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The antibacterial effects of a selection of volatile fatty acids (acetic, propionic, butyric, valeric, and caproic acids) relevant to anaerobic digestion were investigated at 1, 2 and 4 g/L. The antibacterial effects were characterised by the dynamics of Enterococcus faecalis NCTC 00775, Escherichia coli JCM 1649 and Klebsiella pneumoniae A17. Mesophilic anaerobic incubation to determine the minimum bactericidal concentration (MBC) and median lethal concentration of the VFAs was carried out in Luria Bertani broth at 37 °C for 48 h. Samples collected at times 0, 3, 6, 24 and 48 h were used to monitor bacterial kinetics and pH. VFAs at 4 g/L demonstrated the highest bactericidal effect ( p < 0.05), while 1 g/L supported bacterial growth. The VFA cocktail was the most effective, while propionic acid was the least effective. Enterococcus faecalis NCTC 00775 was the most resistant strain with the VFAs MBC of 4 g/L, while Klebsiella pneumoniae A17 was the least resistant with the VFAs MBC of 2 g/L. Allowing a 48 h incubation period led to more log decline in the bacterial numbers compared to earlier times. The VFA cocktail, valeric, and caproic acids at 4 g/L achieved elimination of the three bacteria strains, with over 7 log 10 decrease within 48 h.

The effects of the inoculum (anaerobic digestion effluent) to substrate (simulated food waste) ratio (ISR) 4.00 to 0.25 on putative pathogens and microbial kinetics during batch mesophilic anaerobic digestion were investigated. Red fluorescent protein labelled (RFPAKN132) Escherichia coli JM105 was introduced as a marker species, and together with the indigenous Clostridium sp., Enterococcus sp., Escherichia coli , and total coliforms were used to monitor pathogen death kinetics. Quantitative polymerase chain reaction was also used to estimate the bacterial, fungal, and methanogenic gene copies. All the ISRs eliminated E. coli and other coliforms (4 log 10 CFU/mL), but ISR 0.25 achieved this within the shortest time (≤2 days), while ISR 1.00 initially supported pathogen proliferation. Up to 1.5 log 10 CFU/mL of Clostridium was reduced by acidogenic conditions (ISR 0.25 and 0.50), while Enterococcus species were resistant to the digestion conditions. Fungal DNA was reduced (≥5 log 10 copies/mL) and was undetectable in ISRs 4.00, 2.00, and 0.50 at the end of the incubation period. This study has demonstrated that ISR influenced the pH of the digesters during batch mesophilic anaerobic digestion, and that acidic and alkaline conditions achieved by the lower (0.50 and 0.25) and higher (4.00 and 2.00) ISRs, respectively, were critical to the sanitisation of waste.

Mycobacterium avium subspecies paratuberculosis ( Map ) was monitored by quantitative PCR over a range of temporal and spatial scales in the River Tywi catchment. This study shows the persistence of Map over a 10-year period with little change, which correlates with the recognised levels of Johne's disease in British herds over that period (aim 1). Map was quantified within the river at up to 10 8 cell equivalents L -1 and was shown to be consistently present when monitored over finer timescales (aim 4). Small wastewater treatment plants where the ingress of human-associated Map might be expected had no significant effect (aim 2). Map was found for the first time to be located in natural river foams providing another route for spread via aerosols (aim 5). This study provides evidence for the environmental continuum of Map from the grazing infected animal via rain driven runoff through field drains and streams into main rivers; with detection at a high frequency throughout the year. Should Map need to be monitored in the future, we recommend that weekly or monthly sampling from a fixed location on a river will capture an adequate representation of the flow dynamics of Map in a catchment (aim 3). The human exposure to Map during this process and its impact on human health remains unquantified., Competing Interests: The authors declare no conflict of interest.

Three variants of the multidrug-resistant plasmid pLUH01 were assembled by deep sequencing from nasopharyngeal swabs. All have a 21-bp deletion in the RS14515 hypothetical gene. Variants 1 through 3 have 2, 6, and 3 nucleotide substitutions, respectively, compared to the pLUH01 reference genome. We named the new plasmid variants pLUH01/Lancaster/2015/1 to pLUH01/Lancaster/2015/3.

Microbial communities, associated with almost all metazoans, can be inherited from the environment. Although the honeybee ( Apis mellifera L.) gut microbiome is well documented, studies of the gut focus on just a small component of the bee microbiome. Other key areas such as the comb, propolis, honey, and stored pollen (bee bread) are poorly understood. Furthermore, little is known about the relationship between the pollinator microbiome and its environment. Here we present a study of the bee bread microbiome and its relationship with land use. We estimated bacterial community composition using both Illumina MiSeq DNA sequencing and denaturing gradient gel electrophoresis (DGGE). Illumina was used to gain a deeper understanding of precise species diversity across samples. DGGE was used on a larger number of samples where the costs of MiSeq had become prohibitive and therefore allowed us to study a greater number of bee breads across broader geographical axes. The former demonstrates bee bread comprises, on average, 13 distinct bacterial phyla; Bacteroidetes , Firmicutes , Alpha-proteobacteria , Beta-proteobacteria, and Gamma-proteobacteria were the five most abundant. The most common genera were Pseudomonas , Arsenophonus , Lactobacillus , Erwinia, and Acinetobacter . DGGE data show bacterial community composition and diversity varied spatially and temporally both within and between hives. Land use data were obtained from the 2007 Countryside Survey. Certain habitats, such as improved grasslands, are associated with low diversity bee breads, meaning that these environments may be poor sources of bee-associated bacteria. Decreased bee bread bacterial diversity may result in reduced function within hives. Although the dispersal of microbes is ubiquitous, this study has demonstrated landscape-level effects on microbial community composition.

