Journal of Bacteriology, August 2000, p. 4343-4347, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Construction and Use of Derivatives of Transposon
Tn4001 That Function in Mycoplasma pulmonis and
Mycoplasma arthritidis
Kevin
Dybvig,*
C.
Todd
French, and
LeRoy
L.
Voelker
Department of Comparative Medicine,
University of Alabama at Birmingham, Birmingham, Alabama 35294
Received 16 February 2000/Accepted 18 April 2000
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ABSTRACT |
Previous attempts to introduce transposon Tn4001 into
Mycoplasma pulmonis and Mycoplasma arthritidis
have not been successful, possibly due to functional failure of the
transposon's gentamicin resistance determinant. Tn4001C
and Tn4001T were constructed, respectively, by insertion of
a chloramphenicol acetyltransferase gene and the tetM
tetracycline resistance determinant into Tn4001. Both
Tn4001C and Tn4001T transposed in M. pulmonis, and Tn4001T transposed in M. arthritidis. The incorporation of a Tn4001T
derivative that contained lacZ into either
Mycoplasma species resulted in transformants with readily
detectable LacZ activity. Tn4001T may be of general utility
for use as a mycoplasma cloning vehicle because tetM
functions in all species of Mycoplasma examined thus far.
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TEXT |
Few strategies exist to genetically
manipulate mycoplasmas. There are no plasmids known to replicate in
Mycoplasma species other than Mycoplasma mycoides
and Mycoplasma capricolum (15, 16). The
gram-positive bacterial transposon Tn916 can transpose into
the genome of virtually all species of Mycoplasma for
which transformation methods have been described, and Tn4001
can transpose into a number of Mycoplasma species,
including Mycoplasma pneumoniae, Mycoplasma
genitalium, and Mycoplasma gallisepticum
(10). Transformation of these species with plasmids
containing either transposon results in insertion of the transposon
into the mycoplasmal genome at any of numerous sites. Tn916
is large (18 kb) and not amenable to use as a cloning vehicle. In
contrast, Tn4001 (4.7 kb) is small enough to be used as a
cloning vehicle to insert genes into the mycoplasmal chromosome.
SmaI and BamHI cloning sites have been introduced
into Tn4001 to create Tn4001mod, which has been
used as a vector in M. gallisepticum and M. pneumoniae (17, 20).
Tn4001 does not function in several species of
Mycoplasma. Earlier reports of transposition of
Tn4001 in Mycoplasma pulmonis were incorrect, and
transformation of this species with plasmids containing
Tn4001 has not previously been achieved (6, 18). The inability to introduce Tn4001 into M. pulmonis by transformation could result from either lack of
transposase activity or failure of the transposon's gentamicin
resistance determinant to serve as a selectable marker. In the present
study, we inserted alternative antibiotic resistance determinants into
Tn4001 to construct transposon derivatives that function in
M. pulmonis and in Mycoplasma arthritidis. One of
the transposon derivatives (Tn4001T) contains the
tetM gene and may be used as a broad-host-range mycoplasma
vector, as indicated by the successful expression of a lacZ
fusion gene in both M. pulmonis and M. arthritidis.
Transformation of M. pulmonis and M. arthritidis with transposons Tn4001C and
Tn4001T.
To determine whether Tn4001 would
function in M. pulmonis if an appropriate antibiotic
resistance marker was provided and to develop additional antibiotic
resistance markers for this species, a chloramphenicol
acetyltransferase gene (cat) was developed for use in
M. pulmonis. One reason a cat determinant was
chosen was that growth of M. pulmonis was found to be highly
susceptible to the inhibitory effects of chloramphenicol at a
concentration of 15 µg/ml. A chimeric gene consisting of the coding
region of the cat gene of Escherichia coli
plasmid pACYC184 (5) and the promoter region from the
expression locus of the M. pulmonis vsa genes (2)
was constructed. The cat coding region was amplified by
PCR using the cat forward primer
(5'-GGAAGGTACCATGGAGAAAAAAATCAC-3') and the
cat reverse primer
(5'-CACTTCTCGAGGCGTAGCACCAGG-3'). A 460-bp
portion of the vsa locus encompassing the promoter and the Shine-Dalgarno (SD) sequence was amplified by PCR using the vsa forward primer (5'-CGTTTCTGCAGTTTTTTTGAACC-3')
and the vsa reverse primer
(5'-TGCATGGTACCTCCTATTTTAAAATTATG-3'). PCR amplification conditions were as described previously (11). The
