Download Different susceptibility of two animal species infected with isogenic

Microbiology (2003), 149, 3203–3212
DOI 10.1099/mic.0.26469-0
Different susceptibility of two animal species
infected with isogenic mutants of Mycobacterium
bovis identifies phoT as having roles in
tuberculosis virulence and phosphate transport
Desmond M. Collins, R. Pamela Kawakami, Bryce M. Buddle,
Barry J. Wards and Geoffrey W. de Lisle
Correspondence
Desmond M. Collins
AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand
desmond.collins@agresearch.
co.nz
Received 8 May 2003
Revised
15 July 2003
Accepted 28 July 2003
The Mycobacterium tuberculosis complex includes Mycobacterium bovis, which causes
tuberculosis in most mammals, including humans. In previous work, it was shown that M. bovis
ATCC 35721 has a mutation in its principal sigma factor gene, sigA, causing a single amino acid
change affecting binding of SigA with the accessory transcription factor WhiB3. ATCC 35721
is avirulent when inoculated subcutaneously into guinea pigs but can be restored to virulence by
integration of wild-type sigA to produce M. bovis WAg320. Subsequently, it was surprising to
discover that WAg320 was not virulent when inoculated intratracheally into the Australian brushtail
possum (Trichosurus vulpecula), a marsupial that is normally very susceptible to infection with
M. bovis. In this study, an in vivo complementation approach was used with ATCC 35721 to
produce M. bovis WAg322, which was virulent in possums, and to identify the virulence-restoring
gene, phoT. There are two point deletions in the phoT gene of ATCC 35721 causing frameshift
inactivation, one of which is also in the phoT of BCG. Knockout of phoT from ATCC 35723, a
virulent strain of M. bovis, produced M. bovis WAg758, which was avirulent in both guinea pigs and
possums, confirming that phoT is a virulence gene. The effect on virulence of mode of infection
versus animal species susceptibility was investigated by inoculating all the above strains by aerosol
into guinea pigs and mice and comparing these to the earlier results. Characterization of PhoT
indicated that it plays a role in phosphate uptake at low phosphate concentrations. At least in vitro,
this role requires the presence of a wild-type sigA gene and appears separate from the ability
of phoT to restore virulence to ATCC 35721. This study shows the advantages of using different
animal models as tools for the molecular biological investigation of tuberculosis virulence.
INTRODUCTION
Tuberculosis continues to be one of the most common
causes of human death due to bacterial infection and is also
a widespread cause of animal morbidity and mortality. In
humans, the primary cause is Mycobacterium tuberculosis,
although in many parts of the world a significant amount of
disease is also due to infection with the very closely related
organisms Mycobacterium africanum and the major animal
pathogen Mycobacterium bovis. Together with a few less
important species, these organisms are collectively referred
to as the M. tuberculosis complex. Recent genomic studies
have shown that the vast majority of genes within these
species are identical, or nearly so, and that the most obvious
differences between species and also between strains within
species are DNA insertions and deletions encoding one or a
small number of genes (Cole, 2002). While the functional
Abbreviation: PPD, purified protein derivative.
0002-6469 G 2003 SGM
significance of these differences as well as the many nonsynonymous nucleotide substitutions that exist between
strains (Fleischmann et al., 2002) are presently unknown,
some of them must account for the differences in pathogenicity observed between different species and strains.
Since the identification of the first genes involved in
mycobacterial virulence (Collins et al., 1995; Wilson et al.,
1995), further development and improvement of molecular genetic techniques has enabled the discovery of an
increasing number of such genes (de Mendonca-Lima et al.,
2003; Glickman & Jacobs, 2001). While there is continuing
discussion about what constitutes a true virulence gene
(Barry, 2001; Wassenaar & Gaastra, 2001), it is undoubtedly
helpful to know if inactivation of a gene affects the virulence
of an organism in an animal model, as this potentially
enables further study of the host–pathogen interactions
which are affected directly or indirectly by that gene. In most
cases, virulence of the parent and a mutant daughter strain
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
Printed in Great Britain
3203
D. M. Collins and others
has been determined in a single model of animal virulence,
usually in mice or guinea pigs. This use of a single animal
model reflects practicalities in terms of cost and time of
using more than one animal host under biological containment for investigating a chronic disease, as well as an
underlying assumption that the features of tuberculosis in
different animal hosts are sufficiently similar that most
virulence mechanisms will be the same in all hosts.
Nevertheless, there is long-standing evidence that different
species of the M. tuberculosis complex, as well as different
strains within a species, have different pathogenicities for
different animal hosts. For example, strains of M. tuberculosis are not as virulent as strains of M. bovis in rabbits
but both species appear similarly virulent in guinea pigs
(Dannenberg & Collins, 2001); and South Indian strains
of M. tuberculosis appear of similar virulence to other
M. tuberculosis strains for humans but are less virulent in
guinea pigs (Balasubramanian et al., 1992). Even when
a single animal host is used to determine the level of
virulence, the result obtained depends on the particular
indices that are measured. Growth rate in mice is often
used to assess virulence of strains of the M. tuberculosis
complex but this only indicates a subset of virulence
mechanisms. Commonly used strains of M. tuberculosis
and M. bovis grow at similar rates in mice and by that
criterion would have similar virulence, but mice infected
with M. bovis die much sooner that those infected with
M. tuberculosis, so by that criterion M. bovis would be
more virulent (North et al., 1999). These different ways
of assessing virulence have recently been emphasized by
the production of a number of single-gene mutants of
M. tuberculosis that have similar growth to their parents
in animals but differ markedly when compared on the basis
of animal survival (Kaushal et al., 2002; Steyn et al., 2002).
In earlier work (Collins et al., 1995), it was shown that
M. bovis ATCC 35721 carries a point mutation in the
principal sigma factor gene, sigA (designated rpoV in the
earlier work), that causes attenuation of its virulence in
guinea pigs when it is inoculated subcutaneously (subcutaneous guinea pig model). That work used an in vivo
complementation approach in which ATCC 35721 was
complemented with a wild-type sigA gene (to produce
M. bovis WAg320) that restored virulence in this guinea
pig model. Recently, it was found that the arginine to
histidine change this mutation encoded at position 515 in
SigA (SigA-R515H) reduced the interaction of this sigma
factor with a secondary transcription factor WhiB3 (Steyn
et al., 2002). It was also shown that inactivation of whiB3
in M. tuberculosis H37Rv had no effect on its ability to
grow and persist in mice infected intravenously or in a
subcutaneous guinea pig model but mice infected with this
mutant had significantly longer mean survival times than
those infected with the parent H37Rv strain. In contrast,
inactivation of whiB3 in M. bovis completely attenuated its
growth in a subcutaneous guinea pig model. These results
indicate a different role for some M. tuberculosis and M. bovis
genes in pathogenesis generated in different animal models.