Sufficiently diverse and abundant resources are essential for generalist consumers, and form an important part of a suite of conservation strategies for pollinators. Honey bees are generalist foragers and are dependent on diverse forage to adequately meet their nutritional needs. Through analysis of stored pollen (bee bread) samples obtained from 26 honey bee (Apis mellifera L.) hives across NW-England, we quantified bee bread nutritional content and the plant species that produced these stores from pollen. Protein was the most abundant nutrient by mass (63%), followed by carbohydrates (26%). Protein and lipid content (but not carbohydrate) contributed significantly to ordinations of floral diversity, linking dietary quality with forage composition. DNA sequencing of the ITS2 region of the nuclear ribosomal DNA gene identified pollen from 89 distinct plant genera, with each bee bread sample containing between 6 and 35 pollen types. Dominant genera included dandelion (Taraxacum), which was positively correlated with bee bread protein content, and cherry (Prunus), which was negatively correlated with the amount of protein. In addition, proportions of amino acids (e.g. histidine and valine) varied as a function of floral species composition. These results also quantify the effects of individual plant genera on the nutrition of honey bees. We conclude that pollens of different plants act synergistically to influence host nutrition; the pollen diversity of bee bread is linked to its nutrient content. Diverse environments compensate for the loss of individual forage plants, and diversity loss may, therefore, destabilize consumer communities due to restricted access to alternative resources.

Nasopharyngeal swabs were taken from volunteers attending a general medical practice and a general hospital in Lancaster, UK, and at Lancaster University, in the winter of 2014-2015. 51 swabs were selected based on high RNA yield and allocated to deep sequencing pools as follows: patients with chronic obstructive pulmonary disease; asthmatics; adults with no respiratory symptoms; adults with feverish respiratory symptoms; adults with respiratory symptoms and presence of antibodies against influenza C; paediatric patients with respiratory symptoms (2 pools); adults with influenza C infection (2 pools), giving a total of 9 pools. Illumina sequencing was performed, with data yields per pool in the range of 345.6 megabases to 14 gigabases after removal of reads aligning to the human genome. The data were deposited in the Sequence Read Archive at NCBI, and constitute a resource for study of the viral, bacterial and fungal metagenome of the human nasopharynx in healthy and diseased states and comparison with other metagenomic studies on the human respiratory tract.

The genome sequence of human papillomavirus type 20 (HPV-20; family Papillomaviridae , genus Betapapillomavirus , species Betapapillomavirus 1 , type 20) was assembled by deep sequencing from nasopharyngeal swabs. The assembled genome is 0.37% divergent over its full length from the single complete genome of HPV-20 in GenBank (U31778). We named the strain HPV-20/Lancaster/2015., (Copyright © 2017 Atkinson et al.)

The genome of human papillomavirus type 23 (HPV-23; family Papillomaviridae , genus Betapapillomavirus , species Betapapillomavirus 2 , type 23) was assembled by deep sequencing from nasopharyngeal swabs. The assembled genome is 2.7% divergent over its full length from the single complete genome of HPV-23 in GenBank (accession no. U31781). We named the strain HPV-23/Lancaster/2015., (Copyright © 2017 Atkinson et al.)

Influenza C is not included in the annual seasonal influenza vaccine, and has historically been regarded as a minor respiratory pathogen. However, recent work has highlighted its potential role as a cause of pneumonia in infants. We performed nasopharyngeal or nasal swabbing and/or serum sampling (n = 148) in Lancaster, UK, over the winter of 2014-2015. Using enzyme-linked immunosorbent assay (ELISA), we obtain seropositivity of 77%. By contrast, only 2 individuals, both asymptomatic adults, were influenza C-positive by polymerase chain reaction (PCR). Deep sequencing of nasopharyngeal samples produced partial sequences for 4 genome segments in one of these patients. Bayesian phylogenetic analysis demonstrated that the influenza C genome from this individual is evolutionarily distant to those sampled in recent years and represents a novel genome constellation, indicating that it may be a product of a decades-old reassortment event. Although we find no evidence that influenza C was a significant respiratory pathogen during the winter of 2014-2015 in Lancaster, we confirm previous observations of seropositivity in the majority of the population. (170 words).

The genome of human rhinovirus A22 (HRV-A22) was assembled by deep sequencing RNA samples from nasopharyngeal swabs. The assembled genome is 8.7% divergent from the HRV-A22 reference strain over its full length, and it is only the second full-length genome sequence for HRV-A22. The new strain is designated strain HRV-A22/Lancaster/2015., (Copyright © 2017 Atkinson et al.)

