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Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22
f~) INSTITUT PASTEUR/ELSEVIER
Rev. Microbi+'l. t 996, 147, 619-624
Pards 1996
Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22 P.S. Cocconcelli tl" 21 (*), M. Elli ~-~, B. Riboli ti~ at~d L. Morelli (t' 2)
tl~ Centro Ricerche Biotecnoiogiche, Universitt'z Cat~olica. via Milano 24. 26100, Cremona (Italy) and ~z~ istituto di Microbiologia, Universith Cattolica, via Emilia Parmensc 84, 29100 Pitwenza (ltaly~
SUMMARY
The sequence and genetic organization of the 1,600-bp replication region of the Lac~ tobacillus vector pPSC22, a plasmid derived from a 7.kb cryptic plasmid of L. plantarum used for the cloning of heterologous genes in several lactobacilli, were determined. Sequence analysis revealed the presence of a plus origin of replication containing the two functional elements n/c and bind, required for initiation of the leading strands typical of the rolling circle (RC)-replicating plasmids belonging to the pLSl family, Two open reading frames (copA and repA) were located within the Lactobacillus portion of pPSC22. The repA gene product, a 234-amino acid protein, showed homologies with the Rep protein of the streptocor.cal plasmid pLSl and contained the three conserved domains detected in most Rep proteins of RC-replicating plasmids and ss.coliphages. The genetic organization of the replication region of pPSC22 shared relevant homologies with the lactococcal plasmids pWVO1 and pFX2.
Key-words: Plasmid, Lactobacillus; Plasmid vectors, Origin of replication, RC, Nucleotide sequence.
INTRODUCTION Lactobacilli have been used in food production for centuries and, because of their health properties, were introduced into drugs decades ago. This group of bacteria, because of their industrial significance, has become the focus of a rapidly increasing number of genetic studies especially devoted to the application of recombinant DNA techniques for the genetic modification of L a c t o b a c i l l u s strains. Plasmids (!.5 to 60 kb in size) are frequently found in various strains of l.xwtobacillus
Submitted November 29, 1995, accepted April 9. 1996. (*) Corre~pondin,., author.
and recently, the mode of replication of some of them has been studied in detail. DNA sequence analysis of small multicopy replicons from L. plantarum, (Bates and Gilbert, 1989; Bouia et aL, 1989; Skaugen, 1989; Leer et aL, 1992; Vujcic and Topisirovic, 1993), L. pentosus (Leer et aL, 1992) and L. hilgardii (Josson et aL, 1989) has shown that they belong to a family of highly interrelated plasmids replicating via a similar mechanism, termed the rolling circle (RC) (Novick, 1989; Gruss and Ehrlich, 1989), already observed in a number of Gram-positive multicopy plasmids.
620
P.S. COCCONCELLI ET AL.
A 1.6-kb fragment of a 7-kb cryptic plasmid of L. plantarum, containing the functions for autonomous replication, was used to construct the plasmid vector pPSC22 for lactobacilli (Cocconcelli et al., 1991). This vector was shown to replicate and was used for the cloning of heterologous genes in a variety of Lactobaeillus species, in Lactococcus lactis subsp, iactis, in Bacillus subtilis, in Ruminococcus albus and in Gram-negative bacteria, including Escherichia coli (Cocconcelli et al., 1991, 1992). It was also demonstrated that pPSC22 replicates via a single-stranded DNA intermediate and shows homologies with the origin of replication of other Gram-positive RCR plasmids such as pE194 (Horinouchi and Weisblum, 1982). The genetic organization of several RC-replicating plasmids is now well established (Gruss and Ehrlich, 1989; del Solar et aL, 1993b; Espinosa et aL, 1995), and on the basis of homologies in the leading strand initiation and control region, four families have been established (Novick, 1989).
and transformed in E. coil JM109. Overlapping templates were obtained by exonuclease Ill digestion using the Erase-a-Base TM system (Promega). The complete nucleotide sequence of both strands was determined using the Taq dye primer cycle and Taq DyeDeoxy TM terminator cycle kits (Perkin Elmer) following the supplier's instructions. Sequence analysis was obtained with the Applied Biosystem 373A DNA sequencer. The software package of the University of Wisconsin Genetic Group was used for the DNA sequence analyses. Plasmid transformation Plasmid DNA was transformed in E. coil and in Lactobacillus and by means of electroporation as already reported (Dower et al., 1988; Cocconcelli et aL, 1991). General DNA manipulations Plasmid DNA was extracted from lactobacilli as previously reported (Morelli et al., 1987). Other methods of DNA manipulation were as reported by Sambrook et al. (1989).