3204
In New Zealand, it has been difficult to eradicate bovine
tuberculosis from cattle in some parts of the country because
of continual reinfection from tuberculous populations of
the Australian brushtail possum (Trichosurus vulpecula), an
animal introduced into New Zealand in the nineteenth
century (de Lisle et al., 2001). In the course of tuberculosis
studies on possums, we discovered that both M. bovis
ATCC 35721 and M. bovis WAg320, its complement with
a wild-type sigA gene, are attenuated when inoculated
intratracheally into these animals. This result was in stark
contrast to the situation in guinea pigs, where ATCC 35721
itself is attenuated but WAg320 has virulence approaching
that of a wild-type strain. In this study, we describe this
discovery, its use to identify an ABC transporter gene,
phoT, and the virulence properties of phoT in different
animal models of tuberculosis.
METHODS
Bacterial strains and culture conditions. The M. bovis strains
used in this work are listed in Table 1. Liquid culture of all M. bovis
strains was performed in Tween-albumin broth (Kent & Kubica,
1985) or supplemented Middlebrook 7H9 (Difco) medium (Collins
et al., 2002). Solid culture was performed on supplemented
Middlebrook 7H11 (Difco) medium (Collins et al., 2002). Where
appropriate, media also contained 50 mg hygromycin ml21 or 20 mg
kanamycin ml21. Culturing of M. bovis strains in low phosphate
concentrations was performed in Sauton medium (Darzins, 1958).
For culture of M. bovis from guinea pigs, half the spleen and the left
apical lobe of the lung was used and for culture from mice, all the
spleen and all the lungs. For culturing from possums, 1 g tissue
from the caudal portion of the spleen was used as well as 1 g tissue
from the lung that included a lesion or, if no lung lesion was present, from the right cardiac lobe. Samples from the same animal
were homogenized separately with 20 ml water for 1 min. This was
filtered through sterile cheese cloth and centrifuged at 3500 g for
20 min. The pellet was resuspended in 0?5 ml water and aliquots
were plated onto Middlebrook 7H11 supplemented with 0?6 ml
oleic acid l21, 50 g BSA l21, 20 g glucose l21, 7?7 g NaCl l21, 0?4 %
sodium pyruvate, 0?5 % lysed sheep red blood cells, 10 % bovine
serum, 10 mg fungizone ml21, 200 IU polymyxin B sulphate ml21,
100 mg tarcarcillin ml21 and 10 mg trimethoprim ml21. Escherichia
coli strains were grown with appropriate antibiotics in L broth at
37 uC for XL-1 Blue MR and 30 uC for x2764.
Identification of the virulence-restoring gene by in vivo
complementation of ATCC 35721 in possums. Genomic DNA
was extracted from M. bovis WAg200, partially digested with Sau3AI
and fragments of 30–50 kb were prepared using sucrose gradient
centrifugation as described previously (Collins et al., 1995). The
fragments were ligated to BclI-digested pUHA8, a cosmid shuttle
vector containing a kanamycin resistance gene and a mycobacteriophage integration sequence, that had been constructed from
pYUB178 (Pascopella et al., 1994) by positioning PacI recognition
sites on either side of the BclI cloning site. The fragments were also
ligated into a second vector, pUHA28, that had been constructed by
ligating sigA into the KpnI site in pUHA8. Constructs were packaged
into lambda heads (GigaPack II Gold, Stratagene) and transduced
into E. coli x2764; kanamycin-resistant clones were pooled and
cosmid DNA was prepared as described previously (Collins et al.,
1995). The cosmid DNA was electroporated into ATCC 35721
(Wards & Collins, 1996) and kanamycin-resistant colonies were
pooled into separate ATCC 35721(pUHA8 : : WAg200) and ATCC
35721(pUHA28 : : WAg200) libraries and inoculated into possums.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
Microbiology 149
M. bovis phoT in phosphate transport and virulence
Table 1. Bacterial strains and plasmids
Strain or plasmid
M. bovis
ATCC 35721
ATCC 35723
WAg200
BCG
WAg320
WAg322
WAg324
WAg325
WAg758
E. coli
XL-1 Blue MR
x2764
Plasmids
pYUB178
pUHA8
pBluescript II KS
pUHA9
pUHA28
pUHA30
pUHA37
pUHA38
pUHA39
pUHA43
Relevant properties
Source or reference
Low virulence for guinea pigs; sigA-R515H
Moderate virulence for guinea pigs
Wild-type New Zealand cattle isolate; high virulence for guinea pigs
Pasteur strain 1173P2
ATCC 35721(pYUB178 : : sigA); virulent for guinea pigs
ATCC 35721(pUHA8 : : WAg200 cosmid : : phoT)
ATCC 35721(pUHA8 : : phoT)
ATCC 35721(pUHA8 : : phoT)
ATCC 35723 phoT : : hygr
ATCC*
ATCC*
Collins et al. (1995)
ATCC*
Collins et al. (1995)
This study
This study
This study
This study
Laboratory K-12 strain for in vitro cloning
HB101 lysogen with l cI857 b2 redb3 S7; cosmid cloning
Stratagene
Jacobs et al. (1986)
Mycobacterial integrating cosmid shuttle vector; Kanr
pYUB178 with PacI sites either side of BclI cloning site
E. coli phagemid cloning vector; Ampr
pBluescript II KS with PacI–BclI–PacI cassette inserted at BamHI cloning site
pUHA8 : : sigA
pUHA8 containing virulence-restoring cosmid insert from WAg322
pUHA8 containing 1973 bp of phoT locus
pUHA8 containing 2311 bp of phoT locus
pUHA9 containing phoT–phoY2 locus from ATCC 35721
pUHA9 containing pUHA37 insert interrupted with hygr; suicide plasmid
Pascopella et al. (1994)
Collins et al. (1995)
Stratagene
Collins et al. (2002)
This study
This study
This study
This study
This study
This study
*American Type Culture Collection.
