conservation of transposon structures in soil bacteria

FEMS Microbiology Ecology 30 (1999) 25^37 Conservation of transposon structures in soil bacteria Robert J Holt, Kenneth D Bruce, Peter Strike * School of Biological Sciences, Donnan Laboratories, University of Liverpool, Liverpool L69 7ZD, UK Received 12 January 1999; revised 13 April 1999 ; accepted May 1999 Abstract The presence of Class II transposon genes related to Tn21 and Tn501, and their structural arrangements have been determined in a collection of 124 mercury resistant Gram-negative bacteria Seventy-five of the 124 isolates contained a tnpA (transposase) gene related to Tn21 and Tn501 and in all 64 isolates that contained both tnpA genes and plasmids, the tnpA gene was plasmid borne The relative orientation of the tnp genes and the mer operon (encoding mercury resistance) was also studied and revealed the presence of two distinct structural groups The merC gene was present in 44 isolates Five isolates were found to carry integrase genes and these contained inserted gene cassettes varying in size from 1.1 kb to 4.5 kb The structural arrangement of the tnpA and tnpR (resolvase) genes within the isolates was determined Sixty-nine of the 75 tnpA containing isolates had an arrangement of tnpA and tnpR genes similar to that found in the Tn21 subgroup of transposons Four strains did not produce a PCR product using tnpR primers The remaining two isolates had undetermined arrangements of tnpA and tnpR genes No Tn3-like arrangements of tnpA and tnpR genes were present in these isolates, despite being detected in DNA extracted directly from the isolation sites This suggests that Tn3-like arrangements of tnpA and tnpR genes are not commonly associated with mercury resistance genes in these environments It was also apparent that the recombination events which have previously been observed in these strains have not significantly affected the diversity of the transposon structures within the isolates ß 1999 Federation of European Microbiological Societies Published by Elsevier Science B.V All rights reserved Keywords : Gene diversity ; Transposon; Soil bacteria; tnpA; tnpR; mer operon Introduction The study of genes contained within the indigenous bacterial community in the natural environment is important as it provides an opportunity to assess the e¡ects of selective pressure, e.g due to pollutants, on bacterial gene diversity and therefore * Corresponding author Tel.: +44 (151) 794 3620; Fax: +44 (151) 794 3655; E-mail: strike@liverpool.ac.uk facilitates the development of predictive tools [1^5] A wide range of mechanisms exist that can alter genetic diversity These include mutation, recombination, transposition, transformation and conjugation [1,6^10] In natural environments, transposition mediated transfer of DNA represents a major mechanism for increased mobility of genes contained within the transposon and also a potential mechanism for genetic rearrangement [7,10,11] There are four classes of transposable elements [11], amongst which the Class II transposons have been extensively studied [4,7,11^13] The archetypal 0168-6496 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies Published by Elsevier Science B.V All rights reserved PII: S - ( 9 ) 0 - FEMSEC 1049 20-8-99 26 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 members of this class contain two genes, encoding functions involved in transposition: the transposase gene, tnpA, and the resolvase gene, tnpR [14^18] A resolution (res) site is present in most Class II transposons, at which site speci¢c recombination occurs in order to resolve the cointegrate transposon structures which arise during the transposition process Several transposon structures have been described which have more diverse transposition mechanisms, including Tn4652 and Tn4430 which have di¡erent resolution systems, Tn2610 which has two functional copies of the tnpR gene and Tn5271 which has no resolution system [19^22] Amongst the most widely studied Class II transposons are the mercury resistance elements Tn21 and Tn501 [7,13,14,16] Other members of this class di¡er from Tn21 and Tn501, not only in the transposon associated genes, but also in the structural arrangement of the