High extracellular levels of Mycobacterium tuberculosis glutamine synthetase and superoxide dismutase in actively growing cultures are due to high expression and extracellular stability rather than to a protein-specific export mechanism

Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of My cobacterium tuberculosis, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leade r peptide, their presence in the extracellular medium during early stages o f growth suggested that they might be actively secreted. To understand thei r mechanism of export, we cloned the homologous genes (glnA1 and sodA) from the rapid-growing, nonpathogenic Mycobacterium smegmatis, generated glnA1 and sodA mutants of M. smegmatis by allelic exchange, and quantitated expre ssion and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs from M. tuberculosis. When each of the genes was express ed from a multicopy plasmid, AL smegmatis exported comparable proportions o f both the Al. tuberculosis and M. smegmatis GSs (in the glnA1 strain) or S ODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant Al. smegmatis and M. tuberculosis strai ns even exported nonmycobacterial SODs. To determine the extent to which,ex port of these large, leaderless proteins is expression dependent, we constr ucted a recombinant M. tuberculosis strain expressing green fluorescent pro tein (GFP) at high levels and a recombinant M. smegmatis strain coexpressin g the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB (bacterio ferritin) at high levels. The recombinant M. tuberculosis strain exported G FP even in early stages of growth and at proportions very similar to those of the endogenous M. tuberculosis GS and SOD. Similarly, the recombinant Al . smegmatis strain exported bacterioferritin, a large (similar to 500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. I n contrast, high-level expression of the large, leaderless, multimeric prot ein malate dehydrogenase did not lead to extracellular accumulation because the protein was highly unstable extracellularly. These findings indicate t hat, contrary to expectations, export of M. tuberculosis GS and SOD in acti vely growing cultures is not due to a protein-specific export mechanism, bu t rather to bacterial leakage or autolysis, and that the extracellular abun dance of these enzymes is simply due to their high level of expression and extracellular stability. The same determinants likely explain the presence of other leaderless proteins in the extracellular medium of actively growin g M. tuberculosis cultures.