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Five Acetobacter isolates from lactic acid fermented meat food for pets were characterized by 177 morphological, physiological and biochemical traits. Four isolates were identified as A. pasteurianus, one as A. aceti. It is emphasized that access of such bacteria to lactic acid fermented foods should be avoided.
A new Type II restriction endonuclease ApaCI purified from Acetobacter pasteurianus is an isoschizomer of BamHI that cleaves at the nucleotide sequence 5'-G/GATCC-3' of double-stranded DNA. The single restriction activity present in this strain permits rapidly purified 30,000 units of cleavage activity from 10 g of freshly harvested cells. The resulting ApaCI preparation is free of contaminant nuclease activities that might interfere with in vitro manipulation of DNA.
New cloning vectors were prepared with the aid of a large plasmid isolated from Acetobacter pasteurianus and from plasmids pBR322 and pUC4-KAPA. Of the prepared cloning vectors, pACK5 contains a gene coding for kanamycin resistance, pACT7 and pACT71 contain a gene coding for tetracycline resistance and vector pACG3 with a gene coding for both kanamycin and tetracycline resistance. The vectors prepared only contained the beginning of replication from the pAC1 plasmid and possessed the ability to replicate within E. coli and A. pasteurianus. The vectors are highly stable in both strains and during the 5-d cultivation under nonselective conditions are not eliminated.
A bacterial strain with morphological and biochemical properties close to Acetobacter xylinum has been cultured in nonagitated, inverted sucrose- and yeast water-based medium for the production of thick, smooth, and floating cellulosic pellicles. The cellulose content (greater than 90%, dry weight, depending on the efficiency of water washing) and the beta-D-homopolyglucan nature of these pellicles were assessed by physical, chemical, and enzymatic methods. The apyrogenic bacterial biomass, a minor component of the dried biofilm (BioFill), is inactivated by ethylene dioxide. Once applied on exudating or bloody tissues, this biofilm displays several advantages as a biological dressing, and hence, it is valuable as a temporary skin substitute in the treatment of skin wounds, such as burns, ulcers, grafts, and as an adjuvant in dermal abrasions.
16S-23S rDNA internal transcribed spacer regions (ITS) similarities were determined in 8 Acetobacter and 1 Gluconacetobacter strains. ITS-PCR amplification of the 16S-23S spacers showed 2 products of similar size in 7 strains; only 1 product of similar size was found in the 2 remaining strains. Analysis of the PCR products using restriction endonucleases HaeIII, HpaII and AluI revealed 3 different restriction groups of A. pasteurianus for AluI and HaeIII, and 4 restriction groups for HpaII. ITS nucleotide sequences of all studied strains exhibited a 52-98% similarity.
The aldehyde dehydrogenase complex, which catalyzes the oxidation of acetaldehyde to acetic acid, was purified to apparent homogeneity from the membrane fraction of the industrial vinegar-producing strain Acetobacter europaeus. The determined Km for acetaldehyde was 2.1 mM. SDS-PAGE of the enzyme complex showed the presence of three different subunits with molecular masses of 79, 46, and 17 kDa, respectively. The two larger subunits contained heme. The difference spectrum indicated a cytochrome c, a heme B, and a [2Fe-2S] cluster. The nucleotide sequence of several cloned fragments of a 6-kb chromosomal DNA segment from A. europaeus was determined. It contains three consecutive open reading frames that correspond to proteins with calculated molecular masses of 84.1, 49.0, and 16.7 kDa; these were assigned to the purified proteins and named aldH, aldF, and aldG, respectively. The N-terminal sequence of the 79-kDa subunit was detected within the predicted amino acid sequence of AldH, which indicated the presence of a leader peptide. Cotranscription of the three genes was shown by Northern hybridization. Sequence analysis and experimental evidence allowed the assignment of the following cofactors to the respective subunits of the aldehyde dehydrogenase complex: heme C to AldF, [2Fe-2S] cluster to AldG, and heme B and a molybdopterin cofactor to AldH. Part of an open reading frame, gdhA, was detected upstream of the operon that showed high similarities to the C-terminal part of several pyrroloquinoline-chinone-dependent glucose dehydrogenases.
