Biomedical subjects

Philip L Bond

Publications and source records attributed to Philip L Bond.

15 recordsLinked to original sources

PubMed2006

Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon "Ferroplasma acidarmanus" Fer1.

Analysis of the genome of "Ferroplasma acidarmanus" Fer1, an archaeon that is an extreme acidophile, identified an open reading frame encoding a putative ATP-dependent DNA ligase, which we termed FaLig. The deduced amino acid sequence of FaLig contains 595 amino acids, with a predicted molecular mass of 67.8 kDa. "F. acidarmanus" Fer1 is classified as a Euryarchaeote, but phylogenetic analysis using amino acid sequences showed that FaLig is more similar to DNA ligases from Crenarchaeota, suggesting that lateral transfer of these genes has occurred among archaea. The gene sequence encoding FaLig was cloned into a bacterial expression vector harbouring an upstream His-tag to aid purification. Conditions for expression and purification from Escherichia coli were identified and recombinant FaLig was confirmed to be an ATP-dependent DNA ligase. Optimal conditions for nick-joining by the protein were pH 6-7, 0.5 mM ATP, in the presence of either Mg(2+) or Mn(2+). Using a range of nicked, double-stranded nucleic acids, ligation was detected with the same substrates as previously determined for other DNA ligases. Although FaLig is the DNA ligase from one of the most extreme acidophilic organism yet studied, this characterization suggests that its biochemical mechanism is analogous to that of enzymes from other cellular systems.

Adenosine Triphosphate↗

PubMed2006

Metaproteomics: studying functional gene expression in microbial ecosystems.

The recent availability of extensive metagenomic sequences from various environmental microbial communities has extended the postgenomic era to the field of environmental microbiology. Although still restricted to a small number of studies, metaproteomic investigations have revealed interesting aspects of functional gene expression within microbial habitats that contain limited microbial diversity. These studies highlight the potential of proteomics for the study of microbial consortia. However, the application of proteomic investigations to complex microbial assemblages such as seawater and soil still presents considerable challenges. Nonetheless, metaproteomics will enhance the understanding of the microbial world and link microbial community composition to function.

Computational Biology↗

PubMed2005

Molecular insight into extreme copper resistance in the extremophilic archaeon 'Ferroplasma acidarmanus' Fer1.

'Ferroplasma acidarmanus' strain Fer1 is an extremely acidophilic archaeon involved in the genesis of acid mine drainage, and was isolated from copper-contaminated mine solutions at Iron Mountain, CA, USA. Here, the initial proteomic and molecular investigation of Cu(2+) resistance in this archaeon is presented. Analysis of Cu(2+) toxicity via batch growth experiments and inhibition of oxygen uptake in the presence of ferrous iron demonstrated that Fer1 can grow and respire in the presence of 20 g Cu(2+) l(-1). The Fer1 copper resistance (cop) loci [originally detected by Ettema, T. J. G., Huynen, M. A., de Vos, W. M. & van der Oost, J. Trends Biochem Sci 28, 170-173 (2003)] include genes encoding a putative transcriptional regulator (copY), a putative metal-binding chaperone (copZ) and a putative copper-transporting P-type ATPase (copB). Transcription analyses demonstrated that copZ and copB are co-transcribed, and transcript levels were increased significantly in response to exposure to high levels of Cu(2+), suggesting that the transport system is operating for copper efflux. Proteomic analysis of Fer1 cells exposed to Cu(2+) revealed the induction of stress proteins associated with protein folding and DNA repair (including RadA, thermosome and DnaK homologues), suggesting that 'Ferroplasma acidarmanus' Fer1 uses multiple mechanisms for resistance to high levels of copper.

Copper↗

PubMed2005

Analysis of differential protein expression during growth states of Ferroplasma strains and insights into electron transport for iron oxidation.

To investigate the metabolic biochemistry of iron-oxidizing extreme acidophiles, a proteomic analysis of chemomixotrophic and chemo-organotrophic growth, as well as protein expression in the absence of organic carbon, was carried out in Ferroplasma species. Electron transport chain inhibitor studies, spectrophotometric analysis and proteomic results suggest that oxidation of ferrous iron may be mediated by the blue copper-haem protein sulfocyanin and the derived electron passes to a cbb3 terminal electron acceptor. Despite previous suggestions of a putative carbon dioxide fixation pathway, no up-regulation of proteins typically associated with carbon dioxide fixation was evident during incubation in the absence of organic carbon. Although a lack of known carbon dioxide fixation proteins does not constitute proof, the results suggest that these strains are not autotrophic. Proteins putatively involved in central metabolic pathways, a probable sugar permease and flavoproteins were up-regulated during chemo-organotrophic growth in comparison to the protein complement during chemomixotrophic growth. These results reflect a higher energy demand to be derived from the organic carbon during chemo-organotrophic growth. Proteins with suggested function as central metabolic enzymes were expressed at higher levels during chemomixotrophic growth by Ferroplasma acidiphilum Y(T) compared to 'Ferroplasma acidarmanus' Fer1. This study addresses some of the biochemical and bioenergetic questions fundamental for survival of these organisms in extreme acid-leaching environments.