Colonies recovered from possum lesions were characterized by
junction fragment analysis (Collins et al., 1995). Briefly, DNA
extracted from ATCC 35721(pYUB8 : : WAg200) and ATCC
35721(pYUB28 : : WAg200) clones was characterized by restriction
digestion and Southern blot hybridization using a probe of pUHA8
to reveal the size of fragments at the junction sites where the integrated vector arms join the host chromosomal DNA. The cosmid
insert in a selected clone of ATCC 35721(pYUB8 : : WAg200) was
recovered by digesting its chromosomal DNA with PacI, which has
no sites in the chromosome of M. bovis, and cloning of the PacI
fragment into pUHA9, a derivative of pBluescript II KS that contains PacI cloning sites. This fragment was partially digested with
Sau3AI and fragments of 2–4 kb were prepared by sucrose gradient
fractionation and ligated into the BclI cloning site of pUHA8. This
mini-library was inoculated into possums, M. bovis colonies were
cultured from the lesions and the inserts in two of these virulencerestoring clones were sequenced. DNA sequences were analysed
using the programs of the Genetics Computer Group and compared
to the GenBank (http://www.ncbi.nlm.nih.gov) and Sanger (http://
www.sanger.ac.uk) databases.
et al., 1991). DNA from allelic-exchange mutants in which the phoT
gene had been deleted was identified by restriction digestion and
Southern blot hybridization and confirmed by PCR analysis using
primers on each side of the phoT site into which the hygromycin
resistance gene had been ligated.
Animal models of virulence. All animal work was approved by
the institution’s Animal Ethics Committee. For the subcutaneous
guinea pig model, the virulence of each M. bovis strain was tested in
3–6 female Dunkin-Hartley guinea pigs, as described previously
(Wilson et al., 1995). Animals were inoculated by injecting 106 c.f.u.
subcutaneously into their flank and were killed at 8 weeks. The
Knockout of phoT in M. bovis. Knockout of the phoT gene from
ATCC 35723 was performed in a similar way to that described
previously (Wards et al., 2000). Briefly, a suicide plasmid was constructed by inserting a hygromycin resistance gene into the EcoRV
site of phoT (Fig. 1) in the 1973 bp insert from pUHA37. The
phoT : : hygr fragment was transferred into pUHA9 to produce
pUHA43, which was electroporated into ATCC 35723 using a highefficiency electroporation technique (Wards & Collins, 1996). Cells
were plated onto medium containing hygromycin and resistant colonies were subcultured and their DNA was extracted (van Soolingen
http://mic.sgmjournals.org
Fig. 1. Annotation of the phoT locus in M. tuberculosis and
M. bovis showing the identity of the fragments from pUHA37
and pUHA38 that restored virulence to M. bovis ATCC 35721
in possums. The EcoRV site denotes where the hygr gene was
inserted into the fragment from pUHA37 in producing a suicide
plasmid, pUHA43, for knockout of phoT.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
3205
D. M. Collins and others
assessment of virulence in early work was based on the presence of
visible lesions of tuberculosis in the spleen and, in later comparative
work, on the enumeration of M. bovis c.f.u. in samples from the
spleen. All animals were tested for infection by determining their
delayed-type hypersensitivity to tuberculin using intradermal injection of 4 units (0?1 ml) of bovine purified protein derivative (PPD;
AgriQuality, New Zealand) immediately prior to inoculation with
M. bovis and immediately prior to the animals being killed. For
the possum model, animals were inoculated intratracheally with
105 c.f.u. of M. bovis in 200 ml. The organisms were inoculated
through a 2 mm diameter vinyl cannula per os into the trachea of
anaesthetized animals as described previously (Pfeffer et al., 1994)
and killed after 6 weeks. The assessment of virulence in early work
was based on the presence of visible lesions of tuberculosis in the
lungs and, in later comparative work where groups of four animals
were used, on the enumeration of M. bovis c.f.u. in samples from
the lung and spleen. All possums were tested for infection before
inoculation and before being killed, by using a lymphocyte stimulation assay as described previously (Skinner et al., 2002). For the
aerosol guinea pig and mouse models, single cell suspensions of
each M. bovis strain were prepared by sonication for 30 s and filtration through an 8 mm filter. Groups of three Dunkin-Hartley guinea
pigs and four BALB/c mice were infected as described previously
(Aldwell et al., 2003; McMurray et al., 1985) using an aerosol
chamber which produces droplet nuclei of the size appropriate
for entry into alveolar spaces. The aerosolized solution contained
approximately 56106 c.f.u. ml21; based on previous empirical
observations and the time of animal exposure this would have
resulted in the inhalation and retention in the lungs of approximately 500 c.f.u. for guinea pigs and 30 c.f.u. for mice. Animals
were killed after 8 weeks (except for guinea pigs inoculated with
WAg320) and the assessment of virulence was based on the
enumeration of M. bovis c.f.u. in samples from the lung and spleen.
Guinea pigs were tested for infection by determining their delayedtype hypersensitivity to tuberculin using intradermal injection of
4 units of bovine PPD immediately prior to inoculation with
M. bovis and immediately prior to the animals being killed. In all
cases of M. bovis enumeration, statistical analyses by ANOVA were
performed on log10 transformations of M. bovis c.f.u.
M. bovis culture in ciprofloxacin and low-phosphate
medium. For determination of the MIC of strains in ciprofloxacin,
10-fold serial dilutions of each strain were cultured in duplicate
on supplemented Middlebrook 7H11 medium containing no ciprofloxacin and on the same medium containing doubling dilutions of
0?6–0?0375 mg ciprofloxacin ml21. For culture in low-phosphate
medium, approximately 36106 c.f.u. of each strain in 50 ml Tweenalbumin broth were inoculated into 5 ml Sauton medium containing
serial dilutions of phosphate. The final concentration of phosphate
for each culture was calculated taking into account the phosphate
added in the 50 ml inoculum.
RESULTS
Virulence in possums of M. bovis ATCC 35721
complemented with wild-type sigA (WAg320)
M. bovis ATCC 35721 and its complement with wildtype sigA (WAg320) were assessed for their ability to cause
disease in comparison to a wild-type M. bovis strain
(WAg200) in an intratracheal possum model of virulence.
The results are given together with the genotypes of the
strains in Table 2, in comparison to the results previously
obtained with a subcutaneous guinea pig model (Collins
et al., 1995). Using an assessment of virulence based on
the ability of a strain to produce visible tuberculous
lesions, the complementation of ATCC 35721 with wildtype sigA restored a moderate level of virulence for guinea
pigs but had no ability to restore virulence for possums.
Subsequently, this result was confirmed by repeating the
experiment and enumerating the c.f.u. in the lungs and
spleen of possums and the spleens of guinea pigs (Table 3).
