First description of natural and experimental conjugation between Mycobacteria mediated by a linear plasmid

Abstract

Background: In a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. The transfer mechanism of this insertion sequence to M. kansasii was investigated here.

Methodology/principal findings: A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. The linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau.

Conclusions/significance: Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.

Figure 1. PFGE and Southern blot hybridization with IS1245-derived probe of

M. avium and M. kansasii colonies. (A) PFGE with undigested DNA; (B) Southern blot hybridization with IS1245-derived probe. Open arrow indicates pMA100; closed arrow indicates the uncharacterized smaller hybridization band. 88.1 to 88.4 = M. avium; 88.5 to 88.75 = PCR−IS1245-negative M. kansasii; 88.8 to 88.15 = PCR−IS1245-positive M. kansasii. On the left, Lambda Ladder PFG Marker (NewEngland BioLabs) molecular size markers.

Figure 2. PFGE of DNA genomic preparations.

(A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.

Figure 3. Gene features of the IS1245 flanking regions in pMA100.

The similarities identified in the 7,525-bp sequenced fragment of pMA100 are indicated. Similarities at nucleotide levels were identified using BLASTn and the regions of similarity are depicted as hatched bars; identities at amino acid level were identified using BLASTx and each region is shown as a white arrow, indicating the direction of transcription. Black bar localizes the IS1245 element. KpnI restriction sites are indicated.

Figure 4. Analysis of transconjugants isolated in mating experiments using M. avium 88.3 as donor strain.

(A) PFGE with undigested DNA; (B) Southern blot of PFGE gels with undigested DNA and hybridization with IS1245-derived probe; (C) PFGE-DraI; (D) Southern blot of PFGE-DraI gels and hybridization with pMA100-derived probe. Open arrows indicate the linear plasmid pMA100. 1: M. avium 88.3; 2: M. kansasii 88.6; 3: M. kansasii IAL 413; 4: M. kansasii IEC 6805; 5: M. bovis BCG Moreau; W: wild-type colony; T: transconjugant colony.