQR is the main sodium pump in Vibrio cholerae.

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Angiostrongylus cantonensis and Angiostrongylus mackerrasae are metastrongyloid nematodes that infect various rat species. Terrestrial and aquatic molluscs are intermediate hosts of these worms while humans and dogs are accidental hosts. Angiostrongylus cantonensis is the major cause of angiostrongyliasis, a disease characterised by eosinophilic meningitis. Although both A. cantonensis and A. mackerrasae are found in Australia, A. cantonensis appears to account for most infections in humans and animals. Due to the occurrence of several severe clinical cases in Sydney and Brisbane, the need for epidemiological studies on angiostrongyliasis in this region has become apparent. In the present study, a conventional PCR and a TaqMan assay were compared for their ability to amplify Angiostrongylus DNA from DNA extracted from molluscs. The TaqMan assay was more sensitive, capable of detecting the DNA equivalent to one hundredth of a nematode larva. Therefore, the TaqMan assay was used to screen molluscs (n=500) of 14 species collected from the Sydney region. Angiostrongylus DNA was detected in 2 of the 14 mollusc species; Cornu aspersum [14/312 (4.5%)], and Bradybaenia similaris [1/10 (10%)], which are non-native terrestrial snails commonly found in urban habitats. The prevalence of Angiostrongylus spp. was 3.0% ± 0.8% (CI 95%). Additionally, experimentally infected Austropeplea lessoni snails shed A. cantonensis larvae in their mucus, implicating mucus as a source of infection. This is the first Australian study to survey molluscs using real-time PCR and confirms that the garden snail, C. aspersum, is a common intermediate host for Angiostrongylus spp. in Sydney.

Pentachlorophenol and other chlorinated phenols are highly toxic ubiquitous environmental pollutants. Using gas chromatographic analysis we determined that Dehalococcoides mccartyi strain JNA in pure culture dechlorinated pentachlorophenol to 3,5-dichlorophenol (DCP) via removal of the ortho and para chlorines in all of the three possible pathways. In addition, JNA dechlorinated 2,3,4,6-tetrachlorophenol via 2,4,6-trichlorophenol (TCP) and 2,4,5-TCP to 2,4-DCP and 3,4-DCP, respectively, and dechlorinated 2,3,6-TCP to 3-chlorophenol (CP) via 2,5-DCP. JNA converted 2,3,4-TCP to 3,4-DCP and 2,4-DCP by ortho and meta dechlorination, respectively. 2,3-DCP was dechlorinated to 3-CP, and, because cultures using it could be transferred with a low inoculum (0.5 to 1.5% vol/vol), it may act as an electron acceptor to support growth. Using PCR amplification with targeted and degenerate primers followed by cloning and sequencing, we determined that JNA harbors at least 19 reductive dehalogenase homologous (rdh) genes including orthologs of pcbA4 and pcbA5, pceA, and mbrA, but not tceA or vcrA. Many of these genes are shared with D. mccartyi strains CBDB1, DCMB5, GT, and CG5. Strain JNA has previously been shown to extensively dechlorinate the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1260. Collectively the data suggest that strain JNA may be well adapted to survive in sites contaminated with chlorinated aromatics and may be useful for in situ bioremediation.

This study reviews the charopid snails of Gyrocochlea s.l. This genus has been used as a convenient ‘catch-all’ for thosespecies characterised by chiefly biconcave brown shells that have a strong radially-ribbed teleoconch. The genuscurrently comprises 34 species but could include many more undescribed species that reside in museum collections usingthis broad conchological definition. The study aims to establish a framework for defining natural monophyletic groupswithin the genus and its relatives based on both morphological and molecular data. In doing so, a number of new generaand species that must be characterised to circumscribe this new definition are also described. The study utilisesqualitative and quantitative conchological data, scanning electron microscopy—primarily of the shell protoconch,anatomical studies of the male reproductive system and DNA sequences of cytochrome c oxidase subunit I and theinternal transcribed spacer 2 region of the ribosomal RNA cistrons.This study reaffirms Gyrocochlea s.s. (type species: Helix vinitincta Cox, 1868) as a geographically restricted genuscomprising only a handful of species occurring in the Border Ranges of NSW and Qld following Stanisic (1990) but withthe additional exclusion of Gyrocochlea curtisiana (Hedley, 1912) which will require re-assignment to a new genus.Gyrocochlea vinitincta (Cox) is re-examined and new anatomical details presented. Eight new genera comprisingCumberlandica n. gen., Planorbacochlea n. gen., Barringtonica n. gen., Comboynea n. gen., Cancellocochlea n. gen.,Dictyoropa n. gen., Richmondaropa n. gen. and Macphersonea n. gen. are diagnosed to accommodate species rangingfrom mid-eastern to north-eastern NSW. Seventeen new species are described comprising Cumberlandica wilsoniana n.sp., Cu. wombeyanensis n. sp., P. dandahra n. sp., P. manningensis n. sp., P. nambucca n. sp., P. watagan n. sp., P.graemei n. sp., P. reticulata n. sp., P. yessabahensis n. sp., P. parriwiensis n. sp., Barringtonica polblue n. sp., B.montana n. sp., Comboynea boorganna n. sp., Co. mountaineer n. sp., Co. winghamensis n. sp., Cancellocochleacoolongolook n. sp. and Ca. heatherae n. sp. Gyrocochlea impressa Hedley, 1924, G. planorbis Hedley, 1924, G.conferta Hedley, 1924, G. prava Hedley, 1924, G. eurythma Hedley, 1924, G. conjuncta (Iredale, 1941), G. ponderiStanisic, 2010, G. hawkesburyana Stanisic, 2010 and G. canalis Stanisic, 2010 are variously reassigned to the newgenera. Neotypes are erected for Gyrocochlea impressa Hedley, 1924 (holotype crushed) and Roblinella conjunctaIredale, 1941 (holotype presumed lost). Lectotypes are designated for Gyrocochlea conferta Hedley, 1924 andGyrocochlea eurythma Hedley, 1924. The anatomy of the Sydney Basin Diphyoropa saturni (Cox, 1864) is figured forthe first time and comparisons made between the shell morphology of this species and the general Gyrocochlea shellform. A number of species from other genera are also investigated using molecular techniques to provide a broader viewof the east coast charopid radiation. The biogeographical implications of generic ranges and the conservation status of species are discussed.