APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 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 and Department of Biotechnology,
Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657,2 Japan
Received 22 July 2005/Accepted 18 October 2005

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. Intro-
duction 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.

Acetic acid bacteria, especially strains classified in the gen-
era 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 microbi-
ology, it is important to understand the mechanisms that de-
termine 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 de-
hydrogenase was associated with reduction in acetic acid resis-
tance; 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 ace-
tic acid resistance in Acetobacter aceti, which includes aarA
encoding a citrate synthase, aarB encoding a functionally un-
known 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 as-
similation 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
* Corresponding author. Mailing address: Central Research Insti-
tute, Mizkan Group Co., Ltd., Handa-shi, Aichi 475-8585, Japan. Phone:
81 (569) 24-3331. Fax: 81 (569) 24-5024. E-mail: snakano1@mizkan.co.jp.
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 ob-
tain 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 pos-
sibly functioned as an exporter of acetic acid. To our knowl-
edge, this is the first report of a putative ABC transporter
responsible for acetic acid resistance. We also found that over-
expression 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.
MATERIALS AND METHODS
Bacterial strains and plasmids. The bacterial strains and plasmids used are
listed in Table 1.
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

497
498 NAKANO ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 1. Bacterial strains and plasmids sulfate-polyacrylamide gel (12.5% acrylamide) in the second dimension, accord-
ing to the manufacturer. The proteins were stained with Coomassie brilliant blue
Strain or Reference R250. Protein concentrations were determined with the DC protein assay kit
Relevant characteristics a
plasmid or source (Bio-Rad Laboratories) with bovine serum albumin as a standard.
Strains NH2-terminal amino acid sequencing. After two-dimensional gel electro-
A. aceti phoresis of the membrane fraction, the proteins were blotted on a polyvinylidene
10-8S2 pro; Smr; thermotolerant 28 difluoride membrane (Millipore) with the graphite electroblotter system (Sarto-
m60k-1 10-8S2 aatA::neo This work blot II-S; Sartorius). A 60-kDa protein whose production was enhanced in re-
A. pasteurianus Wild type DSMZ sponse to acetic acid was cut and directly analyzed by Edman degradation on an
DSM 3509 Applied Biosystems model 492cLC protein sequencer. An amino acid sequence
G. polyoxogenes Wild type 10 homology search was performed at the National Center for Biotechnology In-
NBI 1060 formation using the BLAST network service (1).
G. intermedrius Wild type DSMZ DNA manipulation. Total DNA from Acetobacter and Gluconacetobacter was
DSM 11804 prepared as described by Okumura et al. (28). Restriction endonucleases, T4
G. oboediens Wild type DSMZ polynucleotide kinase, and T4 DNA ligase were purchased from TaKaRa BIO
DSM 11826 (Kyoto). Acetobacter strains were transformed by the electroporation method
G. europaeus Wild type DSMZ (44). For hybridization analysis and cloning of the gene encoding the 60-kDa
DSM 6160 protein, oligonucleotides (Espec-oligo Service and Sigma-Aldrich) were used.
E. coli JM109 recA1 endA1 gyrA96 thi hsdR17 46 Nucleotide sequences were determined by the dideoxy chain termination method
supE44 relA1 ⌬(lac-proAB)/ combined with the M13 cloning system on a Shimadzu DSQ1000 DNA se-
F⬘ [traD36 proAB⫹ laclq quencer. The DNA sequence was analyzed by using the Genetyx sequence
lacZ⌬M15] analysis program (Software Development).
Cloning of aatA. To clone aatA, an oligonucleotide (5⬘-TACCGIGTIGGIGGI
Plasmids ITIITIGT-3⬘) was labeled with the 5⬘-end-labeling kit (Amersham Biosciences)
pUC19 Apr; lacZ⬘ 46 and used for the 32P-labeled probe for Southern hybridization against the PstI-
pMV24 Apr; lacZ⬘; Acetobacter/ 13 digested chromosomal DNA from A. aceti 10-8S2. After standard DNA manip-
Gluconacetobacter-E. coli ulation including colony hybridization, a 3.3-kb PstI fragment containing aatA
shuttle vector was cloned. To place the aatA coding region under the control of the E. coli lac
pABC100 Apr; 3.3-kb PstI fragment This work promoter, a 2.2-kb fragment containing aatA was amplified by PCR with primer
containing the whole aatA 1 (5⬘-CTTGCTGTTGCAACGTATCAGGCAGTAAGC-3⬘) and primer 2 (5⬘-