vsa promoter was chosen for its presumed strength, given
that the vsa gene products are major surface proteins of
M. pulmonis (i.e., the V-1 antigens) (2, 14, 21).
The vsa reverse primer and the cat forward primer
were designed with KpnI restriction sites incorporated into
their 5' ends to facilitate cloning such that the correct spacing
between the vsa SD sequence and the cat
translation start codon would be maintained. Accordingly, the
vsa and cat PCR products were digested with
KpnI and ligated together to form a hybrid vsa-cat gene. This gene was PCR amplified using the
vsa forward primer (which contained an internal
PstI site) and the cat reverse primer (which
contained an internal XhoI site). The PCR product was
digested with PstI and XhoI and inserted into the
PstI/XhoI site of plasmid pZErO-1
(Invitrogen). Transformation of E. coli strain JM109
resulted in chloramphenicol-resistant transformants (selected at 25 µg of chloramphenicol/ml), indicating that the vsa-cat
gene functions in E. coli. To incorporate the cat
determinant into Tn4001, the vsa-cat gene was
excised from pZErO-1 by digestion with PstI and
XhoI, and the ends of the DNA fragment were made flush by
digestion with T4 DNA polymerase. BamHI linkers were attached, and the vsa-cat gene was inserted into the
BamHI site of plasmid pISM2062 (17), encoding
chloramphenicol resistance, to generate plasmid pIVC-1 (Fig.
1).
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FIG. 1.
Schematic diagram of Tn4001mod in plasmid
pISM2062, Tn4001C in pIVC-1, Tn4001T in pIVT-1,
and Tn4001T-lac in pIVT-lac. Dark regions indicate
Tn4001mod sequences. Unshaded regions indicate the
cat, tetM, and arcA-lacZ genes. The
hatched region of Tn4001C indicates the vsa
promoter region. Thin lines in Tn4001T and
Tn4001T-lac indicate streptococcal sequences originating
from pJI3 that flank tetM. Arrows represent direction of
gene transcription. Abbreviations: S, SmaI; B,
BamHI; H, HindIII; K, KpnI; Term,
putative transcription terminator derived from M. arthritidis sequences upstream of arcA. The bar
represents the 1.5-kb PCR product used to probe transformants
containing Tn4001T or Tn4001T-lac.
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Transformation of M. pulmonis with pIVC-1 resulted in
chloramphenicol-resistant transformants, indicating that
vsa-cat functions as a selectable marker in this species of
Mycoplasma. M. pulmonis strain KD735-15
(1) was propagated in mycoplasma broth medium consisting of
2.1% PPLO broth without crystal violet (Difco Laboratories, Detroit,
Mich.) supplemented with 20% whole horse serum (Gibco BRL Life
Technologies, Grand Island, N.Y.), 0.5% IsoVitaleX (VWR), 0.02% degraded free-acid DNA (Sigma), 100 µg of ampicillin/ml, and 0.5% glucose. M. pulmonis was transformed with 10 µg
of pIVC-1 by the polyethylene glycol method as described previously
(8). After incubation of transformed cells at 37°C for
2 h in nonselective medium, transformants were selected on
mycoplasma agar (mycoplasma broth medium supplemented with 1.4% agar)
containing 15 µg of chloramphenicol/ml. Transformants were obtained
at a frequency of 2 × 10
7 per CFU, which is about
10-fold less efficient than transformation of M. pulmonis
with the Tn916-containing plasmid pAM120 (7).
As expected, M. pulmonis cells transformed with pIVC-1 had
Tn4001C inserted in the mycoplasmal chromosome. Total DNA
was isolated from strains of Mycoplasma as described
previously (7). Extrachromosomal plasmid was not detected in
mycoplasmal DNA because pIVC-1 does not replicate in mycoplasmas.
