Biomedical subjects

Mark J Bailey

Publications and source records attributed to Mark J Bailey.

At least 19 recordsLinked to original sources

PubMed2006

The histidine utilization (hut) genes of Pseudomonas fluorescens SBW25 are active on plant surfaces, but are not required for competitive colonization of sugar beet seedlings.

The ability to monitor the spatial and temporal distribution of signals in complex environments is necessary for an understanding of the function of bacteria in the wild. To this end, an existing recombinase-based transcriptional reporter strategy (recombinase-based in vivo expression technology, RIVET) has been extended and applied to the plant-colonizing bacterium Pseudomonas fluorescens SBW25. Central to the project was a rhizosphere-inducible locus, rhi14, which functional analyses show is hutT, a histidine-inducible gene that is required for histidine utilization. A transcriptional fusion between hutT and a promoterless site-specific recombinase (tnpR(mut168)) results in excision of a chromosomally integrated tetracycline-resistance cassette in a histidine-dependent manner. The dose- and time-responsiveness of the promoterless recombinase to histidine closely mirrored the histidine responsiveness of an identical hutT fusion to promoterless lacZ. To demonstrate the effectiveness of the strategy, the activity of hutT was monitored on sugar beet seedlings. Low levels of transcriptional activity were detected in the phyllosphere, rhizosphere and in plant extract, but not in vermiculite devoid of seedlings. The histidine concentration in the rhizosphere was estimated to be 0.6 microg ml(-1). The ecological significance of the hut locus was examined by competing a hutT deletion mutant against the wild-type during colonization of sugar beet seedlings. No impact on competitive fitness was detected, suggesting that the ability to utilize plant-derived histidine is not essential for bacterial colonization.

Bacterial Proteins↗

PubMed2006

Bacterial diversity is determined by volume in membrane bioreactors.

It has been proposed that established models and theories developed in classical ecology could be employed to greatly improve the optimization of wastewater treatment plants (WWTP) by placing the microbiological component onto a model-predictive basis. In particular, this could be achieved by better understanding bacterial community assembly and development. The species-area relationship is one of the oldest biological laws and has been used to describe spatial diversity patterns in contiguous habitats and on islands. In the current study, bacterial communities in seven membrane bioreactors (MBR), of increasing size, located across the UK were sampled. A significant linear relationship between bacterial taxa richness and reactor size was observed and was similar to classical species-area relationships of larger organisms colonizing oceanic islands. Rank-abundance plots revealed a gradient of greater evenness in community structure as MBR volume increased. Application of the Raup and Crick probability-based similarity index indicated a strong role for dispersal in MBR colonization and community structure. Our findings demonstrate that the MBR sampled behaved like islands with respect to bacterial colonization in accordance with the theory of island biogeography. In addition this study provides further evidence that biodiversity at the bacterial level is more similar to that of animals and plants than previously postulated.

Bacteria↗

PubMed2005

Life in earth: the impact of GM plants on soil ecology?

The impact of changes incurred by agricultural biotechnology has led to concern regarding soil ecosystems and, rightly or wrongly, this has focused on the introduction of genetically modified (GM) crops. Soils are key resources, with essential roles in supporting ecosystems and maintaining environmental quality and productivity. The complexity of soils presents difficulties to their inclusion in the risk assessment process conducted for all GM plants. However, a combined approach, informed by both soil ecology and soil quality perspectives, that considers the impacts of GM crops in the context of conventional agricultural practices can provide a regulatory framework to ensure the protection of soils without being overly restrictive.

Biodiversity↗

PubMed2005

A platform for high-throughput molecular characterization of recombinant monoclonal antibodies.

We describe quantitative characterization of a sample preparation platform for rapid and high-throughput analysis of recombinant monoclonal antibodies (MAbs) and their post-translational modifications. MAb capture, desalting and in situ reduction/alkylation were accomplished by sequential adsorption of analyte to solid phase beads (protein A, reverse-phase) suspended in microtiter plate wells. Following elution and rapid tryptic digestion in the presence of acid-labile surfactant (RapiGest), peptides were fractionated by stepwise elution from reverse-phase pipet tips and the fraction containing Fc N-glycopeptides isolated. Direct quantitative analysis of the relative abundance of peptide glycoforms by MALDI-TOF MS in linear mode closely correlated with normal phase HPLC analysis of fluorophore labeled N-glycans released by PNGaseF.