Background: Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging infection control problem in hospitals worldwide. Identifying carriers may help reduce potential spread and infections., Aim: To assess whether testing hospital wastewater for CPE can supplement patient-based screening for infection prevention purposes in a hospital without a recognized endemic CPE problem., Methods: Wastewater collected from hospital pipework on 16 occasions during February to March 2014 was screened for CPE using chromID(®) CARBA agar and chromID(®) CPS agar with a 10μg ertapenem disc and combination disc testing. Minimum inhibitory concentrations were determined using British Society for Antimicrobial Chemotherapy methodology and carbapenemase genes detected by polymerase chain reaction or whole-genome sequencing. Selected isolates were typed by pulsed-field gel electrophoresis., Findings: Suspected CPE were recovered from all 16 wastewater samples. Of 17 isolates sent to the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, six (four Citrobacter freundii and two Enterobacter cloacae complex) were New Delhi metallo-β-lactamase (NDM) producers and the remaining 11 (six Klebsiella oxytoca and five Enterobacter cloacae complex) were Guiana-Extended-Spectrum-5 (GES-5) producers, the first to be described among Enterobacteriaceae in the UK. The four NDM-producing C. freundii, two NDM-producing E. cloacae complex, and four out of five GES-5-producing E. cloacae complex were each indistinguishable isolates of the same three strains, whereas the six GES-5-producing K. oxytoca overall shared 79% similarity., Conclusion: CPE are readily isolated from hospital wastewater using simple culture methods. There are either undetected carriers of CPE excreting into the wastewater, or these CPE represent colonization of the pipework from other sources. Surveillance of hospital wastewater for CPE does not appear helpful for infection control purposes within acute hospitals., (Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Assessing metal bioavailability in soil is important in modeling the effects of metal toxicity on the surrounding ecosystem. Current methods based on diffusive gradient thin films (DGTs) and Gel-Integrated Microelectrode are limited in their availability and sensitivity. To address this, Shewanella oneidensis, an anaerobic iron reducing bacterium, was incorporated into a thin layer of agarose to replace the polyacrylamide gel that is normally present in DGT to form biologically mobilizing DGT (BMDGT). Viability analysis revealed that 16-35% of the cells remained viable within the BMDGTs depending on the culturing conditions over a 20 h period with/without metals. Deployment of BMDGTs in standardized metal solutions showed significant differences to cell-free BMDGTs when cells grown in Luria Broth (LB) were incorporated into BMDGTs and deployed under anaerobic conditions. Deployment of these BMDGTs in hematite revealed no significant differences between BMDGTs and BMDGTs containing heat killed cells. Whether heat killed cells retain the ability to affect bioavailability is uncertain. This is the first study to investigate how a microorganism that was incorporated into a DGT device such as the metal reducing bacteria, S. oneidensis, may affect the mobility of metals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Very little is known about the normal gastrointestinal flora of wild birds, or how it might affect or reflect the host's life-history traits. The aim of this study was to survey the species richness of bacteria in the feces of a wild population of blue tits Cyanistes caeruleus and to explore the relationships between bacterial species richness and various life-history traits, such as age, sex, and reproductive success. Using PCR-TGGE, 55 operational taxonomic units (OTUs) were identified in blue tit feces. DNA sequencing revealed that the 16S rRNA gene was amplified from a diverse range of bacteria, including those that shared closest homology with Bacillus licheniformis, Campylobacter lari, Pseudomonas spp., and Salmonella spp. For adults, there was a significant negative relationship between bacterial species richness and the likelihood of being detected alive the following breeding season; bacterial richness was consistent across years but declined through the breeding season; and breeding pairs had significantly more similar bacterial richness than expected by chance alone. Reduced adult survival was correlated with the presence of an OTU most closely resembling C. lari; enhanced adult survival was associated with an OTU most similar to Arthrobacter spp. For nestlings, there was no significant change in bacterial species richness between the first and second week after hatching, and nestlings sharing the same nest had significantly more similar bacterial richness. Collectively, these results provide compelling evidence that bacterial species richness was associated with several aspects of the life history of their hosts.

Declines in insect pollinators in Europe have been linked to changes in land use. Pollinator nutrition is dependent on floral resources (i.e., nectar and pollen), which are linked to landscape composition. Here, we present a stratified analysis of the nutritional composition of beebread in managed honeybee hives with a view to examining potential sources of variation in its nutritional composition. Specifically, we tested the hypothesis that beebread composition correlates with local land use and therefore available floral resources. The results demonstrated that the starch, lipid, and moisture contents of beebread are all highly conserved across hives, whereas levels of protein and nonreducing sugar increased as the year progressed, reducing sugars, however, decreased during the first half of the year and then increased toward the end. Local land use around hives was quantified using data from the Countryside Survey 2007 Land Cover Map. Bee-bread protein content was negatively correlated with increasing levels of arable and horticultural farmland surrounding hives and positively correlated with the cover of natural grasslands and broadleaf woodlands. Reducing sugar content was also positively correlated with the amount of broad-leaved woodland in a 3 Km² radius from the hives. Previous studies on a range of invertebrates, including honeybees, indicate that dietary protein intake may have a major impact on correlates of fitness, including longevity and immune function. The finding that beebread protein content correlates with land use suggests that landscape composition may impact on insect pollinator well-being and provides a link between landscape and the nutritional ecology of socially foraging insects in a way not previously considered.

