Cloning and sequencing the recA+ genes of Acetobacter polyoxogenes and Acetobacter aceti: construction of recA− mutants of by transformation-mediated gene replacement

Gene, 127 (1993) 47-52 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/%06.00

47

GENE 07028

Cloning and sequencing the recA + genes of Acetobacter polyoxogenes and Acetobacter aceti: construction of recA- mutants of by transformationmediated gene replacement (Recombination; acetic acid bacteria; RecA protein; PCR)

Kenji Tayama”, Masahiro Fukaya”, Hiroshi Takemuraa, Hajime Okumuraa, Yoshiya Kawamuraa, Sueharu Horinouchib and Teruhiko Beppub “Nakano Central Research Institute. Nakano Vinegar Co., Ltd., Handa, Aichi 475, Japan; and bDepartment of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. Tel. (81-3) 3812-2111, ext. 5123

Received by A. Nakazawa: 9 September 1992; Revised/Accepted: 11 November/26 November 1992; Received at publishers: 6 January 1993

SUMMARY

The recA+ gene of Acetobacter polyoxogenes was cloned as a gene that conferred methyl methanesulfonate resistance (MMSR) on the RecA- Escherichia coli HBlOl. The cloned recA+ gene also conferred (i) resistance to UV irradiation, (ii) enhanced intrachromosomal recombination, and (iii) permitted prophage 4 80 induction in E. coli recA- lysogens. Nucleotide sequence determination revealed that the recA product consists of 348 amino acids (aa) corresponding to 38 kDa, and shows significant similarity to RecA proteins from other Gram- bacteria. Next, a portion of recA from Acetobacter aceti was cloned by using polymerase chain reaction with oligodeoxyribonucleotide primers design based on the A. polyoxogenes recA sequence. Due to availability of efficient host-vector and transformation systems in A. aceti, recA mutants of A. aceti were obtained by transformation-mediated gene replacement with the cloned A. aceti recA gene which was inactivated by insertion of the kanamycin-resistance-encoding gene from pACYC177. The recA mutants obtained in this way showed similar phenotypes to those of E. coli recA strains, such as increased sensitivity to MMS and to UV irradiation, and decreased homologous recombination.

INTRODUCTION

Genus Acetobacter comprises Gram-, strictly aerobic bacteria used for producing vinegar (acetic acid fermentation) as well as bacterial cellulose (De Ley et al., 1984; Correspondence to: Dr. M. Fukaya, Nakano Central Research Institute, Nakano Vinegar Co., Ltd. Nakamura-cho 2-6, Handa, Aichi 475, Japan. Tel. (81-569) 24-5030; Fax (81-569) 24-5028.

Abbreviations: A., Acetobacter; aa, amino acid(s); Ap, ampicillin; bp, base pair(s); E., Esherichia; kb, lcilobase(s) or 1000 bp; Km, kanamycin; LB, Luria-Bertani (medium); MMS, methyl methanesulfonate; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; R, resistance/resistant; RecA, recombination enzyme; recA, gene encoding RecA, SD, Shine-Dalgarno (sequence); UV, ultraviolet light; YPG, 0.5% yeast extract/0.2% peptone/3% glucose; [ 1, denotes plasmid-carrier state.

Yamanaka et al., 1989). We have developed efficient hostvector systems (Okumura et al., 1985; 1988; Fukaya et al., 1985a, b) important for strain improvement (Fukaya et al., 1989), but also encountered the instability of cloned genes probably due to homologous recombination between chromosomal DNA and plasmid DNA or between plasmids (Valla et al., 1987). We decided therefore to develop recombination-deficient hosts. In E. coli, homologous recombination is governed by several Ret functions, one of best studied being RecA. Interspecies functional complementation among RecAlike proteins in several Gram- and Gram+ bacteria have indicated a strong conservation of these recA-encoded proteins (Ramesar et al., 1989; Miller and Kokjohn, 1990; Zhao and McEntee, 1990; Dybvig et al., 1992). Since various bacterial recA mutants show extremely reduced fre-

48 quencies of homologous recombination (Goldberg and Mekalanos, 1986; Liao and Liu, 1989; Ridder et al., 1991), it was conceivable that an Acetobacter recA mutant might be useful in ensuring the stability of vectors as well as cloned genes. We therefore first cloned and characterized the recA gene from A. polyoxogenes (Entani et al., 1985). Next the recA gene was cloned from A. aceti by the polymerase chain reaction (PCR) method using the primers designed on the basis of information of the A. polyoxogenes recA gene. By gene replacement using the cloned gene a recA mutation was constructed in the chromosome of A. aceti, in which genetic manipulation can be carried out more easily than in A. po~yo~ogenes.