Archaeal Proteins↗

PubMed2005

Screening a wide host-range, waste-water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes.

A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.

Cosmids↗

PubMed2005

A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase.

Using DNA obtained from the metagenome of an anaerobic digestor in a waste water treatment plant, we constructed a gene library cloned in the wide host-range cosmid pLAFR3. One cosmid enabled Rhizobium leguminosarum to grow on ethanol as sole carbon and energy source, this being due to the presence of a gene, termed adhEMeta. The AdhEMeta protein most closely resembles the AdhE alcohol dehydrogenase of Clostridium acetobutylicum, where it catalyses the formation of ethanol and butanol in a two-step reductive process. However, cloned adhEMeta did not confer ethanol utilization ability to Escherichia coli or to Pseudomonas aeruginosa, even though it was transcribed in both these hosts. Further, cell-free extracts of E. coli and R. leguminosarum containing cloned adhEMeta had butanol and ethanol dehydrogenase activities when assayed in vitro. In contrast to the well-studied AdhE proteins of C. acetobutylicum and E. coli, the enzyme specified by adhEMeta is not inactivated by oxygen and it enables alcohol to be catabolized. Cloned adhEMeta did, however, confer one phenotype to E. coli. AdhE- mutants of E. coli fail to ferment glucose and introduction of adhEMeta restored the growth of such mutants when grown under fermentative conditions. These observations show that the use of wide host-range vectors enhances the efficacy with which metagenomic libraries can be screened for genes that confer novel functions.

Alcohol Dehydrogenase↗

PubMed2004

First use of two-dimensional polyacrylamide gel electrophoresis to determine phylogenetic relationships.

Methods for microbial classification are not always capable of distinguishing between isolates at the species level. We have previously characterised four Ferroplasma isolates that were >98.9% similar at the 16S rDNA level, the isolates showed marked phenotypic differences, and one isolate was borderline on the 70% species boundary from DNA-DNA similarity data. In this study we have used statistical comparisons of two-dimensional polyacylamide gel electrophoresis gels for classification of closely related isolates. From the protein profile similarities an un-rooted tree was constructed that was congruent with a tree derived from DNA-DNA similarities.

Archaea↗

PubMed2004

The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms.

Summary In the post-genomic era, the focus of numerous researchers has moved to studying the functional products of gene expression. In microbiology, these "omic" approaches have largely been limited to pure cultures of microorganisms. Consequently, they do not provide information on gene expression in a complex mixture of microorganisms as found in the environment. Our method enabled the successful extraction and purification of the entire proteome from a laboratory-scale activated sludge system optimized for enhanced biological phosphorus removal, its separation by two-dimensional polyacrylamide gel electrophoresis and the mapping of this metaproteome. Highly expressed protein spots were excised and identified using quadrupole time-of-flight mass spectrometry with de novo peptide sequencing. The proteins isolated were putatively identified as an outer membrane protein (porin), an acetyl coenzyme A acetyltransferase and a protein component of an ABC-type branched-chain amino acid transport system. These proteins possibly stem from the dominant and uncultured Rhodocyclus-type polyphosphate-accumulating organism in the activated sludge. We propose the term "metaproteomics" for the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time.

Acetyl-CoA C-Acyltransferase↗

PubMed2004

Metabolically active eukaryotic communities in extremely acidic mine drainage.

Acid mine drainage (AMD) microbial communities contain microbial eukaryotes (both fungi and protists) that confer a biofilm structure and impact the abundance of bacteria and archaea and the community composition via grazing and other mechanisms. Since prokaryotes impact iron oxidation rates and thus regulate AMD generation rates, it is important to analyze the fungal and protistan populations. We utilized 18S rRNA and beta-tubulin gene phylogenies and fluorescent rRNA-specific probes to characterize the eukaryotic diversity and distribution in extremely acidic (pHs 0.8 to 1.38), warm (30 to 50 degrees C), metal-rich (up to 269 mM Fe(2+), 16.8 mM Zn, 8.5 mM As, and 4.1 mM Cu) AMD solutions from the Richmond Mine at Iron Mountain, Calif. A Rhodophyta (red algae) lineage and organisms from the Vahlkampfiidae family were identified. The fungal 18S rRNA and tubulin gene sequences formed two distinct phylogenetic groups associated with the classes Dothideomycetes and Eurotiomycetes. Three fungal isolates that were closely related to the Dothideomycetes clones were obtained. We suggest the name "Acidomyces richmondensis" for these isolates. Since these ascomycete fungi were morphologically indistinguishable, rRNA-specific oligonucleotide probes were designed to target the Dothideomycetes and Eurotiomycetes via fluorescent in situ hybridization (FISH). FISH analyses indicated that Eurotiomycetes are generally more abundant than Dothideomycetes in all of the seven locations studied within the Richmond Mine system. This is the first study to combine the culture-independent detection of fungi with in situ detection and a demonstration of activity in an acidic environment. The results expand our understanding of the subsurface AMD microbial community structure.

Ascomycota↗

PubMed2004

Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments.

Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum Y(T) by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate "Ferroplasma acidarmanus" Fer1(T) required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42 degrees C and the pH optima were 1.0 to 1.7, and "F. acidarmanus" Fer1(T) was capable of growing at pH 0. The optimum yeast extract concentration for "F. acidarmanus" Fer1(T) was higher than that for the other three isolates. Phenotypic results suggested that isolate "F. acidarmanus" Fer1(T) is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and Y(T) group as a single species. "F. acidarmanus" Fer1(T) groups separately, and we propose the new species "F. acidarmanus" Fer1(T) sp. nov.

DNA, Archaeal↗

PubMed2003

Arsenic resistance in the archaeon "Ferroplasma acidarmanus": new insights into the structure and evolution of the ars genes.

Arsenic resistance in the acidophilic iron-oxidizing archaeon " Ferroplasma acidarmanus" was investigated. F. acidarmanus is native to arsenic-rich environments, and culturing experiments confirm a high level of resistance to both arsenite and arsenate. Analyses of the complete genome revealed protein-encoding regions related to known arsenic-resistance genes. Genes encoding for ArsR (arsenite-sensitive regulator) and ArsB (arsenite-efflux pump) homologues were found located on a single operon. A gene encoding for an ArsA relative (anion-translocating ATPase) located apart from the arsRB operon was also identified. Arsenate-resistance genes encoding for proteins homologous to the arsenate reductase ArsC and the phosphate-specific transporter Pst were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance. Phylogenetic analyses of ArsA-related proteins suggest separate evolutionary lines for these proteins and offer new insights into the formation of the arsA gene. The ArsB-homologous protein of F. acidarmanus had a high degree of similarity to known ArsB proteins. An evolutionary analysis of ArsB homologues across a number of species indicated a clear relationship in close agreement with 16S rRNA evolutionary lines. These results support a hypothesis of arsenic resistance developing early in the evolution of life.

Amino Acid Sequence↗

PubMed2003

Characterization of a nitrate-respiring bacterial community using the nitrate reductase gene (narG) as a functional marker.

Bacterial cultures capable of reducing nitrate to nitrite, or of complete denitrification, were established from 5, 10, 15 and 20 cm depths of a freshwater sediment. Taxonomic analysis of the 56 isolates using 16S rRNA gene sequences revealed an unexpected species richness, which included representatives of the gamma-Proteobacteria, Bacillus spp., Staphylococcus spp. and members of the Actinobacteria. Gram-positive species tended to predominate in the lower depths of the sediment, where there was evidence of active sulphate respiration. Sequences (from the narG gene) potentially encoding the catalytic subunit of the membrane-associated nitrate reductase were successfully amplified from 46 of the isolates, using a nested PCR with four degenerate primers. NarG sequences clustered into three major groupings that were supported by alternative phylogenetic analyses. The NarG sequences from Gram-positive isolates (according to rRNA gene phylogeny) clustered together within sequences from the low-G+C Gram-positive bacteria. However, this cluster also included two sequences from members of the genus Pseudomonas. Another group contained mostly NarG sequences from the Proteobacteria (according to rRNA gene phylogeny), but also included five sequences from Gram-positive species. The third group of NarG sequences contained three sequences from Gram-positive species. Thus, the NarG-derived phylogeny is not entirely consistent with 16S rRNA-based taxonomy, precluding the use of the narG gene as a taxonomically useful tool for the characterization of nitrate-respiring bacteria. Total DNA was also extracted from the four depth intervals of the sediment sample and used in similar narG amplifications. Most sequences amplified directly from environmental DNA clustered in the Gram-negative group, and none was in the predominantly Gram-positive group. The study also revealed a degree of spatial organization of a nitrate-respiring community in terms of both microbiology and narG sequences.

Bacteria↗

PubMed2003

Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic micro-organisms.

Acidophilic micro-organisms inhabit some of the most metal-rich environments known, including both natural and man-made ecosystems, and as such are ideal model systems for study of microbial metal resistance. Although metal resistance systems have been studied in neutrophilic micro-organisms, it is only in recent years that attention has been placed on metal resistance in acidophiles. The five metal resistance mechanisms identified in neutrophiles are also present in acidophiles, in some cases utilizing homologous proteins, but in many cases the degree of resistance is greater in acidophiles. This review summarizes the knowledge of acidophile metal resistance and presents preliminary in silico studies on a few known metal resistance systems in the sequenced acidophile genomes.

Amino Acid Sequence↗

PubMed2002

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants.

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called 'Candidatus Accumulibacter phosphatis' was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge 'biography' be well understood--i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked.

Betaproteobacteria↗

PubMed2002

Glycogen-accumulating organisms in laboratory-scale and full-scale wastewater treatment processes.

Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic-aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-beta-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1.8% P (sludge Q) and 1.5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99.7% identical, forming a cluster in the gamma-Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OP10 (39% of the library). One OTU (two clones, of which one was sequenced) was in the gamma-Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the gamma-Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92% of the Q sludge bacteria and 28% of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel gamma-Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two full-scale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the gamma-Proteobacteria radiation be named 'Candidatus Competibacter phosphatis'.

Bacteria↗