Biofilm structures (EPS and bacterial communities) in drinking water distribution systems are conditioned by hydraulics and influence discolouration.

High-quality drinking water from treatment works is degraded during transport to customer taps through the Drinking Water Distribution System (DWDS). Interactions occurring at the pipe wall-water interface are central to this degradation and are often dominated by complex microbial biofilms that are not well understood. This study uses novel application of confocal microscopy techniques to quantify the composition of extracellular polymeric substances (EPS) and cells of DWDS biofilms together with concurrent evaluation of the bacterial community. An internationally unique, full-scale, experimental DWDS facility was used to investigate the impact of three different hydraulic patterns upon biofilms and subsequently assess their response to increases in shear stress, linking biofilms to water quality impacts such as discolouration. Greater flow variation during growth was associated with increased cell quantity but was inversely related to EPS-to-cell volume ratios and bacterial diversity. Discolouration was caused and EPS was mobilised during flushing of all conditions. Ultimately, biofilms developed under low-varied flow conditions had lowest amounts of biomass, the greatest EPS volumes per cell and the lowest discolouration response. This research shows that the interactions between hydraulics and biofilm physical and community structures are complex but critical to managing biofilms within ageing DWDS infrastructure to limit water quality degradation and protect public health.

Impacts of bioturbation on temporal variation in bacterial and archaeal nitrogen-cycling gene abundance in coastal sediments.

In marine environments, macrofauna living in or on the sediment surface may alter the structure, diversity and function of benthic microbial communities. In particular, microbial nitrogen (N)-cycling processes may be enhanced by the activity of large bioturbating organisms. Here, we study the effect of the burrowing mud shrimp Upogebia deltaura upon temporal variation in the abundance of genes representing key N-cycling functional guilds. The abundance of bacterial genes representing different N-cycling guilds displayed different temporal patterns in burrow sediments in comparison with surface sediments, suggesting that the burrow provides a unique environment where bacterial gene abundances are influenced directly by macrofaunal activity. In contrast, the abundances of archaeal ammonia oxidizers varied temporally but were not affected by bioturbation, indicating differential responses between bacterial and archaeal ammonia oxidizers to environmental physicochemical controls. This study highlights the importance of bioturbation as a control over the temporal variation in nitrogen-cycling microbial community dynamics within coastal sediments.

Bioturbation determines the response of benthic ammonia-oxidizing microorganisms to ocean acidification.

Ocean acidification (OA), caused by the dissolution of increasing concentrations of atmospheric carbon dioxide (CO2) in seawater, is projected to cause significant changes to marine ecology and biogeochemistry. Potential impacts on the microbially driven cycling of nitrogen are of particular concern. Specifically, under seawater pH levels approximating future OA scenarios, rates of ammonia oxidation (the rate-limiting first step of the nitrification pathway) have been shown to dramatically decrease in seawater, but not in underlying sediments. However, no prior study has considered the interactive effects of microbial ammonia oxidation and macrofaunal bioturbation activity, which can enhance nitrogen transformation rates. Using experimental mesocosms, we investigated the responses to OA of ammonia oxidizing microorganisms inhabiting surface sediments and sediments within burrow walls of the mud shrimp Upogebia deltaura. Seawater was acidified to one of four target pH values (pHT 7.90, 7.70, 7.35 and 6.80) in comparison with a control (pHT 8.10). At pHT 8.10, ammonia oxidation rates in burrow wall sediments were, on average, fivefold greater than in surface sediments. However, at all acidified pH values (pH ≤ 7.90), ammonia oxidation rates in burrow sediments were significantly inhibited (by 79-97%; p < 0.01), whereas rates in surface sediments were unaffected. Both bacterial and archaeal abundances increased significantly as pHT declined; by contrast, relative abundances of bacterial and archaeal ammonia oxidation (amoA) genes did not vary. This research suggests that OA could cause substantial reductions in total benthic ammonia oxidation rates in coastal bioturbated sediments, leading to corresponding changes in coupled nitrogen cycling between the benthic and pelagic realms.

Bacterial water quality and network hydraulic characteristics: a field study of a small, looped water distribution system using culture-independent molecular methods.

