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Applied and Environmental Microbiology, January 2006, p. 497-505, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.497-505.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Putative ABC Transporter Responsible for Acetic Acid Resistance in Acetobacter aceti

Shigeru Nakano,^1* Masahiro Fukaya,¹ and Sueharu Horinouchi²

Central Research Institute, Mizkan Group Co., Ltd., Handa-shi, Aichi 475-8585,¹ Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan²

Received 22 July 2005/ Accepted 18 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two-dimensional gel electrophoretic analysis of the membrane fraction of Acetobacter aceti revealed the presence of several proteins that were produced in response to acetic acid. A 60-kDa protein, named AatA, which was mostly induced by acetic acid, was prepared; aatA was cloned on the basis of its NH₂-terminal amino acid sequence. AatA, consisting of 591 amino acids and containing ATP-binding cassette (ABC) sequences and ABC signature sequences, belonged to the ABC transporter superfamily. The aatA mutation with an insertion of the neomycin resistance gene within the aatA coding region showed reduced resistance to acetic acid, formic acid, propionic acid, and lactic acid, whereas the aatA mutation exerted no effects on resistance to various drugs, growth at low pH (adjusted with HCl), assimilation of acetic acid, or resistance to citric acid. Introduction of plasmid pABC101 containing aatA under the control of the Escherichia coli lac promoter into the aatA mutant restored the defect in acetic acid resistance. In addition, pABC101 conferred acetic acid resistance on E. coli. These findings showed that AatA was a putative ABC transporter conferring acetic acid resistance on the host cell. Southern blot analysis and subsequent nucleotide sequencing predicted the presence of aatA orthologues in a variety of acetic acid bacteria belonging to the genera Acetobacter and Gluconacetobacter. The fermentation with A. aceti containing aatA on a multicopy plasmid resulted in an increase in the final yield of acetic acid.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acetic acid bacteria, especially strains classified in the genera Acetobacter and Gluconacetobacter (6, 39, 45), are used for industrial vinegar production because of their great ability to oxidize ethanol and high resistance to acetic acid. From the viewpoints of industrial vinegar production and basic microbiology, it is important to understand the mechanisms that determine these two phenotypes. The ethanol oxidation system has been well characterized by biochemical approaches (14, 15, 41, 43) and by genetic approaches (13, 21, 42). With regard to acetic acid resistance, defect in membrane-bound alcohol dehydrogenase was associated with reduction in acetic acid resistance; however, the mechanism has not been elucidated (4, 29, 40). Resistance to acetic acid does not always result from resistance to low pH, since strains capable of growing at low pHs cannot grow when the pH is adjusted with acetic acid. A genetic approach identified a gene cluster responsible for acetic acid resistance in Acetobacter aceti, which includes aarA encoding a citrate synthase, aarB encoding a functionally unknown protein, and aarC encoding a protein probably involved in acetic acid assimilation (16, 17). A biochemical approach to determine changes in protein profiles in response to acetic acid showed that the production of many proteins was changed (27, 38). One of the proteins whose production was enhanced in response to acetic acid was identified as aconitase (27). These results suggested that the enzymes involved in acetic acid assimilation confer acetic acid resistance on the host cell. On the other hand, it is likely that there is some other machinery conferring acetic acid resistance that is located in the cell membrane, because part of acetic acid toxicity is caused by serving as an uncoupling agent, which disturbs the proton motive force (2, 7, 9, 36). Recently, the presence of a proton motive efflux system for acetic acid in A. aceti cultured on the glycerol medium has been reported (24); however, it seems unclear if it contributes to acetic acid resistance in acetic acid fermentation.

We performed two-dimensional gel electrophoresis to obtain a clue to the putative machinery in the membrane fraction. The production profile of many proteins in the membrane fraction was changed in response to acetic acid. We chose one protein with a molecular mass of 60 kDa, which was produced in response to acetic acid. This protein, named AatA, was a putative ATP-binding cassette (ABC) transporter, which possibly functioned as an exporter of acetic acid. To our knowledge, this is the first report of a putative ABC transporter responsible for acetic acid resistance. We also found that overexpression of aatA improved the yield of acetic acid as a result of enhanced acetic acid resistance of the host cell. This paper deals with genetic characterization of aatA and improvement of acetic acid fermentation by means of overexpression of aatA.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and plasmids.
The bacterial strains and plasmids used are listed in Table 1.

