Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and an altered cell wall lipid composition

Abstract

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 α-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 ΔotsAB 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.

The glgX gene product of Corynebacterium glutamicum is required for glycogen degradation and for fast adaptation to hyperosmotic stress

“…Lanes S1 and S2 represent separation of the standard sugars: standard solution S1 contained trehalose (tre), and standard solution S2 contained glucose (glc), maltose (m2), maltotriose (m3), maltoheptaose (m7) and glycogen (glyc). inactivated in C. glutamicum (Tzvetkov et al, 2003;Wolf et al, 2003). The same phenotype has been observed when glycogen synthesis is abolished by disruption of the glycogen synthase gene glgA in C. glutamicum DotsAB (Tzvetkov et al, 2003).…”

“…inactivated in C. glutamicum (Tzvetkov et al, 2003;Wolf et al, 2003). The same phenotype has been observed when glycogen synthesis is abolished by disruption of the glycogen synthase gene glgA in C. glutamicum DotsAB (Tzvetkov et al, 2003). Moreover, overexpression of the treYZ genes in C. glutamicum causes a decrease in the intracellular glycogen content (Carpinelli et al, 2006).…”

“…When these compunds are not available, C. glutamicum synthesizes compatible solutes such as proline and trehalose (Morbach & Krämer, 2003, 2005. For trehalose synthesis, C. glutamicum possesses the OtsAB and the TreYZ pathway (Tzvetkov et al, 2003;Wolf et al, 2003). Absence of intracellular trehalose has been observed when both the OtsAB and the TreYZ pathways are Fig.…”

“…Previous studies have revealed a link between glycogen and trehalose synthesis in C. glutamicum, suggesting a role of glycogen metabolism for fast adaptation to hyperosmotic growth conditions (Padilla et al, 2004;Seibold et al, 2007;Tzvetkov et al, 2003;Wolf et al, 2003). We therefore examined the effects of salt addition in the exponentialgrowth phase on growth, substrate consumption, glycogen level and intracellular trehalose formation of C. glutamicum WT, using minimal medium with 1.5 % glucose as a carbon source.…”

“…before complete consumption of the sugar substrate (Seibold et al, 2007). Therefore, it seems unlikely that glycogen in this organism serves as a long-term carbon storage compound, and in fact, glycogen metabolism in C. glutamicum has previously been suggested to be important for trehalose synthesis (Padilla et al, 2004;Seibold et al, 2007;Tzvetkov et al, 2003;Wolf et al, 2003). It has been shown that linear maltodextrins (formed as intermediates during glycogen synthesis and degradation) serve as substrates for the two-step trehalose biosynthetic pathway via maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) (Carpinelli et al, 2006;De Smet et al, 2000).…”