The DNA adenine methyltransferase-encoding gene (dam) of Vibrio cholerae

Gene, 140 (1994) 67771 0 1994 Elsevier Science B.V. All rights reserved.

GENE

67

0378-l 119/94/$07.00

07683

The DNA adenine methyltransferase-encoding

gene (dam) of

Vibrio cholerae (Spontaneous sequence)

mutation;

DNA mismatch

Rupa Bandyopadhyay* Biophysics

Dirision,

Indiun lnsriture

Received by S.R. Kushner:

repair; strand

and Jyotirmoy ofChemical

discrimination;

cloning;

expression;

base analogs;

nucleotide

Das

Biology Calcuttu 7OOOJ2. India

5 May 1993; Revised/Accepted:

7 September/22

September

1993; Received at publishers:

4 November

1993

SUMMARY

The DNA adenine methyltransferase (MTase)-encoding gene (dam) of Vibrio cholerae, an organism belonging to the family Vibrionaceae, has been cloned and the complete nucleotide (nt) sequence determined. I’. cholerae dam encodes a 21.5kDa protein and is directly involved in methyl-directed DNA mismatch repair. It can substitute for the Escherichia coli enzyme

and can suppress the phenotypic traits associated with E. coli dam mutants. Overproduction Dam MTase does not result in hypermutability in either I/. cholerae or E. coli cells. Overproduction of I/. in a pUC plasmid, however, fails to suppress the 2-aminopurine (2-AP)-sensitive phenotype of E. coli Homology between the nt and deduced amino acid (aa) sequences of the E. coli and I/ cholerae dam 30-35%.

INTRODUCTION

The dam gene of Escherichia coli encodes an MTase which methylates the adenine residue at the N6 position in the sequence S-GATC-3’ (Lacks and Greenberg, 1977; Hattman et al., 1978). Mutation in the dam gene results in hypermutability, hyperrecombination, sensitivity to DNA base analogs such as 2-AP and inviability in the Correspondence to: Dr. J. Das, Biophysics Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700032, India, Tel. (91-33) 473-0350; Fax: (91-33) 473-0284; e-mail: iicb%[email protected] *Present address: The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA. Abbreviations: aa, amino acid(s); Ap, ampicillin; 2-AP, 2-aminopurine; Bla, B-lactamase; hla (ApR). gene encoding Bla; bp, base pair(s); cfu, colony-forming unit; Cm, chloramphenicol; Dam, DNA adenine MTase; dam, gene encoding Dam; kb, kilobase or 1000 bp; LB-agar, LuriaBertani medium containing 1.5% agar; MTase, methyltransferase; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; R, resistance/resistant; ‘, sensitive/sensitivity; Tc, tetracycline; Tn. transposon UV, ultraviolet light; V., Vihrio. SSDI

0378-l

119( 93)E0679-8

of I’. cholerae Dam dam mutants. genes is only

cholerae

presence of a second mutation the recA, recB or recC genes (Marinus and Konrad, 1976; Glickman et al., 1978; Bale et al., 1979; Craig et al., 1984). The role of Dam in DNA mismatch repair has now been established (Claverys and Lacks, 1986; Radman and Wagner, 1986; Modrich, 1987). Because of the lag between replication and methylation by Dam, the newly synthesized DNA strand is transiently undermethylated. The repair enzymes, for which unmethylated or undermethylated strands are the best substrates, can remove mismatched bases from the newly synthesized strand. Over- and underproduction of the dam gene product result in hypermutability (Herman and Modrich, 1981; Marinus et al., 1984). Dam methylation has so far been reported only in enterobacteriaceae such as E. coli, Salmonella typhimurium and Streptococcus pneumoniae. The T4 dam gene product (Schlagman and Hattman, 1983) cannot suppress the hypermutable phenotype of E. coli dam mutants but does suppress all the other phenotypic traits associated with such mutations. Schlagman et al. (1986) con-

68 eluded that the T4 Dam could not fully substitute for the E. coli enzyme. The presence of Dam in 1/. cholerue, an organism

belonging

to the family Vibrionaceae

and the

causative agent of the diarrhoeal disease cholera, has been reported (Bandyopadhyay et al., 1989). Mutants sensitive isolated.

to 2-AP with intact -GATC- Dam activity These mutants exhibit normal spontaneous

tation frequencies

but are sensitive

agents and 9-amino can be suppressed into the mutant sensitivity cholerae.

acridine.

cells. Thus,

and the Dam

to UV light, alkylating

All the mutant

by introducing unlike

activity

were mu-

phenotypes

the E. coli dam gene in E. coli, the 2-AP are not coupled

EcoRI ,/SomHI

in I/.