transposition genes, most notably the orientation of the tnpA and tnpR genes, and the position of the res site [7] The arrangement of these genes has important implications for the study of the evolutionary relationships between di¡erent transposons The structures of four transposons, Tn21, Tn501, Tn3 and Tn5036, are shown schematically in Fig The major di¡erence lies in the arrangement of the tnpA and tnpR genes The tnpA and tnpR genes in Tn3-like transposons are in a di¡erent orientation to that found in the Tn21 subgroup [7] The res site of Tn3 lies between the tnpA and the tnpR gene, whereas in the Tn21-like elements the res site is outside both genes The Tn3-like arrangement is also found on transposons such as Tn2501 and Tn1331 [23,24] Tn4556 is believed to contain a di¡erent arrangement of genes, with the res site being present between the tnpA and tnpR genes which are transcribed in the same direction [25] The potential for transposon mobility is increased if the transposon is present on a conjugative or mobilisable plasmid Transposons found on plasmids include Tn21, Tn501, Tn917, Tn1721, Tn3926 and Tn5422 [14,16,26^29] Variation also exists amongst the transposon associated genes which encode non-essential functions that may confer a selective advantage to the host bacterium Tn21 and Tn501 are members of a large group of related transposons that can confer resist- ance to mercurial compounds, and the mercury resistance (mer) operons of these elements show considerable variation, whilst retaining a number of common features [30,31] Some genes (merR, merP, merT and merA) are present in the majority of mer operons which have been described to date [30,31] However some genes are not, including merB (organomercurial lyase), merC (transport protein) and merF (function unclear) [30,31] Fig shows that Tn21 has a merC gene inserted between the merP and merA genes [16] The merF gene is also commonly found inserted at this point In addition, integron elements have also been identi¢ed in a number of transposons These structures insert and excise speci¢c gene cassettes into a recombination hot spot (rhs) contained within the integron structure, e¡ectively providing a means by which the transposon can acquire novel genetic material [10,32] The number of bacteria in estuarine environments which contain integrons has been estimated at 5% (Young, H.K and Rosser, S.J., personal communication) This represents an enormous potential for gene transfer in the natural environment Previous studies on a diverse collection of 39 Gram-negative mercury resistant bacteria ('93 isolates) have concentrated on the study of sequence diversity The diversity of tnpA, tnpR and merR genes has been studied by RFLP analysis and by DNA sequencing [1,3,4,33] A signi¢cant conclusion from these studies was that recombination between transposon genes and between transposon and mer genes, was common Prior to this study no information was available regarding the structural diversity of the transposon genes carried in these isolates The presence of plasmids had not been studied and as such, the location of the transposons contained within these isolates was also not known In this study, the relationship between the transposition genes and their associated mer operons has been investigated further The number of strains studied was expanded to include 85 new isolates (P96 isolates) These mercury resistant strains were cultured from the same sampling sites as the P93 isolates using the same extraction protocol and were also Gram-negative The identity of the P93 isolates has been determined by Osborn et al [33] using API A range of species were identi¢ed including Enterobacter cloacae, Alcaligenes faecalis, Acinetobacter calcoceticus, Klebsiella oxyto- FEMSEC 1049 20-8-99 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 ca, Agrobacterium radiobacter, Aeromonas spp and Pseudomonas spp [33] The identity of the P96 isolates was not determined in this study The presence of transposon associated genes has been studied in all 124 isolates, and PCR reactions have been carried out to determine the presence of integrons, and the size of any genes present within the rhs of those integrons The presence and structural