AIMS: To determine the spatial and temporal variability in the abundance, structure and composition of planktonic bacterial assemblages sampled from a small, looped water distribution system and to interpret results with respect to hydraulic conditions. METHODS AND RESULTS: Water samples were collected from five sampling points, twice a day at 06:00 h and 09:00 h on a Monday (following low weekend demand) and a Wednesday (higher midweek demand). All samples were fully compliant with current regulated parameter standards. This study did not show obvious changes in bacterial abundance (DAPI count) or community structure Denaturing gradient gel electrophoresis analysis with respect to sample site and hence to water age; however, the study did show temporal variability with respect to both sampling day and sample times. CONCLUSIONS: Data suggests that variations in the bacterial assemblages may be associated with the local system hydraulics: the bacterial composition and numbers, over short durations, are governed by the interaction of the bulk water and the biofilm influenced by the hydraulic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates general stability in bacterial abundance, community structure and composition within the system studied. Trends and patterns supporting the transfer of idealized understanding to the real world were evident. Ultimately, such work will help to safeguard potable water quality, fundamental to public health.

Molecular assays reveal the presence and diversity of genes encoding pea footrot pathogenicity determinants in Nectria haematococca and in agricultural soils.

AIM: The aim of this study was to develop molecular assays for investigating the presence and diversity of pathogenicity genes from the pea footrot pathogen Nectria haematococca (anamorph Fusarium solani f.sp. pisi) in soils. METHODS AND RESULTS: Polymerase chain reaction (PCR) assays were developed to amplify four N. haematococca pathogenicity genes (PDA, PEP1, PEP3 and PEP5) from isolates and soil-DNA from five agricultural fields with a prior footrot history. A collection of 15 fungi isolated on medium selective for Fusarium spp. exhibited variation in their virulence to peas as assessed via a disease index (DI: 0-5; no virulence to the highest virulence). PCR analyses showed that three isolates in which all four pathogenicity genes were detected resulted in the highest DI (>3.88). All four pathogenicity genes were detected in soil-DNA obtained from all five fields with a footrot disease history, but were not amplified from soils, which had no footrot history. Denaturing gradient gel electrophoresis and/or sequence analysis revealed diversity amongst the pathogenicity genes. CONCLUSION: The PCR assays developed herein enable the specific detection of pathogenic N. haematococca in soils without recourse to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular assays that specifically target pathogenicity genes have the capacity to assess the presence of the footrot-causing pathogen in agricultural soils.

Azoxystrobin and soil interactions: degradation and impact on soil bacterial and fungal communities.

AIMS: To provide an independent assessment of azoxystrobin effects on nontarget soil bacteria and fungi and generate some baseline information on azoxystrobin's persistence in soil. METHODS AND RESULTS: Plate based assay showed that azoxystrobin exhibited differential toxicity upon cultured fungi at different application rates. While (14)C labelled isotopes experiments showed that less than 1% of azoxystrobin was mineralized, degradation studies revealed over 60% azoxystrobin breakdown over 21 days. PCR DGGE analysis of 16S and 18S rRNA genes from different soil microcosms showed that azoxystrobin had some effects on fungal community after 21 days (up to 84 days) of incubation in either light or dark soil microcosms. Light incubations increased fungal diversity while dark incubations reduced fungal diversity. Bacterial diversity was unaffected. CONCLUSIONS: Significant biotic breakdown of parent azoxystrobin occurred within 21 days even in the absence of light. Azoxystrobin under certain conditions can reduce fungal soil diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the few independent assessments of azoxystrobin (a widely used strobilurins fungicide) effects on soil fungi when used at the recommended rate. Azoxystrobin and metabolites may persist after 21 days and affect soil fungi.

Soil microbial community response to land use change in an agricultural landscape of western Kenya.

Tropical agroecosystems are subject to degradation processes such as losses in soil carbon, nutrient depletion, and reduced water holding capacity that occur rapidly resulting in a reduction in soil fertility that can be difficult to reverse. In this research, a polyphasic methodology has been used to investigate changes in microbial community structure and function in a series of tropical soils in western Kenya. These soils have different land usage with both wooded and agricultural soils at Kakamega and Ochinga, whereas at Ochinga, Leuro, Teso, and Ugunja a replicated field experiment compared traditional continuous maize cropping against an improved N-fixing fallow system. For all sites, principal component analysis of 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles revealed that soil type was the key determinant of total bacterial community structure, with secondary variation found between wooded and agricultural soils. Similarly, phospholipid fatty acid (PLFA) analysis also separated wooded from agricultural soils, primarily on the basis of higher abundance of monounsaturated fatty acids, anteiso- and iso-branched fatty acids, and methyl-branched fatty acids in the wooded soils. At Kakamega and Ochinga wooded soils had between five 5 and 10-fold higher levels of soil carbon and microbial biomass carbon than agricultural soils from the same location, whereas total enzyme activities were also lower in the agricultural sites. Soils with woody vegetation had a lower percentage of phosphatase activity and higher cellulase and chitinase activities than the agricultural soils. BIOLOG analysis showed woodland soils to have the greatest substrate diversity. Throughout the study the two functional indicators (enzyme activity and BIOLOG), however, showed lower specificity with respect to soil type and land usage than did the compositional indicators (DGGE and PLFA). In the field experiment comparing two types of maize cropping, both the maize yields and total microbial biomass were found to increase with the fallow system. Moreover, 16S rRNA gene and PLFA analyses revealed shifts in the total microbial community in response to the different management regimes, indicating that deliberate management of soils can have considerable impact on microbial community structure and function in tropical soils.