gene on pUC19 AGCATGCCAAAACATAGGCATTGCACCAC-3⬘) and cloned in the SmaI site
pABC101 Apr; 2.2-kb PCR fragment This work of pMV24. The amplified fragment corresponded to the region from nucleotide
containing the whole aatA positions ⫺108 to 1872. To clone the aatA homologue from G. polyoxogenes in the
gene on pMV24 SmaI site of pMV24, a 2.1-kb fragment was amplified by PCR with primer 3 (5⬘-ATT
pABCK Apr Neor; 3.5-kb fragment This work GCCAACCGTACGGCCCTTGGCTGGGGG-3⬘) and primer 4 (5⬘-CCTTGA
containing the neo gene at the TGGCGCGCAAGTGCTGGTGGACGCC-3⬘). The amplified fragment corre-
BalI site of 2.2-kb PCR sponded to the region from nucleotide positions ⫺121 to 1958.
fragment subcloned on pUC19 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 determi-
a
Apr, ampicillin resistance; Neor, neomycin resistance; Smr, streptomycin nant from Tn5 (3) was then inserted in the BalI site within the aatA coding
resistance. 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 frag-
ment and the neomycin resistance gene as the 32P-labeled probes (see Fig. 2C).
(3% [wt/vol]) and acetic acid (1% [wt/vol]) in a 5-liter minijar fermentor and Southern hybridization to determine distribution of aatA. DNA-DNA hybrid-
further cultured at 30°C with agitation at 400 rpm and aeration at a rate of 0.20 ization for detection of aatA homologues in Acetobacter and Gluconacetobacter
volumes per volume per minute, until the acetic acid concentration reached 3% was performed as follows. Total DNA from Acetobacter and Gluconacetobacter
(wt/vol). A portion (700 ml) of this culture was left, and 1.8 liters of fresh YPG was digested with PstI and applied to agarose gel electrophoresis. The probe was
medium supplemented with 4% (wt/vol) ethanol and 3% (wt/vol) acetic acid was prepared by PCR with primer 1 (5⬘-ATGGCGCATCCTCCCCTTCTTCATCTT
added and further cultured. The ethanol concentration was automatically main- CAG-3⬘) and primer 2 (5⬘-TCATTTCCAGTTCCAGCCAGCGTTCTTCAG-3⬘)
tained at 1% (wt/vol) by the addition of ethanol during the cultivation. Acetate and labeled with the AlkPhos Direct Labeling and Detection system (Amersham
concentrations in culture broths were determined by titration with 1 N sodium Biosciences) according to the instruction manuals of the manufacturer. The
hydroxide or use of the enzyme assay kit (Roche). Acetic acid production rate, hybridization conditions were as described previously (27).
specific growth rate (change in optical density at 660 nm [OD600] per hour), and Tests of sensitivity to drugs and various organic acids. A. aceti strains were
maximal acetate concentration were presented as the means of four experiments first cultured in 5 ml of YPG in a test tube with shaking for 24 to 40 h at 30°C.
with standard deviations. A portion (50 ␮l) was inoculated into 5 ml of YPG medium (pH 6.5) or YPG
Escherichia coli was cultured at 37°C in LB medium (10-g/liter Bacto tryptone medium (pH 6.5) supplemented with drugs or organic acids and cultured with
[Difco], 5-g/liter yeast extract, and 10-g/liter NaCl, pH 7.0). Ampicillin and shaking at 30°C. Sensitivity was determined by following the growth by measuring
kanamycin were used at a final concentration of 100 ␮g/ml or 50 ␮g/ml, respec- OD660 values. Specific growth rates (changes in OD660 values per hour) were
tively, when necessary to maintain plasmids. presented as the means of four experiments with standard deviations.
Preparation of the membrane fraction from A. aceti. A. aceti 10-8S2 was grown Nucleotide sequence accession numbers. The nucleotide sequences of aatA and
in YPG medium with and without 1% (wt/vol) acetic acid. The cells were its G. polyoxogenes homologue have been deposited in the DDBJ, EMBL, and
harvested by centrifugation at the mid-exponential phase (14 h of culture in YPG GenBank databases under accession no. AB214909 and AB218699, respectively.
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 RESULTS
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 ⫻ Changes of protein profile of A. aceti during exposure to
g for 1 h at 4°C. The pellets were used as membrane fractions. The membrane acetic acid. Membrane fractions were prepared from A. aceti
fractions were suspended in ReadyPrep Reagent 3 (Bio-Rad Laboratories).
Two-dimensional gel electrophoresis was carried out using the PROTEAN IEF
grown to the exponential and stationary phases in the presence
Cell (Bio-Rad Laboratories) with immobilized pH gradients (precast IPG and absence of 1% (wt/vol) acetic acid and were analyzed by
ReadyStrip gel, pH 3 to 10, 11 cm) in the first dimension and a sodium dodecyl two-dimensional gel electrophoresis (Fig. 1). Several proteins
VOL. 72, 2006 ABC TRANSPORTER FOR ACETIC ACID RESISTANCE IN A. ACETI 499

