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Presence of a Family of Plasmids (29 to 65


Kilobases) with a 26-Kilobase Common Region
in Different Strains of the...
Article in Applied and Environmental Microbiology August 2008
DOI: 10.1128/AEM.00864-08 Source: PubMed

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2008, p. 43004308


0099-2240/08/$08.000 doi:10.1128/AEM.00864-08
Copyright 2008, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 14

Presence of a Family of Plasmids (29 to 65 Kilobases) with a


26-Kilobase Common Region in Different Strains of the
Sulfur-Oxidizing Bacterium Acidithiobacillus caldus
Leonard J. van Zyl, Shelly M. Deane, Lilly-Ann Louw, and Douglas E. Rawlings*
Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa
Received 16 April 2008/Accepted 21 May 2008

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.

Acidithiobacillus caldus is a moderately thermophilic, acidophilic, sulfur-oxidizing, autotrophic, gram-negative bacterium


(14). It is closely related to another bacterium, Acidithiobacillus thiooxidans, except that whereas A. caldus grows at up to
50C, A. thiooxidans has an upper temperature limit of about
35C. As both bacteria have overlapping temperature ranges,
A. caldus has frequently been mistaken for A. thiooxidans and
for several years was known as A. thiooxidans type II (11). Both
bacteria are frequently found in acid mine drainage waters or
in heap reactors used to extract metals such as copper from
ores, whereas A. caldus has been reported to be the dominant
sulfur oxidizer in commercial arsenopyrite concentrate biooxidation tanks that operate at 40C and pH 1.6 (23). These
vigorously aerated tanks are used in a pretreatment process
where they decompose gold-bearing arsenopyrite concentrates
to facilitate the extraction of the gold by cyanide (25).
Plasmids are pieces of extrachromosomal, self-replicating
DNA that are commonly found in bacteria. They gain DNA
efficiently by transposition or recombination, and many plasmids are capable of moving themselves and the DNA that they
carry horizontally between bacteria by means of conjugation.
Because of this, they constitute a highly fluid part of the bacterial genome and are major contributors to the horizontal
gene pool (30). We have been studying plasmids from highly
acidophilic, autotrophic bacteria because these bacteria occupy
a unique ecological niche and also because of their industrial
importance. One of the objectives of these studies has been to
determine whether plasmids from these bacteria possess plas-

MATERIALS AND METHODS


Bacterial strains, culture conditions, and growth media. Acidithiobacillus caldus strains f (nickel pilot plant, Billiton, South Africa), #6 (Fairview mine,
Barberton, South Africa), and MNG (arsenopyrite pilot plant, University of
Cape Town, Cape Town, South Africa) have been described previously (23, 31).
A. caldus strain C-SH12 (DSM 9466) is from a continuous bioreactor, Brisbane,
Australia (11). A. caldus was grown at 37C in potassium tetrathionate medium
(5 mM), sterilized, and adjusted to pH 2.5 as reported previously (23). Escherichia coli strain MX3004 [thi-1 gdh-1 pro(lacU169)hutC gltD227::MudIIPR]
was a gift from F. Bolivar (3).
Transalternating field electrophoresis. A. caldus cells, washed and resuspended in 500 l SET buffer (50 mM Tris, 2 mM EDTA, 25% sucrose, pH 8.0)
were set in an equal volume of 2% LMP agarose (SeaPlaque; FMC Bioproducts).
Cells were lysed with 1% sodium dodecyl sulfate in the presence of proteinase K
(1 mg ml1) at 50C overnight. Proteinase K was deactivated by Pefabloc SC
(Roche Biochemicals) according to the manufacturers instructions, and the
embedded DNA was washed for several hours in successive volumes of TrisEDTA (TE) buffer, pH 7.6. DNA was digested in situ by preincubation of the
agarose plug in the relevant restriction endonuclease buffer, followed by the
addition of the restriction endonuclease and overnight incubation at the appropriate temperature. DNA fragments were separated in a 1% agarose (Seakem
LE; FMC Bioproducts) gel at 100 mA and 12C for 16 h with a 7-s pulse interval,
using a Beckman GeneLine apparatus.

* 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/.

