Previous Article | Next Article 
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
 |
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.
| |
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).
View larger version (16K):
[in this window]
[in a new window]
|
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.
|
|
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 Tn
916-containing plasmid pAM120 (
7).
As expected,
M. pulmonis cells transformed with pIVC-1 had
Tn
4001C 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, Tn
4001C-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 Tn
4001C 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 Tn
4001C 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 Tn
4001 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-Tn
4001 junction region (K.
Dybvig,
unpublished data). No two transformants had the transposon
inserted at
the same site.
View larger version (59K):
[in this window]
[in a new window]
|
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.
|
|
Whether Tn
4001 could transpose in
M. arthritidis
was examined. Attempts to transform
M. arthritidis with
pISM2062 (which contains
Tn
4001mod) and pIVC-1 (which
contains Tn
4001C) 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 Tn
4001 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 Tn
4001 that might function in
M. arthritidis, the
tetM gene was inserted
into
Tn
4001 to generate Tn
4001T. 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 × 10
9 and 3 × 10
7, respectively. Southern blot analysis indicated that
Tn
4001T 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 Tn
4001C and Tn
4001T have an intact
gentamicin resistance determinant, transformants of
M. pulmonis containing either
transposon and transformants of
M. arthritidis containing Tn
4001T 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 Tn
4001T could be
transformed with pIVC-1 at a relatively high frequency, 3 × 10
6 transformants per CFU. Therefore, the presence of one
endogenous
copy of Tn
4001 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
Tn
4001C insertion into
the genome of cells that already
contained Tn
4001T 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.
View larger version (24K):
[in this window]
[in a new window]
|
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.
|
|
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
pISM2062.2
lac
(
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,
Tn
4001T should be used as a general mycoplasmal
cloning
vector.
View larger version (102K):
[in this window]
[in a new window]
|
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.
|
|
Nucleotide sequence accession number.
The arcA
nucleotide sequence of M. arthritidis has been deposited in
GenBank under accession no. AF182646.
| |
ACKNOWLEDGMENTS |
This work was supported by Public Health Service grants GM51126 and AR44252.
| |
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.
| |
REFERENCES |
| 1.
|
Bhugra, B., and K. Dybvig.
1992.
High-frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching.
Mol. Microbiol.
6:1149-1154[CrossRef][Medline].
|
| 2.
|
Bhugra, B.,
L. L. Voelker,
N. Zou,
H. Yu, and K. Dybvig.
1995.
Mechanism of antigenic variation in Mycoplasma pulmonis: interwoven, site-specific DNA inversions.
Mol. Microbiol.
18:703-714[CrossRef][Medline].
|
| 3.
|
Blanchard, A.,
A. Yáñez,
K. Dybvig,
H. L. Watson,
G. Griffiths, and G. H. Cassell.
1993.
Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction.
J. Clin. Microbiol.
31:1358-1361[Abstract/Free Full Text].
|
| 4.
|
Burdett, V.,
J. Inamine, and S. Rajagopalan.
1982.
Multiple tetracycline resistance determinants in Streptococcus, p. 155-158.
In
D. Schlessinger (ed.), Microbiology 1982. American Society for Microbiology, Washington, D.C.
|
| 5.
|
Chang, A. C. Y., and S. N. Cohen.
1978.
Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.
J. Bacteriol.
134:1141-1156[Abstract/Free Full Text].
|
| 6.
|
Dybvig, K.
1993.
The genetics and basic biology of Mycoplasma pulmonis: how much is actually Acholeplasma?
Plasmid
30:176-178[CrossRef][Medline].
|
| 7.
|
Dybvig, K., and J. Alderete.
1988.
Transformation of Mycoplasma pulmonis and Mycoplasma hyorhinis: transposition of Tn916 and formation of cointegrate structures.
Plasmid
20:33-41[CrossRef][Medline].
|
| 8.
|
Dybvig, K.,
G. E. Gasparich, and K. W. King.
1995.
Artificial transformation of mollicutes via polyethylene glycol- and electroporation-mediated methods, p. 179-184.
In
S. Razin, and J. G. Tully (ed.), Molecular and diagnostic procedures in mycoplasmology, vol. I. Academic Press, Orlando, Fla.
|
| 9.
|
Dybvig, K.,
R. Sitaraman, and C. T. French.
1998.
A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements.
Proc. Natl. Acad. Sci. USA
95:13923-13928[Abstract/Free Full Text].
|
| 10.
|
Dybvig, K., and L. L. Voelker.
1996.
Molecular biology of mycoplasmas.
Annu. Rev. Microbiol.
50:25-57[CrossRef][Medline].
|
| 11.
|
Dybvig, K., and A. Woodard.
1992.
Cloning and DNA sequence of a mycoplasmal recA gene.
J. Bacteriol.
174:778-784[Abstract/Free Full Text].
|
| 12.
|
Hahn, T.-W.,
E. A. Mothershed,
R. H. Waldo III, and D. C. Krause.
1999.
Construction and analysis of a modified Tn4001 conferring chloramphenicol resistance in Mycoplasma pneumoniae.