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.

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.
Cloning and nucleotide sequence of aatA. The 60-kDa pro-
tein 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 deter-
mined 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 se-
quence from position 1 to position 8, was used for the 32P-
labeled probe for Southern hybridization against the chromo-
somal 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 pro-
tein by colony hybridization. One of the candidates contained
pABC100, which contained a 3.3-kb PstI fragment. The restric-
tion 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 corre-
sponding to the synthetic oligonucleotides used for cloning.
The deduced amino acid sequence at positions 1 to 12 com-
pletely 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 in-
verted repeat sequence was found 53 bp downstream of aatA,
which might form a stem-loop structure acting as a transcrip-
tion terminator.
A computer-aided homology search predicted that AatA
belonged to an ABC transporter, because it contained se-
quences 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 resis-
tance 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
500 NAKANO ET AL. APPL. ENVIRON. MICROBIOL.

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).

(see below). Therefore, AatA had a structure common to the
ABC superfamily proteins.
Physiological characteristics of an aatA mutant. To eluci-
date 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 recom-
bination (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 ori-
entation opposite to that of aatA.
The amino acid sequence of AatA suggested that this pro-
tein 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).
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 concen-
tration 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 trans-
porter of acetic acid. We examined the sensitivity of this mu-
tant to various organic acids because of the rather broad sub-
strate 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.
Since acetic acid is assimilated by this strain, the growth of
mutant m60k-1 and its parental strain in the medium contain-
ing acetic acid as the main carbon source was compared. As
shown in Fig. 6, almost the same growth rate and the same
VOL. 72, 2006 ABC TRANSPORTER FOR ACETIC ACID RESISTANCE IN A. ACETI 501

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.