Published ahead of print on 30 May 2008.


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PLASMID FAMILY WITH 26-KB COMMON REGION IN A. CALDUS

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

port. The remaining approximately 38-kb region consists of


what appears to be a composite transposon that carries arsenic resistance genes, mobilization genes, and a putative endonuclease and its modification enzyme.
Restriction endonuclease mapping and Southern hybridization with specific regions of pTcM1::Tn11 established that this
construct was unstable when propagated in the E. coli host.
Both pTcM1 and the pTcM1::Tn12 derivative lacked a copy of
the ISAtc1-like insertion at position 9256, suggesting that the
insertion element had undergone transposition in E. coli in the
Tn11 derivate only. The Tn11 derivative also underwent spontaneous deletion of approximately 13 kb of DNA between
nucleotide positions 37789 and 51053, resulting in a morestable version of the rescued plasmid. The instability is possibly
a result of recombination between two insertion sequence (IS)
elements flanking this region. This instability in E. coli was
more marked at 37C than at 30C, and was not observed in
any other version of the various captured plasmids.
Comparison of A. caldus plasmids pTcM1, pTcF1, and pCSH12. Four EZ::TN-captured versions of pC-SH12 were obtained and named pC-SH12::Tn1, -Tn5, -Tn17, and -Tn39.
Extensive mapping with restriction endonucleases, subcloning,
and partial sequencing revealed that pC-SH12 has approximately 26 kb in common with pTcM1 (Fig. 1). The approximately 38-kb region between and including the inverted repeats (IRs) of the composite transposon of pTcM1 is missing
in pC-SH12. A single, 1.7-kb XhoI fragment subcloned from
pC-SH12::Tn17 spanned this region, and sequence analysis
revealed that the CTGGA target site at pC-SH12 position
28809 (duplicated by the Tn insertion in pTcM1) is present in
single copy, confirming the absence of the composite transposon rather than its deletion.
Furthermore, Southern hybridization using an internal
301-bp BglII-StuI fragment of ISAtc1 (12) confirmed that pCSH12 did not contain the ISAtc1-like element present in the 1to 27-kb region of pTcM1. A further difference is that pCSH12 contains a 2.7-kb DNA fragment that is absent from
pTcM1. This DNA occurs within orf13, situated between the
glutamate synthase-like and ABC-type transporter genes and
was fully sequenced. It contains two potential coding sequences, for a transposase (56% identity to a transposase [IS66
family; pfam03050] found on plasmid pPGH1 of Pseudomonas
putida strain H; GenBank/EMBL accession no. AF052749.1)
and a transposase helper protein (IS66 orf2 family protein
[pfam05717] found on pPGH1 and pMAQU02; GenBank/
EMBL accession no. YP_957168), respectively.
A map of pTcF1 (approximately 39 kb) from A. caldus strain
f was reconstructed by comparison to pTcM1 following plasmid subcloning, restriction endonuclease mapping, partial sequencing, and Southern hybridization (Fig. 1). pTcF1 was
found to occur together with the 14-kb plasmid pTC-F14 which
belongs to the broad-host-range IncQ family and has been
extensively studied (6, 10, 33). Five EZ::TN-captured versions
of pTcF1 were obtained and named pTcF1::Tn1 to pTcF1::
Tn5. Extensive mapping with restriction endonucleases and
partial sequencing revealed that pTcF1 has the same approximately 26-kb region in common with pC-SH12 and pTcM1,
followed by a shorter composite transposon located in a position identical to that of pTcM1 (confirmed by sequencing).

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ZYL ET AL.

APPL. ENVIRON. MICROBIOL.

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.