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TABLE 1. Bacterial strains and plasmids

Media and cultural conditions.
YPG medium (pH 6.5) consisted of 5 g of yeast extract (Wako Pure Chemicals), 2 g of polypeptone (Wako Pure Chemicals), and 30 g of glucose in 1 liter of water. Acetobacter and Gluconacetobacter strains, except for Gluconacetobacter polyoxogenes, were first cultured in 5 ml of YPG medium in a test tube with shaking for 24 to 40 h at 30°C. A portion (1 to 5 ml) was inoculated into 100 ml of YPG medium supplemented with ethanol (3% [wt/vol]) and acetic acid (1 to 3% [wt/vol]) in a 500-ml shaking flask and further cultured with shaking at 30°C. G. polyoxogenes was cultured as described by Entani et al. (10).

For acetic acid fermentation tests, Acetobacter strains were first cultured in 5 ml of YPG medium in a 50-ml test tube with shaking for 24 h at 30°C. A portion (10 ml) was inoculated into 2.5 liters of YPG medium supplemented with ethanol (3% [wt/vol]) and acetic acid (1% [wt/vol]) in a 5-liter minijar fermentor and further cultured at 30°C with agitation at 400 rpm and aeration at a rate of 0.20 volumes per volume per minute, until the acetic acid concentration reached 3% (wt/vol). A portion (700 ml) of this culture was left, and 1.8 liters of fresh YPG medium supplemented with 4% (wt/vol) ethanol and 3% (wt/vol) acetic acid was added and further cultured. The ethanol concentration was automatically maintained at 1% (wt/vol) by the addition of ethanol during the cultivation. Acetate concentrations in culture broths were determined by titration with 1 N sodium hydroxide or use of the enzyme assay kit (Roche). Acetic acid production rate, specific growth rate (change in optical density at 660 nm [OD₆₀₀] per hour), and maximal acetate concentration were presented as the means of four experiments with standard deviations.

Escherichia coli was cultured at 37°C in LB medium (10-g/liter Bacto tryptone [Difco], 5-g/liter yeast extract, and 10-g/liter NaCl, pH 7.0). Ampicillin and kanamycin were used at a final concentration of 100 µg/ml or 50 µg/ml, respectively, when necessary to maintain plasmids.

Preparation of the membrane fraction from A. aceti.
A. aceti 10-8S2 was grown in YPG medium with and without 1% (wt/vol) acetic acid. The cells were harvested by centrifugation at the mid-exponential phase (14 h of culture in YPG medium and 20 h of culture in YPG medium supplemented with acetic acid) and the stationary phase (48 h of culture in YPG medium and 60 h of culture in YPG medium supplemented with acetic acid). The harvested cells were suspended in 10 mM potassium phosphate buffer (pH 6.0) and disrupted by passage through a French pressure cell (20,000 lb/in²). The cell lysates were centrifuged at 100,000 x g for 1 h at 4°C. The pellets were used as membrane fractions. The membrane fractions were suspended in ReadyPrep Reagent 3 (Bio-Rad Laboratories). Two-dimensional gel electrophoresis was carried out using the PROTEAN IEF Cell (Bio-Rad Laboratories) with immobilized pH gradients (precast IPG ReadyStrip gel, pH 3 to 10, 11 cm) in the first dimension and a sodium dodecyl sulfate-polyacrylamide gel (12.5% acrylamide) in the second dimension, according to the manufacturer. The proteins were stained with Coomassie brilliant blue R250. Protein concentrations were determined with the DC protein assay kit (Bio-Rad Laboratories) with bovine serum albumin as a standard.

NH₂-terminal amino acid sequencing.
After two-dimensional gel electrophoresis of the membrane fraction, the proteins were blotted on a polyvinylidene difluoride membrane (Millipore) with the graphite electroblotter system (Sartoblot II-S; Sartorius). A 60-kDa protein whose production was enhanced in response to acetic acid was cut and directly analyzed by Edman degradation on an Applied Biosystems model 492cLC protein sequencer. An amino acid sequence homology search was performed at the National Center for Biotechnology Information using the BLAST network service (1).