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.
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-␤-D-thiogalactopyranoside) (Fig. 7). pABC101 was
composed of the aatA coding sequence and E. coli-Acetobacter/
Gluconacetobacter shuttle vector pMV24. E. coli JM109 har-
boring 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 concen-
trations 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 pres-
ence 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 differ- FIG. 4. Hydropathicity profiles of putative ABC transporters. AatA
of A. aceti (A), AatA of G. polyoxogenes (B), and MsrA (C) are shown.
ence in their growth was observed between pH 4.5 and pH 7.0 The hydrophilicity-hydrophobicity plot was calculated according to
(data not shown). Therefore, pABC101 did not affect the Kyte and Doolittle (22) with a sliding window of 20 residues. MsrA is
growth at various pHs, adjusted with HCl. These results clearly composed of 488 amino acid residues.
502 NAKANO ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 2. Sensitivity to drugs of A. aceti strains

MIC (␮g/ml)
Drug
A. aceti 10-8S2 A. aceti m60k-1

Puromycin ⬍15 ⬍15

Erythromycin 20 20
Pyronine Y 0.2 0.2
Rhodamine 6G 25 25
TPPC a 20 20
a
TPPC, tetraphenyl phosphonium chloride.

showed that AatA conferred resistance to acetic acid and the

related acids not only in A. aceti but also in E. coli.
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 FIG. 6. Assimilation of acetic acid by A. aceti strains. The A. aceti
presence of up to 15-g/liter acetic acid. strains 10-8S2 (A) and m60k-1 (B) were cultured at 30°C in the pres-
The acetic acid fermentation profiles of the parental strain ence of 1% (wt/vol) acetic acid in YPG medium. Solid circles, acetic
harboring the vector pMV24 and the transformant harboring acid concentrations; open circles, the growth measured by OD660 values.
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 parental strain harboring the vector pMV24 was able to grow
40-g/liter and 90-g/liter acetic acid in the culture was almost the up to 73.4 ⫾ 2.2 g of acetic acid/liter, whereas the growth of
same between the two strains. The rates of acetic acid produc- transformant continued even at 80.4 ⫾ 3.8 g of acetic acid/liter
tion were also almost the same (0.875 ⫾ 0.116 and 0.888 ⫾ as expected. The final yield of acetic acid (111.7 ⫾ 3.3 g/liter)
0.099 g of acetic acid/liter/h for the parental strain harboring produced by the transformant was higher than that (103.7 ⫾
the vector pMV24 and the transformant, respectively). The 1.8 g/liter) by the parental strain harboring the vector pMV24.
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 Gluco-

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.
FIG. 7. Effect of acetic acid, formic acid, and propionic acid on
growth of E. coli transformants. The E. coli transformants were cul-
tured 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; cir-
cles, acetic acid; diamonds, propionic acid. Error bars indicate stan-
dard deviations from four experiments.
VOL. 72, 2006 ABC TRANSPORTER FOR ACETIC ACID RESISTANCE IN A. ACETI 503

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 pas-
teurianus DSM3509; lane 3, G. polyoxogenes NBI 1060; lane 4, Gluco-
FIG. 8. Acetic acid fermentation by A. aceti strains. Strain 10-8S2
nacetobacter intermedius DSM11804; lane 5, Gluconacetobacter oboediens
(A) and the transformant (B) were cultured as described in Materials
DSM11826; and lane 6, Gluconacetobacter europaeus DSM6160.
and Methods. Diamonds, OD660 values; circles, acetic acid concentra-
tion in the medium.

AatA is classified as one of the type B ABC transporters (23)

