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Diversity amongst Bacillus merA genes amplified from mercury resistant isolates and directly from mercury polluted soil
FEMS Microbiology Ecology 27 (1998) 73^84
Diversity amongst Bacillus merA genes ampli¢ed from mercury resistant isolates and directly from mercury polluted soil Mark C. Hart 1 , Geo¡ N. Elliott, A. Mark Osborn, Donald A. Ritchie, Peter Strike * School of Biological Sciences, Donnan Laboratories, University of Liverpool, Liverpool L69 7ZD, UK Received 4 December 1997; revised 23 April 1998; accepted 8 June 1998
Abstract Mercury resistant (HgR ) Bacillus were isolated from soil at a mercury contaminated site on the banks of the River Mersey, in Northwest England. The frequency of HgR bacteria in the culturable Bacillus population in soil samples ranged from 4.4% to 5.4%. No HgR Bacillus could be isolated from a sediment sample taken close to the contaminated soil sample in 1994. DNA sequences homologous to a merA probe from the HgR isolate Bacillus sp. strain RC607 were found to be chromosomally located in 98% of the HgR Bacillus isolates from the soil site. Oligonucleotide primers designed to the merA gene of RC607 were used to amplify the sequences present in the isolates, and also merA determinants present in bacterial DNA directly extracted from soil. Classification of cultured Bacillus merA products and of 40 merA determinants amplified directly from extracted soil bacterial DNA was based on restriction fragment length polymorphism patterns. This technique revealed a total of 22 classes of amplification products. Unweighted paired group mean analysis was used to examine the relationships between these classes and indicated the presence of microsites in Fiddlers Ferry soil containing distinct HgR populations. API identification of the HgR Bacillus isolates revealed that diversity of merA between these microsites is not simply due to the divergent evolution of differing Bacillus species. Lack of a more substantial class correlation between cultured samples taken at different times indicated significant temporal variation in the genetic composition of Bacillus merA in soil. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bacillus; merA ; Mercury resistance; Restriction fragment length polymorphism ; Soil; Diversity
1. Introduction Mercuric compounds are found widely in the natural environment as a result of processes such as leaching of natural ores, weathering of rocks, and industrial pollution [1]. Resistance to these toxic * Corresponding author. Tel: +44 (151) 794 3620; Fax: +44 (151) 794 3655; E-mail: [email protected] 1
Co-authors.
compounds can be found in a diverse range of bacterial species, which have been isolated from di¡erent environments across the globe [2^4]. The presence of such mercury resistant (HgR ) bacteria is often correlated with the level of mercury contamination in an environment [5,6], although HgR bacteria have been isolated from `uncontaminated' environments [7]. A variety of bacterial HgR mechanisms exist, the most commonly encountered occurring via enzymatic reduction of toxic ionic Hg2 to its less toxic
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 5 8 - 0
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metallic form. This resistance mechanism is conferred by expression of a series of genes arranged in the mer operon. The vast majority of information on the mer operon derives from studies of resistance determinants from Gram-negative bacteria. Within the Gram-negative bacteria the mer operons are commonly found on transposons, for example Tn501 and Tn21, and/or on plasmids such as pDU1358 [8], indicating the potential for high mobility of these elements. Despite the widespread distribution and diversity of Gram-negative mer operons as determined by several studies [5,7,9^12], they all, with the notable exceptions of Thiobacillus [13] and pMERPH mer determinants [14,15], have a broadly similar operon organisation. Signi¢cant similarities are also observed at the sequence level, particularly with respect to the merR, T, P and A genes. By comparison, much less research has been undertaken on mercury resistance in Gram-positive bacteria. To date three HgR determinants have been sequenced from Gram-positive bacteria: the plasmid borne determinant found in Staphylococcus aureus pI258 [16], and the chromosomal determinants of Bacillus sp. strain RC607 [17] and Streptomyces lividans 1326 [18]. These three determinants are divergent from mer operons in Gram-negative bacteria at both the sequence and organisational level, and are in turn quite divergent from each other [19]. Chromosomal resistance to mercury in Bacillus was ¢rst identi¢ed in the strain RC607 [20], isolated from a heavy metal polluted aquatic site in Boston Harbor, USA. Later studies suggested the possible global distribution of the RC607 mer operon, with RC607-like sequences found in 74 Bacillus spp. isolated from Minimata Bay sediment (Japan) [21] and in several Russian environmental isolates [4,22]. Plasmids bearing the Bacillus mer operon were found in several of the strains analysed in the Russian study. Cloning and sequence analysis of the RC607 determinant [17,23] revealed that the Bacillus mer operon starts with an operator/promoter region, clearly homologous to those present in Gram-negative operons. The polypeptide from the ¢rst open reading frame (ORF) [23] shows signi¢cant homology to the MerR of both Tn501 [24] and S. aureus pI258 [16], and has a regulatory function [25]. The merR gene of Bacillus, in contrast to the Gram-negative mer operons, is transcribed in the same direction as
the structural genes. A region (ORF2) with 30% homology to the transmembrane mercuric ion transport merT gene of the Gram-negative systems follows merR, and has therefore been associated with transport of Hg2 ions into the cell, as has ORF4 [23]. To date no known function has been found for ORF3. Following ORF4 is the mercuric reductase gene merA and, located 2.5 kb further downstream [17], the gene that encodes for the enzyme organomercurial lyase, merB, conferring broad spectrum resistance to organic mercury compounds. The DNA sequence between merA and merB genes appears not to be associated with any HgR sequences, although the presence of a strong termination signal between the two suggests that merB may be transcribed independently of the rest of the mer operon [23]. The enzyme responsible for detoxi¢cation of Hg2 ions to the less-toxic volatile Hg0 is mercuric reductase (MerA). Extensive analysis of this intracellular enzyme, puri¢ed from both Gram-negative and Gram-positive bacteria, has revealed it to be a member of the £avin containing NADPH dependent dithiol oxidoreductase family [2]. Although the presence of mercuric reductase is essential for enzymatic detoxi¢cation, and hence resistance to inorganic mercury, expression of the merA gene (and merR), has been reported in a high proportion of Gram-positive environmental strains sensitive to mercury, suggesting the presence of non-functional mer operons in which the mercury transport genes are absent or non-functional [26]. Mercury resistance determinants provide an ideal system to evaluate the e¡ects of stress on genetic distribution and diversity, gene transfer and evolution, given their global distribution on transposons, plasmids or chromosomes in a diverse range of bacteria isolated from a range of clinical and natural environments [3]. Given the key role played by the merA gene in mercury detoxi¢cation in many di¡erent systems, we have employed this gene to undertake a study of diversity in Gram-positive HgR determinants. In this study we report on the presence and diversity of Bacillus RC607-like merA sequences from both culturable and non-culturable bacteria, using a range of molecular techniques including Southern and dot-blot hybridisation, polymerase chain reaction (PCR), and restriction fragment
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length polymorphism (RFLP). The aim of the study was to identify the presence of mercury resistant Bacillus, and to provide an analysis of the extent of variation within these Gram-positive HgR determinants. Comparisons between Bacillus merA genes derived from cultured isolates and those ampli¢ed from directly extracted soil DNA are shown, enabling a comparison of Gram-positive mer diversity with previous studies on Gram-negative mer sequences ampli¢ed from the same environmental site.
