Project description:Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylated amines as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methanol vs methylamine. Growth on methanol results in activation of mdh2 and a number of known accessory proteins. As expected, all genes for N-methylglutamate pathway were induced during growth on methylated amine. Among other functions, responding to a switch from methanol to methylated amines, are a heme-containing amine dehydrogenase (QHNDH), a PQQ-dependent methanol dehydrogenase homologue, a distant homologue of formaldehyde activating enzyme (fae3), molybdenum containing formate dehydrogenase, a set of transporters homologues to urea/ammonium transporters and amino-acid permeases. Genes encoding the PQQ-dependent methanol dehydrogenase and associated cytochrome, the enzymes from the assimilatory H4F-dependent pathway, and the tungsten-containing aldehyde oxidoreductase were down-regulated during growth on methylamine. Genes essential for carbon assimilation (serine cycle) and H4MTP-pathway for formaldehyde oxidation show similar level of expression on both C1-carbon sources. Phenotypic analysis of mutants lacking functional QHNDH had no growth defect on C1-compounds. M. universalis FAM5 strain with the methylene-tetrahydrofolate dehydrogenase lesion, a key enzyme of the H4-folate pathway, were not able to use any C1-compound, methanol or methylated amines. Methyloversatilis universalis FAM5 possesses three homologs of the formaldehyde activating enzymes. The relative expression of two of the formaldehyde activating enzyme (fae1) and fae2 did not change after the shift from methanol to methylamine growth. The relative expression of the third homologs, fae3, was significantly upregulated by methylamine. Single and double fae 2 and fae 3 mutants display similar to wild type growth M-BM- on methanol or methylamine. Strains lacking fae1 lost the ability to grow on both C1-compounds. However upon incubation on methylated amines the fae1-mutant produce revertants (fae1R ). The revertant strains displayed an impaired growth on methylamine but were not able to use methanol. Double mutations in fae1RM-BM- / fae3 or fae1R/fae2 and triple mutant fae1R/fae2/fae3 showed similar to fae1R phenotype. The metabolic pathways for utilization methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed. Methyloversatilis universalis FAM5 grown on methanol and methylamine with two biologial replicates for each condition. RNA-Seq was used for transcriptomics.
Project description:Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylated amines as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methanol vs methylamine. Growth on methanol results in activation of mdh2 and a number of known accessory proteins. As expected, all genes for N-methylglutamate pathway were induced during growth on methylated amine. Among other functions, responding to a switch from methanol to methylated amines, are a heme-containing amine dehydrogenase (QHNDH), a PQQ-dependent methanol dehydrogenase homologue, a distant homologue of formaldehyde activating enzyme (fae3), molybdenum containing formate dehydrogenase, a set of transporters homologues to urea/ammonium transporters and amino-acid permeases. Genes encoding the PQQ-dependent methanol dehydrogenase and associated cytochrome, the enzymes from the assimilatory H4F-dependent pathway, and the tungsten-containing aldehyde oxidoreductase were down-regulated during growth on methylamine. Genes essential for carbon assimilation (serine cycle) and H4MTP-pathway for formaldehyde oxidation show similar level of expression on both C1-carbon sources. Phenotypic analysis of mutants lacking functional QHNDH had no growth defect on C1-compounds. M. universalis FAM5 strain with the methylene-tetrahydrofolate dehydrogenase lesion, a key enzyme of the H4-folate pathway, were not able to use any C1-compound, methanol or methylated amines. Methyloversatilis universalis FAM5 possesses three homologs of the formaldehyde activating enzymes. The relative expression of two of the formaldehyde activating enzyme (fae1) and fae2 did not change after the shift from methanol to methylamine growth. The relative expression of the third homologs, fae3, was significantly upregulated by methylamine. Single and double fae 2 and fae 3 mutants display similar to wild type growth on methanol or methylamine. Strains lacking fae1 lost the ability to grow on both C1-compounds. However upon incubation on methylated amines the fae1-mutant produce revertants (fae1R ). The revertant strains displayed an impaired growth on methylamine but were not able to use methanol. Double mutations in fae1R / fae3 or fae1R/fae2 and triple mutant fae1R/fae2/fae3 showed similar to fae1R phenotype. The metabolic pathways for utilization methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed.
Project description:Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylamine as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methylamine vs methanol. As expected, all genes for the N-methylglutamate pathway were induced during growth on methylamine. Among other functions responding to the aminated source of C?-carbon, are a heme-containing amine dehydrogenase (Qhp), a distant homologue of formaldehyde activating enzyme (Fae3), molybdenum-containing formate dehydrogenase, ferredoxin reductase, a set of homologues to urea/ammonium transporters and amino-acid permeases. Mutants lacking one of the functional subunits of the amine dehydrogenase (?qhpA) or ?fae3 showed no growth defect on C?-compounds. M. universalis FAM5 strains with a lesion in the H?-folate pathway were not able to use any C?-compound, methanol or methylamine. Genes essential for C?-assimilation (the serine cycle and glyoxylate shunt) and H?MTP-pathway for formaldehyde oxidation showed similar levels of expression on both C?-carbon sources. M. universalis FAM5 possesses three homologs of the formaldehyde activating enzyme, a key enzyme of the H?MTP-pathway. Strains lacking the canonical Fae (fae1) lost the ability to grow on both C?-compounds. However, upon incubation on methylamine the fae1-mutant produced revertants (?fae1(R)), which regained the ability to grow on methylamine. Double and triple mutants (?fae1(R)?fae3, or ?fae1(R)?fae2 or ?fae1(R)?fae2?fae3) constructed in the revertant strain background showed growth similar to the ?fae1(R) phenotype. The metabolic pathways for utilization of methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed based on these gene expression and phenotypic data.