Development and application of molecular tools in the study of IncN-related plasmids from lakewater sediments.

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.

An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics.

A systematic evaluation of the value and potential of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure has been undertaken. The reproducibility and robustness of the method has been assessed using environmental DNA samples isolated directly from PCB-polluted or pristine soil, and subsequent polymerase chain reaction (PCR) amplification of total community 16S rDNA. An initial investigation to assess the variability both within and between different polyacrylamide gel electrophoresis (PAGE) runs showed that almost identical community profiles were consistently produced from the same sample. Similarly, very little variability was observed as a result of variation between replicate restriction digestions, PCR amplifications or between replicate DNA isolations. Decreasing concentrations of template DNA produced a decline in both the complexity and the intensity of fragments present in the community profile, with no additional fragments detected in the higher dilutions that were not already present when more original template DNA was used. Reducing the number of cycles of PCR produced similar results. The greatest variation between profiles generated from the same DNA sample was produced using different Taq DNA polymerases, while lower levels of variability were found between PCR products that had been produced using different annealing temperatures. Incomplete digestion by the restriction enzyme may, as a result of the generation of partially digested fragments, lead to an overestimation of the overall diversity within a community. The results obtained indicate that, once standardized, T-RFLP analysis is a highly reproducible and robust technique that yields high-quality fingerprints consisting of fragments of precise sizes, which, in principle, could be phylogenetically assigned, once an appropriate database is constructed.

Description of Pseudaminobacter gen. nov. with two new species, Pseudaminobacter salicylatoxidans sp. nov. and Pseudaminobacter defluvii sp. nov.

An aerobic bacterium, strain BN12T, which degrades substituted naphthalenesulfonates and substituted salicylates was isolated from a 6-aminonaphthalene-2-sulfonate-degrading microbial consortium originating from the River Elbe, Germany. Chemotaxonomic investigations of quinones, polyamines and polar lipids allowed allocation of this strain to the alpha-subclass of the Proteobacteria and revealed similarity to species of the genera Aminobacter, Chelatobacter and Mesorhizobium. This was confirmed by typing with 16S rRNA-targeted oligonucleotide probes and 16S rDNA sequencing and phylogenetic analysis, indicating that BN12T clusters most closely with a strain 'Thiobacillus' THI 051T and with the above genera but comprising a separate branch. DNA-DNA hybridizations demonstrated that strain BN12T is different from all species of Aminobacter currently described and recognized. The fatty acid patterns, substrate utilization profile and biochemical characteristics displayed no obvious similarity to the characteristics of Aminobacter and Chelatobacter species. 'Thiobacillus' THI 051T, however, revealed phenotypic similarities to BN12T. Furthermore, 16S rRNA sequences of Chelatobacter heintzii showed a high similarity to the 16S rRNA sequences of all currently recognized Aminobacter species. On the basis of these and previously published results, the new genus Pseudaminobacter is proposed, harbouring the two new species Pseudaminobacter salicylatoxidans sp. nov. and Pseudaminobacter defluvii sp. nov. The type strains are BN12T (= DSM 6986T) and THI 051T (= IFO 14570T), respectively.

Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon.

Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistance to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the mer operon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram-negative and Gram-positive bacteria from both natural and clinical environments. The presence of mer genes in bacteria from sediment cores suggest that mer is an ancient system. Analysis of DNA sequences from mer operons and genes has revealed genetic variation both in operon structure and between individual genes from different mer operons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non-functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic mer operon and for the Tn21-related family of mer operons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect mer is a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.

Characterization of antibiotic resistance plasmids from Bordetella bronchiseptica.