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1-FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1-4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the 'variable side' with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILE(FH1) and ILE(FH5)) and the NR-II virulence region of genomic island PAGI-5 (ILE(FH4)). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In the first comprehensive geographical survey of distribution in Great Britain, Mycobacterium avium ssp. paratuberculosis (MAP) was detected in 115 of 1092 (10.5%) soil cores, in the range of 5 × 10(2) to 3 × 10(6) MAP cell equivalents (CE) g(-1) wet weight soil with the majority of the positive PCR reactions (n = 75; 65%) occurring around the limit of detection (500-5000 CE g(-1) wet weight soil). The distribution of MAP significantly increased from North to South and was significantly correlated with increasing cattle numbers over the same longitudinal axis. Similarly MAP occurrence significantly increased towards easterly latitudes although none of the parameters measured were associated. Comparisons of land use indicated that MAP was widely distributed in both farming and non-farming areas. Soil core samples taken from the rivers Wyre and Douglas catchments (Lancashire, UK) and river Tywi (South Wales) were negative for MAP. However, river monitoring showed a consistent presence of MAPs throughout those catchments over a 6-month period. We concluded that MAP is widely distributed within and outside the confines of the farming environment; its geographical distribution is wider than originally anticipated and; monitoring rivers describes the MAP status of catchment better than individual soil samples., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Polluted aquifers contain indigenous microbial communities with the potential for in situ bioremediation. However, the effect of hydrogeochemical gradients on in situ microbial communities (especially at the plume fringe, where natural attenuation is higher) is still not clear. In this study, we used culture-independent techniques to investigate the diversity of in situ planktonic and attached bacterial communities in a phenol-contaminated sandstone aquifer. Within the upper and lower plume fringes, denaturing gradient gel electrophoresis profiles indicated that planktonic community structure was influenced by the steep hydrogeochemical gradient of the plume rather than the spatial location in the aquifer. Under the same hydrogeochemical conditions (in the lower plume fringe, 30 m below ground level), 16S rRNA gene cloning and sequencing showed that planktonic and attached bacterial communities differed markedly and that the attached community was more diverse. The 16S rRNA gene phylogeny also suggested that a phylogenetically diverse bacterial community operated at this depth (30 mbgl), with biodegradation of phenolic compounds by nitrate-reducing Azoarcus and Acidovorax strains potentially being an important process. The presence of acetogenic and sulphate-reducing bacteria only in the planktonic clone library indicates that some natural attenuation processes may occur preferentially in one of the two growth phases (attached or planktonic). Therefore, this study has provided a better understanding of the microbial ecology of this phenol-contaminated aquifer, and it highlights the need for investigating both planktonic and attached microbial communities when assessing the potential for natural attenuation in contaminated aquifers.

The composition and dynamics of the gastrointestinal bacterial communities in birds is determined by both host-specific and environmental exposure factors yet these are poorly understood. We selected the zebra finch, Taeniopygia guttata, as the host species to examine the diversity and temporal stability of the faecal microflora in a bird, owing to its importance as a model organism in avian ecology, neuroscience and evolution studies. The stability of the gut bacterial community of individual male and female zebra finches was assessed through repeat faecal sampling via culture and temperature gradient gel electrophoresis and partial sequencing of PCR-amplified eubacterial 16S rRNA gene products. Nineteen bacterial genera were detected across all samples (n = 99), with each bird carrying on average six operational taxonomic units. Using a novel statistical approach, we showed that bacterial assemblages and community richness varied between individual birds but remained stable over time within individuals. Neither the composition nor richness of bacterial communities differed significantly between the sexes. Our results show that zebra finches housed together under controlled conditions show consistent variation between individuals in their gut microflora that is not attributable to differences in host exposure to environmental microbial sources. Future studies could usefully explore the origin of this individual-specific variation and its consequences for host fitness and sexual selection., (© 2010 Blackwell Publishing Ltd.)

The possible effects of changing climate on a southern and a north-eastern English river (the Thames and the Yorkshire Ouse, respectively) were examined in relation to water and ecological quality throughout the food web. The CLASSIC hydrological model, driven by output from the Hadley Centre climate model (HadCM3), based on IPCC low and high CO(2) emission scenarios for 2080 were used as the basis for the analysis. Compared to current conditions, the CLASSIC model predicted lower flows for both rivers, in all seasons except winter. Such an outcome would lead to longer residence times (by up to a month in the Thames), with nutrient, organic and biological contaminant concentrations elevated by 70-100% pro-rata, assuming sewage treatment effectiveness remains unchanged. Greater opportunities for phytoplankton growth will arise, and this may be significant in the Thames. Warmer winters and milder springs will favour riverine birds and increase the recruitment of many coarse fish species. However, warm, slow-flowing, shallower water would increase the incidence of fish diseases. These changing conditions would make southern UK rivers in general a less favourable habitat for some species of fish, such as the Atlantic salmon (Salmo salar). Accidental or deliberate, introductions of alien macrophytes and fish may change the range of species in the rivers. In some areas, it is possible that a concurrence of different pressures may give rise to the temporary loss of ecosystem services, such as providing acceptable quality water for humans and industry. An increasing demand for water in southern England due to an expanding population, a possibly reduced flow due to climate change, together with the Water Framework Directive obligation to maintain water quality, will put extreme pressure on river ecosystems, such as the Thames.

A quantitative real-time 5'-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4x10(1) and 1.9x10(4) cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.

The role of environmental selection in governing the structure of communities of freshwater sulfur bacteria (Achromatium spp) was experimentally tested by mixing sediments from two geographically separated lakes (Rydal Water (RY) and Hell Kettles (HK)) that harboured Achromatium spp. Community profiles of Achromatium spp in sediment microcosms at day 0 and after 60 days were compared to determine whether initial Achromatium community composition or subsequent selection by the sediment environment had greater influence in dictating the final Achromatium community structure. It was found that Achromatium spp from the HK community became established in mixed sediments at the expense of members of the RY community. This selection for the HK Achromatium community was more pronounced when sediment composition was manipulated to resemble HK sediments. Our findings definitively demonstrate that environmental selection is the primary determinant of Achromatium community structure in these sediments.

