Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus

Abstract

The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH 2 -terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.Acetic acid fermentation, in which ethanol is oxidized to acetic acid by acetic acid bacteria, is the most characteristic process in vinegar production (10). This ethanol oxidation is catalyzed by two membrane-bound enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (1-3, 6, 11, 27). The ADHs of Acetobacter spp., a genus of acetic acid bacteria (10), are classified into two types, two component and three component. The former in Acetobacter polyoxogenes consists of a 72-to 78-kDa dehydrogenase subunit (subunit I) and a 44-to 48-kDa cytochrome c subunit (subunit II), and the latter type in Acetobacter aceti and Acetobacter pasteurianus has one more subunit, a 20-kDa subunit of unknown function (subunit III). We recently purified both types of ADHs and cloned the genes encoding the larger two subunits of both types of ADHs. These studies revealed that both subunit I and subunit II were essential for ADH activity (25-27). The genes encoding subunits I and II were clustered (25, 26), but the gene encoding subunit III was not present close to the cluster (25). It had thus been postulated that only the two larger components were required for ADH activity.In subsequent investigations, we found that some spontaneous mutants of A. pasteurianus NCI1452 which lost subunit III showed an ADH activity-deficient phenotype. We therefore began to study how subunit III of the three-component ADH participates in the enzymatic activity and to clarify its function. In the present study, we cloned the gene encoding subunit III from A. pasteurianus NCI1452 and analyzed various ethanol oxidation-defective mutants derived from the parental strain genetically and immunologically. This paper provides evidence that this subunit is indispensable for ADH activity. The function of subunit III in the three-component-type ADH is also discussed. MATERIALS AND METHODSBacteria...

Molecular Properties of Membrane-Bound FAD-ContainingD-Sorbitol Dehydrogenase from ThermotolerantGluconobacter frateuriiIsolated from Thailand

“…21) SldS showed similarity to the small subunits of GADH and 2KGDH, of which the function is also unknown. Kondo et al have suggested that the 20 kDa small subunit of ADH from Acetobacter pasteurianus has a role in the stability of the dehydrogenase subunit and functions as a molecular coupler to the cytochrome c subunit on the cytoplasmic membrane, 30) but the small subunit has no similarity to SldS in amino acid sequence. Although the small subunit of ALDH from acetic acid bacteria contains iron-sulfur clusters, 31) the amino acid sequence of SldS does not have such a binding motif.…”

“…Competent cells of Gluconobacter strains were obtained by the following method: A preculture (1 ml) was transferred into 100 ml of 1% D-sorbitol medium in a 500 ml Erlenmeyer flask with shaking at 30 C for 12 h. Cells were chilled on ice for 30 min before harvesting by centrifugation at 4,000 rpm for 10 min at 4 C, and washed twice with ice-cold 10% (w/v) glycerol. The final cell pellet was resuspended with 0.5-1 ml of ice-cold 10% (w/v) glycerol.…”

Acetic Acid Bacteria: Physiology and Carbon Sources Oxidation

“…The main source of genetic instability has been attributed to mobile genetic elements, mainly transposons, widely distributed in the genome. Insertion sequences (IS) responsible for EtOH oxidation deficiency, like those of the family IS 12528, were found in the chromosome of A. pasteurianus NCIB 7214 (5 copies) [26], A. aceti 1023 (1 copy) and G. oxydans IFO 12528 (10 copies). Likewise 100 copies of IS 1380 occur in A. aceti 1023 and 74 copies in A. pasteurianus NBRC 3283 [1,27].…”

“…The subunit II which molecular size range from 44 to 45 kDa contains three heme c moieties and it is encoded in the adhB gene. The third and smallest subunit (20 kDa), encoded in the adhS, helps the two functional subunits with their association to the membrane protecting the catalytic subunit from proteolysis and it contributes to the correct conformation of the ADH complex for electron transport on the periplasmic surface [26,45]. Oxidation of EtOH occurs at PQQ site that acts as two-electron redox mediator; electrons are initially transferred to UQ, which will be re-oxidized by a membrane-associated oxidase.…”

Novel Enzymatic Method for the Production of Xylitol fromD-Arabitol byGluconobacter oxydans

“…and 5?-GCCTCTAG-AGCAGCCTCAGGGGTGATCCGC-3?) designed from the reported sequence, 20) and cloned into the Pst I-XbaI site in pHSG298 (Takara Shuzo, Japan). Then, the ampR gene of pUC18 (Takara Shuzo, Japan) was ampliˆed with the primers 5?-TTC-AGATCTCATTCAAATATGTATCCGCTC-3?…”