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Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in
vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin
of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A.
caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain f, South
Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence,
pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory
genes plus the plasmid backbone containing the replication region. The two larger plasmids carry, in
addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21
subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for
arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system
occur within these mosaic regions.
mid-associated functions, such as replicons or conjugation or
mobilization and stability systems, that are unique or are similar to those that have been reported before. In addition, we
have sought to identify what types of accessory genes are encoded by these plasmids and whether these genes are likely to
have contributed either to the fitness of the host bacteria in
occupying their unusual ecological niche or to their industrial
usefulness.
We report on a family of plasmids ranging in size from
approximately 29 to 65 kb found in three different A. caldus
strains, two from South Africa and one from Australia. These
plasmids share a common region that includes the plasmid
backbone and some accessory genes, while two of the plasmids
possess additional regions containing different accessory genes.
* Corresponding author. Mailing address: Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602,
South Africa. Phone: 27-21-808 3071. Fax: 27-21-808 3680. E-mail:
der@sun.ac.za.
Supplemental material for this article may be found at http://aem
.asm.org/.
Plasmid isolation from A. caldus (gentle lysis). A. caldus cells grown in 4 liters
of medium were prepared in 10 ml SET buffer as described above. Cells were
lysed with 1% sodium dodecyl sulfate in the presence of proteinase K (1 mg
ml1) at 37C for 15 min, and chromosomal DNA was pelleted by centrifugation
at 48,000 g for 90 min at 4C in a Beckman JA-20 rotor. The plasmidcontaining supernatant was then subjected to CsCl gradient centrifugation (26).
Plasmid cloning, DNA manipulation, and sequencing. Plasmids were rescued and propagated in E. coli EC100D pir cells by using an EZ::TN in vitro
transposition system (EZ::TNR6Kori/KAN-2 transposon; Epicenter, Madison, Wisconsin). A single insertion into each plasmid was confirmed by sequencing from the outward-reading transposon-specific primer KAN-2 FP-1 (5-ACC
TACAACAAAGCTCTCATCAACC-3) or R6KAN-2 RP1 (5-CTACCCTGT
GGAACACCTACATCT-3). The largest plasmid (pTcM1) was mapped for
restriction endonuclease sites and was subcloned using standard protocols (26).
pTcM1 was fully sequenced on both strands except for two regions, XhoI25835 to
ApaI32100 and XbaI45771 to NcoI50641, for which previously published sequences
were available (reference 32 and National Center for Biotechnology Information
[GenBank/EMBL accession number NC_004734/AF325537], respectively). Partial or single-strand sequencing and restriction endonuclease mapping confirmed
the sequence over these two regions. Sequencing was done by the dideoxy
chain-termination method, using an ABI PRISM 377 automated DNA sequencer. Sequences were analyzed by using the Glimmer 2 (www.tigr.org/softlab)
(7) and DNAMAN (Lynnon Biosoft) programs. Comparison searches were performed with the gapped BLAST program (1) and the Conserved Domain Database (20) at the National Center for Biotechnology Information (www.ncbi
.nlm.nih.gov). Southern hybridization was performed using Hybond-N
membrane (Amersham) and 0.4 N NaOH transfer solution according to standard
protocols. Probes were digoxigenin-labeled (Roche). The PCR was carried out in
a PCR Sprint temperature cycling system (Hybaid) using Go-Taq polymerase
(Promega).
Nucleotide sequence accession number. The annotated nucleotide sequence of
pTcM1 is available under the GenBank/EMBL accession number EU421841.
RESULTS
Isolation of cryptic plasmids from A. caldus. Transalternating field electrophoresis of SwaI-digested genomic DNA revealed plasmids in A. caldus strains MNG, f, and #6 of
approximately 65, 40, and 40 kb, respectively (data not shown).
Plasmid pTcM1 was isolated from A. caldus strain MNG by
using an EZ::TN in vitro transposition system by which a transposon containing a replicon capable of replication in E. coli
pir cells and a kanamycin resistance gene is inserted. The
transposon used to rescue pTcM1 was inserted at two different
locations, and these constructs were designated pTcM1::Tn11
(insertion into the HNH endonuclease-like gene) and pTcM1::
Tn12 (insertion into the modification/methylase gene). Construct pTcM1::Tn11 was randomly selected for subcloning and
sequencing. The same technique was used to capture plasmids
pTcF1 and pC-SH12 from A. caldus strains f and C-SH12
(DSM 9466), respectively. The plasmid present in A. caldus
strain #6 was isolated but not transposon captured. Attempts
to isolate kanamycin-resistant pTcM1 transformants by using
E. coli strains lacking the pir gene (required for the replication of the R6K replicon used to clone pTcM1) were unsuccessful. Our conclusion was that the pTcM1 replicon was unable to function in E. coli.