2. Materials and methods 2.1. Soil and sediment samples Soil (SO) and sediment (SE) samples were collected two metres apart, from above and below the tide line of the River Mersey at Fiddlers Ferry, a site with industrial mercury pollution, and the site of previous studies of HgR determinants from Gramnegative bacteria [7,9,12]. SO samples were taken at three separate times, November 1994 (CP94), November 1995 (CP95 and total DNA) and April 1996 (CP96). One SE sample was taken, in November 1994. The total mercury levels for SO and SE were previously determined as 0.44 ppm and 0.16 ppm respectively [7]. 2.2. Bacterial strains and plasmids Escherichia coli AB1157 (SmR ) was the host to all Gram-negative control plasmids used in this study. Gram-negative control plasmids used were: pACYC184: :Tn501 (HgR , CmR ) [24], pSP200 (HgR , CmR ) [15], pMJ100 (HgR , CmR ) [27]. The Gram-positive control was Bacillus sp. strain RC607 mer operon cloned into plasmid pYW33 [23] and subsequently introduced into the host bacterium B. subtilis 168. Ampli¢ed merA genes from total bacterial DNA were cloned into the pGEM-T vector (ApR ) (Promega). E. coli XL-1-Blue (TcR ) (Stratagene) was the host of all Bacillus merA clones. 2.3. Bacillus isolation and identi¢cation Bacillus species were isolated using a modi¢cation of the method of Ramsay [28] which selects speci¢-
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cally for heat-resistant spores. Each sample (10 g wet weight) was heated at 80³C for 30 min to kill vegetative cells, and then suspended in 90 ml of 50 mM Tris-HCl bu¡er (pH 7.5). Following three cycles of 1 min blending and 1 min of cooling on ice, serial dilutions of the homogenate were prepared using 25% Ringers solution (2.25 g NaCl, 0.15 g KCl, 0.12 g CaCl2 , and 0.05 g NaCO3 per litre distilled water). Dilutions were plated in triplicate onto Bacillus germination agar (BAG) [29], and then onto BAG supplemented with 50 WM HgCl2 . The inoculated plates were then incubated at 37³C for at least 2 days. Isolates were examined microscopically for the characteristic Bacillus rod shaped morphology, and con¢rmed as Bacillus strains by identi¢cation using API 50CHB medium and API 50CH strips (Biomerieux) [30], and by 16S rDNA sequencing. Ampli¢cation of 16S rDNA was performed using pA and pHP primers [31]. The PCR ampli¢cation conditions were as described below with the exception that 55³C was used as the annealing temperature. PCR products were puri¢ed for DNA sequencing using Pharmacia Biotech MicroSpin S-400 HR columns in accordance with the manufacturer's instructions. DNA sequencing was carried out on an ABI 373A automated sequencer. 16S rDNA sequences were aligned and compared with sequence databases using the FASTA program in the UWGCG suite of programs [32]. 2.4. Soil DNA extraction Following rigorous UV irradiation and/or autoclaving of all solutions and containers, 2 g wet weight of soil was added to 5 ml 0.12M Na2 PO4 (pH 8.0) together with 2 g of 0.17^0.18-mm Glasperlen beads (B. Braun Biotech International Gmbh). The mixture was beadbeaten for 30 s and DNA was extracted from this suspension following the method of Bruce et al. [33], with the modi¢cation that DNA was precipitated overnight at 320³C with 0.25 vol of 3 M sodium acetate (pH 4.8) and 1 vol isopropyl alcohol. DNA was suspended in 100 Wl of sterile distilled water. 2.5. Dot-blot hybridisation Bacillus isolates were grown overnight in 2 ml of
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Luria broth at 37³C with shaking at 200 rpm. DNA for dot-blot hybridisation was isolated by the method of Wheatcroft and Williams [34], with modi¢cations as described by Osborn et al. [7]. Samples of genomic DNA containing approximately equivalent concentrations, as determined by agarose gel electrophoresis, were denatured by addition of an equal volume of 0.2 M NaOH, and incubated at room temperature for 30 min. The denatured DNA was transferred onto GeneScreen Plus1 hybridisation membranes (Dupont) using a dot-blot manifold. DNA isolated from strains of E. coli containing plasmids pACYC184: :Tn501, pSP200 and pMJ100, bearing HgR determinants from Gram-negative bacteria, were used as negative controls, and clone pYW33 [23] containing the mer operon of Bacillus strain RC607 mer operon as the positive control. The DNA samples were hybridised against two mer probes, a Bacillus merA probe from BglII and SmaI digestion of clone pYW33, and the merRTvP region from Tn501 [9]. Probe DNA was labelled with [K-32 P]dCTP using the Random Primed Labelling Kit (Boehringer Mannheim). Hybridisations with these probes were performed as recommended by the GeneScreen instruction manual with duplicate 100 ml washes in (i) 0.3 M NaCl and 0.03 M Na citrate (2USSC) for 5 min; (ii) 0.3 M NaCl, 0.03 M Na citrate and 1% SDS for 30 min, and a single wash (iii) 0.015 M NaCl and 0.0015 M Na citrate for 30 min. Washes (ii) and (iii) were performed at 65³C instead of 60³C in order to increase the stringency to detect homology greater than 70%. Membranes were exposed to Kodak Scienti¢c XOMAT ¢lm at 370³C, and subsequently developed. 2.6. Isolation of genomic and plasmid DNA To ascertain the location of the Bacillus mer determinants, total DNA from the cultured isolates was extracted using a modi¢ed method of Pitcher [35], with the inclusion of an extra lysozyme step to aid bacterial lysis. Transfer of target DNA onto GeneScreen Plus1 membrane for Southern hybridisation was carried out by using the salt transfer method [36]. Hybridisation conditions, washing and development were performed as previously described for dot-blot hybridisation. Isolation of plasmid DNA was attempted using a number of di¡erent tech-
niques, including those described by Birnboim and Doly [37], Olsen [38] and a modi¢ed version of Birnboim and Doly described by Bogdanova et al. [22] designed speci¢cally for plasmid extraction from Bacillus strains. Control strains were B. subtilis IE60, containing plasmid pGVD1, and plasmidless B. megaterium 7A34, both provided by Dr. D.R. Zeigler of the Bacillus Genetic Stock Centre (Ohio State University). Again, any DNA extracted was Southern blotted using hybridisation conditions as described. 2.7. Oligonucleotide primers Oligonucleotide primers RC607+A (5P-TGG GTG GAA CTT GCG TTA A-3P) and POSAC (5P-GTA [GT]CC [TA]GC ACA [GA]CA AGA TA-3P) were designed to a sequence encoding the active site of RC607 merA, and to a region conserved at the 3P end of both RC607 and S. aureus pI258 merA genes, respectively. These primers amplify a 1.3-kb region of RC607 merA. 2.8. PCR ampli¢cation DNA was isolated from colonies using the boiling lysis method of Gussow and Clackson [39] modi¢ed by boiling for 10 min instead of 5 min. DNA was isolated from clones either using the boiling lysis method with subsequent ampli¢cation as above, or by using the alkaline lysis method [37] with 0.5 Wl of the resultant DNA as target volume. The merA region was ampli¢ed for 30 cycles using a Perkin Elmer DNA Thermal Cycler 480, with each cycle consisting of a denaturation step (94³C, 1 min), an annealing step (53³C, 1 min) and an extension step (72³C, 2 min). After 30 cycles the reaction was completed with a 10 min ¢nal extension stage at 72³C. Each PCR reaction comprised 50 Wl of the boiled bacterial lysate, 20 pmol of both the RC607+A and POSAC primers, 50 WM of each deoxynucleoside triphosphate, 10 Wl of Gibco-BRL 10UTaq DNA polymerase bu¡er, 1.5 mM MgCl2 , and 2.5 U of Taq DNA polymerase. The reactions were made up to a total volume of 100 Wl with sterile distilled water, and overlaid with mineral oil (Sigma). For the control reactions 1 Wl of pYW33 was used as a positive control, and a water control, containing no target DNA was used as a negative control. DNA
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isolated directly from SO95 soil was ampli¢ed as above, except 35 cycles of PCR were used, and the target DNA volume ranged from 0.1 Wl to 10 Wl depending on PCR product yield. 2.9. Cloning of ampli¢ed products The merA products ampli¢ed from DNA extracted directly from soil were cloned into pGEM-T (Promega, UK) following the manufacturer's instructions. Ligation mixtures were subsequently transformed into competent E. coli XL-1 cells, prepared by the method of Nishimura et al. [40]. Clones carrying merA inserts were identi¢ed by digestion with SacI or PstI (Gibco-BRL) of plasmid DNA following alkaline lysis extraction. 2.10. Hybridisation and restriction analysis of PCR products PCR products were identi¢ed by electrophoresis on a 0.7% agarose TBE gel containing 1 Wg ml31 ethidium bromide, and were subsequently hybridised to the RC607 merA probe by Southern blotting. PCR products (10 Wl) of RC607 merA were digested with the following restriction endonucleases: AvaI, ClaI, HaeII, HaeIII, HinfI, PvuII, and VspI (Gibco-BRL), to obtain RFLP pro¢les. Reaction conditions used were those as recommended by the supplier, and the products of each reaction were electrophoresed on a 2% agarose TBE gel containing 1 Wg ml31 of ethidium bromide. 2.11. Numerical analysis The RFLP data were analysed using the Dice coe¤cient of variation [41], where the percentage similarity between two merA determinants was calculated using the formula: % similarity 100U 2Unumber of indistinguishable bands= total number of bands: The similarity coe¤cients calculated were used in the construction of a dendrogram using the unweighted
Fig. 1. Schematic representation of RFLP patterns of ampli¢ed merA fragments from culturable HgR Bacillus isolates and directly ampli¢ed total bacterial DNA. For HinfI digests, bands of double thickness represent doublets.
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paired group mean analysis (UPGMA) program on PHYLIP 3.5C [42].
3.2. Hybridisation DNA extracted from a total of 100 Bacillus strains isolated from the three soil samples was tested by dot-blot hybridisation for homology to the merA gene from Bacillus sp. RC607. Ninety-eight isolates contained sequences with strong homology to the probe, with the negative controls showing no hybridisation. No plasmids could be isolated from the culturable isolates using a variety of extraction methods [37,38] including the Bacillus speci¢c method described by Bogdanova et al. [22]. Plasmid DNA was, however, extracted from the plasmid containing control isolate Bacillus subtilis IE60. Southern hybridisation of total DNA isolated using either the extraction method of Pitcher et al. [35], or the modi¢ed version of Wheatcroft and Williams [34] used by Osborn et al. [7], suggested that merA genes homologous to that from RC607 were located on the chro-
3. Results 3.1. Distribution of cultivable HgR Bacillus in soil and sediment The titres of Bacillus colony forming units (CFU) from SO samples CP94 and CP95 were 7.4U105 and 8.3U106 CFU g31 , respectively. This compares to a total CFU count of 9.6U103 from the SE94 sample. Total CFU counts from SO sample CP96 were not taken. The frequencies of resistance to inorganic mercury (Hg2 ) in the CP94 and CP95 samples were 5.4% and 4.4% respectively. No mercury resistant bacteria were isolated on Bacillus germination agar from sediment.
Table 1 Frequency of each RFLP class of Bacillus merA sequences ampli¢ed from HgR isolates and directly from total bacterial DNA extracted from soil RFLP class
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX XXI XXII a b
Number of clones/isolates Cloned SO95 total DNAa
Isolates '94b
4 ^ ^ ^ ^ ^ ^ 18 10 ^ ^ ^ ^ ^ 1 1 1 1 1 1 1 1 40
1 12 5 1 3 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 22
Class derived from ampli¢ed products of total bacterial DNA. Cultured isolates identi¢ed by year of sampling.
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Isolates '95b
Isolates '96b
1
^ ^ ^ ^ ^
4 1 6 8 2 1 1 1
9 ^ ^ ^ ^ ^ ^ ^ 1 ^ ^ ^ ^ ^ ^ ^ ^ 10
^ ^ ^ ^
^ ^ ^ ^ ^ ^ ^ ^ ^ 25
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mosomes of the environmental Bacillus isolates (data not shown).
