Glucose dehydrogenase

AldF

AldG

AldH

Figure 11-3. Organization of genes coding for ethanol oxidation in Acetobacter. The alcohol dehydrogenase complex (A) consists of three subunits. The dehydrogenase subunit and the cytochrome c subunit are encoded by two co-transcribed genes. The third gene, adhS, is not located near the adh gene cluster. The function of its gene product, AdhS, is not known, although it is required for enzyme activity (except for those strains that apparently contain only subunits I and II). The aldehyde dehydrogenase gene cluster (B) also contains three genes coding for three subunits of aldehyde dehydrogenase. Based on sequence analysis, the gene products all contain domains that bind co-factors involved in electron transport. AldF and AldH are heme-binding proteins, and AldG binds an iron-sulfur cluster. A gene encoding glucose dehydrogenase is upstream of the aldFGH promoter (arrow). The predicted molecular mass (and amino acid residues) are shown for each protein. From Takemura, et al., 1993; Kondo, et al., 1995; and Thurner, et al., 1997.

As noted earlier, the acetic acid pathway is usually considered an incomplete oxidation, since the substrate, ethanol, is only partially oxidized and the acetic acid that is formed is not oxidized further. However, while this is true for some acetic acid bacteria, such as Gluconobac-ter (a member of the so-called suboxydans group), most species of Acetobacter can oxidize acetic acid, provided conditions are suit-able.The latter include bacteria of the oxydans group, represented by the common vinegar-producing species A. aceti and A. pasteurianus. The absence of ethanol in the medium is the main condition necessary for acetic acid oxidation. Ethanol apparently represses synthesis of citric acid cycle enzymes; when ethanol is absent, those enzymes are induced and complete oxidation of acetic acid to CO2 and H2O can occur. Of course, in actual vinegar production, this so-called over-oxidation of acetic acid is particularly undesirable, because it causes the literal disappearance of the end product to the atmosphere.Thus, this is likely one reason why vinegar fermentations have historically been conducted in a semi-continuous mode (see below), in which a minimum amount of ethanol is always present.

Although vinegar-producing cultures are frequently used repeatedly without loss of viability or performance, genetic instability is not uncommon. Spontaneous mutations, at relatively high frequencies, have been reported for several Acetobacter sp., resulting in defects in several important functions. For example, mutants having lost the ability to oxidize ethanol, tolerate acetic acid, and form surface film (see

Figure 11-4. Membrane biology, oxidation of ethanol, and acetate assimilation by Acetobacter aceti. The oxidation reactions (upper panel), catalyzed by the PQQ-dependent enzymes alcohol dehydrogenase (AlDH) and acetaldehyde dehydrogenase (AcDH) occur within the periplasm. Ethanol can also be oxidized by cytoplasmic NAD-dependent dehydrogenases, but at very low rates. Acetic acid assimilation to acetyl Co-A and subsequent oxidation to CO2 via the citric acid cycle occurs only when ethanol is absent. The oxidation reactions are accompanied by transfer of electrons from PQQ to cytochromes of the respiratory chain (lower panel), leading to formation of a proton motive force and synthesis of ATP via oxidative phosphorylation. Oxygen serves as the terminal electron acceptor. Adapted from Matsushita, K., H. Toyama, and O. Adachi. 1994. Respiratory chains and bioenergetics of acetic acid bacteria, p. 247-301. In A.H. Rose and D.W. Tempest (ed.), Advances in Microbial Physiology, vol. 36. Academic Press, Ltd., London.

ethanol acetic acid

Outer membrane

Periplasm

Cytoplasmic membrane

Cytoplasm

Outer membrane ethanol acetaldehyde acetic acid

Cytoplasmic membrane ethanol^^ acetaldehydey^ acetic acid

NAD NADH2

NAD NADH2 acetyl CoA

ethanol acetic acid

2PQQ-H2^ xO2

below) have been obtained. Most industrial strains of Acetobacter contain plasmids,whose loss could conceivably account for phenotypic changes. However, except for the presence of antibiotic resistance genes, functions for other plasmid-borne genes have not been identified, and most plasmids are considered to be cryptic and not essential for acetic acid formation.

In contrast, the presence of insertion sequences (IS) in Acetobacter has been associated with several loss of function mutations. Insertion sequences are small DNA sequences, usually less than 3 kb, that contain inverted repeats and other characteristic features.They can transpose or integrate, randomly or at specific sites within the genome, resulting in the disruption of gene encoding regions. For example, in A. pasteurianus, A. aceti, G. xylinus, and other Acetobacter sp., the high-copy number IS element, IS1380, was found to integrate within the gene coding for cytochrome c, which, along with alcohol dehydrogenase, is essential for ethanol oxidation. In addition, transposition of another insertion sequence, IS1031, in A. xylinum, led to the appearance of mutants that were unable to synthesize the cellulose-containing surface film necessary during surface type fermentations (see below).Thus, it appears that the genetic instability associated with acetic acid bacteria is likely due to inactivation of genes necessary for growth and acetic acid formation by transpos-able IS elements.

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