It has not been possible to isolate darn- V. choThese observations have prompted us to

lerae mutants.

examine

the dam gene of I/. cholerue in greater detail. The

present report describes the identification and cloning of the dum gene of V. cholerue and characterization of its product.

EXPERIMENTAL

AND

DISCUSSION

(a) Cloning of the dam gene To identify the dum gene of I’. cholerue, the DNA from the hypertoxinogenic strain 569B of T/ cholerue was digested with EcoRI and hybridized with a nick-translated E. coli dam gene. Hybridization was observed only at very low stringency and by developing the autoradiogram for more than 72 h. The E. coli dam gene hybridized with EcoRI-digested 1/. cholerue DNA in the 3.5-kb region. Using DNA fragments of this region, a minibank was constructed in the pBR328 plasmid. Since dam-ret double mutants are inviable (Marinus and Morris, 1974; Marinus and Konrad, 1976) the gene bank was maintained in E. coli D1210 (Storz et al., 1990) which is dam+ ret-. CmS ApR TcR colonies were selected as clones harboring the recombinant plasmids. The recombinant plasmids isolated from several hundred clones were individually transferred into E. coli GW38 10 (JM 103 dum::Tn9). Plasmids were isolated from the transformed cells and were digested with Mb01 and DpnI. These enzymes recognize the sequence 5’-GATC but differ in their response to the states of methylation of the adenine residues. The DNA from two recombinant plasmids was resistant to MhoI digestion and sensitive to the enzyme DpnI. Both plasmids carried a 3.2-kb insert which had a single BumHI site producing 2.2- and l.O-kb fragments. The two fragments were separately cloned into EcoRI + BumHI-digested pBR328 plasmid and transformed into E. coli GW3810. Cells carrying the 2.2-kb DNA fragment conferred differential sensitivities to DpnI, Mb01 and Suu3A in E. coli dam mutants. The recombinant plasmid carrying the 2.2-kb I/. cholerue DNA frag-

Fig. 1. The 2.2-kb fragment

of I/ cholerae

DNA carrying

the dam gene

(a), the solid bar represents the 0.7-kb coding region, and construction of recombinant plasmids pRBlO1 and pRB102 carrying the 2.2-kb V. c/derm DNA fragment(b). The thin line represents pBR328 or pUC19, and thick line, chromosomal insert.

ment is referred to as pRBlO1 (Fig. lb). A physical map of the 2.2-kb DNA fragment was constructed using several restriction endonucleases (Fig. la). To confirm that the 2.2-kb DNA contained the dam gene of If. cholerue, the pRBlO1 plasmid was mutagenized in vitro by hydroxylamine. To 50 ul of plasmid suspension (0.2 ug/ul in 1 M Na.acetate buffer pH 4.9), 150 ul of 2 M hydroxylamine in Na.acetate buffer was added and the mixture was incubated at 55°C for 1 h. The pH of the suspension was adjusted to 7.6. The mutagenized plasmid was used to transform GW3810, and 2-AP-sensitive transformants were selected. The plasmids isolated from these cells were susceptible to MhoI digestion and resistant to DpnI. The 2.2-kb I/. cholerue DNA in the plasmids isolated from these sensitive cells were cloned into plasmid pBR328 and transformed into E. coli dam mutant. All transformed cells were sensitive to 2-AP and the plasmids isolated from these cells were resistant to DpnI and susceptible to MhoI digestion. These results eliminate the possibility that mutation in the plasmid vector produced the observed effect. (b) The 2.2-kb DNA fragment can complement E. coli dam mutants The product of the 2.2-kb I/. cholerue DNA can suppress the phenotypic traits associated with the E. coli dam

69 mutants

and can substitute

representative

experiment,

for the E. coli enzyme. 85

spontaneous

In one

a

b

c

d

rifampin-

-bla

resistant clones were recovered from 10’ cfu when cells of strain GW3810 were plated on LB-agar plates containing 10 pg rifampiq’ml. The number of spontaneously mu-