Flow reactors containing quartz sand colonized with biofilm were set up as physical model aquifers to allow degrading plumes of acetate or phenol to be formed from a point source. A noninvasive fluorescent tracer technique was combined with chemical and biological sampling in order to quantify transport and biodegradation processes. Chemical analysis of samples showed a substantial decrease in carbon concentration between the injection and outflow resulting primarily from dilution but also from biodegradation. Two-dimensional imaging of the aqueous oxygen [O2(aq)] concentration field quantified the depletion of O2(aq) within the contaminant plume and provided evidence for microbial respiration associated with biodegradation of the carbon source. Combined microbiological, chemical, and O2(aq) imaging data indicated that biodegradation was greatest at the plume fringe. DNA profiles of bacterial communities were assessed by temperature gradient gel electrophoresis, which revealed that diversity was limited and that community changes observed depended on the carbon source used. Spatial variation in activity within the plume could be quantitatively accounted for by the changes observed in active cell numbers rather than differences in community structure, the total biomass present, or the increased enzyme activity of individual cells. Numerical simulations and comparisons with the experimental data were used to test conceptual models of plume processes. Results demonstrated that plume behavior was best described by growth and decay of active biomass as a single functional group of organisms represented by active cell counts.

The fermentation process is an important component in the biodegradation of organic compounds in natural and contaminated systems. Comparing with terminal electron-accepting processes (TEAPs), however, research on fermentation processes has to some extent been ignored in the past decades, particularly on the persistence of fermentation process in the presence of toxic organic pollutants. Both field and laboratory studies, presented here, showed that microbial processes in a groundwater-based system exhibited a differential inhibitory response to toxicity of phenolic compounds from coal tar distillation, thus resulting in the accumulation of volatile fatty acids (VFAs) and hydrogen. This indicated that fermentation processes could be more resistant to phenol toxicity than the subsequent TEAPs such as methanogenesis and sulfate reduction, thus providing us with more options for enhancing bioremediation processes.

Mycobacterium avium subsp. paratuberculosis from infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km2 containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS900 PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests for M. avium subsp. paratuberculosis in the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive for M. avium subsp. paratuberculosis. Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations.

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).

Viruses are the most abundant biological entities in aquatic systems. Temperate bacteriophages have enormous influences on microbial diversity, genetic exchange and bacterial population dynamics. However, development of molecular tools for their detection in the environment has been problematic. The integrase gene is used here as a molecular marker to analyse the diversity of temperate bacteriophages in a population of freshwater bacteria. Interrogation of the GenBank database revealed 32 non-cryptic enteric phage integrase sequences, leading to the development of a suite of 11 degenerate primer sets specific to the extant sequences elucidated. Application of these primer sets to enterobacterial isolates recovered from a freshwater pond and the temperate phages induced from them revealed a number of diverse integrase genes, including novel integrase-like sequences not represented in the databases. This highlights the potential of utilizing the integrase gene family as a marker for phage diversity.

Temporal temperature gradient electrophoretic (TTGE) analysis of 16S rDNA sequences was optimized to monitor the methanogen population present in water and sediments of a small eutrophic lake, Priest Pot, in the English Lake district. The production of nonrepresentative TTGE profiles due to the generation of polymerase chain reaction (PCR) artifacts initially proved problematical. The use of a proofreading polymerase in the PCR was found to be essential and fully optimized protocols were established and tested to ensure confidence that the TTGE profiles truly reflected sequence diversity. TTGE analysis revealed the methanogen population to be less diverse in water than in sediment. The most genetic diversity was observed in TTGE profiles of sediment DNA isolated in winter and the least was in sediment DNA isolated in summer. DNA sequencing analysis of bands recovered from TTGE gels revealed the presence of two methanogen communities. One clustered with Methanosaeta species and the other with the Methanomicrobiales. Many sequences showed low DNA sequence similarity to known methanogens, suggesting that Priest Pot harbors previously undescribed methanogen species.

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river.

This study presents the first complete sequence of an IncU plasmid, pFBAOT6. This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G. Rhodes, G. Huys, J. Swings, P. McGann, M. Hiney, P. Smith, and R. W. Pickup, Appl. Environ. Microbiol. 66:3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity. pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed. Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid. The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium. The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria. Partitioning and maintenance genes are homologues of genes in IncP plasmids. The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102. The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS630 relative, and other transposable elements in a 43-kb region that may be a novel Tn1721-flanked composite transposon. This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria. The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.

Changes in the abundance of sympatric Achromatium spp. in response to the artificial manipulation of redox conditions in sediment microcosms was determined by fluorescence in situ hybridization (FISH). Adaptation to different redox conditions was shown to be one mechanism that supported the coexistence of functionally similar Achromatium spp. In sediment microcosms, in which the overlying water was oxygenated, Achromatium community size and composition remained unchanged over time. However, imposition of anoxic conditions induced changes in community structure. Anoxia caused a reduction in the relative abundance of Achromatium sp. RY8 (72 +/- 4% to 49 +/- 2%) and an increase in Achromatium sp. RY5 (19 +/- 5% to 32 +/- 3%) and a newly identified Achromatium sp., RYKS (14 +/- 4% to 27 +/- 2%). In anoxic microcosms supplemented with a single addition of nitrate at different initial concentrations the relative decline in Achromatium sp. RY8 was dependent on the initial nitrate concentration. In these experiments nitrate was rapidly removed. In contrast, when high levels of nitrate were maintained by periodic replacement of the overlying water with nitrate supplemented anoxic water, the composition of the Achromatium community remained stable over time. This suggested that all of the coexisting Achromatium spp. are obligate or facultative anaerobes, but, Achromatium sp. RY8 was more sensitive to sediment redox conditions than the other Achromatium species. Given the heterogeneous nature of sedimentary environments, redox-related niche differentiation may promote coexistence of sympatric Achromatium spp.

The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolium salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.

