BMC Infectious Diseases

BioMed Central

Open Access

Research article

Identification of moaA3 gene in patient isolates of Mycobacterium

tuberculosis in Kerala, which is absent in M. tuberculosis H37Rv and
H37Ra
Suma Sarojini, Smitha Soman, Indulakshmi Radhakrishnan and
Sathish Mundayoor*
Address: Mycobacterial Research Group, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India
Email: Suma Sarojini - [email protected]; Smitha Soman - [email protected]; Indulakshmi Radhakrishnan - [email protected];
Sathish Mundayoor* - [email protected]
* Corresponding author

Published: 04 October 2005

BMC Infectious Diseases 2005, 5:81

doi:10.1186/1471-2334-5-81

Received: 30 March 2005

Accepted: 04 October 2005

This article is available from: http://www.biomedcentral.com/1471-2334/5/81

2005 Sarojini et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Tuberculosis is endemic to developing countries like India. Though the whole
genome sequences of the type strain M. tuberculosis H37Rv and the clinical strain M. tuberculosis
CDC1551 are available, the clinical isolates from India have not been studied extensively at the
genome level. This study was carried out in order to have a better understanding of isolates from
Kerala, a state in southern India.
Results: A PCR based strategy was followed making use of the deletion region primers to
understand the genome level differences between the type strain H37Rv and the clinical isolates of
M. tuberculosis from Kerala. PCR analysis of patient isolates using RD1 region primers revealed the
amplification of a 386 bp region, in addition to the expected 652 bp amplicon. Southern
hybridization of genomic DNA with the 386 bp amplicon confirmed the presence of this new region
in a majority of the patient isolates from Kerala. Sequence comparison of this amplicon showed
close homology with the moaA3 gene of M. bovis. In M. bovis this gene is present in the RvD5 region,
an IS6110 mediated deletion that is absent in M. tuberculosis H37Rv.
Conclusion: This study demonstrates the presence of moaA3 gene, that is absent in M. tuberculosis
H37Rv and H37Ra, in a large number of local isolates. Whether the moaA3 gene provides any
specific advantage to the field isolates of the pathogen is unclear. Field strains from Kerala have
fewer IS6110 sequences and therefore are likely to have fewer IS6110 dependent rearrangements.
But as deletions and insertions account for much of the genomic diversity of M. tuberculosis, the
mechanisms of formation of sequence polymorphisms in the local isolates should be further
examined. These results suggest that studies should focus on strains from endemic areas to
understand the complexities of this pathogen.

Background
Tuberculosis remains one of the most life threatening diseases and has been declared as a global emergency in 1993

by the World Health Organization (WHO) [1]. Failure in

adhering to the strict drug regimen has led to the emergence of multi drug resistant isolates of this pathogen.
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PCR
Figure
of clinical
1
isolates of M. tuberculosis for RD1 region
PCR of clinical isolates of M. tuberculosis for RD1
region. Lane 1: 100 bp marker, lane 2: H37Rv, lane 3:
H37Ra, lane 4: M. bovis BCG, lanes 524: Clinical isolates
RGTB 29, 37, 40, 43, 55, 60, 70, 86, 87, 93, 95, 109, 110, 123,
142, 144, 154, 167, 177, 193 respectively.

This, combined with the problem of HIV, worsens the TB

menace in developing countries. However, the main reason for the failure to eradicate tuberculosis lies in the biological properties of the infecting organism and its ability
to persist in a latent state in the macrophages.
Bacterial strains within a single species exhibit variations
in their properties such as pathogenicity, host specificity,
virulence, adaptation to particular habitats and drug
resistance. The passaging of M. tuberculosis H37Rv and M.
bovis BCG for several decades outside the human host
have induced changes in the genome of the pathogen and
have also altered their virulence characteristics. Whole
genome sequences of the type strain M. tuberculosis H37Rv
[2], the clinical strain M. tuberculosis CDC1551 [3] and M.
bovis [4] are already available. Many researchers have used
a number of comparative analysis techniques like subtractive hybridization and microarray to identify differences
in the genomes of laboratory strains and vaccine strains.
Three genomic regions which are absent in M. bovis BCG
but present in M. bovis and M. tuberculosis were first
described using subtractive hybridization [5]. DNA microarray based studies between H37Rv and BCG have shown
that 16 RDs (Regions of Differences) are deleted in BCG
[6]. Similarly, whole genome comparison studies have
shown six deletion regions in M. tuberculosis H37Rv
RvD1 to RvD5 and TbD1 [7].
Our earlier studies on the clinical isolates from Kerala on
IS6110 polymorphism have shown that a large number of
isolates have few or no copy of the sequence [8]. This
made about fifty percent of strains untypable using
IS6110. These results have been corroborated by studies
from other endemic areas in India as well as outside [912]. This has prompted us to speculate on whether there
are major differences in the genome in the field strains
from endemic areas. Therefore, we examined these strains
to see the distribution of the different RD regions. Here we