DNA manipulation.
Total DNA from Acetobacter and Gluconacetobacter was prepared as described by Okumura et al. (28). Restriction endonucleases, T4 polynucleotide kinase, and T4 DNA ligase were purchased from TaKaRa BIO (Kyoto). Acetobacter strains were transformed by the electroporation method (44). For hybridization analysis and cloning of the gene encoding the 60-kDa protein, oligonucleotides (Espec-oligo Service and Sigma-Aldrich) were used. Nucleotide sequences were determined by the dideoxy chain termination method combined with the M13 cloning system on a Shimadzu DSQ1000 DNA sequencer. The DNA sequence was analyzed by using the Genetyx sequence analysis program (Software Development).

Cloning of aatA.
To clone aatA, an oligonucleotide (5'-TACCGIGTIGGIGGIITIITIGT-3') was labeled with the 5'-end-labeling kit (Amersham Biosciences) and used for the ³²P-labeled probe for Southern hybridization against the PstI-digested chromosomal DNA from A. aceti 10-8S2. After standard DNA manipulation including colony hybridization, a 3.3-kb PstI fragment containing aatA was cloned. To place the aatA coding region under the control of the E. coli lac promoter, a 2.2-kb fragment containing aatA was amplified by PCR with primer 1 (5'-CTTGCTGTTGCAACGTATCAGGCAGTAAGC-3') and primer 2 (5'-AGCATGCCAAAACATAGGCATTGCACCAC-3') and cloned in the SmaI site of pMV24. The amplified fragment corresponded to the region from nucleotide positions –108 to 1872. To clone the aatA homologue from G. polyoxogenes in the SmaI site of pMV24, a 2.1-kb fragment was amplified by PCR with primer 3 (5'-ATTGCCAACCGTACGGCCCTTGGCTGGGGG-3') and primer 4 (5'-CCTTGATGGCGCGCAAGTGCTGGTGGACGCC-3'). The amplified fragment corresponded to the region from nucleotide positions –121 to 1958.

Gene disruption.
The 2.2-kb fragment containing aatA was cloned in the SmaI site of pUC19. A 1.1-kb fragment containing the neomycin resistance determinant from Tn5 (3) was then inserted in the BalI site within the aatA coding sequence. This plasmid was introduced in A. aceti 10-8S2, and neomycin-resistant colonies as candidates of mutant aatA (aatA::neo) were selected. Correct aatA-disrupted strains were checked by Southern hybridization with the 2.2-kb fragment and the neomycin resistance gene as the ³²P-labeled probes (see Fig. 2C).

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FIG. 2. Restriction map of the cloned 3.3-kb PstI fragment containing aatA. (A) The PstI fragment on pABC100 was originally cloned in pUC19. pABC101 contained the aatA coding sequence under the control of the E. coli lac promoter in pMV24. The thick arrow indicates the location and direction of three ORFs. ORF3 represents aatA. The thin arrow indicates the direction of the lac promoter in the vector. (B) Structure of the insert of pABCK used for disruption of aatA. pABCK contained the 1.1-kb neomycin resistance gene (neo) (3) in the BalI site of the 2.2-kb aatA coding region on pUC19. (C) Southern blot hybridization to check the correct disruption of the chromosomal aatA gene with probe A (2.2 kb) and probe B (1.1 kb) against the PstI-digested chromosomal DNA from strain 10-8S2 (lanes 1) and mutant m60k-1 (lanes 2).

Southern hybridization to determine distribution of aatA.
DNA-DNA hybridization for detection of aatA homologues in Acetobacter and Gluconacetobacter was performed as follows. Total DNA from Acetobacter and Gluconacetobacter was digested with PstI and applied to agarose gel electrophoresis. The probe was prepared by PCR with primer 1 (5'-ATGGCGCATCCTCCCCTTCTTCATCTTCAG-3') and primer 2 (5'-TCATTTCCAGTTCCAGCCAGCGTTCTTCAG-3') and labeled with the AlkPhos Direct Labeling and Detection system (Amersham Biosciences) according to the instruction manuals of the manufacturer. The hybridization conditions were as described previously (27).

Tests of sensitivity to drugs and various organic acids.
A. aceti strains were first cultured in 5 ml of YPG in a test tube with shaking for 24 to 40 h at 30°C. A portion (50 µl) was inoculated into 5 ml of YPG medium (pH 6.5) or YPG medium (pH 6.5) supplemented with drugs or organic acids and cultured with shaking at 30°C. Sensitivity was determined by following the growth by measuring OD₆₆₀ values. Specific growth rates (changes in OD₆₆₀ values per hour) were presented as the means of four experiments with standard deviations.