General features of pTcM1. Sequence analysis of plasmid
pTcM1 indicated that it is 65,158 bp long and contains 45
complete open reading frames (ORFs) and several disrupted
or incomplete ORFs. All but two of the complete ORFs encode potential proteins with homology to sequences in databases (Fig. 1; see the supplemental material). Plasmid pTcM1
may be divided into two large regions, with the region from
position 1 (an XhoI site) to approximately 27 kb containing
genes putatively involved in replication, metabolism, or trans-
4301
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VAN
ZYL ET AL.
FIG. 1. Genetic maps of the 65,158-bp pTcM1 and the related plasmids pTcF1 and pC-SH12. Coding regions and truncated coding regions
(marked with apostrophes) are indicated by arrows showing their orientation. Solid bars depict the IRs of the composite transposon. ISs are shown
in black, and the unique region on pC-SH12 is unshaded. Position 1 is the first nucleotide of the 6-bp XhoI site.
4303
FIG. 2. Comparison of the replication/partition module of pTcM1 to that of other plasmids that demonstrate conserved synteny over the
replication region. Potential coding sequences are shown as arrows indicating the direction of transcription. Dashed lines join noncontiguous
regions of DNA. Note that in the case of pTcM1, the entire region depicted may be considered contiguous, as its related plasmid pC-SH12 does
not have an interruption between repA and ihfA. Putative oriV regions have been flagged. GenBank/EMBL accession numbers for plasmids are
as follows: pRSB101, AJ698325; pXAC33, NC_003921; pRms149, AJ877225; and pKLC102 integron TNCP23, AY257539.
4304
VAN
ZYL ET AL.
small ORFs (orf18 and -19) whose products share a high level
of sequence identity with pairs of hypothetical proteins from
other plasmids and chromosomes. For example, on pKLC102
and the genomic island TNCP23 of the Pseudomonas aeruginosa clone C strain (17), two genes for conserved hypothetical
proteins (TNCP9 and TNCP10) occur upstream of the pin
gene (also called ORF TNCP11). Similar conserved ORFs
occur in plasmids pRSB101 (orf3 and -4 upstream of pin) and
pRms149 (orf31 and -32, upstream from but not contiguous
with pin). In pTcM1, as in the other cases, the two genes
overlap by 3 nucleotides, suggesting transcriptional coupling.
In pTcM1, the second gene, orf18, codes for a predicted nucleic acid binding protein containing a conserved PIN domain
(COG5611). It shares strong homology (77% identity) with the
predicted proteins from orf3, ORF TNCP9, and orf32 of
pRSB101, pKLC102, and pRms149, respectively. No conserved domains are found for the product of orf19, and it
shares only approximately 65% identity with the predicted
proteins from orf4, ORF TNCP10, and orf31 of pRSB101,
pKLC102, and pRms149, respectively (which are 100% identical to one another). The product of orf4 of pRSB101 belongs
to a group of AbrB homologues (COG2002, Pfam04014) that
act as transcriptional regulators during periods of suboptimal
growth (28).
(ii) Other putative genes encoded by the common 26-kb
region. The remaining genes within the common 26-kb region
consist of seven ORFs that lie in one direction and four ORFs
in the opposite direction (Fig. 1). The seven ORFs represent
the putative genes orf6 (GntR-like transcriptional regulator),
orf7 (SdhA, succinate dehydrogenase-like flavoprotein subunit), orf8 (Fdx-like, 77 aa, 4Fe-4S ferredoxin), orf9 (PBS lyase
HEAT-like repeat, phycobili-like protein), orf10 (second Fdxlike, 107 aa, 4Fe-4S ferredoxin), orf11 (putative bifunctional
glutamate synthase subunit ), and orf12 (no meaningful
BLAST results).
orf7 codes for a 578-aa protein with 68% identity to the
flavoprotein subunit (SdhA/FrdA) of Pseudomonas aeruginosa
succinate dehydrogenase/fumarate reductase. No equivalent of
sdhB (which codes for the electron transfer subunit) or equivalents of sdhC or sdhD occur adjacent to the sdhA gene on the
A. caldus plasmids. Although the gene and amino acid sequences for the C and D membrane anchor polypeptides
of succinate dehydrogenase/fumarate reductase complexes are
poorly conserved, the genes are readily identified due to their
adjacency directly upstream or downstream of the catalytic
subunit genes.
orf11 codes for an 1,160-aa protein that shows domains at
the N terminus of the protein that are conserved in COG0493
(NADPH-dependent subunit of glutamate synthase) and
related oxidoreductases and domains at the C terminus that
are conserved in ferredoxin-NADP() reductase subunit alpha. Glutamate synthases are known to differ widely in molecular weight, subunit composition, and specificity for electron
donor and generally occur in three forms: NADH dependent,
NADPH dependent, and ferredoxin dependent. An analysis of
the functional domains on glutamate synthase (22) highlights
key amino acid features that determine the presence of the
glutamine, flavin mononucleotide, NAD(P)H, flavin adenine
dinucleotide, [3Fe-4S], and [4Fe-4S] clusters and ferredoxinbinding domains. orf11 codes for a product much larger than
Nco I
IR
mobA
ihfA
CTGGA
Nco I
IR
mobA
ihfA
Nco I
Apa I
Nco I
(EcoRI )
TTCTG
tnpA
CBS arsB
TnAtcArs from
A. caldus #6
Nco I
Apa I
NADH ox .