Analysis of the region common to all three plasmids. (i)


Genes likely to be associated with plasmid core functions.
Based on plasmid pC-SH12, the putative replication, partitioning, and possible mobilization functions appear to extend from
the putative mobA-like or mobL-like gene (relaxase gene) in
the direction of the integration host factor (IHF) gene to the
pin-like gene that terminates at nucleotide 5880. In the case of
both pTcM1 and pTcF1, this region has been separated into
two by the insertion of the composite transposon.
The putative RepA-like protein of pTcM1 shows the highest
identity (34%) to a hypothetical protein from the chromosome
of Rhodopseudomonas palustris, although it is much smaller
(437 amino acids [aa] as opposed to 521 aa). The alignment of
pTcM1 RepA with related Rep proteins revealed some conserved regions, mainly in the central part of the protein. The
RepA-like protein of pTcM1 closely matches the size of plasmid-encoded RepA proteins, although these share less amino
acid identity (22% and 18% to the RepA of pRSB101 and
pRms149, respectively). The noncoding regions flanking the
putative repA gene (usually the site of oriV) do not contain any
iteron-type direct repeats, nor a putative DnaA box or A/Trich region as described for pRSB101 (28).
A gene for a putative ParA-like protein was identified by
high amino acid sequence identity of the predicted translation
product to the putative ParAs of pRSB101 and pRSB105-Rep1
(74 and 75%, respectively) (see the supplemental material).
Hayes (15) has identified a number of subgroups within the
ParA superfamily, one of which, the ParF subgroup, includes

the archetype ParF protein of the plasmid TP228, as well as the


ParF-like protein from the pTF5 plasmid of the acidophile
Acidithiobacillus ferrooxidans (8). The ParA-like protein of
pTcM1, together with the more-recently described ParA-like
proteins of pRSB101, pRms149, pXAC33, p49879-2, and
pKLC102-TNCP23, fall within this ParF subfamily and contain
the residues G14, T17, R169, and G179 (in TP228 ParF) that
are uniquely conserved in the ParF subgroup (15). The predicted TGA stop codon of the putative parA gene of pTcM1
overlaps with the ATG start codon of orf3 by two nucleotides,
suggesting possible transcriptional coupling. Similarly, the
ORFs downstream of the parA-like genes of pRSB101 (orf9),
p49879.2 (orf20.2), and pXAC33 (XACa0019) appear transcriptionally linked and have 58%, 38%, and 38% amino acid
identity, respectively, to pTcM1s orf3. No function has been
assigned to orf3 or its homologues, but its positioning with
respect to parA suggests that it may play a role in plasmid
partitioning. orf4 has 51% identity with a hypothetical protein
from plasmid pTT27 from Thermus thermophilus (see the supplemental material) and 48% identity with a hypothetical protein from plasmid p49879.1 from Leptospirillum ferrooxidans
49879 (4). The orf4 product contains a conserved domain
found in an uncharacterized protein conserved in bacteria
(COG4374; function unknown, but thought to act as a PilT N
terminus [PIN] domain nuclease, a component of toxin-antitoxin systems), as well as a related conserved PIN domain of
about 100 aa. This domain has two nearly invariant aspartates
also present in the predicted orf4 protein. The function of the

VOL. 74, 2008

PLASMID FAMILY WITH 26-KB COMMON REGION IN A. CALDUS

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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.

PIN domain is unknown, but a role in signaling appears likely


given the presence of this domain in StbB, a protein proposed
to function in stable inheritance and the autoregulation of the
stability operon, stb (29).
The putative pin-like gene (orf5) of pTcM1 encodes a predicted invertase-like protein with motifs in common with the
PinR family of site-specific recombinases (COG1961) that extend over the whole length (188 aa) of the protein, and not just
the N terminus as is the case for the pRSB101 protein. The
pTcM1 invertase-like protein has 84% and 79% identity to the
invertase-like proteins of pRSB101 and pTF5, respectively.
Whether the pin-like gene has a specific role in plasmid biology
is unknown, but given that it is present proximal to the replication and partitioning genes of pTcM1 (and its related plasmids pTcF1 and pC-SH12), as well as pKLC102, and the plasmids pTCF14 and pTF5, isolated from acidophilic bacteria, it
is likely.
Taking into account gene layout and individual protein homologies, the replication/partition region that is most similar
to that of pTcM1 is that of the 47.8-kb plasmid pRSB101, i.e.,
repA, parA, orf9, and pin (Fig. 2). This is one of several multiple-antibiotic-resistant plasmids isolated from an activatedsludge bacterial community (28). pRSB101 contains a formerly
uncategorized replicon that was shown to have both strong
homology at the amino acid level and the same gene organization as the replicon of pXAC33, a plasmid isolated from the
phytopathogenic bacterium Xanthomonas axonopodis pv. citri