Plasmid
41:120-124[CrossRef][Medline].
|
| 13.
|
Harasawa, R.,
K. Koshimizu,
M. Kitagawa,
K. Asada, and I. Kato.
1992.
Nucleotide sequence of the arginine deiminase gene of Mycoplasma hominis.
Microbiol. Immunol.
36:661-665[Medline].
|
| 14.
|
Horowitz, S. A.,
B. Garret,
J. K. Davis, and G. H. Cassell.
1987.
Isolation of Mycoplasma pulmonis membranes and identification of surface antigens.
Infect. Immun.
55:1314-1320[Abstract/Free Full Text].
|
| 15.
|
King, K. W., and K. Dybvig.
1993.
Mycoplasmal cloning vectors derived from plasmid pKMK1.
Plasmid
31:49-59.
|
| 16.
|
King, K. W., and K. Dybvig.
1994.
Transformation of Mycoplasma capricolum and examination of DNA restriction and modification in M. capricolum and Mycoplasma mycoides subsp. mycoides.
Plasmid
31:308-311[CrossRef][Medline].
|
| 17.
|
Knudtson, K. L., and F. C. Minion.
1993.
Construction of Tn4001lac derivatives to be used as promoter probe vectors in mycoplasmas.
Gene
137:217-222[CrossRef][Medline].
|
| 18.
|
Mahairas, G. G., and F. C. Minion.
1989.
Random insertion of the gentamicin resistance transposon Tn4001 in Mycoplasma pulmonis.
Plasmid
21:43-47[CrossRef][Medline].
|
| 19.
|
Misawa, S.,
M. Aoshima,
H. Takaku,
M. Matsumoto, and H. Hayashi.
1994.
High-level expression of Mycoplasma arginine deiminase in Escherichia coli and its efficient renaturation as an anti-tumor enzyme.
J. Biotechnol.
36:145-155[CrossRef][Medline].
|
| 20.
|
Romero-Arroyo, C. E.,
J. Jordan,
S. J. Peacock,
M. J. Willby,
M. A. Farmer, and D. C. Krause.
1999.
Mycoplasma pneumoniae protein P30 is required for cytadherence and associated with proper cell development.
J. Bacteriol.
181:1079-1087[Abstract/Free Full Text].
|
| 21.
|
Simmons, W. L.,
C. Zuhua,
J. I. Glass,
J. W. Simecka,
G. H. Cassell, and H. L. Watson.
1996.
Sequence analysis of the chromosomal region around and within the V-1-encoding gene of Mycoplasma pulmonis: evidence for DNA inversion as a mechanism for V-1 variation.
Infect. Immun.
64:472-479[Abstract].
|
| 22.
|
Voelker, L. L., and K. Dybvig.
1996.
Gene transfer in Mycoplasma arthritidis: transformation, conjugal transfer of Tn916, and evidence for a restriction system recognizing AGCT.
J. Bacteriol.
178:6078-6081[Abstract/Free Full Text].
|
| 23.
|
Voelker, L. L.,
K. E. Weaver,
L. J. Ehle, and L. R. Washburn.
1995.
Association of lysogenic bacteriophage MAV1 with virulence of Mycoplasma arthritidis.
Infect. Immun.
63:4016-4023[Abstract].
|
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.
This article has been cited by other articles:
-
Saikolappan, S., Sasindran, S. J., Yu, H. D., Baseman, J. B., Dhandayuthapani, S.
(2009). The Mycoplasma genitalium MG_454 Gene Product Resists Killing by Organic Hydroperoxides. J. Bacteriol.
191: 6675-6682
[Abstract]
[Full Text]
-
Bird, D. W., Graber, K., Knutson, A., Washburn, L. R.
(2008). Mutation of Two Mycoplasma arthritidis Surface Lipoproteins with Divergent Functions in Cytadherence. Infect. Immun.
76: 5768-5776
[Abstract]
[Full Text]
-
Dybvig, K., Zuhua, C., Lao, P., Jordan, D. S., French, C. T., Tu, A.-H. T., Loraine, A. E.
(2008). Genome of Mycoplasma arthritidis. Infect. Immun.
76: 4000-4008
[Abstract]
[Full Text]
-
Janis, C., Bischof, D., Gourgues, G., Frey, J., Blanchard, A., Sirand-Pugnet, P.
(2008). Unmarked insertional mutagenesis in the bovine pathogen Mycoplasma mycoides subsp. mycoides SC: characterization of a lppQ mutant. Microbiology
154: 2427-2436
[Abstract]
[Full Text]
-
Lluch-Senar, M., Vallmitjana, M., Querol, E., Pinol, J.
(2007). A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium. Microbiology
153: 2743-2752
[Abstract]
[Full Text]
-
Dybvig, K., Cao, Z., French, C. T., Yu, H.
(2007). Evidence for Type III Restriction and Modification Systems in Mycoplasma pulmonis. J. Bacteriol.
189: 2197-2202
[Abstract]
[Full Text]
-
Simmons, W. L., Bolland, J. R., Daubenspeck, J. M., Dybvig, K.