Fig. 2. Venn diagram to illustrate the sharing of Bacillus merA RFLP classes between three SO HgR isolate samples: CP94 (11/ 94); CP95 (11/95) ; CP96 (4/96), and from directly ampli¢ed total bacterial DNA isolated from SO.
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3.3. PCR ampli¢cation from Bacillus isolates Eighty of the positive Bacillus isolates by dot-blot hybridisation were selected for analysis by PCR, and merA genes were successfully ampli¢ed from 58. Of the ampli¢ed products, 57 showed no size variation from the predicted 1.3-kb fragment based on the RC607 merA. The exception was isolate BA11, from which an 800-bp product was ampli¢ed. No ampli¢cation products were observed from the remainder of the isolates. All 58 ampli¢ed fragments were checked for homology to the RC607 merA probe by Southern hybridisation. Homology was shown for all products, and these were further investigated using RFLP analysis.
Fig. 3. Dendrogram (neighbor-joining UPGMA) of genotypic relationships between merA fragments from cultivable HgR Bacillus isolates and directly ampli¢ed total bacterial DNA derived by PCR/RFLP analysis. Divergence between classes of mer is expressed as percentage similarity. Allocation of individual classes to respective samples is shown on right by di¡erently shaded circles.
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3.4. Extraction of total bacterial DNA and ampli¢cation Total DNA was isolated from the SO95 soil sample as described in Section 2, and visualised by agarose gel electrophoresis. Products of the predicted 1.3kb size were ampli¢ed from this DNA using the RC607+A and POSAC primers, ligated into the pGEM-T vector, and transformed into E. coli. Using the merA primers, PCR products could be ampli¢ed from plasmid DNA extracted from 41 of the resulting transformants, and these fragments were subsequently used for RFLP analysis. 3.5. RFLP analysis of ampli¢ed PCR products Digestion of ampli¢ed merA products derived from both culturable Bacillus isolates and total bacterial DNA, using a number of restriction endonucleases, revealed a variety of di¡erent DNA fragment patterns. From these a total of 22 Bacillus merA Table 2 API identi¢cation of selected Hg resistant Bacillus isolates RFLP class
Isolate
API identi¢cation
I I II II II II II II II III III III III IV V V VI VI VI VII IX IX X XII XIV
BA1 BA34 BA3 BA6 BA22 BA23 BA10 BA20 BA4 BA5 BA7 BA18 BA24 BA26 BA28 BA29 BA33 BA53 BA77 BA35 BA44 BA58 BA45 BA55 BA82
B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B.
cereus (99.2%) sphearicus (54.2%) pantothenticus (100.0%) pantothenticus (94.0%) pantothenticus (94.0%) pantothenticus (91.0%) laterosporus (50.2%) laterosporus (50.2%) subtilis (88.8%) stearothermophilus (98.6%) stearothermophilus (93.5%) amylofaciens (45.8%) subtilis (85.4%) licheniformis (50.0%) subtilis (65.2%) subtilis (65.2%) sphearicus (44.6%) sphearicus (54.2%) sphearicus (54.2%) sphearicus (54.2%) subtilis (84.2%) lentus (99.9%) sphearicus (54.2%) sphearicus (54.2%) ¢rmus (99.6%)
Percentages indicate level of certainty for identi¢cation.
RFLP classes were identi¢ed (Fig. 1, Table 1): 11 from total SO95 DNA and 14 from culturable isolates, with three classes common to both (Fig. 2). Of the 41 clones derived from total SO95 DNA, only one showed no similarity in any digest to RC607 and did not hybridise to the Bacillus merA probe. The nature of this ampli¢ed product is described later. Between culturable isolates obtained from the three SO samples, the numbers of common RFLP classes were limited, with only classes I (RC607) and VI present in samples from more than one year (Table 1). In the limited analysis of CP96, only class VI was found to be common between this and any other year, namely CP95. No similarity was observed between the classes identi¢ed from the CP94 and CP95 samples. The classes derived from clones isolated from the total SO95 DNA showed a degree of similarity to those from the culturable classes, principally those from CP95, with VIII and IX the main classes in both. Again, class I was detected in the cloned merA samples, although the two predominant classes present in the culturable samples as a whole (II and III) were not represented. An additional eight classes distinct from those seen in culturable bacteria were also contained within the fragments obtained from total DNA. Representatives of the classes obtained from total SO95 DNA were all shown, by Southern hybridisation, to have homology to the RC607 merA probe. On the basis of these RFLP results, similarity co-e¤cients were calculated and used to construct a UPGMA dendrogram (Fig. 3). 3.6. API identi¢cation of HgR Bacillus isolates The results of API identi¢cation of the mercury resistant Bacillus isolates are shown in Table 2. A limited number of the Bacillus isolates could be identi¢ed with an excellent degree of certainty (99%) using the Biomerieux 50CH strips, for example BA1 (B. cereus), BA3 (B. pantothenticus) and BA82 (B. ¢rmus). A good level of certainty of identi¢cation (above 90%) included isolates BA6 (B. pantothenticus), and BA5 (B. stearothermophilus), whilst acceptable identi¢cation (above 80%) was achieved for BA24 (B. subtilis). Most of the soil isolates gave either a very doubtful identi¢cation, with the reliability of identi¢cation ranging from 44.6% (BA33, B. sphearicus), to 65.2% (BA28, B. subtilis), or no result.
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Using all the results obtained from these biochemical test strips, including the doubtful identi¢cations, nine di¡erent species of Bacillus were identi¢ed. Species diversity was revealed between and within some RFLP classes, notably classes I, II, and IX, with class II identi¢ed as present in three di¡erent species
4. Discussion The proportion of Bacillus strains that are mercury resistant within Fiddlers Ferry soil, at around 5%, is within the range of mercury resistant bacteria isolated in studies previously cited by Rochelle et al. [6]. With the total culturable Bacillus population, the viable counts from sediment were signi¢cantly lower than those from soil, and so the failure to isolate HgR Bacillus strains from Fiddlers Ferry sediment is therefore not surprising. These lower numbers may re£ect the naturally more anoxic nature of this environment. The vast majority of the SO HgR isolates (98%) hybridised to the RC607 merA probe, indicating the prevalence of volatilisation as the mechanism of Hg resistance in Bacillus. The two remaining HgR isolates may be either su¤ciently divergent from the merA sequence of RC607 to preclude hybridisation, or may simply employ a di¡erent resistance system. These isolates have not as yet been investigated further. The presence in SO samples of sequences homologous to Bacillus merA found in previous studies in Boston Harbor (USA) [20], Minimata Bay (Japan) [21,43] and Russia [4] adds to data on the wide geographical distribution of Bacillus mer operons across the globe. The global distribution of chromosomally encoded Bacillus mer indicates that Hg resistance may have an ancient origin [3]. However, the possible spread of Bacillus mer genes by the horizontal dissemination of mer on plasmids and transposons [4,22] analogous to the dispersal of Gram-negative HgR determinants [3] should not be discounted. The positive hybridisation of the merA probe to chromosomal DNA extracted from all the culturable strains suggests, as with RC607, that the merA in these strains is chromosomally located. Whilst not being absolutely conclusive evidence for the chromosomal location of the mer operon (the presence of large linear or integrated plasmids is always a possi-
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bility), the corresponding failure to isolate plasmid DNA using a number of techniques also supports this conclusion. merA gene fragments were ampli¢ed from 58 of the hybridising isolates with only one size variant from RC607, which ampli¢ed to give an 800-bp product. From sequence analysis (data not shown) this smaller fragment appears to have arisen by annealing of the RC607+A primer to an alternative site 400bp within the normal ampli¢cation product. The 22 hybridising isolates which gave no product presumably re£ect sequence diversity in the Bacillus merA speci¢c primer binding sites and were not further analysed. The Bacillus merA sequences derived from cultured isolates were compared to sequences ampli¢ed from DNA directly extracted from SO95 soil and then cloned. The DNA extraction method used has been shown to extract DNA from both vegetative Bacillus cells and from endospores (data not shown). The potential for the increased frequency of production of chimeric fragments during PCR has been shown not to be a factor in creating spurious diversity [33]. Of the 41 cloned amplimers, 40 were shown to hybridise to the RC607 merA probe. This high percentage is to be expected because of the speci¢city of the merA oligonucleotide primers used. The sequence that did not hybridise to RC607 has since been shown to be 63% identical to the merA gene of Tn501, a Gram-negative mer system, on the basis of DNA sequence comparison (data not shown). The fact that a sequence has been ampli¢ed using oligonucleotide primers designed for Gram-positive mer sequences that is ostensibly of Gram-negative origin may indicate a link between these two seemingly divergent mer systems. Comparison of RFLP data obtained from the 97 PCR products ampli¢ed either from cloned DNA or from cultured isolates initially revealed little in terms of RFLP class correlation between samples. However, division of the 14 classes derived from culturable isolates into the three sampling times gives a clearer picture of the relationships between these classes and those of cloned DNA (Fig. 2). The greatest overlap is found between classes of CP95 and those of cloned SO95 DNA, with classes VIII and IX being the most common in both (Table 1). This is probably due to the fact that both samples were