In this study, we describe the sequence and the genetic organization of the origin of replication of the Lactobaeillus vector pPSC22. RESULTS MATERIALS AND METHODS Piasmids and organisms, and growth conditions The E. coil JM109 strain and the plasmids pUC18 and pUC19 (Yanisch-Perron et al., 1985) were used for subcloning of the Taql-fragments containing the origin of replication of pPSC22. The hosts of pPSC22 were the E. coil strain HB101 (Boyer and Roulland-Dussoix, 1969), and L. reuteri DSM20016. MRS medium (Difco) was used for culturing L. reuteri, Determination of the nucleotide sequence Plasmid pPSC22 was digested with the restriction endonuclease Taql, and 520- and 1080-bp fragments were cloned into the AccI site of plasmid pUC19
ORF RBS
= =
open reading frame, ribosome-binding site,
Nucleotide sequence of the replication region of pPSC22 The nucleotide sequence of the 1,600-bp fragment containing the replication functions of pPSC22 is reported in figure 1. The calculated G+C % of the Lactobacillus portion of pPSC22 is 35.1%, lower than the 44-46% range reported for L. plantarum species (Kandler and Weiss, 1986). Strong DNA sequence homologies were found with the p l a s m i d s b e l o n g i n g to the f a m i l y pLSl/pE194 of RC plasmids (del Solar et al., 1993b). Comparison of the sequence data of the Lactobacillus portion of pPSC22 with the replication regions of the l a c t o c o c c a l p l a s m i d s pWVOI (Leenhouts et al., 1991) and pFX2 (Xu et al., 1991) demonstrated that the replication
RC
=
rolling circle (replication mechanism).
TagLI 1 TL~TCGCTCTATCGTACCCTGGTPa~AAGCGGGC~GAGCGGGCTGCTGGCCCCGG
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Fig. I. Nucleotide sequence of the Lactobacillus portion (Taql-Taql-Taql) of pPSC22. The deduced amino acid sequences of the potenti;fl OP Fs are shown. Double-underlined and asterisks indicate potential promoters and ribosome binding sites. A possible dso site lined) and iterons (inverted letter v) are indicated. The possit~e area coding for the CtRNA is by a dotted line. These sequence data were assigned accession no. X95843 in the EMBL data
regions (undermarked library.
622
P.S. COCCONCELLI ET AL.
functions of these plasmids are nearly identical (96% sequence homology).
pPSC22 double-stranded origin of replication The essential region containing the elements required for in cis initiation of the leading strand synthesis (dso) was identified within the 1,600-bp fragment of pPSC22. The location of the dso in pPSC22, previously identified in the 520-kb Taql fragment through complementation experiments (Cocconcelli et al., 1991), was confirmed by deleting with exonuclease III and transforming into L. reuteri DSM20016 and E. coil HB101. Deletions resulted in the plasrnid no longer being functional in lactobacilli and in the Gram-negative host. By sequence comparison, the two regions, bind and nic, composing the dso of the pLS1 family, were found in the 520-bp TaqI fragment of pPSC22. The nic site was identified as a region of dyad symmetry (positions 243-317) potentially capable of forming a stem-loop structure (AG° = -22 kcal/mol) containing, on the side of the stem, the possible site of DNA nicking TACTACG\A as described for pLSI (de la Campa et al., 1990). Downstream from the palindromic structures of the nic site, iterated sequences, potentially forming the bind locus involved in the binding of the initiation protein (de la Campa et al., 1990; del Solar et al., 1993a), were present. The three iterons (TCGCCAACG) found in the sequences of the dso of pPSC22 showed a high degree of homology with iterated sequences of the pLS1 family, as reported by del Solar et al. (1993a). In addition, the motif GTTT found in pLS1, pWVOI, pFX2 and pKMK1 was located in the direct repeats of the bind site of pPSC22.