Adsorption↗

PubMed2005

Calibration and deployment of custom-designed bioreporters for protecting biological remediation consortia from toxic shock.

We have previously described the development of a panel of site-specific lux-based bioreporters from an industrial wastewater treatment system remediating coking effluents. The Pseudomonad strains carry a stable chromosomal copy of the luxCDABE operon from Photorhabdus luminescens and display proportional responses in bioluminescence decay with increasing phenol concentration up to 800 mg l-1. In this work we describe their deployment to provide a strategic sensing network for protecting bacterial communities involved in the biological breakdown of coking effluents. This evaluation demonstrated the utility of strategic placement of reporters around heavy industry treatment systems and the reliability of the reporter strains under normal operational conditions. Mono-phenol or total phenolic variation within the treatment system accounted for>65-80% of the luminescence response. The reporters exhibited stable luminescence output during normal operations with maximum standard deviations of luminescence over time of c. 5-15% depending on the treatment compartment. Furthermore, deployment of the bioreporters over a 5-month period allowed the determination of an operational range (OR) for each reporter for effluent samples from each compartment. The OR allowed a convenient measure of toxicity effects between treatment compartments and accurately reflected a specific pollution event occurring within compartments of the treatment system. This work demonstrates the utility of genetic modification to provide ecologically relevant bioreporters, extends the sensing capabilities currently obtained through marine derived biosensors and significantly enhances the potential for in situ deployment of reporting agents.

Biodegradation, Environmental↗

PubMed2004

The indigenous Pseudomonas plasmid pQBR103 encodes plant-inducible genes, including three putative helicases.

Plasmid pQBR103 ( approximately 400 kb) is representative of many self-transmissible, mercury resistant plasmids observed in the Pseudomonas community colonising the phytosphere of sugar beet. A promoter trapping strategy (IVET) was employed to identify pQBR103 genes showing elevated levels of expression on plant surfaces. Thirty-seven different plant-inducible gene fusions were isolated that were silent in laboratory media, but active in the plant environment. Three of the fusions were to DNA sequences whose protein products show significant homology to DNA-unwinding helicases. The three helicase-like genes, designated helA, helB and helC, are restricted to a defined group of related Pseudomonas plasmids. They are induced in both the root and shoot environments of sugar beet seedlings. Sequence analysis of the three plasmid-encoded helicase-like genes shows that they are phylogenetically distinct and likely to have independent evolutionary histories. The helA gene is predicted to encode a protein of 1121 amino acids, containing conserved domains found in the ultraviolet (UV) resistance helicase, UvrD. A helA knockout mutant was constructed and no phenotypic changes were found with plasmid-conferred UV resistance or plasmid conjugation. The other 34 fusions are unique with no homologues in the public gene databases, including the Pseudomonas genomes. These data demonstrate the presence of plant responsive genes in plasmid DNA comprising a component of the genomes of plant-associated bacteria.

Adaptation, Physiological↗

PubMed2004

Raman microscopic analysis of single microbial cells.

We demonstrate the utility of the Raman confocal microscope to generate a spectral profile from a single microbial cell and the use of this approach to differentiate bacterial species. In general, profiles from different bacterial taxa shared similar peaks, but the relative abundances of these components varied between different species. The use of multivariate methods subsequently allowed taxa discrimination. Further investigations revealed that the single-cell spectra could be used to differentiate between growth phases of a single species, but these differences did not obscure the overall interspecies discrimination. Finally, we tested the efficacy of the method as a means to identify cells responsible for the uptake of a specific substrate. A single strain was grown in media containing incrementally varying ratios of (13)C(6) to (12)C(6) glucose, and it was found that (13)C incorporation shifted characteristic peaks to lower wavenumbers. These findings suggest that Raman microscopy has significant potential for studies requiring the taxonomic identity and functioning of single microbial cells to be determined.

Bacteria↗

PubMed2004

The tritrophic trinity: a source of pollutant-degrading enzymes and its implications for phytoremediation.