HindIII-digested genomic DNAs isolated from five
independent, Tn4001C-containing M. pulmonis
transformants were analyzed on Southern blots probed with the
cat gene (obtained by PCR amplification of pACYC184 using
the cat forward and reverse primers). Conditions for
Southern hybridization and preparation of 32P-labeled probe
by the random primer method were as described previously
(9). The five transformants exhibited different hybridization banding patterns, indicating that Tn4001C can
insert into the chromosome of M. pulmonis at any of numerous
sites (Fig. 2A). One of the transformants
(Fig. 2A, lane 5) had two DNA fragments that hybridized with the probe,
suggesting either that the initial transformant was simultaneously
transformed with two copies of pIVC-1 or that Tn4001C was
transposed to secondary sites in the chromosome after the initial
insertion event. In related hybridization experiments, it was shown
that the cat probe did not hybridize with M. pulmonis DNA isolated from wild-type cells that had not been
transformed (data not shown). To further explore the issue of whether
derivatives of Tn4001 insert into the M. pulmonis
chromosome at a diversity of sites, the precise transposon insertion
site in about 150 transformants of M. pulmonis was
identified by determining the DNA sequence of the
mycoplasma-Tn4001 junction region (K. Dybvig,
unpublished data). No two transformants had the transposon inserted at
the same site.
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FIG. 2.
Southern analysis of mycoplasmal transformants. (A)
Autoradiogram of a Southern blot hybridized with a
cat-specific probe. Lanes 1 through 5 are
HindIII-digested genomic DNAs from independent
transformants of M. pulmonis containing Tn4001C.
(B) Autoradiogram of a Southern blot hybridized with the probe derived
from sequences upstream of tetM (Fig. 1). Lanes 1 through 5 are HindIII-digested genomic DNAs from five independent
isolates of M. arthritidis transformed with pIVT-lac.
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Whether Tn4001 could transpose in M. arthritidis
was examined. Attempts to transform M. arthritidis with
pISM2062 (which contains Tn4001mod) and pIVC-1 (which
contains Tn4001C) were not successful. It is likely that the
vsa-cat gene does not function as a selectable marker in
this species, suggesting that the vsa promoter may be species specific. In related experiments, attempts to transform M. pulmonis and M. arthritidis with plasmid pKV98
were not successful. pKV98 contains Tn4001 with an
alternative cat gene that has been shown to function in
M. pneumoniae (12), but this cat gene
evidently fails to confer a selectable level of resistance in either
M. pulmonis or M. arthritidis. To develop a
derivative of Tn4001 that might function in M. arthritidis, the tetM gene was inserted into
Tn4001 to generate Tn4001T. tetM was
chosen because this gene functions as a selectable marker in many
Mycoplasma species, including M. arthritidis
(22). A 5-kb HincII fragment from plasmid pJI3 (4) that contained the tetM gene was
isolated and inserted into the SmaI site of pISM2062 to
generate plasmid pIVT-1 (Fig. 1). M. arthritidis strain H606
was propagated in the same medium as was M. pulmonis except
that 0.5% arginine was used instead of glucose. Transformation of
M. arthritidis with 10 µg of pIVT-1 was done as described
previously (22). Transformation of M. arthritidis
(selection at 5 µg of tetracycline/ml) and M. pulmonis (with 3 µg of tetracycline/ml) with pIVT-1 resulted in transformants at frequencies of about 2 × 109 and 3 × 107, respectively. Southern blot analysis indicated that
Tn4001T had transposed into the chromosome of either species
at any of numerous sites, as expected. The results for M. arthritidis transformed with pIVT-lac (i.e., pIVT-1 containing
lacZ, described below) are shown in Fig. 2B. As was the case
for M. pulmonis transformed with pIVC-1, one transformant
(Fig. 2B, lane 1) out of five had two DNA fragments that hybridized
with the probe.
Although Tn4001C and Tn4001T have an intact
gentamicin resistance determinant, transformants of M. pulmonis containing either transposon and transformants of
M. arthritidis containing Tn4001T had no change
in susceptibility to gentamicin compared to wild-type cells lacking the
transposon. The failure of the gentamicin resistance determinant to
confer a selectable level of resistance in M. pulmonis and
M. arthritidis explains why these species could not be
transformed with pISM2062.