The incidence and diversity of methanogens in Priest Pot, a dynamic and active lake, were monitored by analysing mcrA gene sequences generated from total DNA samples obtained at different times of the year and amplified using the polymerase chain reaction. A number of mcrA clones were analysed by developing an RFLP-based protocol to generate a number of restriction patterns that were assigned to a number of classes. The RFLP patterns for each class were compared with published sequence information for mcrA from cultured methanogens as well as with those from other experimental studies. They could be used to assign tentative identification for some of the Priest Pot clones and also revealed the presence of a number of clones that could not be affiliated to any known methanogens. The limitations of using RFLP profiles of mcrA gene sequences for studying methanogen ecology are discussed.

The application of advanced and highly sensitive molecular techniques to the detection of specific bacteria in the freshwater environment is limited, in the first instance, by sampling strategy and sample quality. Further combinations of molecular methods and techniques from apparently unrelated disciplines will ultimately shape the monitoring techniques of the future.

The distillation of acidified coal tars for up to 50 years has given rise to a phenol plume approximately 500 m long, 50 m deep and containing up to 15 g l(-1) dissolved organic carbon (DOC) in the Triassic Sandstones aquifer. A conceptual biogeochemical model based on chemical and microbiological analysis of groundwater samples has been developed as a preliminary to more detailed studies of the controls on natural attenuation. While the development of redox zones and the production of methane and carbon dioxide provide evidence of natural attenuation, it appears that degradation is slow. The existence of sulphate in the plume indicates that this electron acceptor has not been depleted and that consequently methanogenesis is probably limited. Based on a simple estimate of sulphate input concentration, a half-life of about 15 years has been estimated for sulphate reduction. Geochemical modelling predicts that increased alkalinity within the plume has not led to carbonate precipitation, and thus within the limits of accuracy of the measurement, alkalinity may reflect the degree of biodegradation. This implies a loss of around 18% of the DOC over a 30-year period. Despite limited degradation, microbial studies show that there are diverse microbial communities in the aquifer with the potential for both anaerobic and aerobic biodegradation. Microbial activity was found to be greatest at the leading edge of the plume where DOC concentrations are 60 mg l(-1) or less, but activity could still be observed in more contaminated samples even though cells could not be cultured. The study suggests that degradation may be limited by the high phenol concentrations within the core of the plume, but that once diluted by dispersion, natural attenuation may proceed. More detailed studies to confirm these initial findings are identified and form the basis of associated papers.

A range of bacteriological, geochemical process-related and molecular techniques have been used to assess the microbial biodegradative potential in groundwater contaminated with phenol and other tar acids. The contaminant plume has travelled 500 m from the pollutant source over several decades. Samples were obtained from the plume using a multi-level sampler (MLS) positioned in two boreholes (boreholes 59 and 60) which vertically transected two areas of the plume. Activity of the microbial community, as represented by phenol degradation potential and ability to utilise a range of substrates, was found to be influenced by the plume. Phenol degradation potential appeared to be influenced more by the concentration of the contaminants than the total bacterial cell numbers. However, in the areas of highest phenol concentration, the depression of cell numbers clearly had an effect. The types of bacteria present were assessed by culture and DNA amplification by polymerase chain reaction (PCR). Bacterial groups or processes associated with major geochemical processes, such as methanogenesis, sulphate reduction and denitrification, that have the potential to drive contaminant degradation, were detected at various borehole levels. A comparative molecular analysis of the microbial community between samples obtained from the MLS revealed the microbial community was diverse. The examination of microbial activity complemented those results obtained through chemical analysis, and when combined with hydrological data, showed that MLS samples provided a realistic profile of plume effects and could be related to the potential for natural attenuation of the site.

A combination of PCR amplification and oligonucleotide probing was used to investigate the populations of ammonia-oxidisers of the beta-Proteobacteria in the eutrophic and oligotrophic basins of Lake Windermere, a large temperate lake in the English Lake District. Numbers of ammonia-oxidisers (MPN) in the Windermere lakewater were low (< 100 cells ml(-1)) throughout the year with the exception of peaks in August, which coincided with stratification, and November in the South Basin where overturn may have introduced ammonia-oxidising bacteria into the water column. Sediment samples contained larger populations of ammonia oxidisers, usually ca. 10(4) per g. dry weight, which remained relatively constant throughout the seasonal cycle in both Basins. DNA was recovered from lakewater and sediment samples and Nitrosospira and N. europaea-eutropha lineage 16S rRNA genes amplified in a nested PCR reaction, with confirmation of identity by oligonucleotide hybridisation. Nitrosospira 16S rDNA was readily detected in all samples and therefore found to be ubiquitous. In contrast, nitrosomonad DNA of the N. europaea-eutropha lineage could only be detected in the oligotrophic North Basin. Enrichment cultures of lakewater samples only exhibited nitrification at low (0.67 mM) and medium (5 mM) ammonium concentrations, whilst sediment enrichments nitrified at all concentrations tested including high (12.5 mM) ammonium medium. These data suggest that ammonia-oxidiser populations may be physiologically distinguished between lakewater and sediment, and that species distribution in a single lake is non-uniform.

Combined microautoradiography and fluorescence in situ hybridization (FISH) was used to investigate carbon metabolism in uncultured bacteria from the genus Achromatium. All of the Achromatium species identified in a freshwater sediment from Rydal Water, Cumbria, United Kingdom, which were distinguishable only by FISH, assimilated both [(14)C]bicarbonate and [(14)C]acetate. This extends previous findings that Achromatium spp. present at another location could only utilize organic carbon sources. Achromatium spp., therefore, probably exhibit a range of physiologies, i.e., facultative chemolithoautotrophy, mixotrophy, and chemoorganoheterotrophy, similar to other large sulfur bacteria (e.g., Beggiatoa spp.).