-21

kDa

tated cells was reduced to only 2 per 10’ cfu when GW3XlO cells carrying the pRBIO1 plasmid was used. The experiment

was repeated

several times and the results

were highly reproducible. Thus, the dam gene of I/: cholerae can functionally complement the hypermutable phenotype of E. coli dam mutants. The UV-sensitive phenotype was suppressed

in presence

of E. coli dam mutants

of V. choterae Dam. The sur-

vival of GW3810 cells carrying the pRBlO1 plasmid was more than 90% at 10 J/m2 does of UV light, in contrast to only 1% survival

of the mutant

cells not containing

plasmid (Fig. 2a). The sensitivity of E. coli dam mutants towards 2-AP was also suppressed in cells transformed with pRB101. While only 10% of the GW3810 cells survived in presence of 5-10 yg 2-AP/ml, the survival of cells carrying pRBlO1 was almost loo%, even in the presence of 400 pg 2-AP/ml (Fig. 2b). (c) The dam gene product The protein(s) encoded by the 2.2-kb K cholerae DNA fragment were examined in maxicells. Cells of E. coli CSR603 (Sancar et al., 1979) carrying pRBlO1 were irradiated with UV light, labeled with ~35S]methionine for 1 h and the extract of soluble proteins was analysed by SDS-PAGE. The 2.2-kb DNA encodes a 21.5kDa protein (Fig. 3) that was produced in large amounts in maxicells (Fig. 3). Overproduction of the E. co/i Dam protein results in hypermutability (Herman and Modrich, 1981).

Fig. 3. Identification of the “SS-labeled product coded and 0.7-kb T/. cholevae DNA fragments. UV-irradiated

by the 2.2-kb E. coli strain

CSR603 carrying pUC19 (lane a), pRBlO1 (lane b), pRB102 (lane c) or pUC19 containing the 0.7-kb insert (lane d). Equal amount of radioactivity was loaded in each lane. The number represents the molecular mass of the protein in kDa. Methods ~lasmid-coded proteins were examined using the maxicell strain CSR603 of E. cc& (Sancar et al., 1979) carrying the plasmids of interest. Transformed cells were exponentially grown in minimal media containing 1% casamino acids, irradiated with UV light (50 J/m’) and incubated at 37’C. After 1 h r+cycloserine was added per ml and the incubation another 16 h at 37°C. The cells were then harvested, sulfur-depleted medium. incubated at 37-C for 1 h

incubation, 200 pg was continued for suspended in fresh and proteins were

labeled with [“‘Slmethionine (5 $iiml, Amersham). The labeled cells were harvested, washed and the whole cell lysates were analysed by 0.1% SDS-IO% PAGE followed by autoradiography of dried gels.

Overproduction was neither

of the V. cholerne Dam protein in E. coli lethal for the cells nor were they

hypermutable. Similar results were obtained when the cloned K cholerae dam gene was transformed into V. cholerae cells (Panda et al., 1991) and the synthesis of the product was examined under maxicell experimental conditions (data not shown). Dam was synthesized in large amount and the cells were not hypermutable. Thus, besides being smaller than the 31-kDa Dam protein of E. coli (Herman and Modrich, 1981) overproduction of V. cholerae Dam does not result in hypermutability. This property of 5’. choterae Dam protein is similar to that reported for the T4 Dam (Schlagman et al., 1986).

(d) Vector-dependent expression of V. cholerae dam gene

1

1

I

j W&4,,,,.,

Fig. 2. Survival or pRBlO2 (0) of 2-AP (b).

light (J/m’>

2AP(pg/mif

of E. coli GW3810 (A) and cells carrying pRBlO1 (0) following irradiation with UV light (a) or in presence

When the 2.2-kb DNA was cloned into pUC19 between the EcoRI and BanzHI sites, and the recombinant plasmid designated pRB102 (Fig. lb) was used to transform E. coli dam mutants, all transformed cells were sensitive to 2-AP. Less than 2% of transformants survived in the presence of 200 pg 2-AP/ml (Fig. 2b) in contrast to 100% survival when the pRBlO1 plasmid was used for transformation. The 2.2-kb insert from pRB102 was recloned into pBR328 and the recombinant plasmid (pRB103) was used to transform E. coli dam mutants. All transformed cells were 2-AP resistant, thereby eliminating the possibility that the 2.2-kb DNA fragment might have either been modified or mutated. when cloned in pUC vectors. The copy number of pUC vectors is about lo-20-times higher than that of pBR vectors. The 2-APsensitive phenotype of V. cholerae dam gene is, thus, de-

