Nucleotide Sequence of Plasmid p4028, a Cryptic Plasmid fromLeuconostoc oenos

PLASMID

35, 67–74 (1996) 0034

ARTICLE NO.

Nucleotide Sequence of Plasmid p4028, a Cryptic Plasmid from Leuconostoc oenos MANUEL ZU´N˜IGA, ISABEL PARDO,

AND

SERGI FERRER

Facultat de Biologia, Departament de Microbiologia, Universitat de Vale`ncia, E-46100 Burjassot-Valencia, Spain Received February 6, 1996; revised May 6, 1996 The Leuconostoc oenos plasmid p4028 was cloned in pBlueScript (SK/), and its complete nucleotide sequence was determined. The analysis of the nucleotide sequence revealed five open reading frames, all of them located on the same strand and grouped in two clusters separated by a short noncoding stretch. A similarity search against the other sequences deposited in the EMBL and GenBank databases showed that p4028 has no significant similarity with any of the sequences checked. Nevertheless, a putative ATP-binding motif was found in ORF2. A more detailed analysis of this ORF suggests that it could encode for a DNA-dependent ATPase. q 1996 Academic Press, Inc.

Leuconostoc oenos is a lactic acid bacterium (LAB) commonly found in musts and wines which performs the malolactic fermentation (MLF) (Lafon-Lafourcade, 1983). MLF improves the quality of and confers microbiological stability to wines (Davis et al., 1985). Few studies have dealt with the genetics of Leuc. oenos, while dairy leuconostocs have received more attention. Plasmid DNA has been demonstrated in many strains (Orberg and Sandine, 1984), and the conjugal transfer of several plasmids has been reported (Tsai and Sandine, 1987). Gene transfer by electroporation also has been demonstrated in the dairy leuconostocs (Luchansky et al., 1988). However, less is known about the plasmids of Leuc. oenos. It has been reported the presence of plasmid DNA in a few strains of this microorganism (Janse et al., 1987), and one such plasmid (pLo13) has been characterized at the nucleotide sequence level (Fremaux et al., 1993). Analysis of the nucleotide sequence of this plasmid indicated that pLo13 probably replicates through a rolling-circle mechanism (RCR) and belongs to the pC194 family (Novick, 1989) but there was little similarity with other members of this family. We decided to sequence p4028, a small cryptic plasmid of Leuc. oenos to obtain insight into the organization and characteristics of this plasmid with

a view to developing a suitable cloning vector for Leuc. oenos. Strains used in this study were Leuc. oenos CECT 4028 harboring plasmid p4028 and Escherichia coli XL1Blue. Leuc. oenos was grown in MLO medium (Caspritz and Radler, 1983) at 307C. Standard methods were used for growth, transformation, and selection of transformants of E. coli. Plasmid DNAs were purified with the Magic Minipreps System of Promega. Restriction enzymes and T4 ligase were purchased from Boehringer-Manheim and used as recommended by the manufacturer. Plasmid p4028 was inserted in the HindIII site of pBlueScript II (SK/) in both orientations. For nucleotide sequence determinations, a series of nested deletions were obtained with the Erase-a-Base System (Promega). A set of overlapping deletions was selected for each construct. Deletions were sequenced by the dideoxy-chain termination method (Sanger et al., 1977) using the Sequenase 2.0 kit (USB). A universal 17-mer primer (Pharmacia) was used for priming. The University of Wisconsin Genetics Computer Group software package (GCG ver 7.3-APX, 1991) was used for computer-assisted nucleotide sequence analysis. Database searches were performed at the Net Center for Biotechnology Information (NCBI) 67

0147-619X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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using the BLAST network service (Altschul et al., 1990). Prediction of the secondary structure of the potential peptides encoded in p4028 was made through the PHD service (profile network prediction, Heidelberg) from the EMBL-Heidelberg (Rost and Sander, 1993). p4028 contained unique restriction endonuclease sites for EcoRV, HindIII, StuI, StyI, and XbaI. p4028 was digested with HindIII and ligated to pBlueScript II (SK/) digested with the same enzyme. Analysis of the XL1Blue transformants allowed the isolation of two clones harboring the intact p4028 plasmid inserted in opposite orientations. The complete nucleotide sequence of p4028 was determined (Fig. 1; EMBL and GenBank Accession Nos. Z29976 and L28806, respectively). Plasmid p4028 contained 4410 bp and had a calculated G / C content of 31%. The analysis of the nucleotide sequence revealed that p4028 consisted of two domains: one containing five open reading frames (named ORF1 to ORF5), all of them preceded by a putative ribosome binding site (RBS), and a noncoding domain with a high potential for secondary structure. All ORFs were in the same strand, showed the same orientation, and began with the putative start codon ATG. ORF1 and ORF2 partially overlapped. Moreover the last codon of ORF4 and ORF5 start codon shared their last and first bases, respectively. This structure suggested the translational coupling between ORF4 and ORF5 since the RBS preceding ORF5 can form a hairpin structure with part of the coding sequence of ORF5 (positions 3217–3223, DG7 Å 02.8 Kcal/mol; see Fig. 1). This arrangement would make the RBS inaccessible for the ribosome until the pairing was disrupted by the ribosome translating ORF4. In searching several nucleotide and amino acid sequence databases, no significant similarities with p4028 were found. Moreover, we could not establish putative roles for the ORF1, ORF3, ORF4, and ORF5 products. However, a potential P-loop was located in ORF2 (bases 1407–1430; see Figs. 1 and 2). The P-loop or A motif is commonly found in