Barring bioavailability and nutritional limitations, virtually all organic anthropogenic chemicals can be naturally biodegraded. It is to this phenomenon we owe thanks to the long established 'tritrophic trinity' of microbe-plant-insect interactions. Over hundreds of millennia these organisms have coevolved, producing hundreds of thousands of different chemicals that are used to attract, defend, antagonize, monitor and misdirect one another. In comparison, the numbers of truly novel chemicals of anthropogenic origin are negligible. It is only now that we are beginning to appreciate the fortuitous evolution of xenobiotic-degrading enzymes from these interactions. We argue that success in phytoremediation can be hastened through understanding the structure, sources, uses and targets of these secondary metabolites. Owing to recent developments in molecular biology, particularly stable isotope probing, we eagerly anticipate highly significant insights into trophic interactions, particularly in the rhizosphere, providing phytoremediation with a solid mechanistic understanding.

Animals↗

PubMed2004

13CO2 pulse labelling of plants in tandem with stable isotope probing: methodological considerations for examining microbial function in the rhizosphere.

Recently developed 13CO2 pulse labelling and stable isotope probing (SIP) methods offer the potential to track 13C-labelled plant photosynthate into phylogenetic groups of microbial taxa in the rhizosphere, permitting an examination of the link between soil microbial diversity and carbon flow in situ. We tested the feasibility of this approach to detect functional differences in microbial communities utilising recently fixed plant photosynthate in moisture perturbed grassland turfs. Specifically, we addressed two questions: (1) How does moisture perturbation (three treatments; continual wetting, drying, and drying followed by rewetting) affect the assimilation of 13C-labelled exudates carbon into the soil microbial community?; (2) Can 13C deposited in soil from pulse-labelled plants be used to identify microbes utilising plant exudates using SIP methodologies? Net CO2 fluxes showed that prior to 13CO2 pulse labelling, all treatments were photosynthetically active, but differences were observed in night time respiration, indicating moisture treatments had impacted on net CO2 efflux. Measurements of pulse-derived 13C incorporated into soil RNA over 2 months showed that there was only evidence of 13C enrichment in the continuously wetted treatments. However, isotopic values represented only a 0.1-0.2 13C at.% increase over natural abundance levels and were found to be insufficient for the application of RNA-SIP. These findings reveal that in this experimental system, the microbial uptake of labelled carbon from plant exudates is low, and further optimisation of methodologies may be required for application of SIP to natural plant-soil systems where 13C tracer dilution is a consideration.

Carbon Dioxide↗

PubMed2004

The Horizontal Gene Pool: an ESF workshop summary.

The European Science Foundation (ESF) funds a limited number of exploratory workshops each year that enable scientists to meet and develop plans for a program of integrated research which would benefit from a coordinated European effort. In summer 2003, the Standing Committee for Life and Environmental Sciences (LESC) sponsored such a workshop called The Horizontal Gene Pool: The Functional Role of Mobile Genetic Information--How Bacteria Perceive, Sample, and Utilize Genetic Elements in evolution and Local Adaptation. The workshop took place at St. Catherine's College, Oxford, UK. Its purpose was to identify how recent advances in the application of genomics and microbial ecology can be harnessed to determine the genetic mechanisms that underpin the biological role of the horizontal gene pool. Scientific excellence at the workshop was contributed by senior scientists and young investigators from research institutes located in nine European countries.

Bacteria↗

PubMed2004

Functional and phylogenetic analysis of a plant-inducible oligoribonuclease (orn) gene from an indigenous Pseudomonas plasmid.

Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenous Pseudomonas plasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3' to 5' exoribonuclease specific for small oligoribonucleotides. The deduced amino acid sequence of the plasmid-derived orn (orn(pl)) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes. Deletion of orn(pl) generated no observable phenotype, but inactivation of the chromosomal copy caused slow growth in Pseudomonas putida KT2440. This defect was fully restored by complementation with orn from Escherichia coli (orn(E.coli)). Plasmid-derived orn(pl) was capable of partially complementing the P. putida orn mutant, demonstrating functionality of orn(pl). Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria. A survey of orn(pl) from related Pseudomonas plasmids showed a sporadic distribution but no sequence diversity. These data suggest that the orn(pl) was acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of the Proteobacteria.