Nucleotide sequence accession numbers.
The nucleotide sequences of aatA and its G. polyoxogenes homologue have been deposited in the DDBJ, EMBL, and GenBank databases under accession no. AB214909 and AB218699, respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Changes of protein profile of A. aceti during exposure to acetic acid.
Membrane fractions were prepared from A. aceti grown to the exponential and stationary phases in the presence and absence of 1% (wt/vol) acetic acid and were analyzed by two-dimensional gel electrophoresis (Fig. 1). Several proteins were apparently induced by acetic acid, and some proteins almost disappeared. A protein of an apparent molecular mass of 60 kDa, which was later named AatA, was greatly induced by acetic acid and was chosen for further characterization.

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FIG. 1. Two-dimensional gel electrophoresis of the A. aceti proteins induced by acetic acid. The membrane fractions from A. aceti 10-8S2, which was grown in the medium without (A and C) or with 1% (wt/vol) (B and D) acetic acid, were prepared as described in Materials and Methods. The proteins produced at the exponential growth phase (A and B) and at the stationary growth phase (C and D) were analyzed. The proteins were stained with Coomassie brilliant blue R250. Open triangles indicate the proteins produced in response to acetic acid, and solid triangles indicate those that disappeared in the presence of acetic acid.

Cloning and nucleotide sequence of aatA.
The 60-kDa protein was prepared from a polyvinylidene difluoride membrane on which the proteins had been blotted and directly applied to Edman degradation for determination of its NH₂-terminal amino acid sequence. The amino acid sequence was determined to be Met-Ala-His-Pro-Pro-Leu-Leu-His-Leu-Gln-Asp-Ile. For cloning of the gene encoding the 60-kDa protein, an oligonucleotide, 5'-ATGGCICAICCICCIITIITICAIITICA-3', designed on the basis of the NH₂-terminal amino acid sequence from position 1 to position 8, was used for the ³²P-labeled probe for Southern hybridization against the chromosomal DNA of A. aceti 10-8S2 digested with various restriction enzymes. The chromosomal DNA digested with PstI gave a signal at 3.3 kb. We recovered PstI fragments of about 3.3 kb each, cloned them in the PstI site of pUC19, and selected candidates containing the coding sequence of the 60-kDa protein by colony hybridization. One of the candidates contained pABC100, which contained a 3.3-kb PstI fragment. The restriction map of the cloned fragment is shown in Fig. 2.

The nucleotide sequence of the cloned fragment revealed the presence of three open reading frames (ORFs). Of the three ORFs, ORF3 contained the nucleotide sequence corresponding to the synthetic oligonucleotides used for cloning. The deduced amino acid sequence at positions 1 to 12 completely matched that determined by the Edman degradation procedure. Parts of the deduced amino acid sequence of ORF3 is shown in Fig. 3. The calculated molecular mass was 65.5 kDa, which was in good agreement with that determined by the two-dimentional gel electrophoresis. We named this ORF aatA (for acetic acid transporter), because, as described below, it turned out to encode a putative ABC transporter. ORF1 showed similarity in amino acid sequence to RNA polymerase sigma E factor. ORF2, with 248 amino acids, contained helix-turn-helix DNA-binding motifs and showed similarity in amino acid sequence to two-component response regulator. An inverted repeat sequence was found 53 bp downstream of aatA, which might form a stem-loop structure acting as a transcription terminator.

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FIG. 3. Alignment of amino acid sequences of the ATP-binding sites and signature regions in AatA of A. aceti and its homologue in G. polyoxogenes, CarA, MsrA, SrmB, TlrC, OleB, and UUP. Walker A (GXXGXGKST, where X is any amino acid), Walker B (hhhhDEPT, where h indicates hydrophobic residues), ABC signature I (LSGG), and ABC signature II (hhhH+/–, where +/– indicates charged residues) are shown. The amino acid residues conserved in all the proteins are marked with asterisks. AatA from A. aceti contained sequences well conserved in ABC transporter proteins: a Walker A motif (positions from 39 to 47 and from 314 to 322), a Walker B motif (positions from 141 to 148 and from 424 to 431), an ABC signature I (positions from 121 to 124 and from 404 to 407), and an ABC signature II (positions 171 to 175 and 455 to 459). The similarities in amino acid sequence were 24.5%, 24.4%, 18.7%, 22.3%, 23.7%, and 35.4% with CarA, SrmB, MsrA, TlrC, OleB, and Uup, respectively.

