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Mike Manefield

Publications and source records attributed to Mike Manefield.

11 recordsLinked to original sources
PubMed2006

RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater.

Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [(13)C(6)]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the (13)C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [(14)C]benzene showed that the labeled carbon was mostly incorporated into (14)CO(2) within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.

Azoarcus↗
PubMed2005

Unlocking the 'microbial black box' using RNA-based stable isotope probing technologies.

Microbial ecologists have long sought to associate the transformation of compounds in the environment with the microbial clades responsible. The development of stable isotope probing (SIP) has made this possible in many ecological and biotechnological contexts. RNA-based SIP technologies represent a significant leap forward for culture-independent 'functional phylogeny' analyses, where specific consumption of a given compound carrying a (13)C signature can be associated with the small subunit ribosomal RNA molecules of the microbes that consume it. Recent advances have led to the unequivocal identification of microorganisms responsible for contaminant degradation in engineered systems, and to applications enhancing our understanding of carbon flow in terrestrial ecosystems.

Bacteria↗
PubMed2005

Colonial architecture in mixed species assemblages affects AHL mediated gene expression.

Many bacterial species produce metabolites that accumulate in the extracellular environment and induce specific transcriptional responses in producing cells. This phenomenon, most often referred to as quorum sensing, is thought to constitute a self-cell-density sensing mechanism allowing bacterial populations to alter gene expression in response to increases in their own density. Quorum sensing systems involving N-acyl-L-homoserine lactone (AHL) production and response are the most intensively investigated example. In this study we have employed a novel technique, known as dielectrophoresis, to investigate the impact of colonial architecture on the induction of AHL mediated gene expression. Using dielectrophoresis, we constructed artificial mixed species microcolonies with specific architectures. In this way, we were able to show that approximately 1000 Escherichia coli cells layered over an immobilised cluster of approximately 500 AHL responsive cells alters the response of this cluster to AHLs supplied either exogenously or endogenously. These findings lend credence to the hypothesis that the accumulation of extracellular metabolites signifies generic crowding in mixed species assemblages.

4-Butyrolactone↗
PubMed2005

Functional and compositional comparison of two activated sludge communities remediating coking effluent.

The success of engineered microbiological systems is evident in the global application of activated sludge communities to remediate coking effluent. However, there is a lack of understanding of the microbiology underlying treatment efficiency and stability. In this study, two functionally distinct activated sludge pools, treating the same effluent and operating under the same conditions, were examined to establish a relationship between overall diversity and/or functional diversity with respect to process stability. Molecular profiling, sequencing and RNA-based stable isotope probing were used to examine the bacterial diversity, general composition and functional composition of the most abundant members of the two communities. The inferior process stability in one of the pools could not be explained by reduced total bacterial diversity or evenness. RNA-based stable isotope probing revealed that both pools harboured an abundant phenol-degrading Acidovorax species, and that the pool of inferior stability accommodated an additional closely related phenol-degrading Acidovorax species at high abundance. These results are discussed in the context of deterministic and stochastic models of microbial community assembly.

Base Sequence↗
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

Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

Bacteria↗
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

Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors.

Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

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↗
PubMed2002

RNA stable isotope probing, a novel means of linking microbial community function to phylogeny.

Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [13C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus. Our results suggest that this organism is important for the function of this bioreactor.

Carbon Isotopes↗