Of 52 antibiotic-resistant Bordetella bronchiseptica isolates from cats, ten carried plasmids. Only two of these plasmids, pLV1400 and pLV1401, were self-transmissible to Escherichia coli K12; both plasmids encoded resistance to ampicillin, tetracycline, sulphonamides, streptomycin and mercuric chloride, and were of incompatibility group P (IncP). Transferable tetracycline resistance has not been reported in B. bronchiseptica previously. The plasmids were identical in size (c.51 kb), restriction endonuclease digestion pattern and gene sequences (trfA and korA) within the IncP replicon. The trfA and korA sequences differed from those of the archetypal IncP plasmids RP4 and R751. Although the two B. bronchiseptica isolates were from epidemiologically and geographically separated cats, pulsed-field gel electrophoresis of their XbaI- or DraI-digested chromosomal DNA indicated that they were genotypically identical. The plasmid-encoded ampicillin resistance was mediated by a penicillinase of molecular weight 49,000, and pI 8.45 which was inhibited by clavulanate (IC50 = 0.1 mg/L) and tazobactam (IC50 = 0.42 mg/L) but not by parachloromercuribenzoate or EDTA. The high-level tetracycline resistance was mediated by a class C efflux mechanism that has not been described previously in this genus. The presence of transferable multi-drug resistance on a promiscuous plasmid may limit options for therapy of respiratory tract infection in companion and farm animals.

Distribution of class II transposase and resolvase genes in soil bacteria and their association with mer genes.

Southern hybridization was performed on 30 gram-negative, mercury-resistant soil bacteria isolated from three terrestrail sites in Great Britain; two of these sites were mercury polluted (SO and SE), and one was pristine (SB). Most of the isolates (20 of 30) hybridized to probes encoding regions of the transposase (tnpA) and resolvase (tnpR) genes from Tn501 and Tn21. Isolates SE9 and SB3 hybridized to the Tn21 but not the Tn501 tnpA probe; however, they differed in that SB3 hybridized to both Tn501 and Tn21 tnpR probes while SE9 did not hybridize to either tnpR probe. The remaining isolates (7 of 30) did not hybridize to any of the transposon gene probes under the conditions used. tnpA and tnpR regions were PCR amplified from most of the hybridizing isolates and from Tn501 and Tn21, and variation was assessed by restriction fragment length polymorphism analysis. On the basis of these data, tnpA regions were divided into eight restriction fragment length polymorphism classes and tnpR regions were divided into five classes. Similarity coefficients were calculated between classes and used to construct dendrograms showing percent similarity. A compilation of the data from this study on tnpA and tnpR regions and a previous study on merRT delta P regions (A. M. Osborn, K. D. Bruce, P. Strike, and D. A. Ritchie, Appl. Environ. Microbiol. 59:4024-4030, 1993) indicates the presence of hybrid transposons and provides evidence for extensive recombination, both between transposon genes and between transposon and mer genes, within these natural populations of bacteria.

The mercury resistance operon of the IncJ plasmid pMERPH exhibits structural and regulatory divergence from other Gram-negative mer operons.

The bacterial mercury resistance determinant carried on the IncJ plasmid pMERPH has been characterized further by DNA sequence analysis. From the sequence of a 4097 bp Bg/II fragment which confers mercury resistance, it is predicted that the determinant consists of the genes merT, merP, merC and merA. The level of DNA sequence similarity between these genes and those of the mer determinant of Tn21 was between 56 center dot 4 and 62 center dot 4%. A neighbour-joining phylogenetic tree of merA gene sequences was constructed which suggested that pMERPH bears the most divergent Gram-negative mer determinant characterized to date. Although the determinant from pMERPH has been shown to be inducible, no regulatory genes have been found within the Bg/II fragment and it is suggested that a regulatory gene may be located elsewhere on the plasmid. The cloned determinant has been shown to express mercury resistance constitutively. Analysis of the pMERPH mer operator/promoter (O/P) region in vivo has shown constitutive expression from the mer PTCPA promoter, which could be partially repressed by the presence of a trans-acting MerR protein from a Tn21-like mer determinant. This incomplete repression of mer PTCPA promoter activity may be due to the presence of an extra base between the -35 and -10 sequences of the promoter and/or to variation in the MerR binding sites in the O/P region. Expression from the partially repressed mer PTCPA promoter could be restored by the addition of inducing levels of Hg2+ ions. Using the polymerase chain reaction with primers designed to amplify regions in the merP and merA genes, 1 center dot 37 kb pMERPH-like sequences have been amplified from the IncJ plasmid R391, the environmental isolate SE2 and from DNA isolated directly from non-cultivated bacteria in River Mersey sediment. This suggests that pMERPH-like sequences, although rare, are nevertheless persistent in natural environments.