(GenBank/EMBL accession no. NC_003921). The replication/


partition module (parA, orf9, parC, repA, and kfrA-like coding
sequences) (Fig. 2) of pRSB101 exhibits a conserved synteny
with that of pXAC33, pRms149 (an IncP-6 plasmid isolated
from a Pseudomonas aeruginosa clinical strain) (13), and the
class I transposon TNCP23 of the P. aeruginosa clone C plasmid pKLC102 (17).
Downstream of the putative repA gene of pC-SH12 is
a region that appears to encode few ORFs. Two ORFs that
it does encode are orf22, predicted to be a 77-aa protein
with 48% amino acid sequence identity to a 64-aa putative membrane protein from Dehalococcoides ethenogenes
(AAW39104.1), and a gene (orf21) for a 112-aa putative IHF
alpha subunit with 63% amino acid sequence identity to an
IHF from Neisseria gonorrhoeae. Present in the vicinity of the
putative IHF-encoding gene is a large coding sequence (orf20)
for a protein predicted to be 928 aa long. It has a conserved
domain of the MobA/MobL family (pfam03389) that includes
the MobA protein from the E. coli plasmid RSF1010 and the
MobL protein from the Acidithiobacillus ferrooxidans plasmid
pTF1 (9). The mobL gene encodes a relaxase that nicks the
plasmid DNA at the oriT site. The N-terminal approximately
227-aa sequence encoded by orf20 has strong homology (46%
identity) to the MobA (709 aa) of pRSF1010. However, the
C-terminal region shows little homology to any protein in the
database. No oriT site was identified within the common 26-kb
region. The mobA-like or mobL-like gene is followed by two

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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

APPL. ENVIRON. MICROBIOL.

the subunit of bacterial glutamate synthases yet smaller than


the NADH-glutamate synthases and contains domains for
NADPH, flavin adenine dinucleotide, and [4Fe-4S] binding.
However, the product of orf11 does not contain a glutaminebinding domain (a Cys-Asp-His catalytic triad found within the
N-terminal region of the NADH- and Fd-glutamate synthase
proteins and the -subunit of the NADPH-glutamate synthase
proteins; 22) and, therefore, would not be expected to have
glutamate synthase activity. Attempts to complement the E.
coli glutamate synthase mutant MX3004 with the plasmid
genes for the ability to grow on minimal medium without
glutamate were unsuccessful (data not shown). Instead, the
predicted amino acid sequence of orf11 has 67% identity over
almost its entire sequence to a hypothetical protein found in a
variety of organisms (e.g., Nitrosococcus oceani) (see the supplemental material).
The four ORFs reading in the opposite direction encode three
putative ABC-type membrane transport-like proteins, orf16 (445
aa, substrate-binding/periplasmic component of nitrate/sulfonate/
bicarbonate transporter; disrupted by ISAtc1-like element in
pTcM1), orf15 (230 aa, permease component of binding-proteindependent transporter), orf14 (259 aa, ATPase component of
nitrate/sulfonate/bicarbonate transporter), and a putative orf13,
the product of which gave no meaningful similarity using a
BLAST search.
Composite transposons and ISs. Besides the 2.7-kb IS66like element that has been inserted into the hypothetical orf13,
plasmid pC-SH12 contains only the approximately 26-kb region common to all three plasmids, and no other transposons
or IS elements are present. In contrast, both pTcM1 and
pTcF1 contain a composite transposon and numerous ISs.
Plasmids pTcM1 and pTcF1 have approximately 38 and 12
kb of additional DNA, respectively, that represent highly mosaic areas bordered by the 40-bp imperfect IRs of a transposon
identical to that of TnAtcArs (31). Within these IR sequences
are several other genes, transposons, and IS elements. Some of
the genes in this mosaic area have been extensively studied in
other contexts (6, 10, 31, 33), while others are of unknown
origin and function. Of particular interest are the genes coding
for arsenic resistance, as these have previously been found only
on the chromosome of A. caldus strains isolated from biomining environments known to contain high levels of arsenic (31).
pTcF1 contains some of the Tnars genes (Fig. 3) and yet is
different from the TnAtcArs from A. caldus strain #6 (described in reference 31), as well as from the pTcM1 ars region.
TnAtcArs contains a repeat of the arsDA genes, which is not
found either in the other versions of the arsenic operon that
appear on plasmids pTcF1 and pTcM1 or the plasmid-like
operon found in A. caldus strain #6 (32). The CTGGA 5-bp
direct repeats found on the flanking regions of the transposon
IRs also differ from those in the previously reported TnAtcArs
(TTCTG). Mapping and Southern blotting confirmed that the
7.4-kb NcoI-NcoI region from mobA to arsA, as well as the
1.2-kb NcoI-NcoI region from arsA to the NADH oxidase-like
gene, is identical in all the plasmid-like arsenic operons, including the uncloned plasmid from A. caldus strain #6. The
region downstream of the putative NADH oxidase (orf28) in
the plasmid-like arsenic operons of pTcM1 and A. caldus #6
contains two ORFs (orf29 and orf30) coding for proteins with
low homology to the TrbJ and TrbL components of the type IV