because it contains two ABCs in tandem on a single polypep-
nacetobacter was examined by Southern blot analysis using
tide. It is interesting that this feature of AatA, including two
aatA as the hybridization probe; positive signals were detected
ABC motifs, has a structure common to the family of macro-
in all the strains examined (Fig. 9).
lide antibiotics transporters (25), suggesting that AatA has the
We cloned a DNA fragment giving a positive signal from G.
same function as they have. The macrolide transporters are
polyoxogenes NBI1060 that is an industrial strain used for high-
considered to function as an efflux pump because active efflux
acidity vinegar production (10). The cloning procedure was
of the drug from cells has been demonstrated (30, 33). Re-
similar to that for cloning aatA from A. aceti. The DNA frag-
cently, it was also speculated that macrolide transporters play
ment prepared by PCR in Southern blot analysis described
a role in some other cellular function and that resistance is
above was used as a probe for hybridization against the partial
conferred as a secondary effect, such as ribosomal protection
PstI-digested chromosomal DNA, and a 3.0-kb PstI fragment
by competitive binding with the macrolide antibiotics (33). An
giving the positive signal was cloned and sequenced, confirm-
alternative hypothesis for the function of AatA is that it res-
ing that this DNA fragment contained an aatA homologue.
cues some cell functions from the damage occurred by acetic
The aatA homologue encoded a 608-amino-acid protein show-
acid. However, a mutation and overexpression of the aatA gene
ing end-to-end similarity to AatA (66.6% of similarity), includ-
affected resistance to formic and propionic acids and to acetic
ing the Walker and signature sequences characteristic of ABC
acid simultaneously, and a similar profile of acetate assimila-
transporters (Fig. 3), and showed a hydropathy profile similar
tion was observed with mutant m60k-1 as with the parental
to that of AatA (Fig. 4B).
strain. These results seem to support the idea that AatA func-
Introduction of aatA homologue from G. polyoxogenes into
tions as an efflux pump of acetic acid.
A. aceti 10-80S2 by use of the vector pMV24 enabled the host
AatA protein had no apparent hydrophobic membrane
to grow at a higher acetic acid concentration; a maximum
spanning domains. In the previous study, we analyzed the sol-
concentration of acetic acid that allowed the growth was 15
uble proteins by two-dimentional gel electrophoresis and did
g/liter for strain 10-80S2 harboring the vector pMV24 and 17.5
not detect a protein corresponding to AatA when induction
g/liter for the transformant, and the specific growth rate of the
with acetic acid was carried out (27). It was reported that OleB
transformant in the presence of 15 g of acetic acid/liter was
protein was found both in the soluble and the membrane
0.0167 ⫾ 0.0022, which was higher than that of strain 10-80S2
fractions (30). This was explained by assuming that its normal
harboring the vector pMV24 (0.0130 ⫾ 0.0015).
physiological location would be the cytoplasmic face of the
membrane, interacting with the membrane component. The
DISCUSSION cellular localization of AatA suggests that it also tightly bound
with a membrane protein in a cell and that the complex of
Among the proteins induced by acetic acid in A. aceti, we AatA with a membrane protein was not lost during cell dis-
found a putative ABC transporter named AatA. Higher sen- ruption and fractionation.
sitivity to acetic acid of mutant m60k-1 and enhancement in It is noteworthy that AatA conferred the resistance to short-