RESULTS AND DISCUSSION

(a) CIoning of the recA gene from Acetobucter ~of~oxoge~es

Using pHC79 cosmid vector we constructed a genomic library of the chromosomal DNA from A. polyoxogenes in RecA- E. coli HBlOl (see legend for Fig. 1). RecAf transformants of HBlOl were selected on LB plates containing Ap (50 ~g/ml) and MMS (1.3 mM). Ten MMSR colonies among approximately 1600 ApR transformants were isolated. Restriction analysis of the plasmid DNAs purified from these transformants showed that all contained an approximate 30-kb insert exhibiting a similar restriction pattern. One of them (pMVRH2) was used for further studies. Plasmid pMVRH2 was found to confer also UV resistance on E. coli HBlOl, in addition to MMS resistance (Table I). We then subcloned the region conferring resistance to MMS and UV in the pMV330 vector (Fukaya et al., 1985b). The resultant plasmid (pMVRP1)

contained a 4.0-kb PstI-fragment (Fig. 1), which conferred UV resistance (Table I). It was confirmed by Southern hybridization that the inserted DNA fragment originated from A. polyoxogenes (data not shown). To confirm that this PstI fragment contains a gene which complements E. coli recA, we characterized the E. coEi[pMVRPl]. It is well known that the E. coli RecA is a key protein for homologous gene recombination, DNA repair, and a protease-iike activity including prophage induction. Restoration of homologous recombination was assayed as (i) the frequency of chromosomal recombination and (ii) spontaneous induction of the 4 80 prophage. The frequency of intrachromosomal recombination was determined by ~-complementation of two separate j3-galactosidase-encoding genes located on the same chromosomal DNA, using E. coli GY7066 {&MS286 (4 80dIIlacBKl) ArecA306) (Dutreix et al., 1989). The frequency of complementation was enhanced over a thousand times upon introduction of pMVRP1; the ratio of Lac+ colonies/Lac- colonies was 21.2% in the case of E. coli GY7066[pMVRPl], whereas that of GY7066[pMV330] was below 0.05%. Restoration of homologous recombination was also tested by the frequency of Leu’ colonies obtained by a direct transfo~ation (Hanahan, 1985) of Leu- E. coli HBlOl (1 x lo9 cells) with genomic DNA (10 pg) of Leu+ E. coli K-12. When

TABLE I UV resistance of Escherichia coli HBlOl transformants Plasmid”

pMV330 pMVRH2 pMVRP1

Number of viable cells after UV irradiation (~lls~ml)b None (0 min)

40 J/m” (1 min)

100 J/m2 (2.5 min)

3.6 x 10’ 1.6 x IO8 2.6 x lo*

2.0 x 10’ 7.5 x IO5 4.6 x 106


“Plasmid pMV330 (Fukaya et al., 1985a); pMVRH2 and pMVRP1 (section a). bTo determine UV survival, logarithmically growing E. coli cells were collected, suspended in 1 ml of distilled water (1 x 10s cells/ml), spotted on sterile dishes, and then exposed to ge~icidal UV lamp (254nm, 60 W) at a constant distance (60 cm). After irradiation (0.1 and 2.5 min), 0.1 ml aliquots were diluted and plated on LB plates, incubated at 37”C, and surviving cells were counted.

Fig. 1. Restriction map of the 4-kb PstI fragment and the results of subcloning and insertional inactivation. Open triangles (a), sites of insertion of KmR; broken line ( - - - ), putative coding region of the recA gene (based on sequence shown in Fig. 2). Methods: The 4-kb PsrI fragment was cloned as follows. A genomic library of A. polyoxogenes NBI1028 (Entani et al., 1985) was constructed by using cosmid vector pHC79 (ApR, TcR,cos) (Hohn and Collins, 1980) essentially as described by Goldberg and Ohman (1984). Total DNA of A. polyoxogenes NBI1028, prepared as described by Okumura et al. (1985), was partially digested with &mHI and the digest was ligated to ~~HI-~gested pHC79 DNA. The ligation mixture was then introduced into E. coli HBlOl (Boyer and Roullard-Dussoix, 1969) as described previously (Fukaya et al., 1989). Plasmid pMV330 (ApR, E. coli/Acetobacter shuttle vector; Fukaya et al., 1985a) was used in the subcloning of a fragment encoding recA gene. E. coli HBlOl was transformed as described by Hanahan (1985). The Hue11 fragment containing the KmR gene derived from E. coli plasmid pACYCl77 (Chang and Cohen, 1978) was used for inactivation of the target gene by insertion at the EcoRI-1, EcoRI2 and Sal1 sites (Fukaya et al., 1990). Km and Ap were added at final concentrations of 100 pg/ml and 50 pg/ml, respectively.