Identification of a wild-type gene
complementing ATCC 35721 to virulence for
possums
In order to identify a gene or genes that might restore
virulence to ATCC 35721 for possums, two integrated
cosmid libraries of recombinant M. bovis ATCC 35721
were prepared: ATCC 35721(pUHA8 : : WAg200) and ATCC
35721(pUHA28 : : WAg200). The only difference between
the two libraries was the presence of sigA in pUHA28.
Each library was inoculated into the trachea of two
possums. In each case, one of the two possums developed
Table 2. Virulence and sigA genotype of M. bovis strains in guinea pigs inoculated subcutaneously and in possums inoculated intratracheally
Strain
Genotype
sigA-R515HD
WAg200
ATCC 35721
WAg320
WAg322
WAg324+325
ATCC 35723
WAg758
+
+
+
+
Virulence*
sigA
Guinea pig spleen lesions
Possum lung lesions
+
+++
2
++
++
++
++
+
+++
2
2
+++
+++
+++
+
+
+
+
*Virulence: 2, no lesions; +, 1–5 lesions; ++, 6–50 lesions; +++, >50 lesions or (in possums)
consolidated lung lobe.
DsigA-R515H: mutant allele of sigA encoding histidine instead of arginine at position 515 in SigA.
3206
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
Microbiology 149
M. bovis phoT in phosphate transport and virulence
Table 3. Virulence of M. bovis strains in various animal models
The results are means±SE; the number of animals per group (n) is shown for each model. Values in each column with the same superscripts (a,b,c) are not significantly different (P<0?05).
Strain
Genotype*
sigA
phoT
Virulence
Guinea pig
(log10 c.f.u. per organ)
Subcutaneous
(n=3–6)
Spleen
ATCC 35721
R515H
2
WAg320
R515H, +
2
WAg324
R515H
2, +
ATCC 35723
+
+
WAg758
+
2
2?4b±0?1
4?9a±0?4
4?7a±0?4
5?2a±0?4
2?3b±0?5
Possum, intratracheal
(log10 c.f.u. g”1) (n=4)
Mouse, aerosol
(log10 c.f.u. per organ) (n=4)
Aerosol
(n=3)
Lung
Spleen
6?1b±0?4 3?7b±0?5
7?9a±0?1 5?5a±0?4
3?8c±0?7 3?0b±0?5
5?2bc±0?5 4?0b±0?3
2?2d±0?4 1?0cD±0?0
Lung
Spleen
Lung
Spleen
1?8b±0?3
2?0b±0?1
5?4a±0?8
6?6a±0?2
3?0b±0?4
1?8b±0?1
1?7bD±0?0
4?7a±1?0
4?9a±0?3
1?7bD±0?0
5?9b±0?1
6?6a±0?1
5?5b±0?2
3?6c±0?3
3?1c±0?2
5?1a±0?1
5?0a±0?2
4?7a±0?1
2?2b±0?3
2?0b±0?2
*R515H, mutant allele of sigA encoding histidine instead of arginine at position 515 in SigA; +, wild-type allele; 2, inactive phoT.
DThis is the maximum log10 c.f.u. possible as no organisms were isolated for these animals.
a small lung lesion. Subsequent analysis of the recombinants from these lesions showed that their cosmid inserts
largely overlapped. Reinoculation of one of the ATCC
35721(pUHA8 : : WAg200) recombinants (WAg322) into a
possum caused multiple lung lesions and WAg322 was
also moderately virulent in guinea pigs (Table 2). The
cosmid-sized insert in WAg322 was recovered from
genomic DNA of this strain as an E. coli plasmid,
pUHA30. The insert was subcloned, using partial Sau3AI
digestion, back into the same mycobacterial integrating
shuttle vector, pUHA8, and this mini-library was electroporated into ATCC 35721 to produce ATCC 35721
(pUHA8 : : pUHA30). Inoculation of this library into a
single possum produced numerous lesions from which
individual colonies were isolated. From 70 isolates analysed
by restriction digestion and Southern blot hybridization,
the two most common fragment patterns were found in
32 and 10 isolates respectively. Strains with both these
predominant patterns were isolated from lesions in both
the lung and spleen. Representative cultures (WAg324 and
WAg325) with each of these patterns were combined
together and inoculated into two possums. This mixed
inoculum produced extensive lesions in both animals, and
subsequent culture followed by DNA extraction, restriction
digestion and Southern blot hybridization showed that
both WAg324 and WAg325 were present in the lesions
(Table 2). Subsequently, this result with WAg324 was
confirmed in four possums by enumerating the c.f.u. in
the lungs and spleen (Table 3). The subcloned fragments
of pUHA30 in WAg324 and WAg325 were recovered by
PacI digestion of genomic DNA, cloned into the E. coli
sequencing vector pUHA9 to produce pUHA37 and
pUHA38 respectively, and sequenced. The sequences were
1973 bp and 2311 bp in length and overlapped by 1973 bp
(Fig. 1). Comparison of this 1973 bp sequence to that of
http://mic.sgmjournals.org
M. tuberculosis strains H37Rv and CDC1551 and M. bovis
strain AF2122/97 revealed that WAg200 had an identical
sequence to that of M. bovis AF2122/97 and that this
sequence differed from that of the two M. tuberculosis strains
by two nucleotides. Annotation of the 1973 bp region
into ORFs was identical in the genome sequences of both
M. tuberculosis H37Rv and CDC1551 and is shown in Fig. 1.
The sequence contains only two complete ORFs, for genes
designated phoT and phoY2 in H37Rv. The two nucleotide
differences between M. bovis and M. tuberculosis were in
the coding region of phoT (Fig. 2); one change was neutral
and the other caused a change from phenylalanine to
leucine at position 35 on PhoT.
The equivalent region to this 1973 bp sequence was
cloned from M. bovis ATCC 35721 to produce pUHA39.
Sequencing of the insert in this plasmid revealed no
mutations in phoY2 but two well-separated point deletions
in the phoT ORF as shown in the alignment of phoT ORFs
in Fig. 2. Subsequent sequencing of part of phoT in M. bovis
BCG revealed that it too has one of the same frameshift
mutations (Fig. 2). PhoT is an ABC transporter with a high
similarity to proteins of the phosphate transport system,
usually designated PstB. There are close homologues of
M. bovis PhoT in other slow-growing mycobacteria (Mycobacterium leprae, 91 % identical; Mycobacterium avium,
89 % identical) as well as a pstB gene in M. tuberculosis that
is 49 % identical to M. bovis PhoT. Recent sequencing of
the genome of M. bovis AF2122/97 (Garnier et al., 2003)
has revealed that, compared to M. tuberculosis pstB, M. bovis
pstB has a frameshift mutation, the functional significance
of which has yet to be elucidated. Because of the possible
functional relatedness of phoT and pstB, the pstB genes in
the M. bovis strains used for this study were also sequenced.