The distribution of insertion sequence 1 (IS1)-containing bacteria was investigated in Windermere (Cumbria, UK), a freshwater body impacted by treated sewage discharge and run-off from the surrounding catchment. Culturable IS1-containing bacteria were recovered from the water column at three depths in Windermere North Basin (WNB) and South Basin (WSB), and from sediment at both sites (at the sediment surface in WSB and to a depth of 12-13 cm in WNB). Polymerase chain reaction amplification of IS1 and the Escherichia coli/Shigella sp. specific gene uidA, from community DNA from shallow sediments, extended the detection limit beyond that of culture at both sites. This detection was extended further into deep sediment extracted from WNB as IS1 and uidA were detected in sub-samples to a depth of 4.7 and 2.3 m, respectively. Analysis of a representative subset of 90 IS1-carrying isolates recovered from water and sediment at both sites demonstrated 21 heterogeneous IS1 profiles with estimated copy numbers ranging from 1 to 16. Identification of the host bacteria showed that the element was confined mainly to Enterobacter spp. However, this study showed IS1 to be present in Citrobacter freundii for the first time. Plasmids were carried by 75.3% of enterobacterial isolates and four plasmids (2.6%) carried IS1. DNA sequence analysis of five IS1 clones demonstrated that IS1 isoforms from this study were similar (>89% nucleotide identity) to known IS1 isoforms. Two isoforms of IS1 from a single Enterobacter cloacae isolate differed by 6.7% at the nucleotide level suggesting that they had been acquired independently.

Oxytetracycline-resistant (OT(r)) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England. The transfer of OT(r) plasmids from these isolates was investigated. Using Escherichia coli J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OT(r) plasmids (designated pFBAOT1 to 17). Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either Aeromonas hydrophila HG3 (eight isolates), A. veronii b.v. sobria HG8 (six isolates), and A. caviae HGB5 (one isolate). One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level. Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G. Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7 tet gene determinants remaining unclassified. In all cases, Tet A was located on a 5.5-kb EcoRI restriction fragment. Hybridization with inc-rep probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids. Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OT(r) plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from A. salmonicida strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital E. coli strain). In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other. The presence of tetracycline resistance transposon Tn1721 in its entirety or in a truncated form in these plasmids was demonstrated. These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different Aeromonas species and E. coli and between the human and aquaculture environments in distinct geographical locations. Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment.

Microorganisms in natural environments have often been treated as 'black box' systems. Researchers have measured the inputs and outputs of the box, and have made bulk measurements on cell behaviour. However, unravelling the details of the diversity and interactions that exist within these microbial populations has proven exceptionally difficult. The information gained from the black box approach has been invaluable, and has allowed models of global foodwebs to be generated and tested. However, there is still little information about the interactions of individual microbial cells within natural populations. Such studies are essential to fully understand the integrated functioning of ecosystems. To achieve this goal, researchers need to be able to identify individual cells within a population, enumerate them, estimate both viability and activity, and monitor changes in response to relevant parameters. Due to the diversity, heterogeneity and numbers of cells that make up these populations, these measurements require automation and speed. At present, the use of flow cytometry in conjunction with nucleic acid probes provides an excellent method with which to pursue such studies.

The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HIN:dIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HIN:dIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 72.6% identity to a region of the Escherichia coli chromosome at 43.3', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.

Homology to IncN, P, Q and W inc regions was investigated amongst 114 Hg(2+)-resistant or antibiotic-resistant bacteria isolated from lakewater sediments. No hybridisation signals were found with Inc P, Q and W probes, and only one plasmid, pLV1402, hybridised to the IncN probe. PCR primers designed to conserved regions in the replicon of the IncN plasmid pCU1 and the related beta replicon from pGSH500 were used to amplify a 978-bp fragment from pLV1402, with sequence analysis showing a close relationship (99.2% identity) between their replication genes. A 387-bp region from the pLV1402 rep gene was used to re-screen the isolates and identified another related plasmid, pLV1403. A 3.7-kb probe containing the alpha replicon from pGSH500 hybridised to both pLV1402 and pLV1403, suggesting that both are multi-replicon plasmids. The PCR primers and probes described will be useful in future studies of plasmid diversity.

The diversity and ecology of natural communities of the uncultivated bacterium Achromatium oxaliferum were studied by use of culture-independent approaches. 16S rRNA gene sequences were PCR amplified from DNA extracted from highly purified preparations of cells that were morphologically identified as A. oxaliferum present in freshwater sediments from three locations in northern England (Rydal Water, Jenny Dam, Hell Kettles). Cloning and sequence analysis of the PCR-amplified 16S rRNA genes revealed that multiple related but divergent sequences were routinely obtained from the A. oxaliferum communities present in all the sediments examined. Whole-cell in situ hybridization with combinations of fluorescence-labelled oligonucleotide probes revealed that the divergent sequences recovered from purified A. oxaliferum cells corresponded to genetically distinct Achromatium subpopulations. Analysis of the cell size distribution of the genetically distinct subpopulations demonstrated that each was also morphologically distinct. Furthermore, there was a high degree of endemism in the Achromatium sequences recovered from different sediments; identical sequences were never recovered from different sampling locations. In addition to ecological differences that were apparent between Achromatium communities from different freshwater sediments, the distribution of different subpopulations of Achromatium in relation to sediment redox profiles indicated that the genetically and morphologically distinct organisms that coexisted in a single sediment were also ecologically distinct and were adapted to different redox conditions. This result suggests that Achromatium populations have undergone adaptive radiation and that the divergent Achromatium species occupy different niches in the sediments which they inhabit.