some-binding site (RBS) (GGAG) 8 bp upstream from the translation initiation codon ATG. Ten bp upstream from the ATG of ORb-E, another possible RBS (AAGG) was located. One putative promoter structure (ATrACA-17nucleotides-TATAAA) was present 23 bp upstream from the ATG of ORF1. The putative product of ORFI (copA gene), a peptide containing 53 amino acids, showed a high degree of homology with the Cop transcription repressor proteins of the RC plasmids of the pLS 1 family. The predicted secondary structures showed an o~-helix-turn-ot-helix composed of two helixes from residues 16-27 and between residues 34-52, and the turn motif present in the spacing region (28-33) between the helixes. The 26.8-kDa peptide (RepA), coded by ORF2, showed strong homologies with the replication initiation proteins (Rep) of the pLS 1 family. Five conserved motifs, R-I to R-V, as described by del Solar et al. (1993b), and two conserved H residues were found in the amino acid sequence of RepA.
Possible regulative mechanism of pPSC22 replication in the sequence of the replication region of pPSC22, the two elements involved in the control of replication and copy number present in the pLSl family were found: the CopA protein, as described above, and a small antisense RNA that overlapped the translation initiation signal of the repA gene (positions 750-654). The area coding for the countertranscribed RNA (CtRNA) is shown in figure 1. A p u t a l ~ nromoter from position 750 to position 721 (-35 region TITAGT-17-bp spacing, -10 region TATATT) was fouod. Downt, Zre,tm from the promoter region, inverted repeats were present, forming a possible transcription terminator.
Plasmid replication proteins Downstream from the dso, computer-assisted analysis revealed the presence of two open reading frames, ORFI (positions 504-662) and ORF2 (positions 730-I425), potentially capable of encoding polypeptides of 53 and 234 amino acids, respectively. ORFI was preceded by a typical ribo-
DISCUSSION In the study described here, the nucleotide sequence of the 1600-bp replication region of the Lactobacillus plasmid vector pPSC22 was determined and analysed. On the basis of sequence
THE REPLICATION REGION OF LACTOBACILLUS VECTOR pPSC22
analysis of the leading strand initiation and control region, the Lactobacillus vector pPSC22 was demonstrated to belong to the recently described pLS1 family (del Solar et al., 1993a) of highly interrelated plasmids. The genetic elements involved in the initiation of replication and in the copy number control of the p L S I family, the loci b i n d and nic of dso (del Solar et al., 1993a), the repA gene, the copA gene, coding for the Cop repressor protein (del Solar and Espinosa 1992), and a CtRNA complementary to the cop-rep m R N A (Novick, 1989), were found in the sequence of pPSC22. Comparison of the sequence data of the Lactobacillus portion of pPSC22 with the replication regions of the l a c t o c o c c a l p l a s m i d s p W V O I (Leenhouts et al., 1991) and pFX2 (Xu et aL, 1991), a vector derived from the plasmid pDI25 (Xu et aL, 1990), demonstrated that the replication functions of these plasmids are nearly identical ( 9 6 % sequence h o m o l o g y ) . The presence, in the highly interrelated family of plasmids that replicate via an RC mechanism, of common casseres of genetic information was already reported (Projan and Novick, 1e~88). The high degree of homology among replication regions of plasmids of different molecular mass and isolated from different bacterial hosts (pPSC22 was derived from a 7-kb cryptic plasmid of L. planmnm~, p W V O l was a native 2,2-kb plasrnid of Lactococcus lactis subsp, creraoris, and pFX2 was obtained from p D I 2 5 , a 5,5-kb plasmid of L. lactis subsp, lactis) is an example of blocks of genetic information spread among microorganisms.
Acknowledgements This work was supported by the National Research Council of Italy (CNR), Special Project RAISA, Subproject N. 4 Paper N.