organisation of the mer genes in the isolates has been studied, as have the relative orientations of the tnp genes and mer operons in the isolates The presence and approximate size of plasmids present within the isolates was also determined to ascertain whether any correlation existed between the structural diversity of the transposon and the plasmid diversity The primary aim of this study was to determine the presence and structural diversity of the tnpA and tnpR genes contained within the isolates and to seek evidence, or otherwise, of genetic rearrangements and/or recombination occurring in the transposon associated region Materials and methods 2.1 Extraction of isolates from soil The 39 mercury resistant P93 strains used in this study were previously isolated by Osborn et al [33] from both polluted and pristine sites; these strains were selected by their ability to hybridise to a merRTvP probe All new isolates have been designated P96 in order to distinguish them from the P93 isolates For the P96 collection, bacteria were isolated from soil and sediment samples as previously described [33], and were selected for mercury resistance Unlike the P93 isolates, they were not selected for hybridisation to the merRTvP probe They were isolated from the following sites: P96 SO strains were isolated from soil at a mercury polluted site at Fiddlers Ferry, Merseyside [33] Sediment at this site was used to isolate the P96 SE strains P96 SB strains were isolated from soil at pristine site at Salterbrook Bridge [33] 2.2 Isolation of plasmid DNA Plasmids were isolated from bacteria according to 27 the method described by Olsen et al [34] DNA was visualised on agarose gels (0.7%) using TAE bu¡er (40 mM Tris, 20 mM acetic acid, mM EDTA, pH 8.3), run at 40 V for approximately 24 h, followed by visualisation using ethidium bromide staining (1 Wg ml31 ) 2.3 PCR PCR reactions were carried out under a variety of conditions Typically 50 Wl reactions were used containing Wl of 10UPCR bu¡er, 1.5 mM MgCl2 and 1.25 U of Taq DNA polymerase (GIBCO BRL) DNA template was prepared by the boiling method previously described [33] Twenty pmol of each primer was added to the reaction mix along with dNTPs (Pharmacia Biotech) at a ¢nal concentration of mM Reactions were brought up to 50 Wl using sterile distilled water and then overlaid with mineral oil (Sigma) PCR reactions were carried out in a Perkin Elmer 480 thermal cycler and were typically comprised of at 95³C, followed by 30 cycles of 95³C for min, 62³C for and 72³C for A ¢nal extension step of 10 at 72³C was carried out before fragments were visualised on agarose gels Di¡erent annealing temperatures were used depending on the primers used in each reaction To amplify regions of DNA longer than kb, a long template PCR system was used (Expand PCR, Boehringer Mannheim) in accordance with the manufacturers' instructions [35] 2.4 Primers The primers used in this study are shown in Table The position of the primer within the accession number is also shown All accession numbers listed are for Tn21 sequences, except tn3A and tn3R, which are shown for Tn3, and pos2000 and pos2400, which are shown for Tn4430 2.5 Southern blotting Both Southern blots and dot blots were utilised in this study, depending on the nature of the DNA sample being studied Dot blots were carried out using a Bio-Rad Bio-Dot vacuum manifold in accordance with manufacturers' instructions DNA FEMSEC 1049 20-8-99 28 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 samples for both dot blots and Southern blots were transferred onto positively charged membranes (Appligene) in accordance with manufacturers' instructions Overnight transfer of DNA was set up using 0.4 M NaOH as the transfer bu¡er Southern blots of plasmid DNA di¡ered in that the transfer time was extended to 48 h Prehybridisation of the membrane in both cases was carried out in accordance with the manufacturers' instructions PCR products were used as probes These were prepared for hybridisation by an initial electrophoresis step using low melting point agarose (NuSieve GTG) after which the band was excised using a scalpel and the probe resuspended in sterile distilled water Probes were then radiolabelled with 32 P using