A computer-aided homology search predicted that AatA belonged to an ABC transporter, because it contained sequences well conserved in ABC transporter proteins (23), the Walker A and B motifs, and ABC signatures I and II (Fig. 3). The amino acid sequence alignment includes macrolide resistance deteminants proteins, such as CarA (37), SrmB (37), MsrA (32, 33), TlrC (35), and OleB (30), which are assumed to be efflux pumps, and Uup in E. coli (31), which is assumed to be involved in transposon excision. In addition, the hydropathy profile of AatA was similar to those of the drug transporters and showed that it has no apparent transmembrane domains (Fig. 4). An AatA homologue was found in G. polyoxogenes (see below). Therefore, AatA had a structure common to the ABC superfamily proteins.

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FIG. 4. Hydropathicity profiles of putative ABC transporters. AatA of A. aceti (A), AatA of G. polyoxogenes (B), and MsrA (C) are shown. The hydrophilicity-hydrophobicity plot was calculated according to Kyte and Doolittle (22) with a sliding window of 20 residues. MsrA is composed of 488 amino acid residues.

Physiological characteristics of an aatA mutant.
To elucidate the function of AatA, we disrupted the chromosomal aatA gene by inserting the neomycin resistance gene cassette into the BalI site in the aatA coding region by homologous recombination (Fig. 2B). Correct disruption of aatA was checked by Southern hybridization with the aatA coding region (probe A) and the neomycin resistance gene (probe B) as the ³²P-labeled probes (Fig. 2C). One of the aatA-disrupted mutants obtained in this way was named m60k-1 and was used for further study. The neomycin resistance gene cassette was inserted in an orientation opposite to that of aatA.

The amino acid sequence of AatA suggested that this protein had a different function from that of the drug exporters and Uup because the similarity in amino acid sequence was very low (similarity, 18.7% to 35.4%) and the molecular mass was different from those of the macrolide efflux proteins (55 to 60 kDa) and Uup (72 kDa). As expected, the resistance to various drugs, including erythromycin, of mutant m60k-1 was the same as that of the parental strain (Table 2).

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TABLE 2. Sensitivity to drugs of A. aceti strains

We then examined the growth of mutant m60k-1 at various pHs, adjusted with HCl. Neither the parental strain nor mutant m60k-1 Could grow at pH 3.0; no apparent difference in their growth rates was observed between pH 4.0 and 6.0 (OD₆₆₀ values after 24 h of cultivation at pH 4, 5, and 6 were 0.363, 0.695, and 0.823, respectively, for the parental strain and 0.322, 0.650, and 0.763, respectively, for the mutant). However, the growth of mutant m60k-1 was more repressed as the concentration of acetic acid in the medium increased, compared to the growth of the parental strain (Fig. 5A). The specific growth rate in the presence of 12.5-g/liter acetic acid was 0.0074 ± 0.0011 for the mutant, which was much lower than that of the parental strain (0.0134 ± 0.0010). This finding indicated that AatA is involved in acetic acid resistance, possibly as a transporter of acetic acid. We examined the sensitivity of this mutant to various organic acids because of the rather broad substrate specificity of ABC transporters (11, 23). As shown in Fig. 5B, the resistance of mutant m60k-1 to formic acid, propionic acid, and lactic acid was decreased, although growth was not affected by citric acid, even at high concentrations. Specific growth rates in the presence of formic (1.5 g/liter), propionic (2.5 g/liter), and lactic (7.5 g/liter) acids were 0.0274 ± 0.0002, 0.0416 ± 0.0005, and 0.0396 ± 0.0016, respectively, for the parental strain and 0.0217 ± 0.0006, 0.0144 ± 0.0038, and 0.0232 ± 0.015, respectively, for the mutant. To confirm that AatA conferred the resistance to acetic acid on A. aceti, we constructed pABC101 which containing aatA under the control of the E. coli lac promoter in pMV24 (Fig. 2A) and introduced it in mutant m60k-1. The E. coli lac promoter is constitutively transcribed in Acetobacter (26). Introduction of pABC101 into the mutant restored the acetic acid resistance to the levels (in the presence of acetic acid of up to 15 g/liter) of the parental strain, as measured by growth (data not shown). These results indicated that AatA conferred resistance to acetic acid, formic acid, propionic acid, and lactic acid on A. aceti. But it seemed that AatA is mostly involved in acetic acid resistance because the mutant caused the greatest reduction in resistance to acetic acid.