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PCR
Figure
of clinical
2
isolates of M. tuberculosis for moaA3 gene
PCR of clinical isolates of M. tuberculosis for moaA3
gene. Lane 1: Marker- DNA double digest (EcoR I/Hind III),
lane 2: H37Rv, lane 3: H37Ra, lane 4: M. bovis BCG, lanes 5
24: Clinical isolates RGTB 29, 37, 40, 43, 55, 60, 70, 86, 87,
93, 95, 109, 110, 123, 142, 144, 154, 167, 177, 193 respectively, lane 25: Negative Control.

report the presence of a genomic region in the clinical isolates of M. tuberculosis, which is absent in the type strains
H37Rv and H37Ra.

Methods
Mycobacterial strains and DNA isolation
The type strains used for the study included Mycobacterium
tuberculosis H37Rv, H37Ra and M. bovis BCG. These were
grown in Middlebrook 7H9 Broth (Difco Laboratories)
supplemented with OADC enrichment (Difco Laboratories) and 0.05% glycerol (USB Corporation). Field strains
of Mycobacterium tuberculosis were those isolated from sputum samples of tuberculosis patients from different parts
of Kerala. The strains were biochemically tested for Catalase, Niacin and Nitrate for identification. They were characterised by IS6110 fingerprinting. Drug resistance pattern
was also studied using the four major frontline drugs viz,
isoniazid, rifampicin, ethambutol and streptomycin.

DNA was isolated from cells pelleted from liquid culture

using glass beads in a minibead beater. The DNA was precipitated after phenol:chloroform extraction using 3M
Sodium acetate (pH 5.2) and 100% ethanol and dissolved
in TE buffer, pH 8.0.
PCR amplification of RD1 region
Oligonucleotide primer pairs used for the study were
RD1DLa: 5'-AGA TGA AGA CCG ATG CCG CTA C -3' and
RD1DRa: 5'-CCC GTG TTT CGC TAT TCT ACG C-3'. PCR
was performed in a final volume of 30 l using 1.25 units
of Taq DNA Polymerase (Promega Corporation) for each
reaction. After initial denaturation, amplification was
done using a PCR thermal cycler (BioRad) for 35 cycles of
94C/40 sec, 64C/1 min, 72C/1 min followed by a
final extension of 72C/7 min. To identify the flanking

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sequences of the 386 bp region, another set of primers

were used (moaFP: 5'-CCCATCGTGGTCGTTCACC-3' and
moaRP: 5'-CGATGGCAGCGGTTTACAG-3') which was
expected to amplify a 1254 bp product.
Southern hybridization
Genomic DNA from M. tuberculosis H37Rv, H37Ra, M.
bovis BCG and the clinical isolates digested using EcoR I
(New England Biolabs) was separated on agarose gels,
transferred to nylon membrane (Hybond) and probed
with 32P [dCTP] labelled PCR product. After overnight
hybridization at 65C, the blot was washed with increasing stringency of SSC-SDS and exposed to an activated
Phosphor screen (Kodak). The screen was then scanned
using Personal Molecular Imager FX (BioRad) and the
picture was visualized using the software Quantity One
(BioRad).
Cloning and sequencing
PCR products separated on agarose gel were eluted using
GFX PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech Inc). The eluted DNA was
cloned into pGEMT Easy vector (Promega Corporation).
Plasmid DNA for sequencing was purified using Nucleospin Plasmid kit (Macherey-Nagel) in accordance with
manufacturers' instructions. Plasmids were digested with
EcoR I to check for the presence of inserts. DNA sequencing by cycle sequencing method with the fluorescent dye
terminator (Big Dye Terminator Cycle Sequencing Ready
Reaction Kit, (PE Biosystems)) was carried out with T7
and SP6 promoter primers in an automated sequencer
(ABI Prism 310).