The population of ammonia-oxidizing bacteria in a temperate oligotrophic freshwater lake was analyzed by recovering 16S ribosomal DNA (rDNA) from lakewater and sediment samples taken throughout a seasonal cycle. Nitrosospira and Nitrosomonas 16S rRNA genes were amplified in a nested PCR, and the identity of the products was confirmed by oligonucleotide hybridization. Nitrosospira DNA was readily identified in all samples, and nitrosomonad DNA of the Nitrosomonas europaea-Nitrosomonas eutropha lineage was also directly detected, but during the summer months only. Phylogenetic delineation with partial (345 bp) 16S rRNA gene sequences of clones obtained from sediments confirmed the fidelity of the amplified nitrosomonad DNA and identified two sequence clusters closely related to either N. europaea or N. eutropha that were equated with the littoral and profundal sediment sites, respectively. Determination of 701-bp sequences for 16S rDNA clones representing each cluster confirmed this delineation. A PCR-restriction fragment length polymorphism (RFLP) system was developed that enabled identification of clones containing N. europaea and N. eutropha 16S rDNA sequences, including subclasses therein. It proved possible to analyze 16S rDNA amplified directly from sediment samples to determine the relative abundance of each species compared with that of the other. N. europaea and N. eutropha are very closely related, and direct evidence for their presence in lake systems is limited. The correlation of each species with a distinct spatial location in sediment is an unusual example of niche adaptation by two genotypically similar bacteria. Their occurrence and relative distribution can now be routinely monitored in relation to environmental variation by the application of PCR-RFLP analysis.

Microautoradiography was used to investigate substrate uptake by natural communities of uncultured bacteria from the genus Achromatium. Studies of the uptake of (14)C-labelled substrates demonstrated that Achromatium cells from freshwater sediments were able to assimilate (14)C from bicarbonate, acetate, and protein hydrolysate; however, (14)C-labelled glucose was not assimilated. The pattern of substrate uptake by Achromatium spp. was therefore similar to those of a number of other freshwater and marine sulfur-oxidizing bacteria. Different patterns of radiolabelled bicarbonate uptake were noted for Achromatium communities from different geographical locations and indicated that one community (Rydal Water) possessed autotrophic potential, while the other (Hell Kettles) did not. Furthermore, the patterns of organic substrate uptake within a single population suggested that physiological diversity existed in natural communities of Achromatium. These observations are consistent with and may relate to the phylogenetic diversity observed in Achromatium communities. Incubation of Achromatium-bearing sediment cores from Rydal Water with (35)S-labelled sulfate in the presence and absence of sodium molybdate demonstrated that this bacterial population was capable of oxidizing sulfide to intracellular elemental sulfur. This finding supported the role of Achromatium in the oxidative component of a tightly coupled sulfur cycle in Rydal Water sediment. The oxidation of sulfide to sulfur and ultimately to sulfate by Achromatium cells from Rydal Water sediment is consistent with an ability to conserve energy from sulfide oxidation.

Abstract Methane production and methane oxidation potential were measured in a 30 cm peat core from the Moorhouse Nature Reserve, UK. The distribution of known groups of methanogens and methane oxidizing bacteria throughout this peat core was assessed. Using 16S rRNA gene retrieval and functional gene probing with genes encoding key proteins in methane oxidation and methanogenesis, several major groups of microorganisms were detected. Methane production and oxidation was detected in all depths of the peat core. PCR amplification and oligonucleotide probing experiments using DNA isolated from all sections of the peat core detected methanotrophs from the groups Methylosinus and Methylococcus and methanogens from the groups Methanosarcinaceae, Methanococcaceae, and Methanobacteriaceae. 16S rDNA sequences amplified with the Methylosinus-specific primer were shown to have a high degree of identity with 16S rDNA sequences previously detected in acidic environments. However, no methanogen sequences were detected by the probes available in this study in the sections of the peat core (above 7 cm) where the majority of methanogenesis occurred, either because of low methanogen numbers or because of the presence of novel methanogen sequences.http://link.springer-ny.com/link/service/journals/00248/bibs/38n3p225.html

The relationship of mixotrophic and autotrophic Thiothrix species to morphologically similar chemoorganotrophic bacteria (e.g. Leucothrix species, Eikelboom type 021N bacteria) has been a matter of debate for some years. These bacteria have alternatively been grouped together on the basis of shared morphological features or separated on the basis of their nutrition. Many of these bacteria are difficult to maintain in axenic culture and, until recently, few isolates were available to allow comprehensive phenotypic and genotypic characterization. Several isolates of Thiothrix spp. and Eikelboom type 021N strains were characterized by comparative 16S rRNA sequence analysis. This revealed that the Thiothrix spp. and Eikelboom type 021N isolates formed a monophyletic group. Furthermore, isolates of Eikelboom type 021N bacteria isolated independently from different continents were phylogenetically closely related. The 16S rRNA sequence-based phylogeny was congruent with the morphological similarities between Thiothrix and Eikelboom type 021N. However, one isolate examined in this study (Ben47) shared many morphological features with the Thiothrix spp. and Eikelboom type 021N isolates, but was not closely related to them phylogenetically. Consequently, morphology alone cannot be used to assign bacteria to the Thiothrix/type 021N group. Comparative 16S rRNA sequence analysis supports monophyly of the Thiothrix/type 021N group, and phenotypic differences between the Thiothrix spp. and Eikelboom type 021N bacteria are currently poorly defined. For example, both groups include heterotrophic organisms that deposit intracellular elemental sulfur. It is therefore proposed that the Eikelboom type 021N bacteria should be accommodated within the genus Thiothrix as a new species, Thiothrix eikelboomii sp. nov., and three further new Thiothrix species are described: Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.