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Journal of Clinical Microbiology, May 2005, p. 2481-2484, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2481-2484.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Genomic Variations between Mycobacterium bovis and M. tuberculosis H37Rv

Ariel Amadio,1 María Isabel Romano,2 Fabiana Bigi,2 Ignacio Etchechoury,2 Tanja Kubica,3 Stefan Niemann,3 Angel Cataldi,2* and Karina Caimi2

Institute of Microbiology and Agricultural Zoology,1 Institute of Biotechnology, CICVyA-INTA, Castelar, Argentina,2 Forschungszentrum Borstel, National Reference Center for Mycobacteria, D-23845 Borstel, Germany3

Received 18 June 2004/ Returned for modification 9 August 2004/ Accepted 17 January 2005


   ABSTRACT
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Genetic differences between Mycobacterium bovis and M. tuberculosis were identified. We found (i) a deletion of Rv3479 specific to M. bovis, (ii) that the rpfA gene is shortened to various extents in M. bovis, and (iii) an insertion in Rv0648 and a duplication of lppA common in M. tuberculosis complex isolates.


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Mycobacterium bovis, the agent of bovine tuberculosis, belongs to the M. tuberculosis complex (MTBC), whose members are closely related and show high similarity at the DNA level (2, 5, 8). However, despite this close relationship, they show a large variability in their phenotypic properties, epidemiology, and incidence in human tuberculosis.

The M. tuberculosis H37Rv (4) and M. bovis AF2122/97 (7) genomic sequences were compared using blastn and MSPcrunch (11) software and visualized with Artemis Comparison Tool version 4.0 for Apple (ACT, www.sanger.ac.uk/Software).

The criteria used to select loci for further studies were that they were not IS6110 insertions, PE-PPE family genes (4), single-nucleotide polymorphisms, or previously published regions of difference (1, 2). Four loci were selected: Rv3479, Rv0648, lppA and rpfA. In all these cases, the polymorphisms involved only one gene. The bioinformatic analysis yielded the following information: <OL TYPE = "a">.

(i) Rv3479. The Rv3479 gene encodes a hypothetical transmembrane protein of 3,225 bp in both M. tuberculosis H37Rv and CDC 1551 (6). However, the orthologue gene in M. bovis AF2122/97 carries a 714-bp deletion which starts at base 1254 from the ATG start codon (Fig. 1). This deletion originates two smaller open reading frame with sequences almost identical to Rv3479.



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  		FIG. 1. Schematic representation of gene arrangements at the four loci where differences between M. tuberculosis H37Rv and M. bovis AF2122/97 were found. Genes are indicated as arrowhead boxes, and sizes of genes are shown. Insertions and deletions of one species with respect to the other are represented as diagonal lines.

 
(ii) rpfA (Rv0867). rpfA is a 1,221-bp gene that encodes for a possible resuscitation-promoting factor. The central part of the protein contains APADLAPP repeats (9). The sequenced M. bovis strain lacks part of these repeats (Fig. 1) due to a 240-bp in-frame deletion. These domains are not deleted in M. tuberculosis CDC 1551.

(iii) Rv0648. M. bovis AF2122/97 has a 533-bp in-frame insertion in the Rv0648 gene relative to M. tuberculosis H37Rv (Fig. 1). This characteristic is similar to that of the RvD ele-ments (3) (http://www.pasteur.fr/recherche/unites/Lgmb/Deletion.html). However, the sequence of this insertion indicates that it is not one of the five previously described RvD (3) or TbD1 (2) elements. We propose to refer to this fragment as RvD6. This extra fragment is also present in M. tuberculosis CDC 1551.

(iv) lppA and lppB. The lppA and lppB genes, encoding lipoproteins, are highly similar. lppA is duplicated in M. bovis AF2122/97 with respect to M. tuberculosis H37Rv (Fig. 1). Interestingly, lppA is also duplicated in M. tuberculosis CDC 1551.

The four genes were BLAST searched in the M. smegmatis, M. avium, M. marinum, and M. leprae genomes (data not shown). Orthologues of rpfA genes exist in all of them. However, no Rv3479 and lppA orthologues were found in these mycobacteria. The orthologues of Rv0648 present in M. smegmatis, M. avium, and M. marinum have a size similar to that of M. bovis, suggesting that M. tuberculosis H37Rv suffered a deletion in this gene.

PCRs were applied to M. tuberculosis (20 isolates from Argentina and 10 from The Netherlands), M. bovis (15 isolates from Argentina and 15 from Mexico), M. pinnipedii (2 isolates), and the reference strains M. tuberculosis H37Rv, M. bovis AN5, and M. bovis BCG (five substrains, from the collection of the Institute of Biotechnology, CICVyA-INTA, Castelar, Argentina). M. microti strains (provided by Dick van Soolingen, Rijksinstituut voor Volksgezondheid en Milieu, The Netherlands) and M. bovis (five isolates) and M. bovis subsp. caprae (five isolates) from the National Reference Center for Mycobacteria, Borstel, Germany (10) strains were also used. These strains have been extensively characterized by IS6110 restriction fragment length polymorphism and spoligotyping. PCR amplifications were performed using conventional cycling conditions. The primers used are shown in Table 1.