Results
Screening of RD1 by PCR
RD1, the most significant region of difference between M.
tuberculosis and M. bovis BCG is a 9505 bp long region
absent in all the different BCG substrains. PCR primers
were designed to amplify regions within RD1 to find out
polymorphism between type strains and the clinical isolates. PCR using RD1DLa and RD1DRa primers was
expected to amplify a 652 bp fragment (comprising of
Rv3874 and Rv3875, coding for cfp10 and esat 6) in M.
tuberculosis H37Rv and the clinical isolates. In H37Rv and
H37Ra the expected 652 bp band was observed. In BCG
the 652 bp band was absent as expected, but a 386 bp fragment was amplified. The clinical isolates showed both
652 and 386 bp fragments. A set of twenty patient isolates
from Kerala was used for the initial screening. Of these,
only one isolate (RGTB43), did not have the 386 bp
amplicon. (Fig. 1). Later we screened a total of one hundred isolates from Kerala by PCR and all except three
showed the presence of the 386bp amplicon (Data not
shown). A second PCR using primes designed from the
surrounding regions of moaA3 gene was done to confirm

Figure with
Southern
probed
3 hybridization
radiolabelled
of EcoR
386 bp
I digested
PCR product
genomic DNA
Southern hybridization of EcoR I digested genomic
DNA probed with radiolabelled 386 bp PCR product.
Panel A- Lane1: Marker DNA double digest (EcoR I/Hind
III) lane2: H37Rv, lane 3: H37Ra, lane 4: M. bovis BCG, lane 5:
pool of DNA from local isolates. Panel B Southern hybridization to DNA from individual isolates. Lane 1: Marker
DNA double digest (EcoR I/Hind III), lane 2: M. bovis BCG,
Lanes 322: Isolates RGTB 70, 86, 87, 93, 95, 109, 110, 123,
142, 144, 154, 167, 177, 193, 29, 37, 40, 43, 55, 60
respectively.

the presence of the full ORF in clinical isolates. The

expected amplicon of 1254 bp was obtained in all those
clinical isolates which had the 386 bp fragment (Fig 2).
Southern blot
To confirm the results of the PCR, EcoR I digested genomic
DNA from M. bovis BCG, M. tuberculosis H37Rv and
H37Ra and pooled DNA from all the 20 clinical isolates
(called local pool) were subjected to Southern hybridization using radioactively labelled 386 bp fragment from M.
bovis BCG. Local pool and M. bovis BCG showed a signal
corresponding to about 1.0 kb (Fig. 3A) while H37Rv and
H37Ra were negative. Southern hybridization of each of
the individual clinical isolate was then carried out for confirming the result and all, except RGTB 43, showed a positive signal (Fig. 3B). The strain RGTB 43 was negative by
PCR as well.
DNA sequence homology
Sequencing of the 386 bp amplicon cloned into pGEMT
Easy vector was carried out using T7 and SP6 promoter
primers. The sequence data obtained was compared to the
whole genome of M. bovis [19]M. tuberculosis [20] and
100% sequence homology was obtained with M. bovis
whereas M. tuberculosis H37Rv showed only 61%. The
upstream and downstream sequences of this 386 bp
region were identified by searching the M. bovis genome
database. It was found that the sequenced fragment did
not belong to the RD1 region. Instead, it was found to be
part of RvD5, a deletion in the type strain H37Rv (Fig. 4).
This region corresponds to moaA3 gene in M. bovis which

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3665238

3666302

PCR product
575 to 960 of moaA3
moaA3 gene

moaC3 moaB3 moaA3 Mb3356 Mb3357 embR2 Mb3359 Mb3360 sigJ

M.bovis
moaC3 MT3426 moaA3

MT3427.1 MT3428

MT3429 MT3430 MT3431

Clinical isolate

CDC1551
3705604

3706791

moaC3 Rv3324A Rv3325 Rv3326

Rv3327

sigJ

H37Rv

Figure
A
diagrammatic
4
representation of moaA3 and the surrounding regions
A diagrammatic representation of moaA3 and the surrounding regions. Comparison of the region comprising
moaA3 gene and the surrounding genes in M. bovis, H37Rv and the clinical isolate, CDC1551. All data from references [19, 20,
21, 22]. The coamplified PCR product is shown as a thick line in the moaA3 locus. The amplicon extends from 575 to 960 of
moaA3 gene in M. bovis. The genome coordinates of the moaA3 gene in M. bovis and M. tuberculosis CDC1551 are shown. The
thin dotted lines indicate corresponding genes in M. bovis and CDC1551. The bold dotted lines indicate similar genes in
CDC1551 and H37Rv. Rv3324A is differentially shown since it's a truncated gene and has only partial nucleotide similarity to
CDC1551 moaB3 gene.