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taken at the same time, removing the in£uence of temporal variation within the Bacillus population. The lack of a more substantial correlation between the three sets of cultivated classes seems to indicate changes over time in the genetic composition of Bacillus merA from Fiddlers Ferry soil. Only classes I (CP94 and CP95) and VI (CP95 and CP96) were shared between two cultured samples (Fig. 2), indicating either the presence of microsites within the SO soil, each containing a distinct population of closely related merA genes, or a major change in the Bacillus population in the time periods separating the samples. Closer analysis of the dendrogram (Fig. 3) shows that in many cases, classes from one sample tend to cluster, suggesting that random sampling of isolates or total DNA has not occurred. Such an observation is again consistent with the presence of microsites, each containing a distinct Bacillus subpopulation. Indeed microsites may also provide a plausible explanation for the di¡erences between the CP95 sample and the total SO95 DNA which show little overlap despite being derived from the same large soil sample. In the limited API identi¢cation of isolates (Table 2) species variation can be seen both within and between RFLP classes. The fact that RFLP classi¢cation is not entirely correlated with species indicates that the diversity present within the cultivable merA population is not simply due to the divergent evolution of di¡ering Bacillus species. In a study undertaken on Bacillus isolated from Minimata Bay sediment in Japan [43], a site of methylmercury contamination, seven RFLP classes of mer determinants were identi¢ed [21]. Signi¢cantly the percentage of isolates that hybridised to an RC607 probe, 95% (74 out of 78), is similar to the high percentage observed in our study of cultured bacteria (98%). Comparison of the diversity revealed within the classes obtained in this study with the RFLP classes of our study is di¤cult because of their less detailed RFLP analysis of a larger region of the Bacillus mer operon. The di¡erences observed in classes derived from total DNA and merA fragments ampli¢ed from the culturable isolates may be due to the sampling of di¡erent microsites. Alternatively, it may re£ect the fact that the eight unique merA classes derived from the total DNA might have been ampli¢ed from ac-
tive Bacillus cells present in the soil, which might not be represented as spores in the environment and consequently would not be selected on BAG plates. Restriction classes unique to the cloned DNA may also have arisen from ampli¢cation of non-functional, or cryptic merA genes [9,26]. As with this study, the lack of overlap between classes from cultured isolates and total bacterial DNA was seen within an RFLP analysis of ampli¢ed merRTvP genes of the Gramnegative population, previously studied at the SO and SE sites at Fiddlers Ferry [7,9,12,44], and has been taken to indicate the low proportion of microorganisms that can be isolated by direct culturing techniques. The diversity present in the Bacillus soil population may be due to the very e¡ective capacity of certain Bacillus species for cell-to-cell DNA exchange. Bacterial populations are likely to range from strictly clonal to panmictic [45], but the work of Istock et al. [46] and Duncan et al. [47] showed that genetic exchange within the Bacillus population would reduce the formation of a clonal population structure. The highly sexual nature observed in some Bacillus species [46] may therefore contribute signi¢cantly to the overall level of diversity seen within merA. The large amounts of variation observed between merA classes found in cultured isolates and extracted bacterial DNA highlights the need to extend studies into areas other than cultivation and also emphasises the importance of sample size. Rapid analysis techniques such as denaturing gradient gel electrophoresis are now being used for the analysis of Bacillus merA diversity and will provide alternative means to pro¢le microbial populations. This may enable the £uctuation of closely related bacterial microsite populations to be studied in much more detail.
Acknowledgments This work was supported by NERC Grants GR3/ 9502 and GT4/94196/T. This work was jointly carried out by Geo¡ Elliott and Mark Hart. The probe and primers to the RC607 merA were developed by Dr. A.M. Osborn, who also provided technical advice. The project was supervised by Prof. P. Strike and Prof. D.A. Ritchie. Special thanks also go to Dr.
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K.D. Bruce for his help and support, to I. Mahler for providing the plasmid pYW33 and to Dr. Zeigler of the Bacillus strain stock centre for providing control strains. References [1] Von Burg, R. and Greenwood, M.R. (1991) Mercury. In: Metals and Their Compounds in the Environment (Merian, E., Ed.), pp. 1045^1088. VCH, New York. [2] Foster, T.J. (1987) The genetics and biochemistry of mercury resistance. CRC Crit. Rev. Microbiol. 15, 117^140. [3] Osborn, A.M., Bruce, K.D., Strike, P. and Ritchie, D.A. (1997) Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon. FEMS Microbiol. Rev. 19, 239^262. [4] Yurieva, O., Kholodii, G., Minaklin, L., Gorlenko, Z., Kalyaeva, E., Mindlin, S. and Nikiforov, V. (1997) Intercontinental spread of promiscuous mercury resistance transposons in environmental bacteria. Mol. Microbiol. 24, 321^329. [5] Barkay, T. and Olson, B.H. (1986) Phenotypic and genotypic adaption of aerobic heterotrophic sediment bacterial communities to mercury stress. Appl. Environ. Microbiol. 52, 403^406. [6] Rochelle, P.A., Wetherbee, M.K. and Olson, B.H. (1991) Distribution of DNA sequences encoding narrow and broad spectrum mercury resistance. Appl. Environ. Microbiol. 57, 1581^ 1589. [7] Osborn, A.M., Bruce, K.D., Strike, P. and Ritchie, D.A. (1993) Polymerase chain reaction-restriction fragment length polymorphism analysis shows divergence among mer determinants from Gram-negative soil bacteria indistinguishable by DNA-DNA hybridisation. Appl. Environ. Microbiol. 59, 4024^4030. [8] Gri¤n, H.G., Foster, T.J., Silver, S. and Misra, T.K. (1987) Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinant of plasmid pDU1358. Proc. Natl. Acad. Sci. USA 84, 3112^3116. [9] Bruce, K.D., Osborn, A.M., Pearson, A.J., Strike, P. and Ritchie, D.A. (1995) Genetic diversity within mer genes directly ampli¢ed from communities of noncultivated soil and sediment bacteria. Mol. Ecol. 4, 605^612. [10] Gilbert, M.P. and Summers, A.O. (1988) The distribution and divergence of DNA sequences related to the Tn21 and Tn501 mer operons. Plasmid 20, 127^136. [11] Olson, B.H., Lester, J.N., Cayless, S.M. and Ford, S.J. (1989) Distribution of mercury resistance determinants in bacterial communities of river sediments. Water Res. 23, 1209^1217. [12] Osborn, A.M., Bruce, K.D., Strike, P. and Ritchie, D.A. (1995) Sequence conservation between regulatory resistance genes in bacteria from mercury polluted and pristine environments. Syst. Appl. Microbiol. 18, 1^6. [13] Inoue, C., Sugawara, K. and Kusano, T. (1991) The merR regulatory gene in Thiobacillus ferrooxidans is spaced apart from the mer structural genes. Mol. Microbiol. 5, 2707^2718.
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[14] Osborn, A.M., Bruce, K.D., Ritchie, D.A. and Strike, P. (1996) The mercury resistance operon of the IncJ plasmid pMERPH exhibits structural and regulatory divergence from other Gram-negative mer operons. Microbiology 142, 337^ 345. [15] Peters, S.E., Hobman, J.L., Strike, P. and Ritchie, D.A. (1991) Novel mercury resistance determinants carried by IncJ plasmids pMERPH and R391. Mol. Gen. Genet. 228, 1209^1217. [16] Laddaga, R.A., Chu, L., Misra, T.K. and Silver, S. (1987) Nucleotide sequence and expression of the mercurial-resistant operon from Staphylococcus aureus plasmid pI258. Proc. Natl. Acad. Sci. USA 84, 5106^5110. [17] Wang, Y., Mahler, I., Levinson, H.S. and Halvorson, H.O. (1987) Cloning and expression in Escherichia coli of chromosomal mercury resistance genes from a Bacillus sp. J. Bacteriol. 169, 4848^4851. [18] Sedlmeier, R. and Altenbuchner, J. (1992) Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans. Mol. Gen. Genet. 236, 76^85. [19] Silver, S., and Walderhaug, M. (1992) Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria. Microbiol. Rev. 56, 195^228. [20] Mahler, I., Levinson, H.S., Wang, Y. and Halvorson, H.O. (1986) Cadmium and mercury resistant Bacillus strains from a salt marsh and from Boston harbor. Appl. Environ. Microbiol. 52, 1293^1298. [21] Nakamura, K. and Silver, S. (1994) Molecular analysis of mercury resistant Bacillus isolates from sediment of Minimata Bay, Japan. Appl. Environ. Microbiol. 60, 4596^4599. [22] Bogdanova, E.S., Bass, I.A., Minakhin, L.S., Petrova, M.A., Mindlin, S.Z., Volodin, A.A., Kalyaeva, E.S., Tiedje, J.M., Hobman, J.L., Brown, N.L. and Nikiforov, V.G. (1998) Horizontal spread of mer operons among Gram-positive bacteria in natural environments. Microbiology 144, 609^620. [23] Wang, Y., Moore, M., Levinson, H.S., Silver, S., Walsh, C. and Mahler, I. (1989) Nucleotide sequence of a chromosomal mercury resistance determinant from a Bacillus sp. with broad spectrum mercury resistance. J. Bacteriol. 171, 83^92. [24] Brown, N.L., Ford, S.J., Pridmore, R.D. and Fritzinger, D.C. (1983) Nucleotide sequence of a gene from Pseudomonas transposon Tn501 encoding mercuric reductase. Biochemistry 22, 4089^4095. [25] Helmann, J.D., Wang, Y., Mahler, I. and Walsh, C.T. (1989) Homologous metalloregulatory proteins from both Gram-positive and Gram-negative bacteria control transcription of mercury resistance operons. J. Bacteriol. 171, 222^229. [26] Bogdanova, E.S., Mindlin, S.Z., Pakrova, E., Kocur, M. and Rouch, D.A. (1992) Mercuric reductase in environmental Gram-positive bacteria sensitive to mercury. FEMS Microbiol. Lett. 97, 95^100. [27] Jobling, M.G., Peters, S.E. and Ritchie, D.A. (1988) Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants. Plasmid 20, 106^112. [28] Ramsay, A.J. (1984) Extraction of bacteria from soil: e¤ciency of shaking or ultrasonication as indicated by direct counts and autoradiography. Soil Biol. Biochem. 16, 475^481. [29] Belliveau, B.H., Starodub, M.E. and Trevors, J.T. (1991) Oc-
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M.C. Hart et al. / FEMS Microbiology Ecology 27 (1998) 73^84 currence of antibiotic and metal resistance plasmids in Bacillus strains isolated from marine sediment. J. Can. Microbiol. 37, 513^520. Logan, N.A. and Berkeley, R.C.W. (1984) Identi¢cation of Bacillus strains using the API system. J. Gen. Microbiol. 130, 1871^1882. Edwards, U., Rogall, T., Bloëcker, H., Emde, M. and Boëttger, E.C. (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res. 17, 7843^7853. Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387^395. Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P. and Ritchie, D.A. (1992) Ampli¢cation of DNA from native populations of soil bacteria by using the polymerase chain reaction. Appl. Environ. Microbiol. 58, 3413^3416. Wheatcroft, R. and Williams, P.A. (1981) Rapid methods for the study of both stable and unstable plasmids in Pseudomonas. J. Gen. Microbiol. 124, 433^437. Pitcher, D.G., Saunders, N.A. and Owen R.J. (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8, 151^156. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513^1523. Olsen, J.E. (1990) An improved method for rapid isolation of plasmid DNA from wild type Gram-negative bacteria for plasmid restriction pro¢le analysis. Lett. Appl. Microbiol. 10, 209^212.