49 HBlOl[pMVRPl] was transformed, 21 Leu+ colonies obtained per 10 pg DNA; no Leu+ transformants appeared when the HBlOl[pMV330] control recipient was used.

Spontaneous induction of GY6796 (AllrecA21 (+80atth)} examined using GY4925 as (Devoret et al., 1983) by the

$80 prophage in E. coli (Dutreix et al., 1989) was the indicator bacterium method of Keener et al. 120 29

240 69

360 109

480 149

600 189

720 229

840 269

960 309

1080 349

1200 Fig. 2. The nt sequence of the recA gene from A, po/yoxogenes NBI10’28 and deduced aa sequence, both aligned with the nt and deduced aa sequences of the recA fragment from A. aceti No. 1023. The nt sequences of the recA gene from A. polyoxogenes and recA fragment of A. aceti No. 1023 were determined by the dideoxy chain-termination method (Sanger et al., 1977) using phages M13mp18 and M13mp19 (Messing, 1983). The putative SD sequence of the recA gene from A. polyoxogenes is underlined, and an inverted repeat downstream from the stop codon is indicated by convergent arrows. Amplification of the partial recA gene of No. 1023 was carried out by PCR as follows. Two oligo primers 5’ -GGGGATCCATGTCGACAACCTGCTGATC and 5’ -GGCTGCAGAGGAGAACCACGCGCCGGAC, where the underlined sequence is programmed for the BumHI or PstI site, were synthesized on a DNA synthesizer (model 380B; Applied Biosystems, Foster City, USA) and used for PCR. Total DNA of A. aceti No. 1023 was prepared by the previously described method (Okumura et al., 1985). The conditions for PCR were as described by Tamaki et al. (1991). The nt sequence of the partial recA gene of A. aceti No. 1023 is shown below that of the recA gene from A. potyoxogenes (nt 372-920) with its deduced aa sequence. Sites of two opposing primers which were used for PCR (complementary sequence for one of them) are underlined with broken lines in the nt sequence of the ~plified fragment, but the extrinsic BumHI and PstI sites are omitted. Those aa which are different from that of A. po~yoxogenes NB11028 are indicated. Asterisks indicate the same aa as that from A. polyoxogenes NBI1028. The nt sequence data has been deposited in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession No. D13183 and D13184.

50 (1984). When the host contained the PscI fragment phage induction was enhanced by about 50-fold; 4.3 x 10’ (control) and 2.1 x 104 phages/ml of the culture (1 x 10s cells/ml) were produced in GY6796[pMV330] and GY6796[pMVRPl], respectively. All the above data suggested that the cloned gene corresponded to the recA+ gene of A. polyoxogenes. In addition, the aa sequence of the gene product of the cloned fragment showed significant similarity to that of RecA proteins of other bacteria, as described below. Therefore, we conclude that the cloned gene corresponds to recA of A. polyoxogenes. To our knowledge, this is the first report on cloning of the Acetobacter recA gene,

(b) Nucleotide sequence of recA of Acefobacfer ~o~yo~o~e~e~ To determine the coding region of rec.A in the P&I fragment, we constructed several plasmids carrying various portions of the PstI fragment and tested their ability to restore the recA phenotypes of E. coli HBlOl. Moreover, we inserted the KmR gene of pACYC177 into three restriction sites (see Fig. 1). As shown in Fig. 1, all constructs, with one exception, failed to confer UV resistance on HBlOl, suggesting that the recA gene is located in the left-to-middle portion of the PstI fragment, to the left of the EcoRI-2 site. We then sequenced the corresponding region and found an ORF possibly encoding RecA protein. This ORF consists of 348 aa (Fig. 2). The EcoRI site (nt 8 18-823) and the Sal1 site (nt 470-475) at which the KmR gene insertion abolished the recA function are located in the coding region. The possible SD sequence is present 5 bp upstream from the start codon. An inverted repeat sequence forming a stem-loop structure which possibly functions as a transcription terminator is located downstream from the coding region. The overall G + C content of the coding sequence is 62.9 mol% and those for codon positions 1, 2 and 3 are 67.0 mol%, 42.8 mol% and 79.0 mol%, respectively. The aa sequence deduced from the nt sequence shows high similarity to RecA proteins of E. coli and two other Gram- bacteria (Horii et al., 1980; Sancar et al., 1980; Sano and Kageyama, 1987; Ball et al., 1990) (Fig. 3). The ATP-binding sequence and the aa responsible for the recombination region determined for the E. coli RecA protein (Kawashima et al., 1984; Knight and McEntee, 1985; Wang and Tessman, 1986; Kowalczykowski et al., 1989) are well conserved in the A. ~o~yoxogenes RecA protein (Fig. 3). Therefore, we believe that cloning the reed-like gene from other Acetobacter strains is possible by using PCR.

