This study establishes an efficient specialized transduction system for allelic exchange and transposon mutagenesis in a clinical strain of M. avium
. When combined with the recently completed M. avium
genome sequence, these tools will allow new insights into M. avium
physiology. Several aspects of M. avium
physiology differ substantially from M. tuberculosis
, including colony morphotype switching (22
), environmental persistence (9
), plasmid maintenance (15
), and antibiotic resistance. These distinct properties of M. avium
can now be investigated genetically with the tools presented here.
Unfortunately, we were unsuccessful in generating a leuD
mutant in the SmT colony morphotype (mc2
2500). The high rate of spontaneous hygromycin resistance in both SmD and SmT colony types clearly impaired the efficient selection of recombinants and suggests that this antibiotic marker is not useful for M. avium
. Although mc2
2501 is derived from an original clinical isolate, it is a spontaneous opaque colony variant. Colony morphology in M. avium
is associated with virulence, with SmT types being more virulent for mice than SmD variants (14
). In addition, although opaque types are generally less virulent than SmT types, some opaque strains are isolated from patients and retain the ability to replicate within mice (26
). Thus, it is possible that mgm93 could be used to examine the genetic basis for virulence in M. avium
, depending upon its in vivo characteristics in mice. We plan to test the in vivo growth characteristics of mc2
2501 in immunocompetent mice to assess whether mgm93 derivatives will be useful for pathogenesis studies. In addition, it may be possible to isolate a SmT revertant of mgm93 after in vivo passage that would then be suitable for genetic manipulation.
A minimal set of genetic tools for M. avium
would include efficient transformation, which has not been routinely achieved. The isolation of new mycobacterial plasmid types from M. avium
) may allow efficient transformation vectors to be created, thereby further expanding the set of genetic tools for M. avium.
The efficiency of transposon mutagenesis in the present study is comparable to rates reported previously for M. tuberculosis
by using specialized transduction of a related transposon. This high rate of transposition in M. avium
was achieved despite poor phage entry into M. avium
cells, as measured by luciferase reporter phages. This result suggests that entry of transposon encoding DNA into the host cell is not the limiting factor in efficient transposon mutagenesis in mycobacteria. The leuD
transposon constructed here may insert more efficiently in the chromosome due to its small size, thereby compensating for poor phage entry.
In summary, the present study extends the utility of specialized transduction to include allelic exchange and transposon mutagenesis to a clinical strain of M. avium. Our use of a leucine auxotrophic strain of M. avium as a genetic host eliminated the spontaneous antibiotic resistance that had limited prior genetic studies in M. avium. In conjunction with the recently completed M. avium genome sequence, these genetic tools will allow direct study of M. avium physiology and may allow investigation into M. avium pathogenesis.