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FIG. 5. Effects of various organic acids on growth of A. aceti strains. (A) The A. aceti strains were cultured at 30°C in the presence of acetic acid (circles), lactic acid (squares), or citric acid (triangles) in YPG medium for 120 h, and the growth of the strains was followed by measuring OD660 values. (B) The A. aceti strains were cultured at 30°C in the presence of formic acid (circles) or propionic acid (triangles) in YPG medium for 120 h. Solid symbols, strain 10-8S2; open symbols, mutant m60k-1. Error bars indicate standard deviations from four experiments.

Since acetic acid is assimilated by this strain, the growth of mutant m60k-1 and its parental strain in the medium containing acetic acid as the main carbon source was compared. As shown in Fig. 6, almost the same growth rate and the same maximum growth were observed between them, suggesting that the acetic acid resistance conferred by AatA was probably due to its function as an efflux pump but not as an enhancer to assimilate acetic acid.

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FIG. 6. Assimilation of acetic acid by A. aceti strains. The A. aceti strains 10-8S2 (A) and m60k-1 (B) were cultured at 30°C in the presence of 1% (wt/vol) acetic acid in YPG medium. Solid circles, acetic acid concentrations; open circles, the growth measured by OD660 values.

Expression of aatA in E. coli.
To further confirm that AatA is involved in acetic acid resistance in A. aceti, pABC101 was introduced into E. coli, and the acetic acid resistance of the resultant transformants was examined in the presence of IPTG (isopropyl-ß-D-thiogalactopyranoside) (Fig. 7). pABC101 was composed of the aatA coding sequence and E. coli-Acetobacter/Gluconacetobacter shuttle vector pMV24. E. coli JM109 harboring pABC101 grew in the presence of acetic acid at higher concentrations, up to 1.5 g/liter, than that harboring the vector pMV24, which grew in the presence of acetic acid at concentrations of <0.5 g/liter. In addition, the E. coli transformant with pABC101 exhibited higher levels of resistance to formic acid and propionic acid. The transformant grew in the presence of formic acid and propionic acid up to 1.5 g/liter and 1.0 g/liter, respectively, whereas E. coli harboring the vector pMV24 did not grow at these concentrations. On the other hand, neither the transformant harboring the vector nor that harboring pABC101 grew at pH 4.0, and no apparent difference in their growth was observed between pH 4.5 and pH 7.0 (data not shown). Therefore, pABC101 did not affect the growth at various pHs, adjusted with HCl. These results clearly showed that AatA conferred resistance to acetic acid and the related acids not only in A. aceti but also in E. coli.

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FIG. 7. Effect of acetic acid, formic acid, and propionic acid on growth of E. coli transformants. The E. coli transformants were cultured at 37°C for 90 h in LB medium (pH 5.0) in the presence of various concentrations of acids. Solid symbols, E. coli JM109(pMV24); open symbols, E. coli JM 109(pABC101). Triangles, formic acid; circles, acetic acid; diamonds, propionic acid. Error bars indicate standard deviations from four experiments.

Effects of overexpression of aatA on acetic acid fermentation by A. aceti.
We expected that overexpression of aatA would improve the yield of acetic acid as a result of the growth in the presence of acetic acid at a higher concentration. pABC101 was introduced into A. aceti strain 10-8S2, and its acetic acid resistance was compared. As expected, the transformant grew in the presence of 20-g/liter acetic acid, whereas the parental strain harboring the vector pMV24 was able to grow in the presence of up to 15-g/liter acetic acid.

The acetic acid fermentation profiles of the parental strain harboring the vector pMV24 and the transformant harboring pABC101 were compared (Fig. 8). Although the growth of the transformant was slightly delayed, the average growth rate (measured as the changes in OD₆₆₀ value per hour) between 40-g/liter and 90-g/liter acetic acid in the culture was almost the same between the two strains. The rates of acetic acid production were also almost the same (0.875 ± 0.116 and 0.888 ± 0.099 g of acetic acid/liter/h for the parental strain harboring the vector pMV24 and the transformant, respectively). The parental strain harboring the vector pMV24 was able to grow up to 73.4 ± 2.2 g of acetic acid/liter, whereas the growth of transformant continued even at 80.4 ± 3.8 g of acetic acid/liter as expected. The final yield of acetic acid (111.7 ± 3.3 g/liter) produced by the transformant was higher than that (103.7 ± 1.8 g/liter) by the parental strain harboring the vector pMV24.

