Localisation and distribution of alcohol-cytochrome 553 reductase in acetic acid bacteria

Alcohol-cytochrome 553 reductase was detected in several strains of the mesoxydans and oxydans group ofAcetobacter. A similar enzyme, but with a higher optimum pH, was detected inAcetobacter aceti (liquefaciens) and in two strains ofGluconobacter.Intermittent ultrasonic disruption ofAcetobacter aceti cells, strainsrancens T-5 andmobilis 6428, showed that the alcohol-cytochrome 553 reductase was mainly localized on the cell hull. The NADP-linked aldehyde dehydrogenase appeared to be present as a cytoplasmic component.The oxidation rate of ethanol and acetaldehyde by intact resting cells which have been grown with either glucose or ethanol as a carbon source under either neutral or acidic conditions was nearly identical. The ethanol oxidizing enzyme system thus behaved as constitutive enzymes, as would be expected if they were bound to the cell hull.The results support the hypothesis that the alcohol-cytochrome 553 reductase is one of the important components of the enzyme system responsible for the physiological production of acetic acid from ethanol by acetic acid bacteria.     

Numerical analysis of Frateur's phenotypic data on acetic acid bacteria

The phenotypic data published by J. Frateur on 35 strains of acetic acid bacteria were analyzed numerically. The four clusters obtained correspond very well to Frateur's suboxydans, mesoxydans, oxydans and peroxydans groups, on which the classification of the acetic acid bacteria in the 8th and 9th edition of Bergey's manual is based.     

Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.     

Flagellation and taxonomy ofAcetobacter andAcetomonas

Summary Leifson's findings, that motile, acetate-oxidizing acetic acid bacteria (Acetobacter) have peritrichous flagella, and that motile, non-acetate oxidizing ones (Acetomonas) have polar flagella, of notably short wavelength, are fully confirmed and photographically illustrated. It is not confirmed, however, that the peritrichous flagella ofAcetobacter are always of “orthodox” type with a wavelength of about 2.9 μ, nor that they always tend to be few in number. In one strain ofA. aceti they were numerous, and the wavelength was as short (1.4 μ) as that considered byLeifson to be uniquely confined to the polar flagella ofAcetomonas. Furthermore the polar flagella of the latter genus seem not always to be multitrichous, strains having been found with only a single polar flagellum.     

Are species of bacteria unclassifiable?

Summary Every one of eleven different strains randomly selected from 10 different randomly selected genera have shown the same high frequency of occurrence of colony mutants as did almost all strains ofAcetobacter (previously considered outstanding in this respect). Correlation of other properties with such mutant colony forms was not specifically studied, but in 4 strains correlation was noticed, suggesting its presence in the others, as was so often found inAcetobacter. It is suggested from this, that a similar study of strains of other genera might reveal a similarly high frequency of occurrence of mutants, most so-called pure cultures being thus probably mixtures of different cells with different properties. Also the proportion of each cell-type in the culture may vary from predominance to extinction according to the biochemical and other tests applied for the purpose of the ‘characterization’ of the species for taxonomic purposes. If the classification of such varying mixtures is considered of doubtful use, then it seems to follow that ‘species’ of bacteria are virtually unclassifiable, and that even the conception of a genus should be on a broader basis than is often the case at present.     

Conjugation and heterologous gene expression in Gluconobacter oxydans ssp. suboxydans

Abstract: Conjugal transfer of a series of incompatibility group P and Q plasmids has been studied in the acetic acid bacterium, Gluconobacter oxydans ssp. suboxydans . Transfer frequencies for the IncP/Q vectors ranged from 10⁻⁵−10⁻⁹ exconjugants per recipient cell. It was found in the case of the IncP vector, pRK290, that Bgl II insert constructs displayed increased conjugal transfer frequencies over pRK290 per se, the parent plasmid. A gentamycin-resistant encoding pRK290 vector which was constructed offers considerable potential as a versatile gene delivery system for Gluconobacter . The lactose transposon, Tn951, was used as a model to examine heterologous gene expression in G. oxydans ssp. suboxydans . The expression level of Tn951 encoded β-galactosidase in this strain was found to be less than 5% of that found in the parent Escherichia coli strain, JC3272.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.     