a random prime labelling kit (Boehringer Mannheim) and the probe puri¢ed on a Sephadex G50 column After overnight hybridisation at 65³C, the membrane was washed twice in 2USSC for min, twice in 2USSC/1% SDS for 15 and once in 0.1USSC for 15 Signal detection was carried out on a Molecular Dynamics STORM 860 phosphoimager 2.6 Restriction digests The merC probe used in this study was prepared by digestion of the Tn21 mercP/mercA PCR product with HaeII and StyI to produce a fragment of 500 bp This fragment represents the merC gene and 80 bp of extra sequence at the 5P end of the gene Both restriction enzymes (GIBCO BRL) were used in accordance with manufacturers' recommendations Table Oligonucleotide primers used in this study Primer a 501R1/C 501R2/Ca 1406a 2638a 950b int21Ac int21Bc 4127c 4128c tn3Ad tn3Rd mercPe mercAe mercDf pos2000g pos2400g 2501h 2850h DNA sequence Accession no Position (bp) 5P-GTT CAG CA[GC] CTT CGA CCA G-3P 5P-TA[CG] AGG GTT TC[GC] CG[AG] CTG AT-3P 5P-TGC GCT CCG GCG ACA TCT GG-3P 5P-TCA GCC CGG CAT GCA CGC G-3P 5P-[TC]CT GGA ACT GCT GCT GAT GCT T-3P 5P-GTC AAG GTT CTG GAC CAG TTG C-3P 5P-ATC ATC GTC GTA GCG ACG TCG G-3P 5P-TGA TCC GCA TGC CCG TTC CAT ACA G-3P 5P-GGC AAG CTT AGT AAA GCC CTC GCT AG-3P 5P-GTA TCA GCG CTG CAT GCT CAC-3P 5P-CCC TGC ATC TTT GAG CGC TCT-3P 5P-CCC GAT CAC [AT]GT CAA G[AC]A [ACG]GC-3P 5P-CGC TCG ATC AGC G[AT]G AC[ACG] [CT]G-3P 5P-GTT CGT CGA GCG TCG GCG-3P 5P- GGA ATG AAT ATT GTT CTT ACC AAA ATG-3P 5P-CAG TAT AGC CAG CTG TGT CTG-3P 5P-CAT TGG GAC GAG ATG ATG CGG-3P 5P-GCT CCA TAT ACA CCG TGT TCC-3P X01298 X01298 X04891 X04891 X04891 M33633 M33633 M33633 X12870 V00613 V00613 K03089 K03089 K03089 X13481 X13481 X04981 X04981 539^557 1026^1045 1462^1481 2676^2694 987^1008 1090^1111 219^240 709^733 2321^2346 2721^2741 3270^3290 1075^1095 2116^2135 3733^3750 3466^3493 3445^3466 2490^2501 2829^2851 a These four primers have been previously described [4], having been designed to complement the DNA sequences of the Tn21 and Tn501 tnpR and tnpA sequences respectively b This primer was designed to correspond to the sequence approximately 930 bp from the start codon of the tnpA gene of Tn501/Tn21 c int21A/int21B were used as an indicator of integrase gene presence and 4127/4128 allow the size of the gene cassettes within an integron to be determined (Young, H.K and Rosser, S.J., personal communication) d These primers have been designed to the tnpA and tnpR sequences of Tn3 respectively e These primers complement the DNA sequence in merP and merA respectively allowing the nature of any genes present inbetween merP and merA to be determined f This primer was designed to be homologous to merD Tn21/501 sequence g These primers were designed to consensus sequences from the tnpA genes of Tn917, Tn1546, Tn4430 and Tn5422 [20,26,29,36] h These primers were designed to consensus sequences from the tnpA genes of Tn1, Tn3, Tn21, Tn501, Tn1000, Tn1721, Tn2501, Tn3926, Tn5036 and Tn5401 [14^16,23,27,28,38^41] FEMSEC 1049 20-8-99 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 29 Fig Diagrammatic representation of Tn501, Tn21, Tn3 and Tn5036 and approximate binding position of primers mercP, mercA, mercD, 501R1/C and 501R2/C (not to scale) Results and discussion 3.1 Presence of tnpA genes One hundred and twenty-four Gram-negative mercury resistant strains were examined for the presence of tnpA genes using two sets of primers: pos2000/ pos2400 and 2501/2850 Primers pos2000 and pos2400 were designed to allow the detection of the tnpA genes of Tn917, Tn1546, Tn4430 and Tn5422, which were originally characterised in Gram-positive bacteria [20,26,29,36] No strain produced a PCR product using these primers, despite allowing tnpA ampli¢cation from control strains Given that these primers were designed to amplify Gram-positive transposon sequences, the lack of ampli¢cation from Gram-negative strains is perhaps unsurprising PCR reactions carried out on DNA extracted di- FEMSEC 1049 20-8-99 30 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 Fig Schematic representation of possible arrangements of tnpR and tnpA genes and appropriate primer binding sites (not to scale) rectly from the soil (SO) and sediment (SE) sites yielded products using these