Amino Acid Sequence↗

PubMed2004

N-acyl-l-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge.

The role of intercellular signalling in the regulation of genes and phenotypes in a broad range of bacterial species is now firmly established. In contrast, the impact of intercellular signalling on microbial community parameters, such as species diversity and function, is less well understood. In this study the role of N-acyl-l-homoserine lactones (AHLs) in microbial community dynamics in an industrial wastewater treatment system is addressed. Seven proteobacterial strains producing compounds with AHL-like activity were isolated from the treatment plant. Three of these belong to genera with no previously identified AHL producing species. Addition of AHLs at 2 micro M to sludge samples generated changes in both community function (phenol degradation) and composition as determined by length heterogeneity PCR and denaturing gradient gel electrophoresis. Phenol degradation was more stable as a result of the AHL augmentation. A dominant functional member of the Thauera genus was transiently supplanted by a member of the Comomonas genus in response to AHL addition. This suggests that AHLs can play a role in mediating microbial community parameters and has implications for ecosystem function and industrial wastewater treatment.

4-Butyrolactone↗

PubMed2003

Acyl glucuronide reactivity in perspective: biological consequences.

The metabolic conjugation of exogenous and endogenous carboxylic acid substrates with endogenous glucuronic acid, mediated by the uridine diphosphoglucuronosyl transferase (UGT) superfamily of enzymes, leads to the formation of acyl glucuronide metabolites. Since the late 1970s, acyl glucuronides have been increasingly identified as reactive electrophilic metabolites, capable of undergoing three reactions: intramolecular rearrangement, hydrolysis, and intermolecular reactions with proteins leading to covalent drug-protein adducts. This essential dogma has been accepted for over a decade. The key question proposed by researchers, and now the pharmaceutical industry, is: does or can the covalent modification of endogenous proteins, mediated by reactive acyl glucuronide metabolites, lead to adverse drug reactions, perhaps idiosyncratic in nature? This review evaluates the evidence for acyl glucuronide-derived perturbation of homeostasis, particularly that which might result from the covalent modification of endogenous proteins and other macromolecules. Because of the availability of acyl glucuronides for test tube/in vitro experiments, there is now a substantial literature documenting their rearrangement, hydrolysis and covalent modification of proteins in vitro. It is certain from in vitro experiments that serum albumin, dipeptidyl peptidase IV, tubulin and UGTs are covalently modified by acyl glucuronides. However, these in vitro experiments have been specifically designed to amplify any interference with a biological process in order to find biological effects. The in vivo situation is not at all clear. Certainly it must be concluded that all humans taking carboxylate drugs that form reactive acyl glucuronides will form covalent drug-protein adducts, and it must also be concluded that this in itself is normally benign. However, there is enough in vivo evidence implicating acyl glucuronides, which, when backed up by in vivo circumstantial and documented in vitro evidence, supports the view that reactive acyl glucuronides may initiate toxicity/immune responses. In summary, though acyl glucuronide-derived covalent modification of endogenous macromolecules is well-defined, the work ahead needs to provide detailed links between such modification and its possible biological consequences.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2003

Analysis of the microbial functional diversity within water-stressed soil communities by flow cytometric analysis and CTC+ cell sorting.

Total and active cell counts within soil samples were determined by culture-independent methods using flow cytometry and preparative Nycodenz gradient centrifugation. Whole cells were purified from soil cores and total extractable cell counts assessed by SYBR Green II fluorescence, while active cell counts were determined by 5-cyano-2,3-ditolyl tetrazolium chloride reduction (CTC+ cells). Parallel microcosms, maintained at either field water capacity or subjected to drying, indicated that the total extractable cell count remained between 10(8) and 10(9) g(-1) (dry weight). In contrast, the CTC+ active count fell threefold in dried microcosms (6% of total cell count) when compared to wetted microcosms (18% of total cell count). Specifically, these data highlighted an overall deactivation of microbial biomass during water stress, with 16S rDNA analyses of flow-sorted CTC+ cells demonstrating shifts within the active diversity. Flow cytometry coupled with cell purification techniques represents a significant tool for operationally defining an active and redundant microbial component within soil communities and is demonstrated during water stress.