acetic acid resistance that occurred by overexpression of aatA chain fatty acids C1 (formic acid), C2 (acetic acid), and C3
in A. aceti and E. coli clearly indicated that AatA is involved in (propionic acid) and lactic acid. It is thought that the macrolide
acetic acid resistance in A. aceti. transporters determine the substrate specificity because they
504 NAKANO ET AL.
confer the resistance in the heterologous hosts without simul-
taneous introduction of a gene encoding transmembrane do-
mains (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 reac-
tion. AatA shows no significant similarity in amino acid se-
quence 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 erythro-
mycin 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 ace-
tic 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 Gluconaceto-
bacter. The positive signals, which were detected in all the
acetic acid bacteria tested in the Southern hybridization exper-
iment (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 appar-
APPL. ENVIRON. MICROBIOL.
ently serve to reduce the intracellular acetic acid concentra-
tion. Our speculation as to a possible correlation between the
two mechanisms is that the enhancement of the cytosolic en-
zyme activity to assimilate acetic acid leads to production of
more ATP, which in turn is used for ABC transporter func-
tioning. 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 electro-
phoretic analysis, is also required.
As we expected, overexpression of aatA resulted in improve-
ment 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.
REFERENCES
1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic local alignment search tool. J. Mol. Biol. 215:403–410.
2. Axe, D. D., and J. E. Bailey. 1995. Transport of lactate and acetate through
the energized cytoplasmic membrane of Escherichia coli. Biotechnol. Bioeng.
47:8–19.
3. Beck, E., G. Ludwig, E. A. Auerswald, B. Reiss, and H. Schaller. 1982.
Nucleotides sequence and extract localization of the neomycin phospho-
transferase gene from transposon Tn5. Gene 19:327–336.
4. Chinnawirotpisan, P., G. Theeragool, S. Limtong, H. Toyama, O. Adachi,
and K. Matsushita. 2003. Quinoprotein alcohol dehydrogenase is involved in
catabolic acetate production, while NAD-dependent alcohol dehydrogenase
in ethanol assimilation in Acetobacter pasteurianus. J. Biosci. Bioeng. 96:564–
571.
5. Deguchi, Y., I. Yamato, and Y. Anraku. 1990. Nucleotide sequence of gltS,
the Na⫹/glutamate symport carrier gene of Escherichia coli B. J. Biol. Chem.
265:21704–21708.
6. De Ley, J., J. Swings, and F. Gosselé. 1984. Key to the genera of the family
Acetobacteraceae, p. 268–278. In N. R. Krieg and J. G. Holt (ed.), Bergey’s
manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co.,
Baltimore, Md.
7. Diez-Gonzalez, F., and J. B. Russell. 1997. The ability of Escherichia coli
O157:H7 to decrease its intracellular pH and resist the toxicity of acetic acid.
Microbiology 143:1175–1180.
8. Dong, J. M., J. S. Taylor, D. J. Latour, S. Iuchi, and E. C. C. Lin. 1993. Three
overlapping lct genes involved in L-lactate utilization by Escherichia coli. J.
Bacteriol. 175:6671–6678.
9. Dürre, A. P., H. Bahl, and G. Gottschalk. 1988. Membrane processes and
product formations in anaerobes, p. 187–206. In L. E. Erickson and D. Y.-C.
Fung (ed.), Handbook of anaerobic fermentation. Marcel Dekker, New
York, N.Y.
10. Entani, E., S. Ohmori, H. Masai, and K. Suzuki. 1985. Acetobacter polyoxo-
genes sp. nov., a new species of an acetic acid bacterium useful for producing
vinegar with high acidity. J. Gen. Appl. Microbiol. 31:475–490.
11. Fath, M. J., and R. Kolter. 1993. ABC transporter: bacterial exporters.
Microbiol. Rev. 57:995–1017.