The nitrogen requirements ofGluconobacter,Acetobacter andFrateuria

The nitrogen requirements of 96Gluconobacter, 55Acetobacter and 7Frateuria strains were examined. Only someFrateuria strains were able to grow on 0.5% yeast extract broth or 0.5% peptone broth. In the presence ofd-glucose ord-mannitol as a carbon source, ammonium was used as the sole source of nitrogen by all three genera. With ethanol, only a fewAcetobacter strains grew on ammonium as a sole nitrogen source. Singlel-amino acids cannot serve as a sole source of carbon and nitrogen for growth ofGluconobacter, Acetobacter orFrateuria. The singlel-amino acids which were used by most strains as a sole nitrogen source for growth are: asparagine, aspartic acid, glutamine, glutamic acid, proline and alanine. SomeAcetobacter andGluconobacter strains deaminated alanine, asparagine, glutamic acid, threonine, serine and proline. NoFrateuria strain was able to develop on cysteine, glycine, threonine or tryptophan as a sole source of nitrogen for growth. An inhibitory effect of valine may explain the absence of growth on this amino acid. No amino acid is “essential” forGluconobacter, Acetobacter orFrateuria.     

Molecular identification of Acetobacter isolates from submerged vinegar production, sequence analysis of plasmid pJK2-1 and application in the development of a cloning vector

Three new Acetobacter strains were isolated from vinegar. By plasmid profiling they were recognized as genotypically different from each other. Sequencing of the genes for 16S and 23S rRNA and DNA–DNA hybridization of total DNA against DNA of all type strains of Acetobacter identified Acetobacter strains JK2 and V3 as A. europaeus, and Acetobacter strain JK3 as A. intermedius. In contrast to the type strain of A. europaeus (DSM 6160), A. europaeus JK2 and V3 do not require acetic acid for growth and can be successfully transferred between media with and without acetic acid. This phenotypic characteristic enables convenient handling of both strains in genetic studies. Plasmid pJK2-1 from A. europaeus JK2 was used as the basis for shuttle plasmid construction with the aim of developing an efficient vector system for these strains. The entire nucleotide sequence of pJK2-1 was determined. High amino acid identities were found for three open reading frames: Rep (replication protein); Dinj1 (DNA damage inducible enzyme); and Dinj2 proteins. A recombinant plasmid pUCJK2-1 (5.6 kb) consisting of the entire plasmid pJK2-1 and the entire plasmid pUC18 was successfully used in transformation experiments. Plasmid pJT2 (5.8 kb) was constructed from pUCJK2-1 with the aim of reactivating the lacZ′ gene. Received: 14 June 1999 / Revision received: 27 August 1999 / Accepted: 17 September 1999     

The Phylogeny of Acetic Acid Bacteria Based on the Partial Sequences of 16S Ribosomal RNA: The Elevation of the Subgenus Gluconoacetobacter to the Generic Level

Thirty-six strains of acetic acid bacteria classified in the genera Acetobacter, Gluconobacter, and Acidomonas were examined for their partial base sequences in positions 1220 through 1375, 156 bases, of 16S rRNA. The strains of the Q₁₀-equipped Gluconobacter species examined were divided into two subgroups, which included the type strains of Gluconobacter oxydans, the type species of the genus Gluconobacter, and of a second species, Gluconobacter cerinus, respectively. The base differences numbered four between the two type strains. The strains of the Q₉-equipped species examined classified in the type subgenus Acetobacter of the genus Acetobacter were not very distant phylogenetically from those of the genus Gluconobacter. The calculated number of base differences was 9–6 between the type strains of G. oxydans and G. cerinus and the type strains of Acetobacter aceti and Acetobacter pasteurianus. In contrast, the strains of the Q₁₀-equipped species examined classified in the subgenus Gluconoacetobacter of the genus Acetobacter were very distant phylogenetically from those of the Acetobacter and Gluconobacter species mentioned above. The number of base differences was calculated to be 14-8. Furthermore, the strains of the methanol-assimilating, Q₁₀-equipped species of the genus Acidomonas examined were located in phylogenetically isolated positions. The type strain of Acidomonas methanolica (≡ Acetobacter methanolicus), the type species of the genus Acidomonas, had 16–9 base differences. The data obtained here indicated that the members of the subgenus Gluconoacetobacter of the genus Acetobacter can be distinguished at the generic level. The new genus Gluconoacetobacter was proposed with the type species, Gluconoacetobacter liquefaciens, in recognition of the genus Acidomonas along with the genera Acetobacter and Gluconobacter in the classification of the acetic acid bacteria.     