[39] Gussow, D. and Clackson, T. (1989) Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 17, 4000. [40] Nishimura, A., Morita, M., Nishimura, Y. and Sugino, Y. (1990) A rapid and highly e¤cient method for preparation of competent Escherichia coli cells. Nucl. Acids Res. 18, 6169. [41] Dice, L.R. (1945) Measures of the amount of ecologic association between species. Ecology 26, 297^302. [42] Felsenstein, J. (1993) PHYLIP (Phylogeny Inference Package) Version 3.5C. Department of Genetics, University of Washington, Seattle, WA. [43] Nakamura, K., Sakamoto, M., Uchiyama, H. and Yagi, O. (1990) Organomercurial-volatilizing bacteria in the mercurypolluted sediment of Minimata bay, Japan. Appl. Environ. Microbiol. 56, 304^305. [44] Bruce, K.D., Osborn, A.M., Harrison, J.F., Strike, P. and Ritchie, D.A. (1997) Diversity within the mervPA region of bacterial mercury resistance determinants ; implications for gene evolution in environments. FEMS Microbiol Ecol. (submitted). [45] Maynard-Smith, J., Smith, N.H., O'Rourke, M. and Spratt, B.G. (1993) How clonal are bacteria? Proc. Natl. Acad. Sci. USA 90, 4384^4388. [46] Istock, C.A., Duncan, K.E., Ferguson, N. and Zhou, X. (1992) Sexuality in a natural population of bacteria-Bacillus subtilis challenges the clonal paradigm. Mol. Ecol. 1, 95^103. [47] Duncan, K.E., Ferguson, N., Kimura, K., Zhou, X. and Istock, C.A. (1994) Fine scale genetic and phenotypic structure in natural populations of Bacillus subtilis and B. licheniformis: implications for bacterial evolution and speciation. Evolution 48, 2002^2025.
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Diversity amongst Bacillus merA genes ampli¢ed from mercury resistant isolates and directly from mercury polluted soil Mark C. Hart 1 , Geo¡ N. Elliott, A. Mark Osborn, Donald A. Ritchie, Peter Strike * School of Biological Sciences, Donnan Laboratories, University of Liverpool, Liverpool L69 7ZD, UK Received 4 December 1997; revised 23 April 1998; accepted 8 June 1998
Abstract Mercury resistant (HgR ) Bacillus were isolated from soil at a mercury contaminated site on the banks of the River Mersey, in Northwest England. The frequency of HgR bacteria in the culturable Bacillus population in soil samples ranged from 4.4% to 5.4%. No HgR Bacillus could be isolated from a sediment sample taken close to the contaminated soil sample in 1994. DNA sequences homologous to a merA probe from the HgR isolate Bacillus sp. strain RC607 were found to be chromosomally located in 98% of the HgR Bacillus isolates from the soil site. Oligonucleotide primers designed to the merA gene of RC607 were used to amplify the sequences present in the isolates, and also merA determinants present in bacterial DNA directly extracted from soil. Classification of cultured Bacillus merA products and of 40 merA determinants amplified directly from extracted soil bacterial DNA was based on restriction fragment length polymorphism patterns. This technique revealed a total of 22 classes of amplification products. Unweighted paired group mean analysis was used to examine the relationships between these classes and indicated the presence of microsites in Fiddlers Ferry soil containing distinct HgR populations. API identification of the HgR Bacillus isolates revealed that diversity of merA between these microsites is not simply due to the divergent evolution of differing Bacillus species. Lack of a more substantial class correlation between cultured samples taken at different times indicated significant temporal variation in the genetic composition of Bacillus merA in soil. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bacillus; merA ; Mercury resistance; Restriction fragment length polymorphism ; Soil; Diversity
1. Introduction Mercuric compounds are found widely in the natural environment as a result of processes such as leaching of natural ores, weathering of rocks, and industrial pollution [1]. Resistance to these toxic * Corresponding author. Tel: +44 (151) 794 3620; Fax: +44 (151) 794 3655; E-mail: [email protected] 1
Co-authors.
compounds can be found in a diverse range of bacterial species, which have been isolated from di¡erent environments across the globe [2^4]. The presence of such mercury resistant (HgR ) bacteria is often correlated with the level of mercury contamination in an environment [5,6], although HgR bacteria have been isolated from `uncontaminated' environments [7]. A variety of bacterial HgR mechanisms exist, the most commonly encountered occurring via enzymatic reduction of toxic ionic Hg2 to its less toxic
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 5 8 - 0
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metallic form. This resistance mechanism is conferred by expression of a series of genes arranged in the mer operon. The vast majority of information on the mer operon derives from studies of resistance determinants from Gram-negative bacteria. Within the Gram-negative bacteria the mer operons are commonly found on transposons, for example Tn501 and Tn21, and/or on plasmids such as pDU1358 [8], indicating the potential for high mobility of these elements. Despite the widespread distribution and diversity of Gram-negative mer operons as determined by several studies [5,7,9^12], they all, with the notable exceptions of Thiobacillus [13] and pMERPH mer determinants [14,15], have a broadly similar operon organisation. Signi¢cant similarities are also observed at the sequence level, particularly with respect to the merR, T, P and A genes. By comparison, much less research has been undertaken on mercury resistance in Gram-positive bacteria. To date three HgR determinants have been sequenced from Gram-positive bacteria: the plasmid borne determinant found in Staphylococcus aureus pI258 [16], and the chromosomal determinants of Bacillus sp. strain RC607 [17] and Streptomyces lividans 1326 [18]. These three determinants are divergent from mer operons in Gram-negative bacteria at both the sequence and organisational level, and are in turn quite divergent from each other [19]. Chromosomal resistance to mercury in Bacillus was ¢rst identi¢ed in the strain RC607 [20], isolated from a heavy metal polluted aquatic site in Boston Harbor, USA. Later studies suggested the possible global distribution of the RC607 mer operon, with RC607-like sequences found in 74 Bacillus spp. isolated from Minimata Bay sediment (Japan) [21] and in several Russian environmental isolates [4,22]. Plasmids bearing the Bacillus mer operon were found in several of the strains analysed in the Russian study. Cloning and sequence analysis of the RC607 determinant [17,23] revealed that the Bacillus mer operon starts with an operator/promoter region, clearly homologous to those present in Gram-negative operons. The polypeptide from the ¢rst open reading frame (ORF) [23] shows signi¢cant homology to the MerR of both Tn501 [24] and S. aureus pI258 [16], and has a regulatory function [25]. The merR gene of Bacillus, in contrast to the Gram-negative mer operons, is transcribed in the same direction as
the structural genes. A region (ORF2) with 30% homology to the transmembrane mercuric ion transport merT gene of the Gram-negative systems follows merR, and has therefore been associated with transport of Hg2 ions into the cell, as has ORF4 [23]. To date no known function has been found for ORF3. Following ORF4 is the mercuric reductase gene merA and, located 2.5 kb further downstream [17], the gene that encodes for the enzyme organomercurial lyase, merB, conferring broad spectrum resistance to organic mercury compounds. The DNA sequence between merA and merB genes appears not to be associated with any HgR sequences, although the presence of a strong termination signal between the two suggests that merB may be transcribed independently of the rest of the mer operon [23]. The enzyme responsible for detoxi¢cation of Hg2 ions to the less-toxic volatile Hg0 is mercuric reductase (MerA). Extensive analysis of this intracellular enzyme, puri¢ed from both Gram-negative and Gram-positive bacteria, has revealed it to be a member of the £avin containing NADPH dependent dithiol oxidoreductase family [2]. Although the presence of mercuric reductase is essential for enzymatic detoxi¢cation, and hence resistance to inorganic mercury, expression of the merA gene (and merR), has been reported in a high proportion of Gram-positive environmental strains sensitive to mercury, suggesting the presence of non-functional mer operons in which the mercury transport genes are absent or non-functional [26]. Mercury resistance determinants provide an ideal system to evaluate the e¡ects of stress on genetic distribution and diversity, gene transfer and evolution, given their global distribution on transposons, plasmids or chromosomes in a diverse range of bacteria isolated from a range of clinical and natural environments [3]. Given the key role played by the merA gene in mercury detoxi¢cation in many di¡erent systems, we have employed this gene to undertake a study of diversity in Gram-positive HgR determinants. In this study we report on the presence and diversity of Bacillus RC607-like merA sequences from both culturable and non-culturable bacteria, using a range of molecular techniques including Southern and dot-blot hybridisation, polymerase chain reaction (PCR), and restriction fragment
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length polymorphism (RFLP). The aim of the study was to identify the presence of mercury resistant Bacillus, and to provide an analysis of the extent of variation within these Gram-positive HgR determinants. Comparisons between Bacillus merA genes derived from cultured isolates and those ampli¢ed from directly extracted soil DNA are shown, enabling a comparison of Gram-positive mer diversity with previous studies on Gram-negative mer sequences ampli¢ed from the same environmental site.