ATCC 35721, ATCC 35723 and BCG Pasteur were all
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
3207
D. M. Collins and others
(a)
1 2
3
4
5
6
phoT
knockout
Wild-type
phoT
(b)
1 2
3
4
5
6
phoT
knockout
Wild-type
phoT
Fig. 2. Alignment of phoT from: M. tb, M. tuberculosis H37Rv
and CDC1551; M. bov, M. bovis AF2122/97 and WAg200;
35721, M. bovis ATCC 35721; BCG, M. bovis BCG Pasteur;
* denotes identical nucleotide to M. tb; % denotes point deletion.
found to have the same frameshift mutation in pstB as that
present in AF2122/97. The presence of the pstB and phoT
frameshift mutations in BCG Pasteur has now been confirmed by comparison to recently available sequence from
the genome project for BCG Pasteur (http://www.sanger.
ac.uk/Projects/M_bovis/).
Knockout of phoT in M. bovis
In order to determine the role of phoT in a virulent strain
of M. bovis with a wild-type sigA genotype, a suicide plasmid approach was used to inactivate the phoT gene in
M. bovis ATCC 35723 by allelic exchange. A Southern
blot hybridization representing two successful knockout
recombinants, a single homologous recombinant and three
3208
Fig. 3. (a) Southern blot hybridization after BamHI digestion
and probing with phoT of DNA from six hygromycin-resistant
recombinants: lanes 1–3, illegitimate recombinants; lane
4, single homologous recombinant; lanes 5 and 6, allelicexchange phoT knockout recombinants. The expected size of
wild-type and knockout fragments is indicated by arrows.
(b) Agarose gel of PCR products from phoT knockouts of
M. bovis in comparison to reference products: lane 1, molecular size markers; lane 2, M. bovis ATCC 35723; lane
3, M. bovis phoT knockout (WAg758); lane 4, second M. bovis
phoT knockout; lane 5, pUHA43 plasmid used for knockout of
phoT; lane 6, pUHA37 plasmid with wild-type phoT.
illegitimate recombinants is shown in Fig. 3(a). An agarose
gel of PCR products from these knockouts in comparison
to reference products is shown in Fig. 3(b). Compared to
its moderately virulent parent, M. bovis ATCC 35723,
the phoT knockout strain (WAg758) was found to have
reduced virulence when assessed in the subcutaneous
guinea pig model on the basis of spleen lesions (Table 2)
and confirmed by enumeration of c.f.u. in the spleen
(Table 3).
One possible explanation for some of the differences in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
Microbiology 149
M. bovis phoT in phosphate transport and virulence
virulence observed between the strains used in this study
was their mode of administration to animals: into the lungs
in the intratracheal possum model and subcutaneously
and eventually into the draining lymph circulation in the
subcutaneous guinea pig model. In order to further
investigate the importance of these modes of administration on the assessment of virulence, a range of strains was
administered to guinea pigs and mice by aerosol into the
lungs and the results compared to those obtained when
the same strains were administered subcutaneously into
guinea pigs and intratracheally into possums. Virulence
was assessed by the mean numbers of c.f.u. in the spleen
and lungs of groups of infected animals. In the case of
subcutaneously infected guinea pigs, none of the groups
had lesions in their lungs and lung tissues were not submitted for culture of M. bovis. The results are shown for
all four animal models in Table 3.
sigA, phoT (WAg324) was unable to restore growth in lowphosphate medium. At the lowest phosphate concentration,
strains with wild-type sigA and knockout of phoT (WAg320
and WAg758) did not grow, in contrast to ATCC 35723
with wild types of both sigA and phoT, which did grow.
This indicates that in wild-type strains of M. bovis, phoT
plays a role in acquiring phosphate when it is at very
low concentrations. The MIC of ciprofloxacin for various
M. bovis strains is also given in Table 4. There was no
difference between any of the strains except for WAg758,
which had half the MIC of the other strains. This indicates
that phoT may play a role in phosphate transport in ATCC
35723, as resistance to ciprofloxacin has been shown to
be associated with phosphate transport in Mycobacterium
smegmatis (Bhatt et al., 2000).
DISCUSSION
WAg320, which contains sigA, was significantly more
virulent than its parent, ATCC 35721, in both the subcutaneous and aerosol guinea pig models and in the lungs
of mice but both strains were highly attenuated in possums.
WAg320 was particularly virulent in the aerosol guinea pig
model and the animals were sufficiently clinically affected
after 29 days to be killed on ethical criteria. In contrast,
WAg324, which contains phoT, was significantly more
virulent than its parent in possums and in the subcutaneous
guinea pig model but was significantly less virulent than
its parent in the aerosol guinea pig model and of similar
virulence to its parent in the mouse model. The phoT
knockout WAg758 was significantly less virulent than its
parent ATCC 35723 in all the animal models except mice,
although even in mice the mean c.f.u. was 0?5 log lower.
In this study, we observed that the wild-type principal sigma
factor gene, sigA, which was able to restore virulence to
M. bovis ATCC 35721 in a subcutaneous guinea pig model,
was unable to do so for an intratracheal possum model.
In order to discover a gene or genes that might be important for the virulence of M. bovis in a natural wildlife host
and to identify additional mutations in ATCC 35721, we
exploited this observation by employing an in vivo complementation approach. A genomic cosmid library of a
virulent M. bovis strain was used to identify a virulencerestoring cosmid and then a mini-library of this virulencerestoring cosmid was used to identify a 1973 bp DNA
sequence that restored virulence. Analysis of this wild-type
M. bovis sequence and the homologous sequence in ATCC
35721 showed that a gene in ATCC 35721, phoT, had two
frameshift mutations. Since these frameshift mutations are
in the core conserved domain of this ABC transporter
(Higgins, 2001), it can be concluded that the gene in ATCC
35721 is inactivated.
In vitro growth of M. bovis strains in
ciprofloxacin and low-phosphate medium
The growth of M. bovis strains in liquid medium containing
various low concentrations of phosphate is given in Table 4.