primers (data not shown) [37] The presence of Gram-positive tnpA sequences at these sites was con¢rmed by DNA sequencing (data not shown) Primers 2501 and 2850 were designed to allow the detection of the tnpA genes of Tn1, Tn3, Tn21, Tn501, Tn1000, Tn1721, Tn2501, Tn3926, Tn5036 and Tn5401 [14^16,23,27,28,38^41] Seventy-¢ve strains produced a PCR product using these primers (Table 2) The presence of tnpA genes in 22 out of 30 P93 SO, SE and SB isolates had been previously demonstrated by hybridisation to tnpA probes from both Tn21 and Tn501 [4] Those isolates previously identi¢ed as containing tnpA sequences by probe hybridisation, also produced PCR products using primers 2501 and 2850 [4] To allow the comparison of the P93 and P96 isolates, PCR reactions were carried out on all 124 isolates using the primers 1406 and 2638, used in the characterisation of the P93 isolates [4] All FEMSEC 1049 20-8-99 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 31 Table Presence of tnpA genes and arrangements of tnpA and tnpR genes Isolation group Number in isolation group tnpA gene P93 SO P93 SE P93 SB P93 T2 P96 SO P96 SE P96 SB Total 10 10 10 30 31 24 124 10 6 20 22 75 tnpA/tnpR arrangement Tn21/Tn501 No tnpR Unknown 6 20 18 69 0 0 0 0 2 75 strains produced a PCR product using these primers (Table 2) These PCR products hybridised to the corresponding tnpA PCR products from both Tn21 and Tn501 The high number of isolates in this study containing transposase genes compared to similar studies on marine and clinical isolates [12,13] may have been caused by the initial selection for resistance to HgCl2 The higher numbers of tnpA containing isolates observed in the P93 isolates may be explained by their initial selection for hybridisation to a mer probe [33] Forty-nine of the strains did not produce a tnpA PCR product, 22 of which were in the P96 SB group This may indicate sequence variation at the primer annealing sites or suggest the lack of this gene in these isolates present Results are shown in Fig One hundred of the isolates were found to contain plasmids, with both large plasmids ( s 50 kb) and small plasmids ( 20 kb) being identi¢ed Of the 75 strains containing tnpA genes, 64 were found to contain plasmids, while 11 apparently did not Whilst large and small plasmids were observed in both the P93 and P96 collections, the P96 isolates contained a higher proportion of smaller plasmids (data not shown) No incompatibility group data are available for these plasmids, but a previous study indicates that mercury resistance plasmids isolated from the environment not conform to existing incompatibility groupings [42] 3.2 Plasmid extraction The location of the tnpA genes contained within the 75 positively amplifying isolates was determined by Southern hybridisation (Fig 3) In all 64 plasmid containing strains, the tnpA gene was located on a large plasmid In the 11 apparently plasmid-free Plasmid extractions were carried out on the collection of 124 isolates, and the samples were analysed on agarose gels to determine the size of any plasmids 3.3 Location of tnpA genes Fig Presence of plasmids and location of tnpA genes Numbers in brackets indicate the number of strains FEMSEC 1049 20-8-99 32 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 Table Integron containing isolates Strain Integrase genea Integron insert sizeb P93 T2 37 P96 SE6 P96 SE9 P96 SE15 P96 SE19 Other 119 isolates + + + + + 1.2 kb 1.1 kb 1.4 kb 4.5 kb 1.3 kb n.a a b +, integrase gene present; 3, no PCR product n.a., not applicable strains, the tnpA gene was detected in the chromosomal material Chromosomally located tnpA genes were only found in those isolates which appeared to contain no plasmids 3.4 Structural arrangement of tnpA and tnpR genes The structural arrangement of the tnpA and tnpR genes was determined by the use of PCR Of the four possible arrangements of these genes indicated in Fig 2, the most commonly encountered was that of the Tn21-like elements [7] Initially, PCR reactions were carried out using 950 and 501R1/C primers [4], corresponding to the arrangement of tnpA and tnpR genes found on Tn21-like elements (Table 2) Of the 75 isolates that produced a tnpA PCR product, 69 of these yielded an arrangement