12. Fleischman, R. D., M. D. Adams, O. White, R. A. Clayton, E. F. Kirkness,
A. R. Kerlavage, C. J. Bult, J.-F. Tomb, B. A. Dougherty, J. M. Merrick, K.
Mckenney, and J. C. Venter. 1995. Whole-genome random sequencing and
assembly of Haemophillus influenzae Rd. Science 269:496–512.
13. Fukaya, M., K. Tayama, T. Tamaki, H. Tagami, H. Okumura, Y. Kawamura,
and T. Beppu. 1989. Cloning of the membrane-bound aldehyde dehydro-
genase gene of Acetobacter polyoxogenes and improvement of acetic acid
production by use of the cloned gene. Appl. Environ. Microbiol. 55:171–176.
14. Fukaya, M., K. Tayama, H. Okumura, Y. Kawamura, and T. Beppu. 1989.
Purification and characterization of membrane-bound aldehyde dehydro-
genase from Acetobacter polyoxogenes sp. nov. Appl. Microbiol. Biotechnol.
32:176–180.
15. Fukaya, M., K. Tayama, T. Tamaki, H. Ebisuya, H. Okumura, Y. Kawamura,
S. Horinouchi, and T. Beppu. 1989. Characterization of cytochrome a1 that
functions as a ubiquinol oxidase in Acetobacter aceti. J. Bacteriol. 175:4307–
4314.
16. Fukaya, M., H. Takemura, H. Okumura, Y. Kawamura, S. Horinouchi, and
T. Beppu. 1990. Cloning of genes responsible for acetic acid resistance in
Acetobacter aceti. J. Bacteriol. 172:2096–2104.
VOL. 72, 2006 ABC TRANSPORTER FOR ACETIC ACID RESISTANCE IN A. ACETI
17. Fukaya, M., H. Takemura, K. Tayama, H. Okumura, Y. Kawamura, S.
Horinouchi, and T. Beppu. 1993. The aarC gene responsible for acetic acid
assimilation confers acetic acid resistance on Acetobacter aceti. J. Ferment.
Bioeng. 76:270–275.
18. Gibello, A., M. D. Collins, L. Domı́nguez, J. F. Fernández-Garayzábal, and
P. T. Richardson. 1999. Cloning and analysis of the L-lactate utilization genes
from Streptococcus iniae. Appl. Environ. Microbiol. 65:4346–4350.
19. Halestrap, A. P., and N. T. Price. 1999. The proton-liked monocarboxylate
transporter (MCT) family: structure, function and regulation. Biochem. J.
343:281–299.
20. Hosie, A. H. F., D. Allaway, and P. S. Poole. 2002. A monocarboxylate
permease of Rhizobium leguminosarum is the first member of a new subfamily
of transporters. J. Bacteriol. 184:5436–5448.
21. Kondo, K., T. Beppu, and S. Horinouchi. 1995. Cloning, sequencing, and
characterization of the gene encoding the smallest subunit of the three-
component membrane-bound alcohol dehydrogenase from Acetobacter pas-
teurianus. J. Bacteriol. 177:5048–5055.
22. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the
hydropathic character of a protein. J. Mol. Biol. 157:105–132.
23. Linton, K. J., and C. F. Higgins. 1998. The Escherichia coli ATP-binding
cassette (ABC) proteins. Mol. Microbiol. 28:5–13.
24. Matsushita, K., T. Inoue, O. Adachi, and H. Toyama. 2005. Acetobacter aceti
possesses a proton motive force-dependent efflux system for acetic acid. J.
Bacteriol. 187:4346–4352.
25. Méndez, C., and J. A. Salas. 2001. The role of ABC transporters in anti-
biotic-producing organisms: drug secretion and resistance mechanisms. Res.
Microbiol. 152:341–350.
26. Nakai, T., A. Moriya, N. Tonouchi, T. Tsuchida, F. Yoshinaga, S. Horinouchi,
Y. Sone, H. Mori, F. Sakai, and T. Hayashi. 1998. Control of expression by
the cellulose synthase (bcsA) promoter region from Acetobacter xylinum
BPR2001. Gene 213:93–100.
27. Nakano, S., M. Fukaya, and S. Horinouchi. 2004. Enhanced expression of
aconitase raises acetic acid resistance in Acetobacter aceti. FEMS Microbiol.
Lett. 235:315–322.
28. Okumura, H., T. Uozumi, and T. Beppu. 1985. Construction of plasmid
vector and genetic transformation system for Acetobacter aceti. Agric. Biol.
Chem. 49:1011–1017.
29. Okumura, H., T. Uozumi, and T. Beppu. 1985. Biochemical characteristics of
spontaneous mutants of Acetobacter aceti deficient in ethanol oxidation.