Acetobacter diazotrophicus is a nitrogen-fixing bacterium that growth inside sugar cane plant tissue where the sucrose concentration is approximately 10%. The influence of high sugar content on nitrogenase was measured in the presence of oxygen and of nitrogen added in the form of ammonium and amino acids. In all parameters analyzed, 10% sucrose protected nitrogenase against inhibition by oxygen, ammonium, some amino acids, and also to some extent by salt stress. The oxygen concentration at which inhibition occurred increased from 2 kPa in 1% glucose or gluconic acid, to 4 kPa (0.4 atm) in 10% sucrose. Nitrogenase activity was partially inhibited by increased ammonium levels (2.0, 5.0, and 10.0 mM) in the presence of 1% sucrose, but the cells maintained their nitrogenase activity at 10% sucrose. This could be explained by the slow ammonium assimilation by the cells in the presence of high sucrose concentrations, i.e., independent of its concentration between 2 and 10 mM, the assimilation of ammonium was reduced to one-third in cells grown with 10% sucrose. Some amino acids were also tested in the presence of 1 and 10% sucrose. Cells grown in 1% sucrose had their nitrogenase activity reduced by 50-98% in the presence of glutamic acid, glutamine, alanine, asparagine, or threonine, whereas with 10% sucrose, nitrogenase activity was increased by glutamic acid and was reduced by only 61-73% by the other amino acids. The effect of NaCl concentrations (0.0, 0.25, 0.5, 0.75, or 1.0%) was also studied at the two concentrations of sucrose. Nitrogenase activity and growth of A. diazotrophicus, which was visualized by the pellicle formation in semi-solid medium, showed sensitivity even to low NaCl concentrations, which was somewhat relieved at the higher sucrose level. These observations indicate different osmotolerance mechanisms for sucrose and salt.
Acetobacter pasteurianus, an obligately oxidative bacterium, is the first organism shown to utilize pyruvate decarboxylase (PDC) as a central enzyme for oxidative metabolism. In plants, yeast, and other bacteria, PDC functions solely as part of the fermentative ethanol pathway. During the growth of A. pasteurianus on lactic acid, the central intermediate pyruvate is cleaved to acetaldehyde and CO(2) by PDC. Acetaldehyde is subsequently oxidized to its final product, acetic acid. The presence of the PDC enzyme in A. pasteurianus was confirmed by zymograms stained for acetaldehyde production, enzyme assays using alcohol dehydrogenase as the coupling enzyme, and by cloning and characterization of the pdc operon. A. pasteurianus pdc was also expressed in recombinant Escherichia coli. The level of PDC activity was regulated in response to growth substrate, highest with lactic acid and absent with mannitol. The translated PDC sequence (548 amino acids) was most similar to that of Zymomonas mobilis, an obligately fermentative bacterium. A second operon ( aldA) was also found which is transcribed divergently from pdc. This operon encodes a putative aldehyde dehydrogenase (ALD2; 357 amino acids) related to class III alcohol dehydrogenases and most similar to glutathione-dependent formaldehyde dehydrogenases from alpha-Proteobacteria and Anabeana azollae.