The sigA gene encoding the major sigma factor of RNA polymerase from the marine cyanobacterium Synechococcus sp. strain PCC 7002: cloning and characterization.

The gene encoding the principal sigma factor from Synechococcus sp. strain PCC 7002 was isolated and characterized. The Synechococcus sp. strain PCC 7002 sigA gene encodes a protein of 375 amino acids (43 center dot 7 kDa) that is required for viability under normal growth conditions. The SigA protein was overproduced in Escherichia coli and the purified protein was used to raise polyclonal antiserum in rabbits. This antiserum was used in immunoblot analyses of partially purified RNA polymerase from Synechococcus sp. strain PR6000. The probable in vivo translational start site was identified by a comparison of amino acid sequencing results obtained with SigA proteins overproduced in E. coli with immunoblot analyses of SigA protein in crude preparations of RNA polymerase from the cyanobacterium. The sigA gene is encoded on a transcript of 1700 bases that initiates 496 nucleotides upstream from the probable in vivo translational start site. The abundance of sigA transcripts decreases rapidly after the removal of combined nitrogen from the growth medium.

Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria.

Individual merRT delta P regions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conserved mer sequences of Tn501, Tn21 and pMER419. Soil and sediment samples were taken from four sites in the British Isles; one 'pristine' (SB) and three polluted (SO, SE, T2) with respect to mercury. The sizes of the PCR products amplified (approximately 1 kb) were consistent with their generation from mer determinants related to the archetypal elements found in Gram negative bacteria. Forty-five individual clones of sequences obtained from these four sites were isolated which hybridized (> 70% homology) to a merRT delta P probe from Tn501. The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling. Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites. UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified. The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation. Variation of mer genes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic variation.

Accumulation and intracellular fate of tellurite in tellurite-resistant Escherichia coli: a model for the mechanism of resistance.

The tellurite accumulation properties of three Escherichia coli strains containing different tellurium-resistance determinants of Gram-negative origin, from plasmids pMER610, pHH1508a and RK2, were compared. In all three cases membrane-associated tellurium crystallization was observed, and neither reduced uptake nor increased export contributed to the resistance. Specific membrane-proximal reduction is proposed as the mechanism of resistance to tellurite coded by all three determinants, despite their lack of sequence homology.

Polymerase chain reaction-restriction fragment length polymorphism analysis shows divergence among mer determinants from gram-negative soil bacteria indistinguishable by DNA-DNA hybridization.

Mercury resistant (Hgr) bacteria were isolated from four terrestrial sites: three containing high levels of mercury (sites T2, SE, and SO) and one uncontaminated site (SB). The frequencies of Hgr bacteria in the total cultivable populations were 0.05% (SB), 0.69% (SO), 4.8% (SE), and 25% (T2). Between 35 and 100% of the isolates from the four sites contained DNA sequences homologous to a DNA probe from the mercury resistance (mer) operon of the Tn501 Hgr determinant. The mer sequences of 10 Tn501-homologous Hgr determinants from each site were amplified by the polymerase chain reaction, with primers designed to consensus sequences of the mer determinants of Tn501, Tn21, and pMJ100, and were classified on the basis of the size of the amplified product and the restriction fragment length polymorphism pattern. Two main groups of amplification product were identified. The first, represented by the T2 and SB isolates and one SE isolate, gave an amplification product indistinguishable in size from that amplified from Tn501 (approximately 1,010 bp). The second group, represented by the SO isolates and the majority of the SE isolates, produced larger amplification products of 1,040 or 1,060 bp. Restriction fragment length polymorphism analysis revealed that each amplification product size group could be further subdivided into five subgroups.

Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction.

Specific DNA sequences from native bacterial populations present in soil, sediment, and sand samples were amplified by using the polymerase chain reaction with primers for either "universal" eubacterial 16S rRNA genes or mercury resistance (mer) genes. With standard amplification conditions, 1.5-kb rDNA fragments from all 12 samples examined and from as little as 5 micrograms of soil were reproducibly amplified. A 1-kb mer fragment from one soil sample was also amplified. The identity of these amplified fragments was confirmed by DNA-DNA hybridization.