VOL. 74, 2008

Nco I

IR

mobA

ihfA

CTGGA

tnpR arsR arsCarsD arsA

Nco I

IR

mobA

ihfA

CTGGA tnpR arsR arsCarsD arsA

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

Plasmid-like ars operon


from A. caldus #6

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

TTCTG tnpR arsR arsCarsD arsA

Nco I
Apa I

IR

Nco I

PLASMID FAMILY WITH 26-KB COMMON REGION IN A. CALDUS

Arsenic gene region


from pTcM1

IR

tnpA

CTGGA

Arsenic gene region


from pTcF1

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.

secretory pathway systems of IncP-type plasmids (2; see the


supplemental material). The TrbL homologue of pTcM1, although smaller than the TrbL of IncP-type plasmids, contains
clusters of glycyl residues and has a hydropathy plot consistent
with their transmembrane segments (data not shown).
The arsB gene of pTcF1 is interrupted by an IS which appears to have occurred while the plasmid was in A. caldus, as all
the Tn-captured versions of the plasmid propagated in E.
coli have an IS element in this position. Furthermore, PCR
amplification using primers within arsB 200 bp to either side of
this IS and total DNA from A. caldus strain f yielded only the
IS-containing product (results not shown), implying that this A.
caldus strain does not contain a functional arsB gene. Since the
plasmid-borne copies appear to either lack an arsB or have an
inactivated arsB (in pTcF1), it is probably the chromosomal
copy of TnAtcArs (present in strains MNG and #6 only) that
confers arsenic resistance.
Whereas additional genes on plasmid pTcF1 are limited to a
transposon containing a defective ars operon, plasmid pTcM1
contains ORFs encoding a putative HNH endonuclease and a
modification enzyme (Fig. 1; see the supplemental material).
Also present were all five of the mob genes, an oriT, a pasAB
(plasmid addiction system), a complete mobA-repB (relaxaseprimase) gene, and partial remnants of repA and repC genes
which were, in all cases, identical to those of previously isolated
A. caldus plasmid pTC-F14 (Fig. 1; see the supplemental material). Plasmid pTcM1 was mobilizable using the pTC-F14like mob genes carried on the composite transposon that is
present on pTcM1 but missing from plasmids pTcF1 and pCSH12. Mobilization by the self-transmissible plasmid RP4 be-