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  		TABLE 1. Primers used in this study

 
PCR results. (i) Rv3479. The shorter band was observed in all the M. bovis (wild type and BCG) isolates tested. In all the other MTBC isolates tested, the larger PCR band was detected, including M. bovis subsp. caprae (Fig. 2). These results indicate that this element, as well as RD4 (2), is specifically deleted in classical M. bovis strains, suggesting that the deletion occurred after the divergence of M. bovis and M. bovis subsp. caprae.



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  		FIG. 2. Example of PCR assessment of the presence of the differences in several MTBC isolates. Primers flanking the differential regions were used for PCR. (Rv3479 [primers directed to the Rv3479 gene]) Lanes 1 to 10, M. tuberculosis isolates; lane 11, M. microti; lane 12, M. bovis BCG; lane 13, M. bovis AN5; lane 14, M pinnipedii; lanes 15 to 25, M. bovis isolates; lanes 26 to 30, M. bovis subsp. caprae isolates; lane 31, molecular weight marker. (rpfA) Lanes 1 to 5, M. bovis subsp. caprae isolates; lanes 6 to 14, M. bovis isolates; lane 15, M. bovis AN5; lane 16, M. microti; lanes 17 and 18, M pinnipedii; lanes 19 to 23, M. bovis BCG substrains; lanes 24 to 33, M. tuberculosis isolates; lane 34, molecular weight marker. (Rv0648) Lane 1, M. microti; lanes 2 and 3, M. pinnipedii; lanes 4 to 8, M. bovis subsp. caprae isolates; lanes 9 to 16, M. bovis isolates; lanes 17 to 26, M. tuberculosis isolates; lane 27, molecular weight marker. (lppA) Lane 1, M. microti; lanes 2 and 3, M pinnipedii; lane 4, M. bovis BCG; lane 5, M. bovis AN5; lanes 6 to 11, M. bovis isolates; lanes 12 to 16, M. bovis subsp. caprae isolates; lanes 17 to 25, M. tuberculosis isolates; lane 26, molecular weight marker.

 
(ii) rpfA. The rpfA gene is polymorphic in M. bovis because a larger PCR band was observed in M. bovis BCG, AN5, and 20/35 wild-type isolates, as well as in the other MTBC species tested. A smaller (6/35 isolates) or an intermediate (9/35) band was amplified in other M. bovis isolates (Fig. 2).

(iii) Rv0648. The 3,648-bp Rv0648 gene has high similarity to several {alpha}-mannosidases. M. tuberculosis is polymorphic at this gene because four isolates produced a small amplification band, while the other six produced a larger band. The other MTBC species tested showed only the larger band.

(iv) lppA and lppB. The lppA duplication was found in M. bovis and in 4/10 M. tuberculosis isolates, as suggested by a higher amplification band compared to most M. tuberculosis isolates (Fig. 2). M. bovis BCG and AN5 have a nonduplicated lppA gene. In consequence, lppA is not a species-specific marker. Band sizes from M. pinnipedii and M. microti suggest that there is no lppA duplication.

rpfA amplification products were cloned in pGEM-T easy vector (Promega) and sequenced using Dye Terminator reactions. In M. tuberculosis, there are five perfectly conserved amino acid repeats (APADLAPP) and eight degenerated and shorter repeats. We observed that there is a progressive loss of internal repeats of rpfA in M. bovis. While the described motif is conserved in M. bovis BCG and AN5, a fragment composed of a set of two linkers and four repeats was lost in some local strains (as in the sequenced M. bovis strain). Furthermore, two local M. bovis strains lack the other linker and six contiguous repeats (data not shown). While the expression of the rpfA gene was well demonstrated in M. tuberculosis and M. bovis BCG, we do not know whether the smaller-size gene variants from M. bovis strains are expressed. In any case, rpfA is a novel marker to differentiate and type M. bovis strains. As with Rv3479, the variable shortening of rpfA seems to be specific to classical M. bovis. This gene appears to perform an important role because it is conserved in several mycobacteria (data not shown).

In conclusion, the differences between M. bovis and M. tuberculosis genes described here could be used as markers for the differentiation and species determination of MTBC isolates.


   ACKNOWLEDGMENTS
 
The present study was supported by grants from the Centro Argentino Brasileño de Biotecnología (CABBIO). M.I.R., F.B., A.A., and A.C. are fellows of the National Research Council of Argentina (CONICET). K.C. has a fellowship from Fundación Antorchas.

We are grateful to Haydee Gil and Valeria Rocha for technical help. We acknowledge The Institute for Genomic Research for the use of unpublished genomic sequences of M. smegmatis and M. avium and the Sanger Centre for M. marinum.


   FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Biotecnología, CICVyA/INTA, Los Reseros y Las Cabañas, 1712 Castelar, Argentina. Phone: 54-11-4621-1447. Fax: 54-11-4621-0199. E-mail: acataldi{at}cnia.inta.gov.ar. Back


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Journal of Clinical Microbiology, May 2005, p. 2481-2484, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2481-2484.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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