Analyse g~nttique de la r~gion de rtplication du vecteur plasmidique pPSC22 de LactobacUlus Nous avons dttermin6 la stquence et I'organisation gtnttique de la rtgion de rtplication, de
623
1.600 bp, du vect,~ur plasmidique pPSC22 de /_x+ctoba¢'illt+s, un plasmide dtriv6 d'un plasmide cryptique de 7 kb de I. plantarum utilisd pour le clonage des g~nes h&drelogues chez divers lactobacilleg. L'analyse s6quentielle r~v~le la prtsence d'une oridine de rtplication < contenant les deux ~ltments fonctionnels, nic et bind, requis pour l'initiation obserx,te avec les plasmideg h rdplication , rolling circle >>qui appartiennent "hla famille pLS1. Deux ORFs (copa et repA) ont 6t6 localists darts la portion Lactob¢zcillus de pPSC22. Le produit du g~ne repA, une prottine de 234 acides aminds, a des homologies avecla protdine Rep du plasmide streptococcique pLS1 et contient les trois domaincs c o n s e r v t s que i ' o n dtc~le dans la plupart des protdines Rep des plasmides -3 triplication ¢
References Bates, E.M. & Gilbert, H.J. (1989), Characterization of a cryptic plasmid from l_z+ctob+willusplantarum. Gene, 85. 253-258. Bouia, A., Bringcl, F., Frey, L., Kammer, B,, Belarbi, A., Guyonvarch, A. & hubert. J.C. (1989), Structural orgzmization of pLPI. a cryptic plasmid from Lactobacillus plantarum CCM 1904. Plasmid, 22, 185-192. Boycr, H.W. & Roulland-Dussoix, D. (1969), A complemcntation analysis of the restriction and modification of DNA in L:scherichiacoil J. Mol. BioL, 41,459-472. Cocconceili, P.S., Gasson, M.J., Morelli, L. & Bottazzi, V, (1991), Single-stranded DNA plasmid, vector construction and cloning of Baccih+s strearothermophilus a-amylase in Lactobacillus. Res. MicrobioL, 142, 643-652. Cocconce|li. P-S., Ferrari, E., Rossi, F. & Bottazzi. V. (1992), P~asmid transformation of Ruminococcus albus by means of high-voltage electroporation. FEMS Microbiol. Lett., 94, 203-207. De la Campa. A.G., Del Solar, G.H. & Espinosa, M. (1990), Initiation of replication of plasmid pLS1. The initiatior protein repB acts on two distant DNA regions. J. MoL BioL. 213, 247-262. Del Solar, G. & Espinosa. M. (1992), The copy number of plasmid pLSI is regulated by two trans-acting plasmid products: the antisense RNA and the repressor Rep A. Mol. Microbiol., 6, 83-94. Del Solar, G., Moscoso, M. & Espinosa, M. (1993a), in vivo definition of the functional origin of replication (ori(+)) of the promiscuous plasmid pLS I. Mol. Gen. Genet.. 237, 65-72.
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Del Solar, G., Moscoso, M. & Espinosa, M. (I993b), Rolling circle-replicating plasmids from Gram-positive and Gram-negative bacteria: a wall falls. Mol. Microbiol., 8, 789-796. Dower, W.J., Miller, J.F. & Rangsdale, C.W. (1988), High efficiency transformation of Escherichia coli by high voltage elcctroporation. Nucl. Acids. Res., 16, 61276145. Espinosa, M., Del Solar, G., Rojo, F. & Alonso, J.C. (1995), Plasmid rolling circle replication and its control. FEMS MicrobioL Lett., 130, 111-120. Gruss, A. & Ehrlich, S.D. (1989), The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. MicrobioL Rev., 53, 231-24 i. Horinouchi, S. & Wiesblum, B. (1982), Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J. Bacteriol., 150, 804-814. Josson, K., Soetaert, P., Michielis, F., Joos, J. & Mahillon, J. (1990), LactobaciIlus hilgardii plasmid pLAB 1000 consists of two functional cassettes commonly found in other Gram-positive organisms. J. Bacteriol., 172, 3089-3099. Kandler, O. & Weiss, N. (1986), Regular, non-sporing Gram-positive rods, in "Bergey's manual of systematic bacteriology" (P.H. Sneath Ed.) (pp. 1208-1260). Williams & Wiikins, Baltimore, MD. Leenhouts, K.L, Tolner, B., Bron, S., Kok, J., Venema, G. & Seegers, J.F.M. (1991), Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVOl. Plasmid, 26, 55-66. Leer, R.J., Van Luijk, N., Posno, M. & Powels, P.H.