tween strains of E. coli occurred in spite of the disruption of


the mobE gene by the insertion of an ISAtc1-like element
(data not shown). This observation is consistent with the report
that the deletion of mobE does not eliminate mobilization,
although its absence reduced mobilization approximately 10fold (33).
Interestingly, a distantly related but complete repC is also
present, with the gene product being most similar to the equivalent gene product of a plasmid remnant located on the chromosome of Chlamydia suis (see the supplemental material).
Similar to pTcM1, the large mosaic plasmids pRSB105 (27)
and pRms149 (13) contain a 5-Mob-protein mobilization module that resembles those of the small, mobilizable, broad-hostrange plasmids pTC-F14 and pTF-FC2 (33).
The presence of a putative restriction endonuclease and
modification system (interrupted by an IS situated between the
genes) on pTcM1 might seem unexpected; however, such systems are frequently found on plasmids. They have been reported to serve as toxin-antitoxin postsegregation plasmid stability mechanisms with the restriction endonuclease serving as
the toxin and the modification enzyme as the antitoxin (21).
Other ORFs, such as tniA (complete) and tniB (truncated),
dsbG (truncated), and orf35, appear to be remnants of what
were presumably previous transposition events.
Plasmid pTcM1 contains three copies of an ISAtc1-like insertion element, two of which are 100% identical at the nucleotide level while the third is 95.55% identical (Fig. 4A and B).
The transposase of the IS element found within the arsenic
operon most closely resembles (326/399 amino acids, or 81%
identity) that of the ISAfe1 element of Acidithiobacillus fer-

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VAN

ZYL ET AL.

APPL. ENVIRON. MICROBIOL.


ABC OPERON ISAtc1
BglII(673) NcoI(685)
PstI(936)
PvuI(790)
StuI(974)

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).

rooxidans ATCC 19859 (16), and this element has, therefore,


been designated an ISAfe1-type element. At the nucleotide
level, it is only 77.46% identical to the ISAfe1 sequence. The
two IS elements of pTcF1 were ISAtc1-like. The IS elements of
each plasmid occurred in different places (Fig. 1), suggesting
that transposition had taken place after the plasmids diverged.
At least one transposition event and one DNA rearrangement took place after the plasmids were captured in E. coli.
An ISAtc1 element was inserted between the fdx-like genes
of pTcM1 during capture or replication of pTcM1::11 in E.
coli, and restriction endonuclease digestion of crude plasmid from A. caldus strain MNG indicated that it was not
present in the native DNA (not shown). In addition, a spontaneous deletion of approximately 13 kb of DNA occurred
between ISAfe1(ARS) and ISAtc1(MobE) when pTcM1::
Tn11 was propagated in E. coli. The deletion resulted in the
loss of one of the 8-bp-target-site duplications of ISAtc1
(GAGAAACA) and resulted in the two IS elements lying
tail-to-tail with only the first 5 bp of the ISAfe1-like elements 5 8-bp target site separating them.

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

VOL. 74, 2008

PLASMID FAMILY WITH 26-KB COMMON REGION IN A. CALDUS

three plasmids. All three plasmids analyzed in detail had an


approximately 26-kb region in common that would be expected
to contain the replicon and other functions associated with
plasmid stability and maintenance. Plasmid pC-SH12 consists
of only this 26-kb region plus a 2.7-kb transposon that had been
inserted into this region and which was absent from pTcM1
and pTcF1.
The sequence and layout of putative replicon and partitioning systems (Fig. 2) were partly related to plasmids reported
from other bacteria, in particular, plasmids RSB101 and
RSB105 from uncultured microorganisms present in activated
sludge (27, 28). The plasmid backbone therefore appears be
similar to those found in other environments and is not unique.
Unfortunately, little research has been carried out on the replication regions of these plasmids, and as only a rudimentary
conjugation system exists for A. caldus (19), this inhibited further work on the putative replicon and partition genes.
The presence of identical accessory genes on the common
26-kb region of plasmids from two different continents suggests
that they have been part of this plasmid family for an extended
period of time. However, their function and whether they contribute to host cell fitness remain unclear. Several of the seven
ORFs on the 26-kb fragment, including the putative gntR-like
regulator gene (orf6), the atypical sdhA-like gene (orf7), the
ferredoxin-like genes (orf8 and orf10), the PBS lysase HEATlike gene (orf9), and the atypical glutamate synthetase-like
subunit gene (orf11), have a resemblance to similar collections
of genes in other bacteria. Often these are also linked to ABC
transporter-like genes as found in the 26-kb region. For example, the PBS lyase HEAT-like repeat gene of Nitrobacter winogradskyi occurs immediately downstream of a [4Fe-4S] ferredoxin-like gene and a succinate dehydrogenase flavoprotein
subunit-like gene and in the vicinity of a number of ABC-type
nitrate/sulfonate/bicarbonate transport system genes (GenBank/
EMBL accession no. CP000115.1; Protein Data Bank accession no. ABA03944.1 to ABA03939.1). Similarly, the PBS
lyase HEAT-like repeat gene of Burkholderia multivorans
ATCC1716 is preceded by three ABC-type transport system
genes, a ferredoxin-like, a succinate dehydrogenase-like, and a
GntR-like regulator gene (GenBank/EMBL accession no.
CP000868; Protein Data Bank accession no. ABX14981.1 to
ABX14987.1). Unfortunately, these database matches are to
sequences of whole genomes, and no further information is
available on the roles of the individual gene products. ABC
transporter-like genes are capable of transporting a variety of
substances across cell membranes (5), but the amino acid sequences of such transporters are frequently poorly conserved
and it is difficult to judge by sequence comparisons what substances a particular transporter might transport. The function
of these putative transporters of the three A. caldus plasmids is,
therefore, unknown. Furthermore, given the large differences
in pH across the cell membrane of extreme acidophiles compared with that of neutrophiles, it is uncertain whether transporters from bacteria such as A. caldus will function in a genetically tractable bacterium like E. coli in which the function
of such genes could be tested.
Plasmids pTcM1 and pTcF1 contain a transposon bordered
by 40-bp IR sequences that contain a selection of arsenic resistance genes that are similar to those reported on TnAtcArs
(31). The TnAtcArs present in A. caldus strain #6 was acquired