Growth was clearly associated with the presence of a wildtype sigA gene. In the absence of the wild-type version of
Work to this point had established that phoT was necessary to restore virulence to ATCC 35721 in the intratracheal possum model, that it also restored virulence in
Table 4. In vitro growth of M. bovis strains in low-phosphate medium and in ciprofloxacin
Strain
Genotype*
sigA
ATCC 35721
WAg320
WAg324
ATCC 35723
WAg758
R515H
R515H, +
R515H
+
+
GrowthD at phosphate
concentration (mM) of:
Ciprofloxacin
MIC (mg ml”1)
phoT
4?0
0?62
0?29
0?14
2
2
2, +
+
2
+
+
NG
NG
NG
+
+
NG
NG
NG
NG
NG
+
+
+
+
+
+
NG
+
0?30
0?30
0?30
0?30
0?15
*R515H, mutant allele of sigA encoding histidine instead of arginine at position 515 in SigA; +, wild-type
allele; 2, inactive phoT.
DNG, No growth; +, growth.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
3209
D. M. Collins and others
the subcutaneous guinea pig model and that ATCC 35721
has mutations in both its sigA and phoT genes. Clearly,
ATCC 35721 is substantially different from a wild-type
M. bovis strain and might even have further mutations
perhaps of a compensatory nature, so it was desirable to
confirm the virulence properties of phoT in a virulent
M. bovis strain. In order to do this, the gene was inactivated
by allelic exchange from the moderately virulent M. bovis
strain, ATCC 35723, to produce WAg758. WAg758 was
significantly less virulent than its parent in both the subcutaneous guinea pig and intratracheal possum models,
establishing that phoT activity is necessary to maintain the
virulence of ATCC 35723 in these models.
The ability of phoT to restore virulence to ATCC 35721 for
both the subcutaneous guinea pig and intratracheal possum
models contrasts with the ability of sigA to restore virulence to ATCC 35721 only for the subcutaneous guinea
pig model. This raised the question of whether the difference between the models with respect to sigA was due to
differences in the host response to infection of guinea pigs
and possums or was related to the mode of administration.
It was possible that sigA might be sufficient for restoring
virulence to ATCC 35721 when the strain is inoculated
subcutaneously but not when animals are infected via the
lungs. To investigate this possibility and also the effect of
the presence or absence of phoT, the same group of five
strains was inoculated into two additional animal models:
aerosol models of the mouse and guinea pig. An important
result from this work was the finding that WAg320 (ATCC
35721 complemented with sigA) was significantly more
virulent than ATCC 35721 in both the subcutaneous and
aerosol guinea pig models as well as in the mouse model.
The original finding that WAg320 is virulent in guinea
pigs (Collins et al., 1995) therefore reflects a general host
susceptibility of guinea pigs to this strain that is not
dependent on route of administration. The corollary of this
is that because WAg320 is not virulent when administered
into the lungs of possums, there is a clear difference in
susceptibility to this strain between possums and both
guinea pigs and mice. At the molecular level, it has now been
shown that the mutation in SigA-R515H in ATCC 35721
affects its binding to an accessory transcription factor,
WhiB3, which is thought to regulate genes that influence
the immune response of the host (Steyn et al., 2002). If
this is the case, then regulation of these genes by WhiB3
does not appear to be important for influencing the
immune response of possums.
Inoculation of ATCC 35723 and its phoT knockout strain
(WAg758) in four different models gave consistent results.
In three of the models, WAg758 was significantly less
virulent than its parent while in the mouse model the
trend was in the same direction but the difference was not
significant with the small number of animals used. Clearly,
phoT is important for the virulence of M. bovis in a range
of animals. ATCC 35721 recombinants complemented
with phoT were also found to be virulent when inoculated
3210
in the subcutaneous guinea pig model irrespective of the
presence of the wild-type allele of the principal sigma factor
gene, sigA. In our earlier work in which sigA was identified
in a similar in vivo complementation approach to that used
here for phoT, we analysed the predominant virulencerestoring clone from the library which represented 80 % of
the recombinant M. bovis isolates recovered from guinea
pigs (Collins et al., 1995). In light of the present finding
that phoT also restores virulence to ATCC 35721 in the
subcutaneous guinea pig model, we would now conclude
that some of the remaining 20 % of virulence-restoring
recombinants from the first study probably contained
cosmids incorporating the phoT locus. The most likely
explanation for their unequal representation in the earlier
study is the under-representation of cosmids containing
phoT in the library used in that study. Another possible
explanation is that phoT restores ATCC 35721 (WAg324) to
a slightly lower degree of virulence than does sigA (WAg320)
and is outcompeted by WAg320 when both are inoculated
together. However, far greater numbers of animals per
group would be needed than those used throughout this
study in order to determine if there is a significant degree of
difference in virulence between these two strains.
Sequencing of a PCR product made from genomic DNA of
M. bovis BCG showed that one of the frameshift mutations
in the phoT of ATCC 35721 was also present in BCG. It is
now well established (Wards et al., 2000; Lewis et al., 2003)
that the absence of the RD1 region from BCG contributes
greatly to its attenuation and that much of this attenuation
can be accounted for by the loss of the esat-6 gene (Wards
et al., 2000), one of the nine genes deleted from BCG in the
RD1 region. The identification of a frameshift mutation
causing inactivation of the phoT gene, which has virulence
properties and is very distant from RD1 on the chromosome, indicates that phoT may also be contributing to the
attenuation of BCG.
Comparative DNA analysis of M. bovis PhoT shows that it
is essentially identical to M. tuberculosis PhoT and encodes
an ATP-binding cassette (ABC) protein with a probable
role in phosphate transport (Cole et al., 1998). Bacteria
generally have an active phosphate transport system that
includes two membrane-spanning proteins, a substratebinding protein and an ABC protein designated PstB
which hydrolyses ATP and provides the energy required
for transport (van Veen, 1997; Linton & Higgins, 1998).
Normally, these proteins are in a single operon or at the
same locus on the genome. The M. tuberculosis complex is
unusual in having three putative pst operons that between
them contain three phosphate-binding proteins and four
membrane-spanning proteins (Braibant et al., 2000) and it
is thought that these multiple copies might enable subtle
biochemical adaptations of these organisms to different
phosphate-limiting conditions during the infectious cycle
(Sarin et al., 2001). pstB is the only gene encoding an ABC
protein in these operons, but phoT, which is 130 kb from
pstB on the chromosome, is also an ABC protein gene and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