NADH ox.
trbJ
trbL
NADH ox.
NADH ox.
IR
trbJ
CBS arsB
CTGGA
tnpA
trbL
IS element
(PvuI )
CTGGA tnpR
arsR arsCarsD arsA
Nco I
ihfA
Nco I
mobA
arsD arsA
4305
IR
Nco I
ApaI
IR
Nco I
Nco I
Apa I
IR
Nco I
IR
tnpA
CTGGA
FIG. 3. Schematic of variations in transposon-related arsenic resistance operons and flanking regions found in A. caldus strains. The two arsenic
operons isolated from A. caldus #6 were obtained from a gene bank, whereas the two arsenic operons isolated from A. caldus strains f and MNG,
respectively, were identified from plasmids (pTcF1 and pTcM1) carried within these strains. Slashes at the ends of the plasmid-like ars operon
indicate the limits of the flanking regions cloned and sequenced. The IHF, MobA-like, TrbJ-like, and TrbL-like proteins are usually associated with
plasmid function. Only restriction enzyme sites used in Southern hybridization characterization of the arsenic operon type have been shown.
Parentheses denote loss of a restriction enzyme site. Dashed lines indicate a region of pTcM1 that contains genes not relevant to arsenic resistance
(gray arrows) or plasmid function (black arrows). Genes associated with transposition are shown as white arrows. IR denotes the IRs associated
with transposition, and the 5-bp target site repeats that result from insertion of a transposon are shown as TTCTG and CTGGA in the case of the
chromosome- and plasmid-like transposons, respectively.
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VAN
ZYL ET AL.
EcoRV(351)
EcoRI(1164)
AAAATATA
AAAATATA
1302
ARSENIC OPERON ISAfe1
BglII(673) PstI(681)
SacI(942)
SacII(317)
NcoI(685)
PvuII(94) SphI(290)
StuI(974) StuI(1049) PstI(1194)
NcoI(414) SalI(551)
PvuI(790)
CACAATTC
AACCTGGA
1304
MobE pTcF14 ISAtc1
PstI(936)
BglII(673) NcoI(685)
PvuI(790)
StuI(974) StuI(1049) SmaI(1214)
GAGAAACA
GAGAAACA
1302
RE - MOD ISAtc1
BglII(673) NcoI(685)
PstI(936)
PvuI(790)
StuI(974)
EcoRV (351)
EcoRI (1164)
AACCTGGA
TCGCATGG
1302
A
100%
RE - MOD operon
ABC operon
ISAtc1 AF325537
MobE operon
ISAfe1 U66426
95%
90%
85%
80%
75%
100%
97%
95%
93%
77%
ARS operon
B
FIG. 4. (A) Comparison of ISs on pTcM1. The IS elements have been named after their region of insertion and are shown in the 5 to 3
direction. Enzyme positions include gaps created by optimal sequence alignments using ClustalX in DNAMAN. The flanking sequence depicts the
target site of insertion, which usually becomes duplicated as an 8-bp perfect repeat. (B) Homology tree of ISAtc1- and ISAfe1-like elements.
ISAfe1, GenBank/EMBL accession number U66426, was described by Holmes et al. (16). ISAtc1 from pTC-F14, GenBank/EMBL accession
number AF325537, was described by Goldschmidt et al. (12).
DISCUSSION
Our work on plasmid biology is aimed at addressing two
questions. Are the replicons and other plasmid biology-associated genes found in the highly acidophilic, sulfur-oxidizing
biomining bacteria like A. caldus similar to those of other
bacteria? Since plasmids are an important part of the horizontal gene pool and may contribute to host-cell adaptability and
fitness, what types of accessory genes are present on these
bacterial plasmids?
We have identified four plasmids that have a common DNA
region within strains of A. caldus that were isolated from two
continents, Africa and Australia. Related plasmids might be
more widely spread, as we examined only three strains from
South Africa, one from Australia and two from the United
Kingdom (the type strain KU and strain BC13). All of the
South African strains (f, MNG, and #6) and the single strain
from Australia (C-SH12) had related plasmids. Although the
plasmid in strain #6 was not cloned, Southern hybridization
experiments indicated high levels of homology with the other
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ZYL ET AL.
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