codes for molybdenum cofactor biosynthesis protein A,

MoaA1.
The moaA3 gene was present in 97 of the 100 clinical isolates tested (details not presented). These isolates had varying IS6110 and drug resistance profiles suggesting the
possible absence of a relationship between the moaA3
fragment, IS6110 copy number and drug resistance
profile.

Discussion
Studies using subtractive hybridization [5] and microarrays [6] have identified 16 regions, (ranging in size from
212.7 kb), in M. tuberculosis H37Rv which are absent in
M. bovis BCG. Deletions are also reported in H37Rv
RvD1 to RvD5 and TbD1 [7]. These results suggest that
generation of deletions may be a major mechanism for
creating genetic diversity among the members of the complex. On this basis, we sought to screen the clinical isolates of M. tuberculosis from Kerala for differences in the
RD regions. Initially, we used primers spanning RD1

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Table 1: Details of clinical isolates of M. tuberculosis. The resistance (R)/sensitivity(S) profile of the isolates to the four frontline antituberculosis drugs and their IS6110 copy number are shown below.

RGTB No:

29
37
40
43
55
60
70
86
87
93
95
109
110
123
142
144
154
167
177
193

Drug resistance profile

IS6110 copy no:

Isoniazid

Ethambutol

Rifampicin

Streptomycin

R
S
S
S
R
R
S
R
R
R
S
S
R
S
R
S
R
S
S
R

R
S
R
R
R
R
S
R
R
S
S
S
S
S
R
S
S
R
R
R

S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
R
S
S
R

S
S
S
S
S
R
S
S
S
S
S
S
S
S
R
S
S
S
S
S

region, since RD1 is the most important region of

difference and is deleted in all the substrains of M. bovis
BCG [6]. The loss of RD1 is one major genetic event that
contributes to the attenuation of BCG, and its reintroduction into an attenuated strain resulted in a significant
increase in virulence [13].
Of the nine open reading frames predicted within the 9.5
kb RD1 region, ORFs coding for cfp10 and esat6 are considered to be very important as there is vigorous host
response to these proteins. Amplification using primers
that span this region was expected to give a 652 bp PCR
product. But the PCR results revealed an extra amplicon of
386 bp in the local isolates and BCG. Further characterization by sequencing and homology search indicated that
this region is a part of the moaA3 gene which codes for
molybdopterin cofactor protein A in M. bovis. The
sequence of the 386 bp amplicon obtained from the local
strains showed 100% homology with M. bovis as compared to 61% with M. tuberculosis H37Rv. The PCR primers that we made spanning the RD1 region was similar to
portions of the moaA3 gene in the RvD5 region, which
resulted in the amplification of the 386 bp fragment. This
amplicon spanned the nucleotides 575 to 960 of the
moaA3 gene in M. bovis (Fig 4). The moaA3 gene is absent
in H37Rv, but another gene in the biosynthetic pathway,
moaC3, was the closest to the 386 bp amplicon, with a
homology of 61%. Database searches revealed that the

1
1
0
9
1
2
1
1
3
1
6
12
8
0
10
1
1
14
1
1

moaA3 gene is present in the CDC1551 in the RvD5 region

as well. To confirm the location of the moaA3 gene in our
isolates, a second PCR designed to amplify the flanking
sequences of moaA3 gene was performed. The results confirmed the location of the moaA3 gene in the clinical isolates from Kerala. In M. bovis (Mb3355) the gene is 1065
bp long while in CDC 1551, the moaA3 gene (MT3427) is
1189 bp long, due to an additional 123 bp in the C terminal region. In the overlapping region, CDC1551 has
100% homology with M. bovis moaA3. Genome comparison studies have shown that moaA3 is one among the few
genes that is present in CDC1551 and absent in H37Rv
[3]. Southern hybridization studies done in our lab confirmed that moaA3 gene is absent in the type strains M.
tuberculosis H37Rv and H37Ra and is present in most of
the clinical isolates in Kerala as well as in M. bovis BCG.
Since moaA3 gene has been seen in the RvD5 region in
both M bovis and in CDC1551, we have presumed that
these genes are in the same region in our local strains as
well, but these results need confirmation. The regions surrounding the moaA3 gene and the IS6110 elements flanking the RvD5 region in these local isolates merit further
investigation.
Molybdopterin is a cofactor required for nitrate reductase
and many other enzymes involved in anaerobic metabolism. Genes involved in the molybdopterin cofactor biosynthesis pathway are present in almost all organisms. M.

