Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability

Abstract

Acetobacter pasteurianus NCI1380, a thermophilic strain isolated from the surface culture of acetic acid fermentation, showed genetic instability to produce at high frequency spontaneous mutants which were deficient in ethanol oxidation because of the loss of alcohol dehydrogenase activity. Southern hybridization experiments with the cloned alcohol dehydrogenase-cytochrome c gene cluster as the probe showed insertion of an unknown DNA fragment into a specific position in the cytochrome c gene in most of the mutant strains. Cloning and sequencing analyses revealed that the inserted sequence was 1,665 bp in length and had a terminal inverted repeat of 15 bp. In addition, this inserted sequence was found to generate a 4-bp duplication at the inserted site upon transposition. The target site specificity was not very strict, but a TCGA sequence appeared to be preferentially used. The inserted sequence contains two long open reading frames of 461 and 222 amino acids which are overlapped and encoded by different strands. Although these open reading frames showed no homology to any protein registered in the DNA data bases, the longer open reading frame contained many basic amino acids (87 of 461), as was observed with transposases of so-called insertion sequence (IS) elements. All of these characteristics are typical of IS elements, and the sequence was named IS1380. The copy number of IS1380 in a cell of A. pasteurianus NCI1380 was estimated to be about 100. Several strains of acetic acid bacteria also contained IS1380 at high copy numbers. These results suggest that IS1380 is associated with the genetic loss of ethanol-oxidizing ability as well as the genetic instability of acetic acid bacteria in general.

Microbiological Aspects of Acetate Oxidation by Acetic Acid Bacteria, Unfavorable Phenomena in Vinegar Fermentation

“…20 23) In all experiments so far described in this paper, bacterial growth was followed by reading the turbidity of the culture medium as a reference to cell number. As has been reported by Takemura et al,24) it must be noted that the turbidity does not mean the total viable cells. Different from those used by Takemura et al,24) the acetic acid bacteria used in this study were always exposed to acidic conditions, at least, in the presence of 1 % acetic acid.…”

“…As has been reported by Takemura et al,24) it must be noted that the turbidity does not mean the total viable cells. Different from those used by Takemura et al,24) the acetic acid bacteria used in this study were always exposed to acidic conditions, at least, in the presence of 1 % acetic acid. The viable cell count experiments during the culture showed a good accordance Fermentation period (h) Fermentation period (h) A. rancens SKU 1111 was incubated i'l a medium containing 3% acetic acid as carbon and energy sources.…”

Development of Thermotolerant Acetic Acid Bacteria Useful for Vinegar Fermentation at Higher Temperatures

“…It cannot be accepted that the turbidity means the total viable cells. As has been reported by Takemura et al,22) it must be noted that the majority of the cells began to die in the stationary phase after ethanol was exhausted and acetic acid accumulated to the maximal level.…”

“…It can be readily expected that ADH and ALDH activities go down and lose their physiological roles when ethanol is consumed from the culture medium. Alternatively, the decrease in specific activities of ADH and ALDH in the prolonged culture would reflect the predominated increase of ADH deficient mutants as reported by Takemura et al 22 ) They found a novel insertion sequence, IS 1380, which causes a genetic instability that produces spontaneous ADH deficient :r3~8~O~C--------~~=O==~ To I liter of sake mash medium containing I % acetic acid, ethanol was added to 5% and vinegar fermentation was done at 30°C for the period as indicated under static conditions. Every two days, acetic acid accumulated (0), bacterial growth (.…”

Identification of Insertion Sequence from a γ-Hexachlorocyclohexane Degrading Bacterium,Sphingomonas paucimobilisUT26

“…IS1380 from the acetic acid bacterium A. pasteurianus causes a loss of ethanol oxidation activity by insertion into the cytochrome c gene. 12) IS1427, which also belongs to the IS1380 family, from the 1,2-dibromoethane-degrading bacterium Xanthobacter autotrophicus GJ10, confers monobromoacetate resistance on it by insertion to a site proximal to dhlB encoding a haloacid dehalogenase, resulting in overexpression and mobilization to a plasmid of dhlB.…”