4307

by a different transposition event than those on plasmids


pTcM1 and pTcF1, as the direct repeats flanking the inverted
repeats are different. Furthermore, although the pieces of
many of the ars genes present on plasmids pTcM1 and pTcF1
that have been sequenced are identical to those present on
TnAtcArs, the nucleotide sequences of the tnpA genes are only
95.35% identical. This suggests that two closely related transposons have acquired similar ars genes or that there has been
gene exchange between related transposons such that some
parts of the transposons are more closely related than others.
One can speculate on why the ars operons on pTcM1 and
pTcF1 have been modified so that they would no longer be
able to confer arsenic resistance on their hosts. Although both
A. caldus strain f and A. caldus MNG were derived from
consortia exposed to arsenopyrite in the early 1980s, neither
strain has been exposed to arsenic for at least 15 years. A.
caldus strain f, the source of pTcF1, is from a consortium
treating a nickel ore and no longer has a need for arsenic
resistance, whereas A. caldus strain MNG is the source of
pTcM1, and although it was originally isolated from an
arsenopyrite plant, it has been grown on sulfur or stored on
pyrite and has not been exposed to arsenic for many years. The
gene for the membrane-located arsenite pump (arsB gene
product) is no longer functional in both cases. As ArsB is
known to be harmful to a cell if overexpressed, there might be
selection against the retention of an active arsB gene when
high levels of arsenic resistance are no longer required.
The occurrence of many IS elements almost identical to
ISAtc1 and ISAfe1 previously reported to occur in many copies
per cell in numerous isolates of A. ferrooxidans, A. thiooxidans,
and A. caldus (16, 18, 24) is not surprising. Furthermore, these
IS elements were still highly active and capable of transposition, especially when the plasmids were cloned in E. coli. When
transferred to E. coli, the IS elements would have entered an
environment where the frequency of transposition would not
have been repressed by existing elements of the same type, and
this is probably the reason for the high levels of transposition
in this host.
In summary, the three plasmids reported here belong to a
class of plasmid present in A. caldus isolates from different
parts of the world that have a unique replicon, although it is
related to other, previously reported replicons. These plasmids
have acquired additional genes, many of unknown function,
that are present on a 26-kb common region. The absence,
rather than deletion, of the composite transposon points to
pC-SH12 being the progenitor plasmid from which the plasmids pTcM1 and pTcF1 arose by the acquisition of a composite transposon carrying arsenic resistance and other genes.
ACKNOWLEDGMENTS
This work was funded by grants from the National Research Foundation (Pretoria), BHP-Billiton Johannesburg Technology Centre, and
the BioMinE project 500329 of EU framework 6.
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