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 3 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
1. 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: [email protected]
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 0 1 6 8 - 6 4 9 6 ( 9 9 ) 0 0 0 3 6 - 7
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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. 1.
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. 1 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-
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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.
2. 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, 1 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 5 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 1 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 4 min at 95³C, followed by 30 cycles of
95³C for 1 min, 62³C for 1 min and 72³C for 2 min.
A ¢nal extension step of 10 min 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 3 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
1. 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
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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 5 min,
twice in 2USSC/1% SDS for 15 min and once in
0.1USSC for 15 min. 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 1
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].
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29
Fig. 1. 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).
3. 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-
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Fig. 2. 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
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31
Table 2
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
9
30
31
24
124
10
6
6
9
20
22
2
75
tnpA/tnpR arrangement
Tn21/Tn501
No tnpR
Unknown
9
6
6
9
20
18
1
69
1
0
0
0
0
2
1
4
0
0
0
0
0
2
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. 3. One hundred of
the isolates were found to contain plasmids, with
both large plasmids ( s 50 kb) and small plasmids
( 6 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 do 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. 3. Presence of plasmids and location of tnpA genes. Numbers in brackets indicate the number of strains.
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Table 3
Integron containing isolates
Strain
Integrase genea
Integron insert sizeb
P93 T2 37
P96 SE6
P96 SE9
P96 SE15
P96 SE19
Other 119 isolates
+
+
+
+
+
3
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. 2. 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
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Fig. 4. Nucleotide sequence of region spanning merD and tnpR genes in P96 SE13.
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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 2 kb product, which is the expected size from
Tn501 and a 1.3 kb product. The PCR product
from Tn501 hybridised to all the 2 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 1 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 do
Table 4
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
2 kb
1.3 kb
No product
Yes
No
No product
Unknown
9
0
5
0
0
1a
0
15a
0
3
1
4
0
9a
0
17a
1
3
0
5
20
13
2
44
10
4
0
5
0
16
9
44
0
0
5
0
0
2
0
7
0
5
5
2
30
13
15
70
0
1
0
2
0
0
0
3
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 2 kb and 1.3 kb PCR products.
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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 1 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 do
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.
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