RESUMO
Lanthanides (Ln) are an essential cofactor for XoxF-type methanol dehydrogenases (MDHs) in Gram-negative methylotrophs. The Ln3+ dependency of XoxF has expanded knowledge and raised new questions in methylotrophy, including the differences in characteristics of XoxF-type MDHs, their regulation, and the methylotrophic metabolism including formaldehyde oxidation. In this study, we genetically identified one set of Ln3+- and Ca2+-dependent MDHs (XoxF1 and MxaFI), that are involved in methylotrophy, and an ExaF-type Ln3+-dependent ethanol dehydrogenase, among six MDH-like genes in Methylobacterium aquaticum strain 22A. We also identified the causative mutations in MxbD, a sensor kinase necessary for mxaF expression and xoxF1 repression, for suppressive phenotypes in xoxF1 mutants defective in methanol growth even in the absence of Ln3+. Furthermore, we examined the phenotypes of a series of formaldehyde oxidation-pathway mutants (fae1, fae2, mch in the tetrahydromethanopterin (H4MPT) pathway and hgd in the glutathione-dependent formaldehyde dehydrogenase (GSH) pathway). We found that MxaF produces formaldehyde to a toxic level in the absence of the formaldehyde oxidation pathways and that either XoxF1 or ExaF can oxidize formaldehyde to alleviate formaldehyde toxicity in vivo. Furthermore, the GSH pathway has a supportive role for the net formaldehyde oxidation in addition to the H4MPT pathway that has primary importance. Studies on methylotrophy in Methylobacterium species have a long history, and this study provides further insights into genetic and physiological diversity and the differences in methylotrophy within the plant-colonizing methylotrophs.
RESUMO
Our understanding of the molecular mechanisms of bacterial pathogenesis has been improved especially by the discovery of host cell contact-dependent secretion systems such as the type-III secretion system (T3SS) found in numerous pathogens. Although the identification of pathogen effectors translocated into host cells through T3SS is essential to the understanding of pathogenesis, their general sequence uniqueness confound attempts to identify such proteins by sequence homology. Here we report the development of a functional high-throughput screening system for pathogen effectors in yeast that consists of a Gateway(TM)-compatible Tet-Off inducible expression vector and a yeast strain expressing a reporter, facilitating identification of the effectors affecting host vesicular trafficking pathways. We evaluated this system and optimized the screening condition using several known pathogen effectors. We found this system useful in functional characterization of pathogen effector and it can be adapted to functional high-throughput screening as well.