of genes similar to that found in the Tn21 subgroup of transposons, this fragment being of the size expected from these transposons The nature of these PCR products was veri¢ed by their hybridisation to tnpA/tnpR PCR products from Tn21 and Tn501 The six strains which produced no PCR products were then examined using four combinations of primers to determine whether any other arrangements of genes were present This was carried out using the following primer combinations: 501R1/C and 950, 501R1/C and 1406, 501R2/C and 950, and 501R2/C and 1406, thus covering all four possible arrangements of genes as indicated in Fig No PCR products were produced using these combinations of primers The six strains were also subjected to PCR using primers tn3A and tn3R, which were designed to Tn3 tnpA and tnpR genes respectively No strains had Tn3-like gene arrangements PCR products were however ampli¢ed using primers tn3A and tn3R on DNA extracted from these soil (SO) and sediment (SE) sites indicating that Tn3-like elements were present (data not shown) [37] These data suggest that Tn3-like arrangements of tnpA and tnpR genes are present in these environments but are not commonly associated with mer operons However as the Tn3-like arrangement of tnpA and tnpR genes was detected in DNA extracted directly from the environment, it was not possible to determine the nature of the genes associated with this arrangement Subsequently the presence of tnpR genes in those isolates not producing any tnpA/tnpR PCR products was determined by PCR using primers 501R1/C and 501R2/C Four strains produced no PCR products using these primers, suggesting that these strains either have no tnpR gene or that the speci¢city of the primers used is such that they did not allow the detection of diverse genes (Table 2) Two strains, P96 SE19 and P96 SE30 had both tnpR and tnpA genes, in undetermined con¢gurations Expanded PCR using all ¢ve sets of primers yielded no PCR products for these two strains All strains producing a tnpA PCR product have had their tnpA/tnpR arrangement determined except for P96 SE19 and SE30 These two isolates may contain transposon structures which are distinct in evolutionary terms from the Tn3 group of Class II transposons, despite having gene sequences which are similar to the other transposons studied 3.5 Presence of integrase genes and size of inserted gene cassettes The presence of integrase genes serves as an indicator of the presence of integron elements within the transposon PCR using primers int21A and int21B, designed to allow ampli¢cation from a wide range of integrase genes (Young, H.K and Rosser, S.J., personal communication) was carried out on the collection of isolates Five of the 124 strains produced PCR products of the correct size which hybridised to the corresponding PCR product from Tn21 (Table 3) The size of the gene cassettes inserted into the rhs of the integron elements was determined by PCR using primers 4127 and 4128 (Young, H.K and Rosser, S.J., personal communication) Insert size varied between 1.1 kb and 4.5 kb Although the nature of these inserts is currently undetermined, such FEMSEC 1049 20-8-99 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 Fig Nucleotide sequence of region spanning merD and tnpR genes in P96 SE13 FEMSEC 1049 20-8-99 33 34 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 cassettes commonly encode antibiotic resistance genes With the exception of P93 T2 37, all the isolates found to contain integron structures were members of the P96 SE group, totalling 13% of those isolates This is in contrast to the P93 SE group in which no PCR products were seen using these primers 3.6 Relative arrangement of merD and tnpR genes The arrangement of mer and tnp genes was studied in the 75 isolates that positively ampli¢ed for tnpA by PCR using primers mercD and 501R2/C, corresponding to the Tn21/501 orientation of the merD and tnpR genes All strains failing to produce a PCR product were tested using primers mercD and 501R1/C in order to ascertain whether any structural diversity (i.e inversion of tnpR) was present in the isolates No such arrangements were observed (Table 4) Reactions using the Tn21/Tn501-like primers yielded two sizes of