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FIG. 8. Acetic acid fermentation by A. aceti strains. Strain 10-8S2 (A) and the transformant (B) were cultured as described in Materials and Methods. Diamonds, OD660 values; circles, acetic acid concentration in the medium.

Distribution of homologous gene to aatA in Acetobacter and Gluconacetobacter.
The distribution of aatA gene among acetic acid bacteria belonging to the genera Acetobacter and Gluconacetobacter was examined by Southern blot analysis using aatA as the hybridization probe; positive signals were detected in all the strains examined (Fig. 9).

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FIG. 9. Southern blot analysis to determine distribution of aatA in acetic acid bacteria. Hybridization with the aatA probe (the 2.2-kb PCR fragment) was done with the PstI-digested total DNAs from acetic acid bacteria. Lane 1, A. aceti 10-8S2; lane 2, Acetobacter pasteurianus DSM3509; lane 3, G. polyoxogenes NBI 1060; lane 4, Gluconacetobacter intermedius DSM11804; lane 5, Gluconacetobacter oboediens DSM11826; and lane 6, Gluconacetobacter europaeus DSM6160.

We cloned a DNA fragment giving a positive signal from G. polyoxogenes NBI1060 that is an industrial strain used for high-acidity vinegar production (10). The cloning procedure was similar to that for cloning aatA from A. aceti. The DNA fragment prepared by PCR in Southern blot analysis described above was used as a probe for hybridization against the partial PstI-digested chromosomal DNA, and a 3.0-kb PstI fragment giving the positive signal was cloned and sequenced, confirming that this DNA fragment contained an aatA homologue. The aatA homologue encoded a 608-amino-acid protein showing end-to-end similarity to AatA (66.6% of similarity), including the Walker and signature sequences characteristic of ABC transporters (Fig. 3), and showed a hydropathy profile similar to that of AatA (Fig. 4B).

Introduction of aatA homologue from G. polyoxogenes into A. aceti 10-80S2 by use of the vector pMV24 enabled the host to grow at a higher acetic acid concentration; a maximum concentration of acetic acid that allowed the growth was 15 g/liter for strain 10-80S2 harboring the vector pMV24 and 17.5 g/liter for the transformant, and the specific growth rate of the transformant in the presence of 15 g of acetic acid/liter was 0.0167 ± 0.0022, which was higher than that of strain 10-80S2 harboring the vector pMV24 (0.0130 ± 0.0015).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Among the proteins induced by acetic acid in A. aceti, we found a putative ABC transporter named AatA. Higher sensitivity to acetic acid of mutant m60k-1 and enhancement in acetic acid resistance that occurred by overexpression of aatA in A. aceti and E. coli clearly indicated that AatA is involved in acetic acid resistance in A. aceti.

AatA is classified as one of the type B ABC transporters (23) because it contains two ABCs in tandem on a single polypeptide. It is interesting that this feature of AatA, including two ABC motifs, has a structure common to the family of macrolide antibiotics transporters (25), suggesting that AatA has the same function as they have. The macrolide transporters are considered to function as an efflux pump because active efflux of the drug from cells has been demonstrated (30, 33). Recently, it was also speculated that macrolide transporters play a role in some other cellular function and that resistance is conferred as a secondary effect, such as ribosomal protection by competitive binding with the macrolide antibiotics (33). An alternative hypothesis for the function of AatA is that it rescues some cell functions from the damage occurred by acetic acid. However, a mutation and overexpression of the aatA gene affected resistance to formic and propionic acids and to acetic acid simultaneously, and a similar profile of acetate assimilation was observed with mutant m60k-1 as with the parental strain. These results seem to support the idea that AatA functions as an efflux pump of acetic acid.

AatA protein had no apparent hydrophobic membrane spanning domains. In the previous study, we analyzed the soluble proteins by two-dimentional gel electrophoresis and did not detect a protein corresponding to AatA when induction with acetic acid was carried out (27). It was reported that OleB protein was found both in the soluble and the membrane fractions (30). This was explained by assuming that its normal physiological location would be the cytoplasmic face of the membrane, interacting with the membrane component. The cellular localization of AatA suggests that it also tightly bound with a membrane protein in a cell and that the complex of AatA with a membrane protein was not lost during cell disruption and fractionation.