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adenine and guanine nucleotide-binding proteins (Saraste et al., 1990). The crystalographic analysis of several proteins containing a P-loop revealed that the P-loop connects a b-sheet with an a-helix region (Saraste et al., 1990). The analysis of the secondary structure of the ORF2 product predicted a similar structure for this region, further supporting the existence of a functional P-loop. A more detailed analysis of the amino acid sequence encoded by ORF2 revealed the presence of a motif B (bases 2139–2168, see Figs. 1 and 2). Both motifs are commonly found in numerous helicases and other DNA- or RNA-dependent ATPases (Hodgman, 1988; Koonin, 1993). Koonin (1993) defined a third conserved domain in the helicase superfamily III, the NtrC superfamily, and the DnaA family. This third domain was also found in ORF2 (2229–2252, Figs. 1 and 2). The predicted secondary structure for this domain was also conserved, i.e., a hydrophylic residue preceded by a hydrophobic stretch forming a b-sheet. An alignment among ORF2 and some proteins belonging to these three families is shown in Fig. 2. However, there are important differences among ORF2 and these proteins. Specifically, these three conserved motifs were clustered in a 100-amino-acid region in all the other proteins, while in ORF2 the P-loop was 200 amino acids upstream from the motif B. These large spacers are also found in helicases belonging to superfamilies I and II (Hodgman, 1988) but these helicases contain some additional conserved motifs lacking in ORF2. Thus, this analysis led us to suggest that the product of ORF2 is a DNA-dependent ATPase different than those previously described. A putative s70-like promoter element could be located upstream ORF3 (Fig. 1) but not upstream of ORF1. Nevertheless, a putative TATA box was located upstream ORF1 (Fig. 1) and following an intrinsic curved sequence (see below) that could compensate for the lack of a 035 hexamer (Collis et al., 1989). Moreover, a possible r-independent terminator was located downstream of ORF5 (Fig. 2). The presence of only one possible terminator downstream of ORF5 suggests that all the five

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FIG. 1. p4028 plasmid nucleotide sequence and the deduced amino acid sequences of the protein products. Underlined: putative start codons; double underlined: ribosomal binding site (RBS); bold type: putative promoter elements; bold type and underlined: putative DnaA box. Arrows indicate inverted repeats; Doubleheaded arrows, direct repeats. TRM: r-independent terminator.

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FIG. 1—Continued

ORFs could be transcribed in one mRNA molecule, but data available are insufficient to rule out the possible presence of other protein-dependent terminator sequences. Furthermore,

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the presence of a putative promoter upstream ORF3 would suggest that the three latter ORFs could also be transcribed independently. All of the noncoding regions of p4028 had great

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FIG. 1—Continued

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FIG. 1—Continued

potential to form complex secondary structures and several regions with distinctive characteristics could be observed. A long imperfect inverted repeat was located upstream

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ORF1 (bases 441–534, DG7 Å 031 Kcal/ mol). This inverted repeat was flanked upstream by a T-rich stretch in the coding strand (about 50 bases) and downstream by an A-

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FIG. 1—Continued

rich region (about 100 bases). This A-rich region had a high probability of being curved (not shown), since the stretches of A are phased within the DNA helix (Koo et al., 1986). Data available are insufficient to determine the possible role of these regions, but it could be speculated that they might be involved in the regulation of the transcription of the ORFs since it has been shown that intrinsic curvatures upstream the promoter region can enhance the transcriptional activity (Pe´rezMartı´n et al., 1994). Two 18-bp AT-rich direct repeat sequences were found (bases 3807– 3824 and 3832–3849). These were preceded by a putative DnaA box (bases 3698–3706). DnaA binds to a 9-bp sequence named DnaA box (consensus sequence, TTATC(C/A)A(C/

A)A). The sequence found in p4028 was TTATCTAAA. Bramhill and Kornberg (1988) proposed that DnaA protein is involved in the initiation of the replication of a wide variety of prokaryotic origins taking into account some structural similarities: the presence of DnaA boxes and AT-rich repeats. Thus, some LAB plasmids, such as pCI305 (Hayes et al., 1991) or pWVO2 (Kiewiet et al., 1993), share a similar structural organization with oriC. We suggest that this region could be the replication origin of p4028 and that this plasmid uses u replication. Moreover, we could not detect ssDNA intermediaries of p4028 using the method described by te Riele et al. (1986) (data not shown). Many RCR plasmids are well characterized and can be classified in sev-

FIG. 2. Alignment of the three conserved motifs among ORF2 and DNA-dependent ATPases belonging to the NtrC superfamily, helicase superfamily III, and the DnaA family. The alignment is based on that performed by Koonin (1993). The name of each protein and the abbreviated name of the organism of origin are indicated. Each sequence is accompanied by the GenBank accession number. at: Arabidopsis thaliana; bt: Bacillus thuringiensis; dvb: densonucleosis virus of Bombyx mori; ec: E. coli; fcv: feline calicivirus.

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eral families that share a relatively high homology (Novick, 1989). Plasmid p4028 does not share any significant similarity with any of the RCR plasmids described until now, including pLo13. ACKNOWLEDGMENTS We are very indebted to Drs. Kees Leenhouts, Jan Kok, Gerard Venema, and M. Espinosa for their invaluable assistance and helpful suggestions. This research was supported by the Comisio´n Interministerial de Ciencia y Tecnologı´a (ALI93-0246).

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