M. pulmonis containing Tn4001T could be
transformed with pIVC-1 at a relatively high frequency, 3 × 106 transformants per CFU. Therefore, the presence of one
endogenous copy of Tn4001 in the mycoplasma does not
preclude and may actually stimulate transposition of a second copy into
the chromosome. Stimulation of transposition may result from the
presence of transposase activity in the cell prior to acquisition of
the second copy of the transposon. Whether the mechanism of
Tn4001C insertion into the genome of cells that already
contained Tn4001T was by transposition or by homologous
recombination was not examined. Regardless of the mechanism, however,
the ability to insert both transposons into the genome should make it
possible to complement mutants generated by insertional activation with
one of the transposons by using the other transposon as a vehicle to
introduce a functional wild-type copy of the inactivated gene.
Use of Tn4001T as a vector in M. pulmonis
and M. arthritidis.
A lacZ gene was used to
investigate whether Tn4001T can be used as a vector in
M. pulmonis and M. arthritidis. Little is known about mycoplasmal gene expression signals, and the choice of a promoter
that might drive expression of the lacZ gene was uncertain. Because the vsa promoter evidently did not function in
M. arthritidis (i.e., the vsa-cat gene did not
function as a selectable marker), a promoter originating from M. arthritidis was sought. We reasoned that the arcA gene
(encoding arginine deiminase) promoter might be relatively easy to
isolate and have strong activity.
Degenerate oligonucleotide primers were designed to amplify an internal
region of arcA. The predicted amino acids of the ArcA proteins of Mycoplasma hominis and Mycoplasma
arginini (13, 19), two species of Mycoplasma
that are phylogenetically related to M. arthritidis, were
aligned, and conserved amino acid regions were identified from which
primers with minimal degeneracy could be designed. The arcA
forward primer (5'-CGCTCGAGAYTAYATHACNCCNGC-3') and arcA
reverse primer (5'-GCCTCGAGCRTTNCCCATNCC-3') were used to amplify a PCR
product of the expected size (1.1 kb) from M. hominis strain
PG21 (3) and M. arthritidis strain PG6
(23). The M. arthritidis PCR product was cloned
into the TA cloning vector (Invitrogen), and the amino acids predicted
from its nucleotide sequence were significantly similar to ArcA of
M. hominis and M. arginini, as expected.
To clone the complete M. arthritidis arcA gene, including
the promoter, an inverse PCR strategy was employed using the primers illustrated in Fig. 3. To obtain the 5'
end of arcA, genomic DNA was digested with TaqI,
ligated to generate circular molecules for PCR templates, and amplified
using primers 5'-TGTGTTCTTTTCTTGCATCGTGGC-3' and
5'-AGTTCTATCAGACGAACACCGTGC-3'. To obtain the 3' end of
arcA, genomic DNA was digested with Sau3A,
ligated, and amplified using primers
5'-CGGCTAAATAATGTTTCACGTTGTC-3' and
5'-GAACAAACCTAATGCACTTAGACAC-3'. The resulting PCR products
were cloned into plasmid pGEM-T (Promega) and their nucleotide
sequences were determined, permitting assembly of the sequence of the
complete arcA gene of M. arthritidis. The predicted M. arthritidis ArcA protein (arginine deiminase)
contains 409 amino acids with extensive overall sequence identity, 87 and 80%, to the ArcA proteins of M. arginini (GenBank
accession no. X54141) and M. hominis (GenBank accession no.
D13314), respectively.
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FIG. 3.
Schematic diagrams of the region of the M. arthritidis chromosome containing the arcA gene (top)
and the chimeric arcA-lacZ gene (bottom). Coding regions are
indicated by thick lines, with arrows indicating direction. Regions
shaded in black originate from the M. arthritidis
chromosome. The stippled region is derived from the pZErO vector used
for gene construction. The unshaded region is the lacZ
coding region derived from pISM2062.2lac. The location and
direction of primers used for PCR amplification are illustrated as
follows: +, degenerate primers used for the initial amplification of an
internal portion of arcA, and × and  , primers used
to amplify the 5' and 3' ends, respectively, of arcA by
inverse PCR. The location of the putative transcription terminator
(Term), the 15-nucleotide poly(A) tract, and the arcA SD
sequence are also shown. For clarity, the 3-kb lacZ coding
region is drawn at one-half scale.