We studied the growth characteristics and oxidative capacities of Acetobacter aceti IFO 3281 in batch and chemostat cultures. In batch culture, glycerol was the best growth substrate and growth on ethanol occurred only after 6 days delay, although ethanol was rapidly oxidized to acetic acid. In continuous culture, both glycerol and ethanol were good growth substrates with similar characteristics. Resting cells in a bioreactor oxidized ribitol to L-ribulose with a maximal specific rate of 1.2 g g(-1) h(-1)). The oxidation of ribitol was inhibited by ethanol but not by glycerol. Biomass yield (Y(SX); C-mmol/C-mmol) on ethanol and glycerol was low (0.21 and 0.17, respectively). In the presence of ribitol the yield was somewhat higher (0.25) with ethanol but lower (0.13) with glycerol, with respectively lower and higher CO(2) production. In chemostat cultures the oxidation rate of ribitol was unaffected by ethanol or glycerol. Cell-free extract oxidized ethanol very slowly but not ribitol; the oxidative activity was located in the cell membrane fraction. Enzymatic activities of some key metabolic enzymes were determined from steady-state chemostat with ethanol, glycerol, or ethanol/glycerol mixture as a growth limiting substrate. Based on the measured enzyme activities, metabolic pathways are proposed for ethanol and glycerol metabolism.
Acetobacter diazotrophicus is a diazotrophic bacterium that colonizes sugarcane tissues. Glucose is oxidized to gluconate in the periplasm prior to uptake and metabolism. A membrane-bound glucose dehydrogenase quinoenzyme [which contains pyrroloquinoline quinone (PQQ) as the prosthetic group] is involved in that oxidation. Gluconate is oxidized further via the hexose monophosphate pathway and tricarboxylic acid cycle. A. diazotrophicus PAL3 was grown in a chemostat with atmospheric nitrogen as the sole N source provided that the dissolved oxygen was maintained at 1.0-2.0% air saturation. The biomass yields of A. diazotrophicus growing with glucose or gluconate with fixed N were very low compared with other heterotrophic bacteria. The biomass yields under N-fixing conditions were more than 30% less than with ammonium as the N source using gluconate as the carbon source but, surprisingly, were only about 14% less with glucose. The following scheme for the metabolism of A. diazotrophicus through the different pathways emerged: (1) the respiratory chain of this organism had a different efficiency of ATP production in the respiratory chain (P:O ratio) under different culture conditions; and (2) N fixation was one (but not the sole) condition under which a higher P:O ratio was observed. The other condition appears to be the expression of an active PQQ-linked glucose dehydrogenase.
A Tn5-based transposon bearing the kil gene (killing protein), mediating controlled export of periplasmic proteins into the culture medium, was constructed (Tn5-KIL3). This transposon contained the kil gene of the ColEl plasmid under the growth-phase-dependent promoter of the fic gene (filamentation induced by cAMP) of Escherichia coli, an interposon located upstream of kil, a kanamycin/neomycin-resistance gene, a multiple cloning site and the mob site. The transposition of Tn5-KIL3 to Acetobacter methanolicus showed a moderate transposition frequency (10(-5) -10(-6). By insertion of a Bacillus hybrid beta-glucanase (bgl) as a model protein into the transposon (Tn5-LF3) it was shown that the secretion function as well as the gene of the target protein had been transferred to and stably integrated into the chromosome of A. methanolicus, and that the transposition of Tn5-LF3 was non-specific. beta-Glucanase was highly overexpressed and secreted into the medium during stationary phase. Total and extra-cellular production of beta-glucanase varied depending on the integration site of the transposon. The viability of the bacterial cells was not affected, and cell lysis did not occur.
Acetobacter diazotrophicus SRT4 secretes a constitutive levansucrase (LsdA) (EC 2.4.1.10) that is responsible for sucrose utilization. Immunogold electron microscopical studies revealed that LsdA accumulates in the periplasm before secretion. The periplasmic and extracellular forms of the enzyme were purified to homogeneity. Both proteins exhibited similar physical and biochemical characteristics indicating that LsdA adopts its final conformation in the periplasm. The N-terminal sequence of mature LsdA was pGlu-Gly-Asn-Phe-Ser-Arg as determined by PSD-MALDI-TOFMS (post-source decay-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry). Comparison of this sequence with the predicted precursor protein revealed the cleavage of a 30-residue typical signal peptide followed by the formation of the pyroglutamic acid (pGlu) residue. Thus, in contrast with other Gram-negative bacteria, A. diazotrophicus secretes levansucrase by a signal-peptide-dependent mechanism.
Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110 g 2-furoic acid l(-1) from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2' biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86-91% molar yields.
Bacteria belonging to the genus Acetobacter and Gluconobacter, and enzymes isolated from them, have been extensively used for biosensor construction in the last decade. Bacteria used as a biocatalyst are easy to prepare and use in amperometric biosensors. They contain multiple enzyme activities otherwise not available commercially. The range of compounds analyzable by Gluconobacter biosensors includes: mono- and poly-alcohols, multiple aldoses and ketoses, several disaccharides, triacylglycerols, and complex parameters like utilizable saccharides or biological O2 demand. Here, the recent trends in Gluconobacter biosensors and current practical applications are summarized.
L-Leucine-pyruvate transaminase obtained from Acetobacter suboxydans exhibited absorbance maxima to 280 and 332 nm. The 332 nm peak was derived from the coenzyme bound to the enzyme protein with the epsilon NH2 of a lysine residue. The transaminase showed reactivity against many L-amino acids. The relation between the reactivity and the structure of the amino donor is discussed. The Michaelis constants for L-leucine, pyruvate, L-alanine and alpha-ketoisocaproate were 6.7, 3.1, 7.1 and 0.9 mM, respectively. The equilibrium constant was 5.3. The activation energy at pH 5.0 was 8,800 cal/mol.
The acetate activating system of Acetobacter aceti has been studied. The enzyme responsible, acetyl-CoA synthetase, has been purified about 500-fold from crude cell extracts and was approximately 85% pure as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The purified enzyme showed optimal activity at pH 7.6 in both Tris-HCL and potassium phosphate buffers. In its purest form, the enzyme was stable at 4 degrees-C but denatured upon freezing. The Km values for CoA, ATP and acetate were found to be 0.104 mM, 0.36 mM and 0.25 mM respectively; propionate and acrylate were also activated by the enzyme but not butyrate, isobutyrate or valerate. GTP, UTP, CTP and ADP could not replace ATP in the reaction, and cysteine or pantetheine failed to replace CoA. The cationic requirements were studied and of the divalent cations tested, only Mn2+ could significantly replace Mg2+ in the reaction; K+ and NH4+ stimulated enzyme activity but inhibited at high concentrations; Na+ was a poor activator, but did not inhibit at higher concentrations. The effect of a number of glucose and other metabolites on enzyme activity has been tested.
The capsular polysaccharide (CPS) and the O-side-chain of the lipopolysaccharide (LPS) of Acetobacter methanolicus MB 58/4 (IMET 10945) have been shown to contain the same disaccharide repeating unit, namely, ----2)-beta-D-Galf-(1----3)-beta-D-Galp-(1----. Degradation of the CPS and the LPS with the bacteriophage Acml gave fragments built up of 1-5 repeating units; the octasaccharide preponderated. The phage-associated depolymerase proved to be a beta-D-galactofuranoside hydrolase.
Acetobacter methanolicus MB 70 was shown to be related to the type strain of this species MB 58/4 (IMET 10945) having the same galactan-->2)-beta-D-Gal f-(1-->3)-beta-D-Gal p-(1-->as the capsular polysaccharide (CPS) and the O-side-chain of the lipopolysaccharide (LPS). Additionally, a glucan built up of the disaccharide repeating unit-->6)-alpha-D-Glc p-(1-->2)-alpha-D-Glc p-(1-->was identified in strain MB 70. In the CPS, the polymers were present in the ratio approximately 1:1, whereas the glucan preponderated in the LPS. Bacteriophage Acm6 specific to A. methanolicus MB 70 hydrolysed selectively the glucan component of both CPS and LPS. Structural elucidation of the resulting oligosaccharides led to the identification of the phage-associated depolymerase as an endo-alpha-(1-->6)-D-glucopyranoside hydrolase.