Studies on the Formation of Glucosamine Acid by Acetobacter melanogenum Beijerinck and Pseudomonas fluorescens liquefaciens

d-Glucosaminic acid has recently been found to be an oxidized product of d-glucosamine formed by Ps. fluorescens. It has been revealed that many strains of oxidative bacteria can oxidize glucosamine. The formation of glucosamine acid has been recognized among a large number of strains of Pseudomonas, Acetobacter and Gluconobacter, by means of paper chromatography. Furthermore, one of these strains, A. melanogenum Beijerinck, oxidized glucosamine to glucosaminic acid with the theoretical consumption of oxygen as Ps. fluorescens liquefaciens. Glucosaminic acid was proved by isolation and identification by means of using resting cells.

The experiment of growth shows that Ps. fluorescens liq. could not secure any energy by means of the oxidation of glucosamine.     

Derivation of non-acetifying „quasiacetobacters” from a trueAcetobacter strain,andvice versa

Summary A strain ofAcetobacter rancens gave rise to “quasi-acetobacters” which had lost the essential generic character of oxidation of ethanol to acetic acid, whilst retaining all the other characteristics of the genus. Other quasi-acetobacters with other acetobacter properties were also obtained. Conversely a starch-producing quasi-acetobacter gave rise to two trueAcetobacter strains indistinguishable biochemically fromA. pasteurianus andA. rancens respectively. These phenomena were associated with the presence, in the parent strains, of many swollen filaments and large bodies. It is tentatively suggested that the changes from true to quasi-acetobacter, andvice versa, may be correlated with the production of such bodies, and may indicate some heterodox form of reproduction other than simple fission. The advent of “quasi-acetobacters” seems largely to demolish the genusAcetobacter, whilst failing to indicate an alternative under the existing rigid botanical taxonomic conventions.     

Coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.     

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.     

Studies on the metabolism ofAcetobacter peroxydans

Summary Some physiological and biochemical properties of several strains ofAcetobacter peroxydans have been studied. Their morphology, the aspects of growth on beer-gelatine slants and the colony type are described. The cells are catalase negative and acid resistant. The temperature optimum is 20–25°C. They grow readily on ethanol and on lactate, or on yeast extract alone, but not on carbohydrates and derivatives. They do not consume or oxidize glucose or gluconate. They are overoxidizers. None of these strains was able to grow as hydrogen bacteria. Resting cells oxidize lactate, pyruvate, ethanol, acetate, some Krebs cycle intermediates and several alcohols. Cell-free extracts oxidize glucose-6-phosphate, 6-phosphogluconate and ribose-5-phosphate in suitable conditions. These results favour the taxonomic position of this species as a member of the genusAcetobacter. The results are discussed.     

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden–Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient␣anaerobic fermentation of this pentose. l-Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under ‘academic’ conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.     

Biochemistry and physiology of nitrogen fixation with particular emphasis on nitrogen-fixing phototrophs