(2007). A Stochastic Mechanism for Biofilm Formation by Mycoplasma pulmonis. J. Bacteriol.
189: 1905-1913
[Abstract]
[Full Text]
-
Halbedel, S., Busse, J., Schmidl, S. R., Stulke, J.
(2006). Regulatory Protein Phosphorylation in Mycoplasma pneumoniae: A PP2C-TYPE PHOSPHATASE SERVES TO DEPHOSPHORYLATE HPr(Ser-P). J. Biol. Chem.
281: 26253-26259
[Abstract]
[Full Text]
-
Halbedel, S., Stulke, J.
(2006). Probing In Vivo Promoter Activities in Mycoplasma pneumoniae: a System for Generation of Single-Copy Reporter Constructs. Appl. Environ. Microbiol.
72: 1696-1699
[Abstract]
[Full Text]
-
Pich, O. Q., Burgos, R., Planell, R., Querol, E., Pinol, J.
(2006). Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium: application to studies of the minimal gene complement. Microbiology
152: 519-527
[Abstract]
[Full Text]
-
Glass, J. I., Assad-Garcia, N., Alperovich, N., Yooseph, S., Lewis, M. R., Maruf, M., Hutchison, C. A. III, Smith, H. O., Venter, J. C.
(2006). Essential genes of a minimal bacterium. Proc. Natl. Acad. Sci. USA
103: 425-430
[Abstract]
[Full Text]
-
Janis, C., Lartigue, C., Frey, J., Wroblewski, H., Thiaucourt, F., Blanchard, A., Sirand-Pugnet, P.
(2005). Versatile Use of oriC Plasmids for Functional Genomics of Mycoplasma capricolum subsp. capricolum. Appl. Environ. Microbiol.
71: 2888-2893
[Abstract]
[Full Text]
-
Simmons, W. L., Denison, A. M., Dybvig, K.
(2004). Resistance of Mycoplasma pulmonis to Complement Lysis Is Dependent on the Number of Vsa Tandem Repeats: Shield Hypothesis. Infect. Immun.
72: 6846-6851
[Abstract]
[Full Text]
-
Clapper, B., Tu, A.-H. T., Simmons, W. L., Dybvig, K.
(2004). Bacteriophage MAV1 Is Not Associated with Virulence of Mycoplasma arthritidis. Infect. Immun.
72: 7322-7325
[Abstract]
[Full Text]
-
Clapper, B., Tu, A.-H. T., Elgavish, A., Dybvig, K.
(2004). The vir Gene of Bacteriophage MAV1 Confers Resistance to Phage Infection on Mycoplasma arthritidis. J. Bacteriol.
186: 5715-5720
[Abstract]
[Full Text]
-
Simmons, W. L., Dybvig, K.
(2003). The Vsa Proteins Modulate Susceptibility of Mycoplasma pulmonis to Complement Killing, Hemadsorption, and Adherence to Polystyrene. Infect. Immun.
71: 5733-5738
[Abstract]
[Full Text]
-
Washburn, L. R., Bird, D. W., Dybvig, K.
(2003). Restoration of Cytoadherence to an Adherence-Deficient Mutant of Mycoplasma arthritidis by Genetic Complementation. Infect. Immun.
71: 671-675
[Abstract]
[Full Text]
-
Chen, Q., Wu, H., Fives-Taylor, P. M.
(2002). Construction of a Novel Transposon Mutagenesis System Useful in the Isolation of Streptococcus parasanguis Mutants Defective in Fap1 Glycosylation. Infect. Immun.
70: 6534-6540
[Abstract]
[Full Text]
-
Teachman, A. M., French, C. T., Yu, H., Simmons, W. L., Dybvig, K.
(2002). Gene Transfer in Mycoplasma pulmonis. J. Bacteriol.
184: 947-951
[Abstract]
[Full Text]
-
Prudhomme, M., Turlan, C., Claverys, J.-P., Chandler, M.
(2002). Diversity of Tn4001 Transposition Products: the Flanking IS256 Elements Can Form Tandem Dimers and IS Circles. J. Bacteriol.
184: 433-443
[Abstract]
[Full Text]
-
Chambaud, I., Heilig, R., Ferris, S., Barbe, V., Samson, D., Galisson, F., Moszer, I., Dybvig, K., Wroblewski, H., Viari, A., Rocha, E. P.C., Blanchard, A.
(2001). The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis. Nucleic Acids Res
29: 2145-2153
[Abstract]
[Full Text]
-
Francis, K. P., Yu, J., Bellinger-Kawahara, C., Joh, D., Hawkinson, M. J., Xiao, G., Purchio, T. F., Caparon, M. G., Lipsitch, M., Contag, P. R.
(2001). Visualizing Pneumococcal Infections in the Lungs of Live Mice Using Bioluminescent Streptococcus pneumoniae Transformed with a Novel Gram-Positive lux Transposon. Infect. Immun.
69: 3350-3358
[Abstract]
[Full Text]
Top
Abstract
Text
