Abstract. The enzymology of methanol utilization in thermotolerant methytotrophic Bacillus strains was investigated. In all strains an immunological]y related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent Km values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.

A study has been made in methanol-and ethanol-grown Hyphomicrobium x of the specific activities of the following enzymes : serine hydroxymethyltransferase, serine-glyoxylate aminotransferase, hydroxypyruvate reductase, glycerate kinase, phosphopyruvate hydratase, phosphopyruvate carboxylase, serine dehydratase, malate dehydrogenase (decarboxylating), phosphopyruvate synthase, phosphopyruvate carboxykinase, glycerate phosphomutase, malate dehydrogenase, malate lyase (CoA acetylating-ATP cleaving), citrate synthase, aconitate hydratase, isocitrate lyase and malate synthase. It was concluded that during growth on ethanol the glyoxylate cycle operated, while during growth on methanol, C, units and glyoxylate were converted to malate by the serine pathway. The glyoxylate was regenerated by cleavage of malate into glyoxylate and acetyl-CoA. The acetylCoA was itself oxidized to glyoxylate by some of the reactions of the tricarboxylic acid cycle and isocitrate lyase, thus permitting net synthesis of C3 and C4 compounds from methanol and carbon dioxide. I N T R O D U C T I O NWhen an aerobic bacterium grows on reduced one-carbon compounds such as methanol either of two pathways, the serine pathway or the pentose phosphate cycle of formaldehyde fixation, is likely to be involved in the biosynthesis of cell constituents (Quayle, 1972). The serine pathway has been most extensively investigated in Pseudomonas AMI and is thought to provide the route for synthesis of C, and C4 intermediates during growth on C1 compounds by the following steps : Phosphoenolpyruvate + CO, -+ oxaloacetate + Pi.Hydroxypyruvate + NADH + Hf $ glycerate + NADf, (4)

The metabolic importance of pyruvate oxidase (PoxB), which converts pyruvate directly to acetate and CO 2 , was assessed using an isogenic set of genetically engineered strains of Escherichia coli. In a strain lacking the pyruvate dehydrogenase complex (PDHC), PoxB supported acetate-independent aerobic growth when the poxB gene was expressed constitutively or from the IPTGinducible tac promoter. Using aerobic glucose-limited chemostat cultures of PDH-null strains, it was found that steady-states could be maintained at a low dilution rate (005 h N1 ) when PoxB is expressed from its natural promoter, but not at higher dilution rates (up to at least 025 h N1 ) unless expressed constitutively or from the tac promoter. The poor complementation of PDHdeficient strains by poxB plasmids was attributed to several factors including the stationary-phase-dependent regulation of the natural poxB promoter and deleterious effects of the multicopy plasmids. As a consequence of replacing the PDH complex by PoxB, the growth rate (µ max ), growth yield (Y max ) and the carbon conversion efficiency (flux to biomass) were lowered by 33 %, 9-25 % and 29-39 % (respectively), indicating that more carbon has to be oxidized to CO 2 for energy generation. Extra energy is needed to convert PoxB-derived acetate to acetyl-CoA for further metabolism and enzyme analysis indicated that acetyl-CoA synthetase is induced for this purpose. In similar experiments with a PoxB-null strain it was shown that PoxB normally makes a significant contribution to the aerobic growth efficiency of E. coli. In glucose minimal medium, the respective growth rates (µ max ), growth yields (Y max ) and carbon conversion efficiencies were 16 %, 14 % and 24 % lower than the parental values, and correspondingly more carbon was fluxed to CO 2 for energy generation. It was concluded that PoxB is used preferentially at low growth rates and that E. coli benefits from being able to convert pyruvate to acetylCoA by a seemingly wasteful route via acetate.