It is noteworthy that AatA conferred the resistance to short-chain fatty acids C1 (formic acid), C2 (acetic acid), and C3 (propionic acid) and lactic acid. It is thought that the macrolide transporters determine the substrate specificity because they confer the resistance in the heterologous hosts without simultaneous introduction of a gene encoding transmembrane domains (25, 30, 32). The phenotypes of mutant m60k-1 indicated that AatA was involved in resistance to acetic acid, formic acid, propionic acid, and lactic acid on A. aceti. This suggests that AatA is responsible for recognizing these organic acids. The phenotype of E. coli containing aatA supports this idea. AatA appears to recognize acetic acid most favorably because the aatA mutation caused the severest reduction in resistance to acetic acid among the three fatty acids.

Several transporters for monocarboxylic acids are known; these are the bacterial lactate permease LctP family (8, 12, 18), the eukaryotic proton-linked monocarboxylate transporter MCT family (19), and a monocarboxylate permease having a sodium-binding motif in Rhizobium leguminosarum (20). These three transporters contain no ABC motifs and are considered to transport monocarboxylic acids via a proton-coupled reaction. AatA shows no significant similarity in amino acid sequence to these transporters and contains no sodium-binding motifs (5). Therefore, AatA is different from these known monocarboxylic acid transporters.

Very recently, Matsushita et al. (24) reported the presence of the efflux pump for acetic acid in the other strain of A. aceti. They concluded that the efflux pump is proton motive force dependent because transporter activity was dependent on pH, not on ATP, and was sensitive to a proton uncoupler. Although the protein responsible for the transporter activity has not been identified, it seems that the efflux system found by them is different from AatA.

The mutant became sensitive to acetic acid but was still resistant to some extent. Deletion constructs containing the N- or C-terminal ABC regions of MsrA did not confer erythromycin resistance singly or in combination (34). However, in the case of the OleB, the presence of either the first or the second half of the gene was sufficient to confer the resistance, but disruption or deletion in the interdomain region between the two ABC regions affected resistance (30). In mutant m60k-1, the neomycin resistance gene cassette was integrated in the latter half of the ABC region; thus, the former half of the ABC region and the interdomain region were complete. This might suggest the possibility that AatA had not been completely inactivated in mutant m60k-1. Another explanation is that acetic acid resistance is conferred by several mechanisms and that the mutation in aatA resulted in a partial reduction in acetic acid resistance.

The high level of acetic acid resistance is characteristic of acetic acid bacteria. Consistent with the idea that AatA plays an important role in acetic acid resistance in these bacteria, aatA is distributed in the genera Acetobacter and Gluconacetobacter. The positive signals, which were detected in all the acetic acid bacteria tested in the Southern hybridization experiment (Fig. 9), supposedly represented the aatA orthologue in the individual strains, since the signal in G. polyoxogenes, for example, actually represented the gene that encodes an AatA homologue conferring acetic acid resistance.

The previous and present studies suggest that acetic acid resistance in A. aceti is conferred by at least two mechanisms: one is the assimilation of acetic acid by enzymes, such as citrate synthase (16) and aconitase (27), and the other is the export of acetic acid by the ABC transporter. Both mechanisms apparently serve to reduce the intracellular acetic acid concentration. Our speculation as to a possible correlation between the two mechanisms is that the enhancement of the cytosolic enzyme activity to assimilate acetic acid leads to production of more ATP, which in turn is used for ABC transporter functioning. In this manner, the two mechanisms may be closely related and function coordinately and additionally in response to acetic acid. To elucidate the whole mechanism of acetic acid resistance, identification of other proteins induced by acetic acid, which were detected by two-dimensional gel electrophoretic analysis, is also required.

As we expected, overexpression of aatA resulted in improvement of growth in the presence of a high concentration of acetic acid and an increase in the final yield of acetic acid, probably due to maintenance of a low level of intracellular acetic acid concentration. This finding is very important from the industrial viewpoint because it could provide a clue for finding a new way to breed acetic acid bacteria for vinegar fermentation.

 
* Corresponding author. Mailing address: Central Research Institute, Mizkan Group Co., Ltd., Handa-shi, Aichi 475-8585, Japan. Phone: 81 (569) 24-3331. Fax: 81 (569) 24-5024. E-mail: snakano1{at}mizkan.co.jp

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, January 2006, p. 497-505, Vol. 72, No. 1
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