title: Growth of Bacillus methanolicus in 2 M methanol at 50 °C: the effect of high methanol concentration on gene regulation of enzymes involved in formaldehyde detoxification by the ribulose monophosphate pathway.
abstract: Bacillus methanolicus MGA3 is a Gram-positive aerobic methylotroph growing optimally at 50-53°C. Methylotrophy in B. methanolicus is encoded on pBM19 and by two chromosomal copies of the methanol dehydrogenase (mdh), hexulose phosphate synthase (hps) and phosphohexuloisomerase (phi) genes. However, there are no published studies on the regulation of methylotrophy or the dominant mechanism of detoxification of intracellular formaldehyde in response to high methanol concentration. The µ max of B. methanolicus MGA3 was assessed on methanol, mannitol and glucose. B. methanolicus achieved a µ max at 25 mM initial methanol of 0.65 ± 0.007 h(-1), which decreased to 0.231 ± 0.004 h(-1) at 2 M initial methanol. Slow growth was also observed with initial methanol concentrations of >2 M. The µ max on mannitol and glucose are 0.532 ± 0.002 and 0.336 ± 0.003 h(-1), respectively. Spiking cultures with additional methanol (100 mM) did not disturb the growth rate of methanol-grown cells, whereas, a 50 mM methanol spike halted the growth in mannitol. Surprisingly, growth in methanol was inhibited by 1 mM formaldehyde, while mannitol-grown cells tolerated 2 mM. Moreover, mannitol-grown cells removed formaldehyde faster than methanol-grown cells. Further, we show that methanol oxidation in B. methanolicus MGA3 is mainly carried out by the pBM19-encoded mdh. Formaldehyde and formate addition down-regulate the mdh and hps genes in methanol-grown cells. Similarly, they down-regulate mdh genes in mannitol-grown cells, but up-regulate hps. Phosphofructokinase (pfk) is up-regulated in both methanol and mannitol-grown cells, which suggests that pfk may be a possible synthetic methylotrophy target to reduce formaldehyde growth toxicity at high methanol concentrations.
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA.
authors.forename: Ahmet
authors.initials: A
authors.lastname: Bozdag
authors.forename: Claire
authors.initials: C
authors.lastname: Komives
authors.forename: Michael C
authors.initials: MC
authors.lastname: Flickinger
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media; D005557:Formaldehyde / Q000378:metabolism*; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000378:metabolism; D008353:Mannitol / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D000432:Methanol / Q000378:metabolism*; D012274:Ribulosephosphates / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D012274:Ribulosephosphates; D005557:Formaldehyde; D008353:Mannitol; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D005947:Glucose; D000432:Methanol