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Sequence analysis of the region upstream of the arcA
structural gene failed to reveal a likely promoter candidate
(resembling the consensus 
10 and 35 sequences characteristic of
promoters recognized by 
A). An open reading frame (ORF)
was identified upstream of arcA that may represent the 3'
end of another gene. The amino acids predicted from this ORF have no
significant similarity to sequences deposited in the protein and
nucleotide databases. Immediately downstream of this ORF are sequences
capable of forming a stem-loop structure that may serve as a
transcription terminator. Thus, it seems likely that an arcA
promoter should exist in the area between the putative transcription
terminator and the start of the arcA coding region. The
sequences in this area are interesting and contain a poly(A) sequence
of 15 reiterated nucleotides. Assuming that an arcA promoter
is present in this region, its structure would be unusual and worthy of
further study.
The putative arcA promoter region was isolated from the
cloned inverse PCR product containing the 5' end of the arcA
gene and combined with lacZ (Fig. 3). The plasmid containing
the arcA promoter region was linearized by digestion with
NsiI, the ends were made flush by digestion with T4 DNA
polymerase, XhoI linkers were attached, and a 300-bp region
containing the putative promoter and the beginning of the
arcA structural gene was excised by digestion with
XhoI and EcoRI. This fragment was cloned into the
XhoI/EcoRI site of plasmid pZErO-2.1. The
promoterless lacZ gene from plasmid pISM2062.2lac
(17) was excised as a 3-kb BamHI fragment and inserted into the BamHI site of pZErO-2.1 downstream of the
arcA promoter fragment. This resulted in construction of an
in-frame fusion gene containing the first 33 nucleotides of the
arcA coding region, 31 nucleotides derived from the
pZErO-2.1 polylinker, and lacZ. The arcA-lacZ
gene was excised from pZErO-2.1 by digestion with XhoI and
KpnI, and the ends were made flush by digestion with T4 DNA
polymerase. BglII linkers were attached, cohesive ends were
generated by digestion with BglII, and the gene was inserted
into the BamHI site (BglII and BamHI
ends are compatible) of pIVT-1 to generate pIVT-lac. As
anticipated, E. coli colonies containing either the
arcA-lacZ gene in pZErO-2.1 or pIVT-lac were blue
when assayed on agar supplemented with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal),
indicating that the fusion gene was functional.
Transformation of M. pulmonis and M. arthritidis
with pIVT-lac resulted in tetracycline-resistant colonies
that were found to possess -galactosidase activity when assayed on
mycoplasma agar supplemented with X-Gal at a concentration of 150 µg/ml (Fig. 4). Colonies of wild-type
mycoplasmas that had not been transformed with pIVT-lac were
white, indicating that the -galactosidase activity resulted from the
ArcA-LacZ fusion protein and was not endogenous to the mycoplasmas.
Although it has not been determined whether transcription of
arcA-lacZ is initiated from an arcA promoter or
from outside the arcA promoter region, it is clear that
arcA-lacZ is expressed both in M. pulmonis and in
M. arthritidis, demonstrating the utility of pIVT-1 as a
vector in these species. Because the tetM marker functions
in all species of Mycoplasma in which it has been examined,
Tn4001T should be used as a general mycoplasmal cloning
vector.
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FIG. 4.
Blue (LacZ+) M. pulmonis (A) and
M. arthritidis (C) colonies transformed with pIVT-lac,
assayed on mycoplasma agar plates supplemented with X-Gal. Control
colonies (LacZ) arising from cells that had not been
transformed are shown for M. pulmonis and M. arthritidis in panels B and D, respectively.
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Nucleotide sequence accession number.
The arcA
nucleotide sequence of M. arthritidis has been deposited in
GenBank under accession no. AF182646.
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ACKNOWLEDGMENTS |
This work was supported by Public Health Service grants GM51126 and AR44252.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Comparative Medicine, University of Alabama at Birmingham, Volker Hall, Room 418A, Birmingham, AL 35294-0019. Phone: (205) 934-9327. Fax: (205)
975-4418. E-mail: dybvig{at}uab.edu.
Present address: SmithKline Beecham Pharmaceuticals,
Collegeville, PA 19426.
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Journal of Bacteriology, August 2000, p. 4343-4347, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
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