Multiple roles of trehalose in Mycobacterium smegmatis

Setting. Trehalose is a global stress protectant. It is a major component of mycobacterial cytoplasm and cell wall. Mycobacteria contain all known functional trehalose biosynthesis pathways.;Objective. To study the role of trehalose as a stress protectant and in the cell wall biogenesis in mycobacteria, a genetic approach may be useful.;Design. By using site directed mutagenesis, enzymes involved in trehalose biosynthesis pathways were inactivated. The resulting mutants were analyzed biochemically and also for their growth characteristics. Mutants were subjected to stress conditions such as desiccation, heat stress and hyperosmotic stress. Cell wall glycolipid and mycolate content of a trehalose deficient mutant was detected in the presence and absence of an exogenous trehalose source.;Results. We were able to generate various trehalose biosynthesis mutants of M. smegmatis including a quadruple mutant which lacked all three functional pathways. This mutant rapidly lost viability unless supplied with exogenous trehalose. Loss of viability of trehalose depleted quadruple mutant was due to a defect in cell division associated with the cell wall. Cell wall of this mutant showed rapid loss of trehalose mycolates and mycolic acids when starved of trehalose. Using mutants which did not synthesize trehalose 6 phosphate we showed that trehalose is required for synthesis of trehalose mycolates and mycolic acids. The presence of either the OtsA-OtsB or the TreY-TreZ pathway was sufficient for normal growth as well as comparable cell wall composition. Either of these two pathways was also sufficient for conferring wild type levels of stress protection. TreS did not contribute to trehalose biosynthesis and stress protection and appeared to act as a degradatory pathway.;Conclusion. M. smegmatis has an absolute requirement of trehalose for viability. Trehalose plays a pivotal role in cell wall biogenesis. It is also a major stress protectant in mycobacteria. OtsA-OtsB or TreY-TreZ pathways alone confer wild type trehalose levels and stress resistance. In contrast TreS mediates trehalose degradation.