The hyf locus (hyfABCDEFGHIJ-hyfR-focB) of Escherichia coli encodes a putative 10-subunit hydrogenase complex (hydrogenase-4 [Hyf]); a potential 54 -dependent transcriptional activator, HyfR (related to FhlA); and a putative formate transporter, FocB (related to FocA). In order to gain insight into the physiological role of the Hyf system, we investigated hyf expression by using a hyfA-lacZ transcriptional fusion. This work revealed that hyf is induced under fermentative conditions by formate at a low pH and in an FhlA-dependent fashion. Expression was 54 dependent and was inhibited by HycA, the negative transcriptional regulator of the formate regulon. Thus, hyf expression resembles that of the hyc operon. Primer extension analysis identified a transcriptional start site 30 bp upstream of the hyfA structural gene, with appropriately located ؊24 and ؊12 boxes indicative of a 54 -dependent promoter. No reverse transcriptase PCR product could be detected for hyfJ-hyfR, suggesting that hyfR-focB may be independently transcribed from the rest of the hyf operon. Expression of hyf was strongly induced (ϳ1,000-fold) in the presence of a multicopy plasmid expressing hyfR from a heterologous promoter. This induction was dependent on low pH, anaerobiosis, and postexponential growth and was weakly enhanced by formate. The hyfR-expressing plasmid increased fdhF-lacZ transcription just twofold but did not influence the expression of hycB-lacZ. Interestingly, inactivation of the chromosomal hyfR gene had no effect on hyfA-lacZ expression. Purified HyfR was found to specifically interact with the hyf promoter/operator region. Inactivation of the hyf operon had no discernible effect on growth under the range of conditions tested. No Hyf-derived hydrogenase or formate dehydrogenase activity could be detected, and no Ni-containing protein corresponding to HyfG was observed.Escherichia coli is capable of three alternative modes of energy generation: aerobic respiration, anaerobic respiration and fermentation. In the absence of the appropriate electron acceptors (O 2 , NO 3 Ϫ , NO 2 Ϫ , sulfur or nitrogen oxides, or fumarate), E. coli resorts to mixed-acid fermentation, yielding a maximum of just 3 mol of ATP per mol of glucose consumed (12). During fermentative growth, glycolytic carbon sources are converted to pyruvate that is in turn converted to acetyl coenzyme A and formate by pyruvate formatelyase (22) The formate produced may then be either excreted or further metabolized to H 2 and CO 2 by a membrane-associated, nonenergy-conserving formate hydrogenlyase (Fhl-1) system consisting of formate dehydrogenase H (Fdh-H) and the hydrogenase-3 complex (Hyc) (12,17,37).The genes required for the synthesis of Fhl-1 form the formate regulon, which includes three transcriptional units, namely, the hyc and hyp-fhlA operons and the fdhF gene (13,26,28,37,49). The hyc operon (hycABCDEFGHI) encodes the Hyc complex of Fhl-1 (encoded by hycBCDEFGH), as well as a negative-transcriptional regulator (HycA) of the formate regulon and a protease ...

Acetobacter diazotrophicus, a recently isolated nitrogen-fixing acidotolerant acetic acid bacterium, grew well in simple mineral media and exhibited high rates of gluconic acid formation. Glucose oxidation by the organism was less sensitive to low pH values than glucose oxidation by Gluconobacter oxydans. Growth and glucose oxidation were not affected by high gluconic acid concentrations. These observations indicate that A.diazotrophicus is an interesting organism for the industrial production of gluconic acid. The organism exhibited a high maintenance requirement (ms = 1.0 mmol glucose h -1 (g dry weight) -1) during glucose-limited growth in chemostat cultures at pH 3.5. Enzyme activities in cell-free extracts suggested that glucose metabolism in A. diazotrophicus proceeds exclusively via the hexose monophosphate pathway: the enzymes of the Embden-Meyerhof and Entner-Doudoroff pathways could not be detected. Both the phosphorylative and direct oxidative pathways of glucose metabolism appeared to be operative. In addition to a pyridine nucleotide (strictly NAD)-dependent glucose dehydrogenase, A. diazotrophicus contained a dye-linked, probably pyrrolo-quinoline quinone (PQQ)-dependent, glucose dehydrogenase. The latter activity seemed to be primarily responsible for gluconic acid formation.

The YfiD protein of Escherichia coli has been reported to be an acid-inducible protein.Here it is shown that expression of a yfiD ::lac reporter fusion is enhanced up to 35-fold during acidic growth. The anaerobic transcription factor FNR was confirmed as the major regulator of yfiD expression, and ArcA was found to enhance anaerobic yfiD expression, probably by displacing a repressing FNR dimer in the N935 region of the promoter. Moreover, the pyruvate sensor PdhR was shown to act as a minor anaerobic repressor of yfiD expression. On the basis of its strong homology to the C-terminal region of pyruvate formate-lyase (PFL) it was predicted that YfiD would be a radicalcontaining enzyme. The YfiD radical was found to be introduced by the PFLactivase enzyme, but unlike PFL, AdhE did not deactivate radicalized YfiD. The extent of radical activation of YfiD was enhanced by low intracellular pH, and thus it was concluded that both yfiD expression and YfiD activity are affected by growth at low pH. The yfiD mutant strain JRG4033 excreted increased levels of organic acids compared to the parental strain when grown in chemostat culture under oxygen-starved conditions, consistent with the acid-inducibility of yfiD expression and the recently reported ability of YfiD to rescue the activity of oxygenolytically cleaved PFL.

ATTWOOD, M. M. and HARDER, W. 1972. A rapid and specific enrichment procedure for Hyphomicrobium spp. Antonie van Leeuwenhoek 38: 369-378.An enrichment procedure for the isolation of stalked bacteria of the genus Hyphomicrobium is described. The method is based on the use of an organic C1 compound as carbon and energy source for growth together with anaerobic incubation in the presence of nitrate as an electron acceptor. Optimal conditions for the growth of a numbel of Hyphomicrobium isolates have been investigated. Applying these conditions, Hyphomicrobium spp. have been enriched from a wide range of natural habitats within 1-2 weeks.

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