2. Materials and methods 2.1. Soil and sediment samples Soil (SO) and sediment (SE) samples were collected two metres apart, from above and below the tide line of the River Mersey at Fiddlers Ferry, a site with industrial mercury pollution, and the site of previous studies of HgR determinants from Gramnegative bacteria [7,9,12]. SO samples were taken at three separate times, November 1994 (CP94), November 1995 (CP95 and total DNA) and April 1996 (CP96). One SE sample was taken, in November 1994. The total mercury levels for SO and SE were previously determined as 0.44 ppm and 0.16 ppm respectively [7]. 2.2. Bacterial strains and plasmids Escherichia coli AB1157 (SmR ) was the host to all Gram-negative control plasmids used in this study. Gram-negative control plasmids used were: pACYC184: :Tn501 (HgR , CmR ) [24], pSP200 (HgR , CmR ) [15], pMJ100 (HgR , CmR ) [27]. The Gram-positive control was Bacillus sp. strain RC607 mer operon cloned into plasmid pYW33 [23] and subsequently introduced into the host bacterium B. subtilis 168. Ampli¢ed merA genes from total bacterial DNA were cloned into the pGEM-T vector (ApR ) (Promega). E. coli XL-1-Blue (TcR ) (Stratagene) was the host of all Bacillus merA clones. 2.3. Bacillus isolation and identi¢cation Bacillus species were isolated using a modi¢cation of the method of Ramsay [28] which selects speci¢-
75
cally for heat-resistant spores. Each sample (10 g wet weight) was heated at 80³C for 30 min to kill vegetative cells, and then suspended in 90 ml of 50 mM Tris-HCl bu¡er (pH 7.5). Following three cycles of 1 min blending and 1 min of cooling on ice, serial dilutions of the homogenate were prepared using 25% Ringers solution (2.25 g NaCl, 0.15 g KCl, 0.12 g CaCl2 , and 0.05 g NaCO3 per litre distilled water). Dilutions were plated in triplicate onto Bacillus germination agar (BAG) [29], and then onto BAG supplemented with 50 WM HgCl2 . The inoculated plates were then incubated at 37³C for at least 2 days. Isolates were examined microscopically for the characteristic Bacillus rod shaped morphology, and con¢rmed as Bacillus strains by identi¢cation using API 50CHB medium and API 50CH strips (Biomerieux) [30], and by 16S rDNA sequencing. Ampli¢cation of 16S rDNA was performed using pA and pHP primers [31]. The PCR ampli¢cation conditions were as described below with the exception that 55³C was used as the annealing temperature. PCR products were puri¢ed for DNA sequencing using Pharmacia Biotech MicroSpin S-400 HR columns in accordance with the manufacturer's instructions. DNA sequencing was carried out on an ABI 373A automated sequencer. 16S rDNA sequences were aligned and compared with sequence databases using the FASTA program in the UWGCG suite of programs [32]. 2.4. Soil DNA extraction Following rigorous UV irradiation and/or autoclaving of all solutions and containers, 2 g wet weight of soil was added to 5 ml 0.12M Na2 PO4 (pH 8.0) together with 2 g of 0.17^0.18-mm Glasperlen beads (B. Braun Biotech International Gmbh). The mixture was beadbeaten for 30 s and DNA was extracted from this suspension following the method of Bruce et al. [33], with the modi¢cation that DNA was precipitated overnight at 320³C with 0.25 vol of 3 M sodium acetate (pH 4.8) and 1 vol isopropyl alcohol. DNA was suspended in 100 Wl of sterile distilled water. 2.5. Dot-blot hybridisation Bacillus isolates were grown overnight in 2 ml of
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Luria broth at 37³C with shaking at 200 rpm. DNA for dot-blot hybridisation was isolated by the method of Wheatcroft and Williams [34], with modi¢cations as described by Osborn et al. [7]. Samples of genomic DNA containing approximately equivalent concentrations, as determined by agarose gel electrophoresis, were denatured by addition of an equal volume of 0.2 M NaOH, and incubated at room temperature for 30 min. The denatured DNA was transferred onto GeneScreen Plus1 hybridisation membranes (Dupont) using a dot-blot manifold. DNA isolated from strains of E. coli containing plasmids pACYC184: :Tn501, pSP200 and pMJ100, bearing HgR determinants from Gram-negative bacteria, were used as negative controls, and clone pYW33 [23] containing the mer operon of Bacillus strain RC607 mer operon as the positive control. The DNA samples were hybridised against two mer probes, a Bacillus merA probe from BglII and SmaI digestion of clone pYW33, and the merRTvP region from Tn501 [9]. Probe DNA was labelled with [K-32 P]dCTP using the Random Primed Labelling Kit (Boehringer Mannheim). Hybridisations with these probes were performed as recommended by the GeneScreen instruction manual with duplicate 100 ml washes in (i) 0.3 M NaCl and 0.03 M Na citrate (2USSC) for 5 min; (ii) 0.3 M NaCl, 0.03 M Na citrate and 1% SDS for 30 min, and a single wash (iii) 0.015 M NaCl and 0.0015 M Na citrate for 30 min. Washes (ii) and (iii) were performed at 65³C instead of 60³C in order to increase the stringency to detect homology greater than 70%. Membranes were exposed to Kodak Scienti¢c XOMAT ¢lm at 370³C, and subsequently developed. 2.6. Isolation of genomic and plasmid DNA To ascertain the location of the Bacillus mer determinants, total DNA from the cultured isolates was extracted using a modi¢ed method of Pitcher [35], with the inclusion of an extra lysozyme step to aid bacterial lysis. Transfer of target DNA onto GeneScreen Plus1 membrane for Southern hybridisation was carried out by using the salt transfer method [36]. Hybridisation conditions, washing and development were performed as previously described for dot-blot hybridisation. Isolation of plasmid DNA was attempted using a number of di¡erent tech-
niques, including those described by Birnboim and Doly [37], Olsen [38] and a modi¢ed version of Birnboim and Doly described by Bogdanova et al. [22] designed speci¢cally for plasmid extraction from Bacillus strains. Control strains were B. subtilis IE60, containing plasmid pGVD1, and plasmidless B. megaterium 7A34, both provided by Dr. D.R. Zeigler of the Bacillus Genetic Stock Centre (Ohio State University). Again, any DNA extracted was Southern blotted using hybridisation conditions as described. 2.7. Oligonucleotide primers Oligonucleotide primers RC607+A (5P-TGG GTG GAA CTT GCG TTA A-3P) and POSAC (5P-GTA [GT]CC [TA]GC ACA [GA]CA AGA TA-3P) were designed to a sequence encoding the active site of RC607 merA, and to a region conserved at the 3P end of both RC607 and S. aureus pI258 merA genes, respectively. These primers amplify a 1.3-kb region of RC607 merA. 2.8. PCR ampli¢cation DNA was isolated from colonies using the boiling lysis method of Gussow and Clackson [39] modi¢ed by boiling for 10 min instead of 5 min. DNA was isolated from clones either using the boiling lysis method with subsequent ampli¢cation as above, or by using the alkaline lysis method [37] with 0.5 Wl of the resultant DNA as target volume. The merA region was ampli¢ed for 30 cycles using a Perkin Elmer DNA Thermal Cycler 480, with each cycle consisting of a denaturation step (94³C, 1 min), an annealing step (53³C, 1 min) and an extension step (72³C, 2 min). After 30 cycles the reaction was completed with a 10 min ¢nal extension stage at 72³C. Each PCR reaction comprised 50 Wl of the boiled bacterial lysate, 20 pmol of both the RC607+A and POSAC primers, 50 WM of each deoxynucleoside triphosphate, 10 Wl of Gibco-BRL 10UTaq DNA polymerase bu¡er, 1.5 mM MgCl2 , and 2.5 U of Taq DNA polymerase. The reactions were made up to a total volume of 100 Wl with sterile distilled water, and overlaid with mineral oil (Sigma). For the control reactions 1 Wl of pYW33 was used as a positive control, and a water control, containing no target DNA was used as a negative control. DNA
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isolated directly from SO95 soil was ampli¢ed as above, except 35 cycles of PCR were used, and the target DNA volume ranged from 0.1 Wl to 10 Wl depending on PCR product yield. 2.9. Cloning of ampli¢ed products The merA products ampli¢ed from DNA extracted directly from soil were cloned into pGEM-T (Promega, UK) following the manufacturer's instructions. Ligation mixtures were subsequently transformed into competent E. coli XL-1 cells, prepared by the method of Nishimura et al. [40]. Clones carrying merA inserts were identi¢ed by digestion with SacI or PstI (Gibco-BRL) of plasmid DNA following alkaline lysis extraction. 2.10. Hybridisation and restriction analysis of PCR products PCR products were identi¢ed by electrophoresis on a 0.7% agarose TBE gel containing 1 Wg ml31 ethidium bromide, and were subsequently hybridised to the RC607 merA probe by Southern blotting. PCR products (10 Wl) of RC607 merA were digested with the following restriction endonucleases: AvaI, ClaI, HaeII, HaeIII, HinfI, PvuII, and VspI (Gibco-BRL), to obtain RFLP pro¢les. Reaction conditions used were those as recommended by the supplier, and the products of each reaction were electrophoresed on a 2% agarose TBE gel containing 1 Wg ml31 of ethidium bromide. 2.11. Numerical analysis The RFLP data were analysed using the Dice coe¤cient of variation [41], where the percentage similarity between two merA determinants was calculated using the formula: % similarity 100U 2Unumber of indistinguishable bands= total number of bands: The similarity coe¤cients calculated were used in the construction of a dendrogram using the unweighted
Fig. 1. Schematic representation of RFLP patterns of ampli¢ed merA fragments from culturable HgR Bacillus isolates and directly ampli¢ed total bacterial DNA. For HinfI digests, bands of double thickness represent doublets.