Microbiology 149
M. bovis phoT in phosphate transport and virulence
PhoT has higher homology to PstB in some other organisms
(http://genolist.pasteur.fr/TubercuList) than does PstB of
M. tuberculosis. In particular, PhoT has very high homology
to what appear to be PstB-encoding genes surrounded by
other phosphate transport genes in the as yet unannotated,
unfinished genome sequences of both M. smegmatis and
M. avium (http://www.ncbi.nlm.nih.gov/sutils/genom_table.
cgi?). Comparison of the two M. smegmatis sequences in
TIGR_1772 and AAC15686 to each other and to the PstB
proteins in other organisms indicates that they are almost
certainly from the same gene but that the earlier sequence
(AAC15686) is translated from a gene sequence that has
a small number of sequencing errors (comparisons not
shown). To investigate whether PhoT has a role in phosphate transport, the five strains of this study were cultured
in decreasing concentrations of phosphate. Results of this
work showed that growth at lowered phosphate concentrations required the presence of the wild-type principal sigma
factor SigA but that, even with this factor present, growth
did not occur at the lowest phosphate concentration tested
without the presence of an active PhoT. Recently, it was
shown that disruption of the pst operon in M. smegmatis
reduced its phosphate uptake ability and doubled its
sensitivity to the fluoroquinolone ciprofloxacin (Bhatt
et al., 2000). This group also showed that resistance to
ciprofloxacin involved an efflux system (Banerjee et al.,
2000). Testing of the sensitivity of the strains used in this
study to ciprofloxacin similarly showed that inactivation
of phoT in ATCC 35723 doubled its sensitivity as well as
reducing its ability to scavenge phosphate. Bhatt et al.
(2000) concluded that there is strong evidence that pstB
plays a role in phosphate importing as well as having a
second role in exporting. In this study, PhoT also appears
to have two different functions: a phosphate transporting
activity at low phosphate levels requiring SigA and a
virulence role in the presence of either SigA or, for some
animal models, also SigA-R515H. While the ciprofloxacin
results and analogy to the M. smegmatis system suggest that
this virulence role may involve the export of one or more
substances, this remains to be determined. At this stage,
these conclusions on the role of PhoT in M. bovis must be
accepted with caution as the determination of growth in
low phosphate concentrations measures an in vitro phenotype and virulence is an in vivo phenotype. In better-studied
pathogenic bacteria, it has been shown that uptake of
phosphate has its own regulatory system and that the
relationship of this regulation with virulence is only partly
understood (Rao et al., 2003; von Kruger et al., 1999). An
additional complicating factor in interpreting these results
in the broader context of other species of the M. tuberculosis
complex is the very recent finding that pstB in M. bovis,
including the strains used in this study, has a frameshift
mutation that would be expected to affect the expression
of a functional PstB. Since PstB and PhoT may have
functionally overlapping roles and pstB is not frameshifted
in M. tuberculosis, it is possible that the effects on either
virulence or phosphate scavenging that occur when phoT is
inactivated in M. bovis may not occur in M. tuberculosis.
http://mic.sgmjournals.org
Virulence of pathogenic organisms is a complex phenotype that depends on the function of many genes in the
pathogen as well as on a multitude of responses by the
infected host. In the case of tuberculosis, the importance
of the disease has encouraged the development and use
of many different animal models. No single model exactly
replicates the situation in humans or in important economic species such as cattle, but different models have
advantages for different studies. Mouse models have been
especially useful in immunological studies of tuberculosis
but gave less clear-cut results for the strains used in this
study than did the other models. In particular, ATCC 35721
grew to higher numbers in mice than did ATCC 35723,
even though this latter strain is much more virulent that
ATCC 35721 in guinea pigs and possums. These results
emphasize the caution that should be exercised in using a
single animal model to study gene effects on a phenotype
as complex as virulence even when isogenic strains are
being used. In this study, the susceptibility of different
animals to different strains of M. bovis enabled the discovery of the virulence role of phoT, the likely separation
of this role from its phosphate-scavenging ability, and the
particular importance of this gene for the virulence of
M. bovis in a natural wildlife host. These discoveries
illustrate the advantages of using different animal models
as tools for the molecular biological investigation of
tuberculosis virulence.
ACKNOWLEDGEMENTS
This study was supported by a grant from the New Zealand Foundation
of Research Science and Technology.
REFERENCES
Aldwell, F. E., Tucker, I. G., de Lisle, G. W. & Buddle, B. M. (2003).
Oral delivery of Mycobacterium bovis BCG in a lipid formulation
induces resistance to pulmonary tuberculosis in mice. Infect Immun
71, 101–108.
Balasubramanian, V., Wiegeshaus, E. H. & Smith, D. W. (1992).
Growth characteristics of recent sputum isolates of Mycobacterium
tuberculosis in guinea pigs infected by the respiratory route. Infect
Immun 60, 4762–4767.
Banerjee, S. K., Bhatt, K., Misra, P. & Chakraborti, P. K. (2000).
Involvement of a natural transport system in the process of effluxmediated drug resistance in Mycobacterium smegmatis. Mol Gen
Genet 262, 949–956.
Barry, C. E. (2001). Interpreting cell wall ‘virulence factors’ of
Mycobacterium tuberculosis. Trends Microbiol 9, 237–241.
Bhatt, K., Banerjee, S. K. & Chakraborti, P. K. (2000). Evidence
that phosphate specific transporter is amplified in a fluoroquinolone
resistant Mycobacterium smegmatis. Eur J Biochem 267, 4028–4032.
Braibant, M., Gilot, P. & Content, J. (2000). The ATP binding cassette
(ABC) transport systems of Mycobacterium tuberculosis. FEMS
Microbiol Rev 24, 449–467.
Cole, S. T. (2002). Comparative and functional genomics of the
Mycobacterium tuberculosis complex. Microbiology 148, 2919–2928.
Cole, S. T., Brosch, R., Parkhill, J. & 38 other authors (1998).
Deciphering the biology of Mycobacterium tuberculosis from the
complete genome sequence. Nature 393, 537–544.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
3211
D. M. Collins and others
Collins, D. M., Kawakami, R. P., de Lisle, G. W., Pascopella, L.,
Bloom, B. R. & Jacobs, W. R. (1995). Mutation of the principal
protein-deficient guinea pigs against respiratory challenge with
virulent Mycobacterium tuberculosis. Infect Immun 50, 555–559.
sigma factor causes loss of virulence in a strain of the Mycobacterium
tuberculosis complex. Proc Natl Acad Sci U S A 92, 8036–8040.
North, R. J., Ryan, L., LaCource, R., Mogues, T. & Goodrich, M. E.
(1999). Growth rate of mycobacteria in mice as an unreliable
Collins, D. M., Wilson, T., Campbell, S., Buddle, B. M., Wards, B. J.,
Hotter, G. & de Lisle, G. W. (2002). Production of avirulent mutants
of Mycobacterium bovis with vaccine properties by the use of
illegitimate recombination and screening of stationary phase
cultures. Microbiology 148, 3019–3027.
indication of mycobacterial virulence. Infect Immun 67, 5483–5485.