Bacteria↗

PubMed2003

Development of bespoke bioluminescent reporters with the potential for in situ deployment within a phenolic-remediating wastewater treatment system.

A suite of ecologically relevant, site-specific bioreporters was constructed by transposon mutagenesis of microorganisms isolated from a polluted phenolic-remediating wastewater treatment system. Four Pseudomonad species were engineered to carry a stable chromosomal copy of the lux operon (luxCDABE) derived from Photorhabdus luminescens. These recombinant reporter microorganisms were tested for bioluminescence response to relevant phenol concentrations in the laboratory and to phenolic-containing effluents generated by an industrial wastewater treatment plant. The reporters displayed proportional responses of bioluminescence decay with increasing phenol concentrations up to 800 mg l(-1) of phenol. When deployed against samples from the treatment system, they showed superior operational range and sensing capabilities to that observed for industry standard microorganisms such as Vibrio fischeri. Specifically, the engineered strains accurately predicted toxicity shifts in all the treatment compartments under study (with phenolic concentrations ranging from approximately 10 to 600 mg l(-1)) with a low coefficient of variation of replicate determinations (between 1.16% and 8.32%). This work highlights the utility of genetic modification of native microorganisms from sites of interest to provide robust and ecologically relevant organism-based reagents for toxicity monitoring with the potential for in situ deployment.

Biodegradation, Environmental↗

PubMed2003

Physiological and community responses of established grassland bacterial populations to water stress.

The effects of water stress upon the diversity and culturable activity of bacterial communities in the rhizosphere of an established upland grassland soil have been investigated. Intact monoliths were subjected to different watering regimens over a 2-month period to study community adaptation to moisture limitation and subsequent response to stress alleviation following rewetting. Genetic diversity was analyzed with 16S-based denaturing gradient gel electrophoresis (DGGE) of total soil-extracted DNA (rRNA genes) and RNA (rRNA transcripts) in an attempt to discriminate between total and active communities. Physiological response was monitored by plate counts, total counts, and BIOLOG-GN2 substrate utilization analyses. Controlled soil drying decreased the total number of CFU on all the media tested and also decreased the substrate utilization response. Following rewetting of dried soil, culture-based analyses indicated physiological recovery of the microbial population by the end of the experiment. In contrast, DGGE analyses of community 16S rRNA genes, rRNA transcripts and cultured communities did not reveal any changes relating to the moisture regimens, despite the observed physiological effects. We conclude that the imposed moisture regimen modulated the physiological status of the bacterial community and that bacterial communities in this soil are resistant to water stress. Further, we highlight the need for a reexamination of rRNA transcript-based molecular profiling techniques as a means of describing the active component of soil bacterial communities.

Bacteria↗

PubMed2002

Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro.

Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins [MAPs]). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC(50)) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (> or =2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [14C]-labelled species) showed that adduct formation was much greater for iso-VPA-G. When [14C]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides.

Animals↗

PubMed2002

Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function.

An ongoing challenge within microbial ecology is the development of methodologies that attribute microbial community functions to microbial diversity. One approach, involving the incorporation of stable isotopes from labelled tracer compounds into biological signature molecules (biomarkers), may overcome this current limitation. To examine the potential of RNA as the biomarker in stable isotope probing we have generated a series of atom % (13)C-enriched RNA samples through exploitation of the anabolic abilities of a phenol-degrading environmental isolate. Isotope ratio mass spectrometry was used to determine the atom % (13)C of each RNA sample (ca. 1-100%). The corresponding buoyant density (1.755-1.795 g mL(-1)) was determined by equilibrium density gradient centrifugation and agarose gel electrophoresis. This empirically defined relationship between the atom % (13)C of RNA and its buoyant density suggests ribonucleic acids with atom % (13)C enrichments greater than 10% can be isolated by equilibrium density centrifugation. The processing and analysis of isolated RNA by reverse transcription polymerase chain reaction, denaturing gradient gel electrophoresis, cloning and sequencing are discussed. The RNA-based stable isotope probing protocol presented here will find particular utility in assessing the roles of microbial community members in the biodegradation of natural and anthropogenic xenobiotic compounds.

Biomarkers↗