70

pendent un the number of copies of the 2.2-kb DNA fragment present in pRB102, confer as much UVR (Fig. 2a) and suppress table phenotype of an E. coli dnttz mutant DNA fragment of pRBlO1. It also encodes protein (Fig. 3).

protein. The however, can the hypermuas the 2.2-kb the 21.5kDa

(e) Nocleoride sequence The length of the DNA segment encoding the &na gene was further reduced by digesting the 2%kb DNA with Hi&I which has two sites producing three fragments of 0.9, 0.7 and 0.6 kb (Fig. la). The 0.7-kb fragment complemented the E. coli darn mutation and encoded the 21.5-kDa protein (Fig. 3). A detailed physical map of the 0.7-kb DNA was constructed (Fig. la) and the completc nt sequence of this fragment was determined (Fig. 4), There are two ORFs of 192 and 191 aa, corresponding to 21.3- and 21.2-kDa proteins. The first ORF has the consensus Shine-Dalgarno sequence, AGAAG, eight nt upstream from the start codon. Two GATC sequences are located in tandem between - 30 and - 39 positions upstream from the start codon. The presence of the con-

sensus Shine-Dalgarno sequence is reflected in the high level of expression of the gene product. The upstream sequences of this ORF are similar to E. co& promoters whose expression is inffuenced by adeniae methy~ation. The nt and deduced aa sequences of the V. cholerae dam gene were compared with those of the E. co/i gene. Homology between these two genes both at the nt as well as the aa levels were only about 30-35%. This explains why the Ir. chofe~ar &m gene hybridized with the E. co& gene only at a very tow stringency. The lack sf homology between the E. coli and the K cholerae proteins was surprising in view of the fact that the product of the V. cholerar dam gene can functionalfy complement E. coli dam mutants. When the possible secondary structures of li choferne and E. co& Dam were predicted from the deduced aa sequences by calculating the propensities for each aa to be in z-helix, /?-sheet or fi (reverse) turn, it was observed that residues 58-94 of V. cholerue protein and residues 100-136 of E. co& protein may lead to the same secondary structure. Similarly, the residues 98-152 of ti. cholerae protein and residues 196-249 of E. coti protein may lead to similar structures. These domains might be responsible for the functional complementation. However, structural predictions from primary sequences are highly speculative and the reliability of such predictions is questionable.

We are. grateful to Dr. Chitra Dutta of the Institute for her help in the computation work. The investigation was supported by the Department of Science and Technology (Grant No. SP/SO/D-U/90), Government of India.

REFERENCES Bale. A., d’Atarco* M. and Marinus, M.G.: Characterization adenine methyfation mutants of ~s~~~~j~~~~~coli K-12.

~T~~~~~~Th~~A~~~A~ ~~N~;~~A~~cAcTGG

luiCs&

798 74s

Fig. 4. The nt sequence of the darr! gene of 1/. cholvrae and deduced aa sequence. Sequencing was done by the dideoxynuclcotide chain termination method (Sanger et al., 2977). The aa are numbered on the left margin and aligned with the second nt of each codon; * =stop codon, The nt and deduced aa sequeoce of the t’. ikule?~~e $urn gene has been reported to the EMBL Data Bank under accession No. X67820.

of DNA Mutation

Res. 59 (1979) 157-165. Bandyopadhyay, R., Sengupta. A. and Das, J.: A mutation in the dum gene of I/ibrr’o chnlerae: 2-aminopurine sensitivity with intact GATC methylase activity. Biochem. Wiophys. Res. Commun. 165 (1989) 561-567. Claverys, J.-P. and Lacks, S.A.: Heterodnplex deoxyribonucleic acid base mismatch repair in bacteria. Microbial. Rev. 50 ( 1986) 133-l 56. Craig, R.J., Arraj, J.A. and Marinus, M.G.: Induction of damage inducible (SOS) repair in clum mutants of Escherichia co/i exposed to 2-aminopurine. Mol. Gen. C&net. Glickman, B.W., Van der Elsen, P. and esis in &m mutants of Escherichia residues in mutation avoidance. 307-3 f 2. Hattman,

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regulator of by oxidation.