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paired group mean analysis (UPGMA) program on PHYLIP 3.5C [42].
3.2. Hybridisation DNA extracted from a total of 100 Bacillus strains isolated from the three soil samples was tested by dot-blot hybridisation for homology to the merA gene from Bacillus sp. RC607. Ninety-eight isolates contained sequences with strong homology to the probe, with the negative controls showing no hybridisation. No plasmids could be isolated from the culturable isolates using a variety of extraction methods [37,38] including the Bacillus speci¢c method described by Bogdanova et al. [22]. Plasmid DNA was, however, extracted from the plasmid containing control isolate Bacillus subtilis IE60. Southern hybridisation of total DNA isolated using either the extraction method of Pitcher et al. [35], or the modi¢ed version of Wheatcroft and Williams [34] used by Osborn et al. [7], suggested that merA genes homologous to that from RC607 were located on the chro-
3. Results 3.1. Distribution of cultivable HgR Bacillus in soil and sediment The titres of Bacillus colony forming units (CFU) from SO samples CP94 and CP95 were 7.4U105 and 8.3U106 CFU g31 , respectively. This compares to a total CFU count of 9.6U103 from the SE94 sample. Total CFU counts from SO sample CP96 were not taken. The frequencies of resistance to inorganic mercury (Hg2 ) in the CP94 and CP95 samples were 5.4% and 4.4% respectively. No mercury resistant bacteria were isolated on Bacillus germination agar from sediment.
Table 1 Frequency of each RFLP class of Bacillus merA sequences ampli¢ed from HgR isolates and directly from total bacterial DNA extracted from soil RFLP class
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX XXI XXII a b
Number of clones/isolates Cloned SO95 total DNAa
Isolates '94b
4 ^ ^ ^ ^ ^ ^ 18 10 ^ ^ ^ ^ ^ 1 1 1 1 1 1 1 1 40
1 12 5 1 3 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 22
Class derived from ampli¢ed products of total bacterial DNA. Cultured isolates identi¢ed by year of sampling.
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Isolates '95b
Isolates '96b
1
^ ^ ^ ^ ^
4 1 6 8 2 1 1 1
9 ^ ^ ^ ^ ^ ^ ^ 1 ^ ^ ^ ^ ^ ^ ^ ^ 10
^ ^ ^ ^
^ ^ ^ ^ ^ ^ ^ ^ ^ 25
M.C. Hart et al. / FEMS Microbiology Ecology 27 (1998) 73^84
mosomes of the environmental Bacillus isolates (data not shown).
Fig. 2. Venn diagram to illustrate the sharing of Bacillus merA RFLP classes between three SO HgR isolate samples: CP94 (11/ 94); CP95 (11/95) ; CP96 (4/96), and from directly ampli¢ed total bacterial DNA isolated from SO.
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3.3. PCR ampli¢cation from Bacillus isolates Eighty of the positive Bacillus isolates by dot-blot hybridisation were selected for analysis by PCR, and merA genes were successfully ampli¢ed from 58. Of the ampli¢ed products, 57 showed no size variation from the predicted 1.3-kb fragment based on the RC607 merA. The exception was isolate BA11, from which an 800-bp product was ampli¢ed. No ampli¢cation products were observed from the remainder of the isolates. All 58 ampli¢ed fragments were checked for homology to the RC607 merA probe by Southern hybridisation. Homology was shown for all products, and these were further investigated using RFLP analysis.
Fig. 3. Dendrogram (neighbor-joining UPGMA) of genotypic relationships between merA fragments from cultivable HgR Bacillus isolates and directly ampli¢ed total bacterial DNA derived by PCR/RFLP analysis. Divergence between classes of mer is expressed as percentage similarity. Allocation of individual classes to respective samples is shown on right by di¡erently shaded circles.
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3.4. Extraction of total bacterial DNA and ampli¢cation Total DNA was isolated from the SO95 soil sample as described in Section 2, and visualised by agarose gel electrophoresis. Products of the predicted 1.3kb size were ampli¢ed from this DNA using the RC607+A and POSAC primers, ligated into the pGEM-T vector, and transformed into E. coli. Using the merA primers, PCR products could be ampli¢ed from plasmid DNA extracted from 41 of the resulting transformants, and these fragments were subsequently used for RFLP analysis. 3.5. RFLP analysis of ampli¢ed PCR products Digestion of ampli¢ed merA products derived from both culturable Bacillus isolates and total bacterial DNA, using a number of restriction endonucleases, revealed a variety of di¡erent DNA fragment patterns. From these a total of 22 Bacillus merA Table 2 API identi¢cation of selected Hg resistant Bacillus isolates RFLP class
Isolate
API identi¢cation
I I II II II II II II II III III III III IV V V VI VI VI VII IX IX X XII XIV
BA1 BA34 BA3 BA6 BA22 BA23 BA10 BA20 BA4 BA5 BA7 BA18 BA24 BA26 BA28 BA29 BA33 BA53 BA77 BA35 BA44 BA58 BA45 BA55 BA82
B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B.
cereus (99.2%) sphearicus (54.2%) pantothenticus (100.0%) pantothenticus (94.0%) pantothenticus (94.0%) pantothenticus (91.0%) laterosporus (50.2%) laterosporus (50.2%) subtilis (88.8%) stearothermophilus (98.6%) stearothermophilus (93.5%) amylofaciens (45.8%) subtilis (85.4%) licheniformis (50.0%) subtilis (65.2%) subtilis (65.2%) sphearicus (44.6%) sphearicus (54.2%) sphearicus (54.2%) sphearicus (54.2%) subtilis (84.2%) lentus (99.9%) sphearicus (54.2%) sphearicus (54.2%) ¢rmus (99.6%)
Percentages indicate level of certainty for identi¢cation.
RFLP classes were identi¢ed (Fig. 1, Table 1): 11 from total SO95 DNA and 14 from culturable isolates, with three classes common to both (Fig. 2). Of the 41 clones derived from total SO95 DNA, only one showed no similarity in any digest to RC607 and did not hybridise to the Bacillus merA probe. The nature of this ampli¢ed product is described later. Between culturable isolates obtained from the three SO samples, the numbers of common RFLP classes were limited, with only classes I (RC607) and VI present in samples from more than one year (Table 1). In the limited analysis of CP96, only class VI was found to be common between this and any other year, namely CP95. No similarity was observed between the classes identi¢ed from the CP94 and CP95 samples. The classes derived from clones isolated from the total SO95 DNA showed a degree of similarity to those from the culturable classes, principally those from CP95, with VIII and IX the main classes in both. Again, class I was detected in the cloned merA samples, although the two predominant classes present in the culturable samples as a whole (II and III) were not represented. An additional eight classes distinct from those seen in culturable bacteria were also contained within the fragments obtained from total DNA. Representatives of the classes obtained from total SO95 DNA were all shown, by Southern hybridisation, to have homology to the RC607 merA probe. On the basis of these RFLP results, similarity co-e¤cients were calculated and used to construct a UPGMA dendrogram (Fig. 3). 3.6. API identi¢cation of HgR Bacillus isolates The results of API identi¢cation of the mercury resistant Bacillus isolates are shown in Table 2. A limited number of the Bacillus isolates could be identi¢ed with an excellent degree of certainty (99%) using the Biomerieux 50CH strips, for example BA1 (B. cereus), BA3 (B. pantothenticus) and BA82 (B. ¢rmus). A good level of certainty of identi¢cation (above 90%) included isolates BA6 (B. pantothenticus), and BA5 (B. stearothermophilus), whilst acceptable identi¢cation (above 80%) was achieved for BA24 (B. subtilis). Most of the soil isolates gave either a very doubtful identi¢cation, with the reliability of identi¢cation ranging from 44.6% (BA33, B. sphearicus), to 65.2% (BA28, B. subtilis), or no result.