Pascopella, L., Collins, F. M., Martin, J. M., Lee, M. H., Hatfull, G. F.,
Stover, C. K., Bloom, B. R. & Jacobs, W. R., Jr (1994). Use of in vivo
complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence. Infect Immun 62, 1313–1319.
Dannenberg, A. M., Jr & Collins, F. M. (2001). Progressive pulmonary
Pfeffer, A., Buddle, B. M. & Aldwell, F. E. (1994). Tuberculosis in the
tuberculosis is not due to increasing numbers of viable bacilli in
rabbits, mice and guinea pigs, but is due to a continuous host
response to mycobacterial products. Tuberculosis 81, 229–242.
brushtail possum (Trichosurus vulpecula) after intratracheal inoculation with a low dose of Mycobacterium bovis. J Comp Pathol 111,
353–363.
Darzins, E. (1958). The Bacteriology of Tuberculosis. Minneapolis,
Rao, P. S. S., Lim, T. M. & Leung, K. Y. (2003). Functional genomics
MN: University of Minnesota Press.
de Lisle, G. W., Mackintosh, C. G. & Bengis, R. G. (2001).
approach to the identification of virulence genes involved in
Edwardsiella tarda pathogenesis. Infect Immun 71, 1343–1351.
Mycobacterium bovis in free-living and captive wildlife, including
farmed deer. Rev Sci Tech 20, 86–111.
Sarin, J., Aggarwal, S., Chaba, R., Varshney, G. C. & Chakraborti,
P. K. (2001). B-subunit of phosphate-specific transporter from
de Mendonca-Lima, L., Bordat, Y., Pivert, E., Recchi, C.,
Neyrolles, O., Maitournam, A., Gicquel, B. & Reyrat, J. M. (2003).
Mycobacterium tuberculosis is a thermostable ATPase. J Biol Chem
276, 44590–44597.
The allele encoding the mycobacterial Erp protein affects lung
disease in mice. Cell Microbiol 5, 65–73.
Skinner, M. A., Keen, D. L., Parlane, N. A., Yates, G. F. & Buddle,
B. M. (2002). Increased protection against bovine tuberculosis in the
Fleischmann, R. D., Alland, D., Eisen, J. A. & 23 other authors
(2002). Whole-genome comparison of Mycobacterium tuberculosis
brushtail possum (Trichosurus vulpecula) when BCG is administered
with killed Mycobacterium vaccae. Tuberculosis 82, 15–22.
clinical and laboratory strains. J Bacteriol 184, 5479–5490.
Steyn, A. J. C., Collins, D. M., Hondalus, M. K., Jacobs, W. R.,
Kawakami, R. P. & Bloom, B. R. (2002). Mycobacterium tuberculosis
Garnier, T., Eiglmeier, K., Camus, J-C. & 19 other authors (2003).
The complete genome sequence of Mycobacterium bovis. Proc Natl
Acad Sci U S A 100, 7877–7882.
WhiB3 interacts with RpoV to affect host survival but is dispensable
for in vivo growth. Proc Natl Acad Sci U S A 99, 3147–3152.
Glickman, M. S. & Jacobs, Jr, W. R. (2001). Microbial pathogenesis
van Soolingen, D., Hermans, P. W. M., de Haas, P. E. W., Soll, D. R.
& van Embden, J. D. A. (1991). Occurrence and stability of insertion
of Mycobacterium tuberculosis: dawn of a discipline. Cell 104, 477–485.
Higgins, C. F. (2001). ABC transporters: physiology, structure and
mechanism – an overview. Res Microbiol 152, 205–210.
Jacobs, W. R., Barrett, J. F., Clark-Curtiss, J. E. & Curtiss, R., III
(1986). In vivo repackaging of recombinant cosmid molecules for
analyses of Salmonella typhimurium, Streptococcus mutans, and
mycobacterial genomic libraries. Infect Immun 52, 101–109.
Kaushal, D., Schroede, B. G., Tyagi, S. & 8 other authors (2002).
Reduced immunopathology and mortality despite tissue persistence
in a Mycobacterium tuberculosis mutant lacking alternative sigma
factor, SigH. Proc Natl Acad Sci U S A 99, 8330–8335.
Kent, P. T. & Kubica, G. P. (1985). Public Health Mycobacteriology, a
Guide for the Level III Laboratory. Atlanta, GA: US Department of
Health and Human Service, Centres for Disease Control.
Lewis, K. N., Liao, R., Guinn, K. M., Hickey, M. J., Smith, S., Behr,
M. A. & Sherman, D. R. (2003). Deletion of RD1 from Mycobacterium
tuberculosis mimics bacille Calmette-Guerin attenuation. J Infect Dis
187, 117–123.
sequences in Mycobacterium tuberculosis complex strains: evaluation
of an insertion-sequence dependent DNA polymorphism as a tool in
the epidemiology of tuberculosis. J Clin Microbiol 29, 2578–2586.
van Veen, H. W. (1997). Phosphate transport in prokaryotes:
molecules, mediators and mechanisms. Antonie van Leeuwenhoek
72, 299–315.
von Kruger, W. M., Humphreys, S. & Ketley, J. M. (1999). A role
for the PhoBR regulatory system homologue in the Vibrio cholerae
phosphate-limitation response and intestinal colonization. Microbiology 145, 2463–2475.
Wards, B. J. & Collins, D. M. (1996). Electroporation at elevated
temperatures substantially improves transformation efficiency of
slow-growing mycobacteria. FEMS Microbiol Lett 145, 101–105.
Wards, B. J., de Lisle, G. W. & Collins, D. M. (2000). An esat6
knockout mutant of Mycobacterium bovis produced by homologous
recombination will contribute to the development of a live tuberculosis vaccine. Tubercle Lung Dis 80, 185–189.
Wassenaar, T. M. & Gaastra, W. (2001). Bacterial virulence: can we
Linton, K. J. & Higgins, C. F. (1998). The Escherichia coli ATP-
draw the line? FEMS Microbiol Lett 201, 1–7.
binding cassette (ABC) proteins. Mol Microbiol 28, 5–13.
Wilson, T. M., de Lisle, G. W. & Collins, D. M. (1995). Effect of inhA
McMurray, D. N., Carlomagno, M. A., Mintzer, C. L. & Tetzlaff,
C. L. (1985). Mycobacterium bovis BCG vaccine fails to protect
and katG on isoniazid resistance and virulence of Mycobacterium
bovis. Mol Microbiol 15, 1009–1015.
3212
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 04:15:59
Microbiology 149