(1992), Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014. Mol. Gen. Genet., 234, 265-274. Morelli, L., Cocconcelli, P.S., Bottazzi, V., Damiani, G., Ferretti, L. & Sgaramella, V. (1987), Lactobacillus protoplast transformation. Plasmid, 17, 73-75. Novick, R.P. (1989) Staphylococcal plasmids and their replication. Annu. Rev. MicrobioL 43, 537-565. Projan, S.J. & Novick, R.P. (1988), Comparative analysis of five related staphylococcal plasmids. Plasmid, 19, 203-221. Sambrook, J., Fritsch, E.F. & Maniatis, f. (1989), Molecular cloning: a laboratory manual, 2rid ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Skaugen, M. (I 989), The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum. Plasmid, 22, 175-179. Vujcic, M., Topisirovic, L. (1993), Molecular analysis of the rolling-circle replicating plasmid pAl of Lactobacillus plantarum A! 12. Appl. Environ. Microbiol., 59, 274-280. Yanisch-Perron, C., Vieira, J. & Messing, .I. (1985), Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mpl8 and pUCl9 vectors. Gene, 33, 103-109. Xu, F., Pearce, L.E. & Yu, P.L. (1990), Molecular cloning of a proteinase gene from Lactococcus lactis subsp. cremonis and construction of a new lactococeal vector pFXI. Arch. Microbiol., 154, 99-104. Xu, F, Pearce, LE. & Yu, P.L. (1991), Genetic analysis of a lactococcal plasmid replicon. Mol. G~ i. Genet., 227, 33-39.
Rev. Microbi+'l. t 996, 147, 619-624
Pards 1996
Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22 P.S. Cocconcelli tl" 21 (*), M. Elli ~-~, B. Riboli ti~ at~d L. Morelli (t' 2)
tl~ Centro Ricerche Biotecnoiogiche, Universitt'z Cat~olica. via Milano 24. 26100, Cremona (Italy) and ~z~ istituto di Microbiologia, Universith Cattolica, via Emilia Parmensc 84, 29100 Pitwenza (ltaly~
SUMMARY
The sequence and genetic organization of the 1,600-bp replication region of the Lac~ tobacillus vector pPSC22, a plasmid derived from a 7.kb cryptic plasmid of L. plantarum used for the cloning of heterologous genes in several lactobacilli, were determined. Sequence analysis revealed the presence of a plus origin of replication containing the two functional elements n/c and bind, required for initiation of the leading strands typical of the rolling circle (RC)-replicating plasmids belonging to the pLSl family, Two open reading frames (copA and repA) were located within the Lactobacillus portion of pPSC22. The repA gene product, a 234-amino acid protein, showed homologies with the Rep protein of the streptocor.cal plasmid pLSl and contained the three conserved domains detected in most Rep proteins of RC-replicating plasmids and ss.coliphages. The genetic organization of the replication region of pPSC22 shared relevant homologies with the lactococcal plasmids pWVO1 and pFX2.
Key-words: Plasmid, Lactobacillus; Plasmid vectors, Origin of replication, RC, Nucleotide sequence.
INTRODUCTION Lactobacilli have been used in food production for centuries and, because of their health properties, were introduced into drugs decades ago. This group of bacteria, because of their industrial significance, has become the focus of a rapidly increasing number of genetic studies especially devoted to the application of recombinant DNA techniques for the genetic modification of L a c t o b a c i l l u s strains. Plasmids (!.5 to 60 kb in size) are frequently found in various strains of l.xwtobacillus
Submitted November 29, 1995, accepted April 9. 1996. (*) Corre~pondin,., author.
and recently, the mode of replication of some of them has been studied in detail. DNA sequence analysis of small multicopy replicons from L. plantarum, (Bates and Gilbert, 1989; Bouia et aL, 1989; Skaugen, 1989; Leer et aL, 1992; Vujcic and Topisirovic, 1993), L. pentosus (Leer et aL, 1992) and L. hilgardii (Josson et aL, 1989) has shown that they belong to a family of highly interrelated plasmids replicating via a similar mechanism, termed the rolling circle (RC) (Novick, 1989; Gruss and Ehrlich, 1989), already observed in a number of Gram-positive multicopy plasmids.