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170*

180

&p uAcwG i!c ______ #AID EC HD pa MAID s!

VVAE~LVGPD~~S --___-___-______ ~ST~FSVD~EGV~~DFX KSGPVKADAEEVADAEADX SGGELVMSGDDFEDDEAETSEPFX Fig. 3. Alignment

of

aa

sequences

of

RecA

proteins

from

A. polyoxogenes, A. aceti, E. coli, Pseudomonas aeruginosn and Serratia

rnarcescens. The aa sequences were deduced and analyzed by using the GENETEX sequence analysis program (Software Development Co., Tokyo, Japan). Identical aa are boxed. Numbering is as in Fig. 2, with the last digit of each number aligned with given aa. The aa marked with asterisks are responsible for the recombinase activity (Argifi9, Ilez2s, and Gly30’), protease activity (Glyzo4), prophage induction (IIe’25), ATP-binding (Ty?), and thermostability (Va1246and Ile2’s) in E. coli. Sources are: A. poiyoxogenes (Ap) (Fig. 2); A. aceti (Au) (Fig. 2); E. coii (EC) (Horii et al., 1980; Sancar et al., 1980); P. ueruginosa (Ps) (Sane and Kageyama, 1987); S. marcescens (Sm) (Ball et al., 1990).

(c) Construction of recA mutant of Acetobucter aceti by gene replacement To obtain recA_ mutants, the gene replacement method can be used. A disrupted recA gene is introduced as a linearized plasmid into a RecA+ host and subjected to homologous recombination between the intact chromosomal recA gene and the introduced disrupted gene. We attempted to isolate recA_ mutants of A. polyoxogenes by this method, but we failed because it was nearly impossible to grow colonies of this strain on solid media (Entani et al., 1985). We then changed the target from A. polyoxogenes to A. aceti No. 1023, since efficient host-vector and transformation systems for the latter strain were developed by our group. We amplified an internal part of the recA gene of A. aceti No. 1023 by PCR using two opposing oligo

51 recA

mutants can be easily obtained by the genereplacement method with the cloned mutagenized recA gene; such a strategy is very useful for engineering recA mutants of Acetobacter, which grow normally and thus will be useful for biotechnological applications of gene manipulation in Acetobacter, e.g., improving acetic acid production or cellulose biogenesis.

I

I

I

I

0 5 10 UV

I

I

60

20 irradiation

(min)

Fig. 4. UV survival of KmR recA- mutant of A. aceti IO-80Sl. A. aceti lo-80Sl (Okumura et al., 1985), a pro- mutant from A. aceti No. 1023 (Ohmori et al., 1980), was used as a host. UV resistance was determined as described in footnote b to Table I, except that treated cells (1 x 10’ cells/ml) were incubated on YPG plates at 30°C. UV dose was 42 J/m2/min. Closed circles (O), A. aceti lo-80Sl (RecA* control); open circle (0), KmR mutant of A. aceti lo-80Sl transformed with the partial recA gene from No. 1023 inactivated by insertion of the HaeII fragment of pACYC177 into the MluI site (Fig. 2).

primers encoding the conserved sequences (see legend for Fig. 2). The amplified DNA fragment was about 0.55 kb, which was in good agreement with the size expected from the A. polyoxogenes recA gene. This fragment having a BumHI site at one end and a PstI site at the other end was cloned into M13mp18 and M13mp19. As expected, the aa sequence encoded by the cloned fragment was found to be quite similar to that of the RecA protein of A. polyoxogenes (Fig. 2 and 3). Only eight aa in a total of 183 aa were different, To construct a recA mutant, the Hue11 fragment containing the KmR gene of E. coli plasmid pACYC177 (Chang and Cohen, 1978) was inserted by ligation into the MluI site (nt 767-772 in Fig. 2) in the A. uceti recA gene. The resulting recombinant plasmid was digested with BumHI and PstI in order to remove a DNA fragment containing the replication origin functioning in Acetobucter and to obtain a linear DNA for efficient homologous recombination. The linearized DNA fragment was introduced by transformation into A. uceti lo8OS1, a pro- mutant derived from A. uceti No. 1023, and KmR transformants obtained were tested for their MMS and UV resistance. Resistance to MMS of almost all KmR transformants was reduced (below 0.5 mM) when compared with the parental strain (2.0 mM), as judged by growth on YPG plates containing various concentrations of MMS. KmR transformant also became more sensitive to UV irradiation (Fig. 4), and their complementation of the pro auxotrophy by transformation reduced from 2.9 x lO’/ug DNA to less than l/ug DNA. Such phenotypes of KmR transformants clearly indicated that these were RecA-. In the present study, we have shown that

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