product from di¡erent isolates: a kb product, which is the expected size from Tn501 and a 1.3 kb product The PCR product from Tn501 hybridised to all the kb PCR products indicating that the gene arrangement in these strains was similar to that of Tn501 The DNA sequence of the 1.3 kb PCR product from P96 SE13 was determined (Fig 4) and this sequence has been assigned accession number AF134211 This indicated the presence of a region of DNA corresponding to that found between the tnpR and merD genes of Tn3926, Tn5036, Tn5059 and pMER610, which contains three open reading frames of unknown function [28,40,43] DNA sequence information was used to obtain a probe from the sequenced PCR product, corresponding to the region of DNA unique to these transposons This was used to ascertain that the region of DNA between the tnpR and merD genes in all the strains producing a 1.3 kb PCR product was of a similar nature One isolate, P96 SE9 was seen to produce both sizes of fragment This may represent two distinct mer operons contained within this strain The 44 strains which contain a tnpA gene, but which did not produce a PCR product using the tnpR/merD primers, may contain a mer operon which is not associated with the tnpA gene, or alternatively this may be due to sequence diversity at the primer binding sites 3.7 Presence of merC gene Using PCR primers mercP and mercA, the presence and size of any genes contained within the merP/merA interval was determined for all 124 isolates PCR reactions using these primers produced two sizes of product using Tn21 or Tn501 as templates Tn21 yielded a kb PCR product due to the presence of the merC gene, and Tn501 a 600 bp product PCR products were obtained from 54 of the isolates (Table 4) and all were found to hybridise to both Tn21 and Tn501 mercP/mercA PCR products To distinguish between those strains which produce a PCR product containing merC and those which Table Results of merD/tnpR PCR and merC PCR Isolation group P93 SO P93 SE P93 SB P93 T2 P96 SO P96 SE P96 SB Total merD/tnpR PCR merC PCR kb 1.3 kb No product Yes No No product Unknown 0 1a 15a 9a 17a 20 13 44 10 16 44 0 0 5 30 13 15 70 0 merD/tnpR PCR was carried out on the 75 strains which positively ampli¢ed using tnpA primers merC PCR was carried out on all 124 strains a P96 SE9 produces both kb and 1.3 kb PCR products FEMSEC 1049 20-8-99 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 not, PCR products of both sizes were hybridised to a merC gene probe Forty-four of the 47 kb PCR products hybridised to the merC probe, i.e they contained a merC gene The three strains that did not contain merC genes, P93 SE31, T2 37 and T2 38, apparently contain a gene between the merP and merA genes that is of similar size to merC, but which remains of an undetermined nature Seven isolates were found to produce a 600 bp PCR product, which did not hybridise to the merC probe and were therefore assumed to be Tn501-like All isolates in the P93 SO, SE and T2 group which produced a PCR product (except the three strains with unknown inserts) contained a merC gene, whereas all the P93 SB isolates were Tn501-like, containing no genes between merP and merA The P96 SO isolates produced no PCR products, which correlates well to the merD/tnpR data for this group (data not shown) This suggests that the observed mercury resistance of these isolates may not be due to archetypal Gram-negative mer genes, i.e mercury resistance may be conferred by a non-mer operon system or by a mer operon with a sequence divergent from that of Tn21 and Tn501 All nine of the P96 SB strains which produced a PCR product contained the merC gene, as did all of the P96 SE strains except P96 SE26 and SE28 This is interesting as the P96 SB group did not produce a merD/tnpR PCR product This may be due to the mer genes being located at a position removed from the transposase gene or contained on nontransposon structures There is also a di¡erence in the SB strains in that the P93 SB isolates which gave a PCR product all contained a Tn501-like mer operon while the P96 SB isolates which gave a PCR product, all contained Tn21-like mer operons containing merC The observed frequency of merC genes is higher than previously described and merC genes