Genetic Dissection of Trehalose Biosynthesis in Corynebacterium Glutamicum: Inactivation of Trehalose Production Leads to Impaired Growth and an Altered Cell Wall Lipid Composition

The analysis of the available Corynebacterium genome sequence data led to the proposal of the presence of all three known pathways for trehalose biosynthesis in bacteria, i.e. trehalose synthesis from UDP-glucose and glucose 6-phosphate (OtsA-OtsB pathway), from malto-oligosaccharides or alpha-1,4-glucans (TreY-TreZ pathway), or from maltose (TreS pathway). Inactivation of only one of the three pathways by chromosomal deletion did not have a severe impact on C. glutamicum growth, while the simultaneous inactivation of the OtsA-OtsB and TreY-TreZ pathway or of all three pathways resulted in the inability of the corresponding mutants to synthesize trehalose and to grow efficiently on various sugar substrates in minimal media. This growth defect was largely reversed by the addition of trehalose to the culture broth. In addition, a possible pathway for glycogen synthesis from ADP-glucose involving glycogen synthase (GlgA) was discovered. C. glutamicum was found to accumulate significant amounts of glycogen when grown under conditions of sugar excess. Insertional inactivation of the chromosomal glgA gene led to the failure of C. glutamicum cells to accumulate glycogen and to the abolition of trehalose production in a DeltaotsAB background, demonstrating that trehalose production via the TreY-TreZ pathway is dependent on a functional glycogen biosynthetic route. The trehalose-non-producing mutant with inactivated OtsA-OtsB and TreY-TreZ pathways displayed an altered cell wall lipid composition when grown in minimal broth in the absence of trehalose. Under these conditions, the mutant lacked both major trehalose-containing glycolipids, i.e. trehalose monocorynomycolate and trehalose dicorynomycolate, in its cell wall lipid fraction. The results suggest that a dramatically altered cell wall lipid bilayer of trehalose-less C. glutamicum mutants may be responsible for the observed growth deficiency of such strains in minimal medium. The results of the genetic and physiological dissection of trehalose biosynthesis in C. glutamicum reported here may be of general relevance for the whole phylogenetic group of mycolic-acid-containing coryneform bacteria.