Agric. Biol. Chem. 49:2485–2487.
30. Olano, C., A. M. Rodrı́guez, C. Méndez, and J. A. Salas. 1995. A second ABC
transporter is involved in oleandomycin resistance and its secretion by Strep-
tomyces antibioticus. Mol. Microbiol. 16:333–343.
31. Reddy, M., and J. Gowrishankar. 1997. Identification and characterization of
ssb and uup mutants with increased frequency of precise excision of trans-
poson Tn10 derivatives: nucleotide sequence of uup in Escherichia coli. J.
Bacteriol. 179:2892–2899.
32. Reynolds, E., J. I. Ross, and J. H. Cove. 2003. Msr(A) and related macrolide/
streptogramin resistance determinants: incomplete transporters? Int. J. An-
timicrob. Agents 22:228–236.
505
33. Ross, J. I., E. A. Eady, J. H. Cove, W. J. Cundliffe, S. Baumberg, and J. C.
Wootton. 1990. Inducible erythromycin resistance in staphylococci is en-
coded by a member of the ATP-binding transport super-gene family. Mol.
Microbiol. 4:1207–1214.
34. Ross, J. I., E. A. Eady, J. H. Cove, and S. Baumberg. 1996. Minimal func-
tional system required for expression of erythromycin resistance by msrA in
Staphylococcus aureus RN4220. Gene 183:143–148.
35. Rosteck, P. R., Jr., P. A. Reynolds, and L. J. Babaszak. 1991. Homology
between proteins controlling Streptomyces fradiae tylosin resistance and
ATP-binding transport. Gene 102:27–32.
36. Russel, J. B. 1992. Another explanation for the toxicity of fermentation acids
at low pH: anion accumulation versus uncoupling. J. Appl. Bacteriol. 73:
363–370.
37. Schoner, B., M. Geistlich, P. Rosteck, Jr., R. N. Rao, E. Seno, P. Reynolds,
K. Cox, S. Burgett, and C. Hershberger. 1992. Sequence similarity between
macrolide-resistance determinants and ATP-binding transport proteins. Gene
115:93–96.
38. Steiner, P., and U. Sauer. 2001. Proteins induced during adaptation of
Acetobacter aceti to high acetate concentrations. Appl. Environ. Microbiol.
67:5474–5481.
39. Swings, J. 1992. The genera Acetobacter and Gluconobacter, p. 2268–2286. In
A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.),
The prokaryotes, 2nd ed., vol. 3. Springer-Verlag, New York, N.Y.
40. Takemura, H., S. Horinouchi, and T. Beppu. 1991. Novel insertion sequence
IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing
ability. J. Bacteriol. 173:7070–7076.
41. Takemura, H., K. Kondo, S. Horinouchi, and T. Beppu. 1993. Induction by
ethanol of alcohol dehydrogenase activity in Acetobacter pasteurianus. J.
Bacteriol. 175:6857–6866.
42. Tamaki, T., M. Fukaya, H. Takemura, K. Tayama, H. Okumura, Y.
Kawamura, M. Nishiyama, S. Horinouchi, and T. Beppu. 1991. Cloning and
sequencing of the gene cluster encoding two subunits of membrane-bound
alcohol dehydrogenase from Acetobacter polyoxogenes. Biochim. Biophys.
Acta 1088:292–300.
43. Tayama, K., M. Fukaya, H. Okumura, Y. Kawamura, and T. Beppu. 1989.
Purification and characterization of membrane-bound alcohol dehydro-
genase from Acetobacter polyoxogenes sp. nov. Appl. Microbiol. Biotechnol.
32:181–185.
44. Wong, H. C., A. L. Fear, R. D. Calhoon, G. H. Eichinger, R. Mayer, D.
Amikam, M. Benziman, D. H. Gelfand, J. H. Meade, A. W. Emerick, R.
Burner, A. Ben-Bassat, and R. Tal. 1990. Genetic organization of cellulose
operon in Acetobacter xylinum. Proc. Natl. Acad. Sci. USA 87:8130–8134.
45. Yamada, Y., K. Hoshino, and T. Ishikawa. 1997. The phylogeny of acetic acid
bacteria based on the partial sequences of 16S ribosomal RNA: the elevation
of the subgenus Gluconoacetobacter to the genetic level. Biosci. Biotechnol.
Biochem. 61:1244–1251.
46. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage
cloning vectors and host strains: nucleotide sequences of the M13mp18 and
pUC19 vectors. Gene 33:103–119.