are seen here in a wider range of bacterial species and plasmids [44] 3.8 Conclusions The majority of isolates in this study contained arrangements of tnpA and tnpR genes similar to that found in the Tn21 subgroup of bacteria Those strains which di¡ered from this basic gene arrangement were P96 SE19 and SE30 which had undeter- 35 mined arrangements of tnpA and tnpR genes, the four strains which may not contain a tnpR gene and those strains which did not produce PCR products with the primers used The majority of mer operons contained in the isolates fell into two major structural groups, the Tn21/501-like structures and the shorter Tn3926-like structures The di¡erences observed between the P93 and P96 isolates may be due to the selection of the P93 isolates by their ability to hybridise to a mer probe; such a selection was not carried out on the P96 isolates This may explain the higher numbers of strains in the P96 groups which not appear to contain mer operons related to those of Tn21-like transposons [33] Such novel transposon genes may not be detectable using the PCR primers employed in this study This study shows that the predominant transposon gene structures contained in a collection of mercury resistant isolates were Tn21-like, whereas the region of DNA between the mer operon and the transposase genes fell into two structural groups It is interesting to note that these isolates show a distinct lack of structural diversity compared to that which might be expected from such a study if genetic recombination and rearrangement were common place The Tn3-like arrangement of tnpA and tnpR genes was not observed in any of the strains, despite being detected in DNA extracted directly from the soil (SO) and sediment (SE) sites, suggesting that Tn3-like structures are not associated with mercury resistance genes in this environment However, if recombination is frequent within the bacterial community, it might be expected that recombination between Tn3-like transposons and Tn21-like transposons would give rise to a mercury resistance transposon containing a Tn3-like arrangement of tnpA and tnpR genes The position of the res site in members of the Tn21 subgroup of transposons is such that it may allow recombination to occur with greater frequency between transposition genes and the genes with which they are associated, whereas the Tn3-like arrangement may favour recombination between the transposition genes themselves [7] This may explain the high frequency of Tn21-like mercury resistance transposons in this environment The genetic diversity of the P93 isolates has previously been studied in detail [1,3,4,33] The sequence diversity of both tnpA and tnpR genes has been FEMSEC 1049 20-8-99 36 R.J Holt et al / FEMS Microbiology Ecology 30 (1999) 25^37 studied by RFLP and DNA sequencing [1,4] The previous RFLP study carried out by Pearson et al., 1996, indicated the presence of three classes of tnpR genes and six classes of tnpA genes within the strains isolated from soil and sediment There was no observed linkage between di¡erent classes of the two genes, suggesting that recombination is frequent between the tnpA and tnpR genes and between mer and tnp genes within the P93 isolates [4] This compared with the data presented in this study suggests that recombination may have occurred only between closely related transposons and that this has not signi¢cantly a¡ected the actual structural arrangement of the genes Acknowledgements This work was funded by NERC R.J.H is supported by a NERC Ph.D studentship, (Ref No GT4/ 95/173/T) and K.D.B by a NERC fellowship, (Ref No GT5/94/TLS) This work was also partly supported by a NERC grant awarded to P.S and D.A Ritchie (Ref No GR3/09502) This work bene¢ted from the use of the SEQNET facility, Daresbury We would like to thank Dr Hillary K Young for her assistance with the integron work, Dr Paul Eggleston for his critical reading of the manuscript, Angela Rosin for DNA sequencing and Prof D.A Ritchie for his continued support of this work References [1] Holt, R.J., Strike, P and Bruce, K.D (1996) Phylogenetic analysis of tnpR genes in mercury resistant soil bacteria: the relationships between 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