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Using all the results obtained from these biochemical test strips, including the doubtful identi¢cations, nine di¡erent species of Bacillus were identi¢ed. Species diversity was revealed between and within some RFLP classes, notably classes I, II, and IX, with class II identi¢ed as present in three di¡erent species
4. Discussion The proportion of Bacillus strains that are mercury resistant within Fiddlers Ferry soil, at around 5%, is within the range of mercury resistant bacteria isolated in studies previously cited by Rochelle et al. [6]. With the total culturable Bacillus population, the viable counts from sediment were signi¢cantly lower than those from soil, and so the failure to isolate HgR Bacillus strains from Fiddlers Ferry sediment is therefore not surprising. These lower numbers may re£ect the naturally more anoxic nature of this environment. The vast majority of the SO HgR isolates (98%) hybridised to the RC607 merA probe, indicating the prevalence of volatilisation as the mechanism of Hg resistance in Bacillus. The two remaining HgR isolates may be either su¤ciently divergent from the merA sequence of RC607 to preclude hybridisation, or may simply employ a di¡erent resistance system. These isolates have not as yet been investigated further. The presence in SO samples of sequences homologous to Bacillus merA found in previous studies in Boston Harbor (USA) [20], Minimata Bay (Japan) [21,43] and Russia [4] adds to data on the wide geographical distribution of Bacillus mer operons across the globe. The global distribution of chromosomally encoded Bacillus mer indicates that Hg resistance may have an ancient origin [3]. However, the possible spread of Bacillus mer genes by the horizontal dissemination of mer on plasmids and transposons [4,22] analogous to the dispersal of Gram-negative HgR determinants [3] should not be discounted. The positive hybridisation of the merA probe to chromosomal DNA extracted from all the culturable strains suggests, as with RC607, that the merA in these strains is chromosomally located. Whilst not being absolutely conclusive evidence for the chromosomal location of the mer operon (the presence of large linear or integrated plasmids is always a possi-
81
bility), the corresponding failure to isolate plasmid DNA using a number of techniques also supports this conclusion. merA gene fragments were ampli¢ed from 58 of the hybridising isolates with only one size variant from RC607, which ampli¢ed to give an 800-bp product. From sequence analysis (data not shown) this smaller fragment appears to have arisen by annealing of the RC607+A primer to an alternative site 400bp within the normal ampli¢cation product. The 22 hybridising isolates which gave no product presumably re£ect sequence diversity in the Bacillus merA speci¢c primer binding sites and were not further analysed. The Bacillus merA sequences derived from cultured isolates were compared to sequences ampli¢ed from DNA directly extracted from SO95 soil and then cloned. The DNA extraction method used has been shown to extract DNA from both vegetative Bacillus cells and from endospores (data not shown). The potential for the increased frequency of production of chimeric fragments during PCR has been shown not to be a factor in creating spurious diversity [33]. Of the 41 cloned amplimers, 40 were shown to hybridise to the RC607 merA probe. This high percentage is to be expected because of the speci¢city of the merA oligonucleotide primers used. The sequence that did not hybridise to RC607 has since been shown to be 63% identical to the merA gene of Tn501, a Gram-negative mer system, on the basis of DNA sequence comparison (data not shown). The fact that a sequence has been ampli¢ed using oligonucleotide primers designed for Gram-positive mer sequences that is ostensibly of Gram-negative origin may indicate a link between these two seemingly divergent mer systems. Comparison of RFLP data obtained from the 97 PCR products ampli¢ed either from cloned DNA or from cultured isolates initially revealed little in terms of RFLP class correlation between samples. However, division of the 14 classes derived from culturable isolates into the three sampling times gives a clearer picture of the relationships between these classes and those of cloned DNA (Fig. 2). The greatest overlap is found between classes of CP95 and those of cloned SO95 DNA, with classes VIII and IX being the most common in both (Table 1). This is probably due to the fact that both samples were
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taken at the same time, removing the in£uence of temporal variation within the Bacillus population. The lack of a more substantial correlation between the three sets of cultivated classes seems to indicate changes over time in the genetic composition of Bacillus merA from Fiddlers Ferry soil. Only classes I (CP94 and CP95) and VI (CP95 and CP96) were shared between two cultured samples (Fig. 2), indicating either the presence of microsites within the SO soil, each containing a distinct population of closely related merA genes, or a major change in the Bacillus population in the time periods separating the samples. Closer analysis of the dendrogram (Fig. 3) shows that in many cases, classes from one sample tend to cluster, suggesting that random sampling of isolates or total DNA has not occurred. Such an observation is again consistent with the presence of microsites, each containing a distinct Bacillus subpopulation. Indeed microsites may also provide a plausible explanation for the di¡erences between the CP95 sample and the total SO95 DNA which show little overlap despite being derived from the same large soil sample. In the limited API identi¢cation of isolates (Table 2) species variation can be seen both within and between RFLP classes. The fact that RFLP classi¢cation is not entirely correlated with species indicates that the diversity present within the cultivable merA population is not simply due to the divergent evolution of di¡ering Bacillus species. In a study undertaken on Bacillus isolated from Minimata Bay sediment in Japan [43], a site of methylmercury contamination, seven RFLP classes of mer determinants were identi¢ed [21]. Signi¢cantly the percentage of isolates that hybridised to an RC607 probe, 95% (74 out of 78), is similar to the high percentage observed in our study of cultured bacteria (98%). Comparison of the diversity revealed within the classes obtained in this study with the RFLP classes of our study is di¤cult because of their less detailed RFLP analysis of a larger region of the Bacillus mer operon. The di¡erences observed in classes derived from total DNA and merA fragments ampli¢ed from the culturable isolates may be due to the sampling of di¡erent microsites. Alternatively, it may re£ect the fact that the eight unique merA classes derived from the total DNA might have been ampli¢ed from ac-
tive Bacillus cells present in the soil, which might not be represented as spores in the environment and consequently would not be selected on BAG plates. Restriction classes unique to the cloned DNA may also have arisen from ampli¢cation of non-functional, or cryptic merA genes [9,26]. As with this study, the lack of overlap between classes from cultured isolates and total bacterial DNA was seen within an RFLP analysis of ampli¢ed merRTvP genes of the Gramnegative population, previously studied at the SO and SE sites at Fiddlers Ferry [7,9,12,44], and has been taken to indicate the low proportion of microorganisms that can be isolated by direct culturing techniques. The diversity present in the Bacillus soil population may be due to the very e¡ective capacity of certain Bacillus species for cell-to-cell DNA exchange. Bacterial populations are likely to range from strictly clonal to panmictic [45], but the work of Istock et al. [46] and Duncan et al. [47] showed that genetic exchange within the Bacillus population would reduce the formation of a clonal population structure. The highly sexual nature observed in some Bacillus species [46] may therefore contribute signi¢cantly to the overall level of diversity seen within merA. The large amounts of variation observed between merA classes found in cultured isolates and extracted bacterial DNA highlights the need to extend studies into areas other than cultivation and also emphasises the importance of sample size. Rapid analysis techniques such as denaturing gradient gel electrophoresis are now being used for the analysis of Bacillus merA diversity and will provide alternative means to pro¢le microbial populations. This may enable the £uctuation of closely related bacterial microsite populations to be studied in much more detail.
Acknowledgments This work was supported by NERC Grants GR3/ 9502 and GT4/94196/T. This work was jointly carried out by Geo¡ Elliott and Mark Hart. The probe and primers to the RC607 merA were developed by Dr. A.M. Osborn, who also provided technical advice. The project was supervised by Prof. P. Strike and Prof. D.A. Ritchie. Special thanks also go to Dr.
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K.D. Bruce for his help and support, to I. Mahler for providing the plasmid pYW33 and to Dr. Zeigler of the Bacillus strain stock centre for providing control strains. References [1] Von Burg, R. and Greenwood, M.R. (1991) Mercury. In: Metals and Their Compounds in the Environment (Merian, E., Ed.), pp. 1045^1088. VCH, New York. [2] Foster, T.J. (1987) The genetics and biochemistry of mercury resistance. CRC Crit. Rev. Microbiol. 15, 117^140. [3] Osborn, A.M., Bruce, K.D., Strike, P. and Ritchie, D.A. (1997) Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon. FEMS Microbiol. Rev. 19, 239^262. [4] Yurieva, O., Kholodii, G., Minaklin, L., Gorlenko, Z., Kalyaeva, E., Mindlin, S. and Nikiforov, V. (1997) Intercontinental spread of promiscuous mercury resistance transposons in environmental bacteria. Mol. Microbiol. 24, 321^329. [5] Barkay, T. and Olson, B.H. (1986) Phenotypic and genotypic adaption of aerobic heterotrophic sediment bacterial communities to mercury stress. Appl. Environ. Microbiol. 52, 403^406. [6] Rochelle, P.A., Wetherbee, M.K. and Olson, B.H. (1991) Distribution of DNA sequences encoding narrow and broad spectrum mercury resistance. Appl. Environ. Microbiol. 57, 1581^ 1589. [7] Osborn, A.M., Bruce, K.D., Strike, P. and Ritchie, D.A. (1993) Polymerase chain reaction-restriction fragment length polymorphism analysis shows divergence among mer determinants from Gram-negative soil bacteria indistinguishable by DNA-DNA hybridisation. Appl. Environ. Microbiol. 59, 4024^4030. [8] Gri¤n, H.G., Foster, T.J., Silver, S. and Misra, T.K. (1987) Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinant of plasmid pDU1358. Proc. Natl. Acad. Sci. USA 84, 3112^3116. [9] Bruce, K.D., Osborn, A.M., Pearson, A.J., Strike, P. and Ritchie, D.A. (1995) Genetic diversity within mer genes directly ampli¢ed from communities of noncultivated soil and sediment bacteria. Mol. Ecol. 4, 605^612. [10] Gilbert, M.P. and Summers, A.O. (1988) The distribution and divergence of DNA sequences related to the Tn21 and Tn501 mer operons. Plasmid 20, 127^136. [11] Olson, B.H., Lester, J.N., Cayless, S.M. and Ford, S.J. (1989) Distribution of mercury resistance determinants in bacterial communities of river sediments. Water Res. 23, 1209^1217. [12] Osborn, A.M., Bruce, K.D., Strike, P. and Ritchie, D.A. (1995) Sequence conservation between regulatory resistance genes in bacteria from mercury polluted and pristine environments. Syst. Appl. Microbiol. 18, 1^6. [13] Inoue, C., Sugawara, K. and Kusano, T. (1991) The merR regulatory gene in Thiobacillus ferrooxidans is spaced apart from the mer structural genes. Mol. Microbiol. 5, 2707^2718.
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