Summary This paper presents an overview of aspects of N₂-fixation in phototrophic N₂-fixers. Nitrogenase is little different in phototrophs from other organisms. Evidence suggests that fixed carbon dissimilation rather than direct photoreduction from oxidised inorganic compounds or exogenous photosynthetic electron donors is the major route of reductant supply to nitrogenase in phototrophs; inRhodospirillum rubrum pyruvate is a possible electron donor to nitrogenase; in cyanobacteria the oxidative pentose phosphate pathway is important, although some recent evidence implicates glycolysis and the tricarboxylic acid cycle in reductant supply in heterocystous cyanobacteria. In photosynthetic organisms light modulation of various enzymes occurs-some Calvin cycle enzymes are light activated, some oxidative pentose phosphate pathway and glycolytic enzymes are deactivated and some tricarboxylic acid cycle enzymes are activated. Reduced levels of thioredoxin in heterocysts may contribute to the sustained functioning of the oxidative pentose phosphate pathway in heterocysts in the light and dark. In photosynthetic bacteria such asRhodospirillum rubrum an activating enzyme which removes a modifying group from inactive Fe protein can activate nitrogenase. O₂ and NH ₄ ⁺ both inhibit N₂-fixation and there is some evidence in cyanobacteria that O₂ stability of whole cell nitrogenase can be achieved by prolonged incubation of cultures at high O₂.     

Incapability of Gluconobacter oxydans to produce tartaric acid

The dependence of tartaric acid production by Gluconobacter oxydans ssp. oxydans ATCC 19357 and G. oxydans ssp. suboxydans ATCC 621 on vanadate was investigated. It was found with both organisms that trataric acid could only be produced in a medium containing vanadate (NH(4)VO(3)). A proposed intermediate of the tartaric acid metabolism in G. oxydans, 5-ketogluconic acid, was tested on its reactivity in the presence of the oxidizing catalyst vanadate. It could be shown that 5-ketogluconic acid and the catalyst vanadate, but not the activity of G. oxydans, were responsible for the formation of tartaric acid. G. oxydans was not able to produce tartaric acid by itself. The stereochemical identity of the formed tartaric acid could be identified as the L-(+)-type. Oxalic acid was formed from 5-ketogluconic acid with vanadate in the absence and in the presence of G. oxydans. The ratio of oxalic acid to tartaric acid was 1:1.     

Carbohydrate Metabolism by the Acetic Acid Bacteria

A general review of the acetic acid bacteria belonging to the intermediate type was accomplished physiologically, biochemically and morphologically. Conclusively, it was clarified that these were clearly a specific group and different from both Acetobacter and Gluconobacter, These were intermediate between lactaphilic and glycophilic, besides, on the carbohydrate oxidizability, these were intermediate between Acetobacter and Gluconobacter as mentioned previously.¹⁾ These showed the same result as Acetobacter on the vitamin requirement for the growth, but were closely related to Gluconobacter on the carbohydrate availability. And on the oxidative activity for amino acid, accompanying the deamination, these were also clearly distinguished from both Acetobacter and Gluconobacter, particularly these oxidized strongly l-serine. Differing from the observations by other investigators, these showed single flagellation, with the exception of multi-polar, but never multi-peritrichous.     

Nucleotide sequence analysis of small cryptic plasmid pGP2 from <Emphasis Type="Italic">Acetobacter estunensis</Emphasis>

Complete nucleotide sequence of plasmid pGP2 from Acetobacter estunensis GP2 was identified after initial cloning of EcoRI fragment followed by preparation of deletion derivatives. Its size was defined to 2,797 bp and several sites for several restriction enzymes were revealed by DNA sequencing. Sequence analysis predicts three putative open reading frames (ORFs). ORF1 shows significant identity with the bacterial excinuclease α-subunit, ORF2 is a putative replication protein with low similarity with other Acetobacter plasmid’s replication proteins, and ORF3 encodes a class B acid phosphatase/phosphotransferase. The replication module comprises a DnaA box like sequence, direct repeats, a potential prokaryotic promoter and a rep gene. The rep module is similar with several θ-replicating, iteron-containing modules from plasmids, suggesting pGP2 replication may follow the same course. Any phenotypic character determinant gene is absent in pGP2, suggesting this plasmid to be cryptic. However, a pGP2 derivative plasmid, containing the putative pGP2 rep region, can replicate and is stably maintained in Acetobacter and Escherichia coli strains; it can also carry foreign DNA fragments. Thus, pGP2-X could serve as a cloning shuttle vector between these bacteria. Prepared deletion derivatives of plasmid pGP2 suggested that Rep protein is essential for plasmid replication in host bacteria. In its natural host, A. estunensis GP2, pGP2 maintains a four-times lower copy number than in E. coli.