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BMC Infectious Diseases 2005, 5:81

tuberculosis H37Rv dedicates 21 genes to the biosynthesis

of this cofactor [2]. But there is no gene homologous to
the moaA3 found in M. bovis. This cofactor is thought to be
involved in the biosynthesis of molybdopterin precurser Z
from guanosine in M. bovis. Studies in Escherichia coli have
suggested that these molybdoenzymes have the ability to
hydroxylate or dehydroxylate certain compounds enabling the bacteria to detoxify them [14]. In addition, E.
coli with defective moa show a decrease in the frequency of
adaptive mutations [15]. Thus, one may infer that the
moaA3 gene might have a role in the intracellular survival
of the local M. tuberculosis strains or may provide some
selective advantage to them. A recent study using a promoter trap vector has identified two of the genes, moaX
and moeB1 as upregulated in mouse lungs upon infection
[16]. A systematic study is required to understand the
exact role of this protein in the lifecycle of this pathogen.
At the same time, the effect due to the lack of moaA3 on
M. tuberculosis H37Rv may be difficult to quantify as the
remaining array of moa genes could be expected to complement any lost activity.
The RvD5 region from which the amplicon was generated
is an IS6110 mediated deletion in the type strain H37Rv
[7]. IS6110, a powerful genetic marker for strain differentiation [17] has also been shown to play an important role
in mediating genomic rearrangements and deletions in
mycobacteria. In fact, four of the five genomic deletions in
M. tuberculosis H37Rv (except RvD1) are predicted to be
IS6110 mediated recombinations [7]. But IS6110 mediated alterations may not provide much selective advantage to the bacteria from endemic areas such as Kerala, as
a large percentage of the isolates have very few copies of
IS6110 [8]. Insertions and deletions are important in the
evolution of a bacterial species. M. tuberculosis, considered
an evolutionarily "young" pathogen, would not be
expected to have undergone extensive variations in its
genome [18]. But, in spite of this, differences could be
detected between the laboratory strains and clinical isolates, both by sequence analysis as in the case of
CDC1551 [3] and by PCR as in this study. In a scenario of
few/no copies of IS6110 other insertion sequences or
mobile genetic elements could be involved in these variations. Therefore, a detailed study of the genome of field
strains from different endemic regions would provide
more insights into the diversity of this pathogen.

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So the strains from endemic areas need to be examined in

greater detail to understand the complexities of this pathogen. Such an understanding is essential for us to be able
to plan adequate control measures for tackling what is
appearing to be the world's number one killer.

Abbreviations
RD: Region of Difference
RGTB: Rajiv Gandhi Centre for Biotechnology Tuberculosis isolates
PCR: Polymerase Chain Reaction

Competing interests
The author(s) declare that they have no competing
interests.

Authors' contributions
SS carried out most of the experiments, data analysis and
wrote the manuscript. SS# did part of the experimental
work. IR did the IS6110 work and contributed to the writing of the manuscript. SM conceived and co-designed the
study, provided inputs for writing and supervised the
study. All authors read and approved the final manuscript.

Acknowledgements
Suma Sarojini and Smitha Soman are recipients of Senior Research Fellowship and Junior Research Fellowship respectively from Council for Scientific
and Industrial Research (CSIR), Govt. of India. This study received financial
assistance under program support from the Department of Biotechnology,
Government of India. Rajiv Gandhi Centre for Biotechnology is under the
Kerala State Council for Science, Technology and Environment. We thank
Laiza K. Paul for excellent technical assistance.

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H37Ra. The results obtained suggest that the population
of strains in endemic areas is different from type strains,
as suggested earlier by our analysis of IS6110. The field
strains may also vary between different endemic regions.

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