620
P.S. COCCONCELLI ET AL.
A 1.6-kb fragment of a 7-kb cryptic plasmid of L. plantarum, containing the functions for autonomous replication, was used to construct the plasmid vector pPSC22 for lactobacilli (Cocconcelli et al., 1991). This vector was shown to replicate and was used for the cloning of heterologous genes in a variety of Lactobaeillus species, in Lactococcus lactis subsp, iactis, in Bacillus subtilis, in Ruminococcus albus and in Gram-negative bacteria, including Escherichia coli (Cocconcelli et al., 1991, 1992). It was also demonstrated that pPSC22 replicates via a single-stranded DNA intermediate and shows homologies with the origin of replication of other Gram-positive RCR plasmids such as pE194 (Horinouchi and Weisblum, 1982). The genetic organization of several RC-replicating plasmids is now well established (Gruss and Ehrlich, 1989; del Solar et aL, 1993b; Espinosa et aL, 1995), and on the basis of homologies in the leading strand initiation and control region, four families have been established (Novick, 1989).
and transformed in E. coil JM109. Overlapping templates were obtained by exonuclease Ill digestion using the Erase-a-Base TM system (Promega). The complete nucleotide sequence of both strands was determined using the Taq dye primer cycle and Taq DyeDeoxy TM terminator cycle kits (Perkin Elmer) following the supplier's instructions. Sequence analysis was obtained with the Applied Biosystem 373A DNA sequencer. The software package of the University of Wisconsin Genetic Group was used for the DNA sequence analyses. Plasmid transformation Plasmid DNA was transformed in E. coil and in Lactobacillus and by means of electroporation as already reported (Dower et al., 1988; Cocconcelli et aL, 1991). General DNA manipulations Plasmid DNA was extracted from lactobacilli as previously reported (Morelli et al., 1987). Other methods of DNA manipulation were as reported by Sambrook et al. (1989).
In this study, we describe the sequence and the genetic organization of the origin of replication of the Lactobaeillus vector pPSC22. RESULTS MATERIALS AND METHODS Piasmids and organisms, and growth conditions The E. coil JM109 strain and the plasmids pUC18 and pUC19 (Yanisch-Perron et al., 1985) were used for subcloning of the Taql-fragments containing the origin of replication of pPSC22. The hosts of pPSC22 were the E. coil strain HB101 (Boyer and Roulland-Dussoix, 1969), and L. reuteri DSM20016. MRS medium (Difco) was used for culturing L. reuteri, Determination of the nucleotide sequence Plasmid pPSC22 was digested with the restriction endonuclease Taql, and 520- and 1080-bp fragments were cloned into the AccI site of plasmid pUC19
ORF RBS
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open reading frame, ribosome-binding site,
Nucleotide sequence of the replication region of pPSC22 The nucleotide sequence of the 1,600-bp fragment containing the replication functions of pPSC22 is reported in figure 1. The calculated G+C % of the Lactobacillus portion of pPSC22 is 35.1%, lower than the 44-46% range reported for L. plantarum species (Kandler and Weiss, 1986). Strong DNA sequence homologies were found with the p l a s m i d s b e l o n g i n g to the f a m i l y pLSl/pE194 of RC plasmids (del Solar et al., 1993b). Comparison of the sequence data of the Lactobacillus portion of pPSC22 with the replication regions of the l a c t o c o c c a l p l a s m i d s pWVOI (Leenhouts et al., 1991) and pFX2 (Xu et al., 1991) demonstrated that the replication
RC
=
rolling circle (replication mechanism).
TagLI 1 TL~TCGCTCTATCGTACCCTGGTPa~AAGCGGGC~GAGCGGGCTGCTGGCCCCGG
60
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300
301 TTAAGTGCCGAGTGCCJLATTTTTGTGCCAAAAACGCTCTATCCCAACTGGCTCAAGGGTT 360 361 T A A G G G G T T T T T C A A T C G C C A A C G A A T C G C C A A C G T T T T C G C C A A C G T T T T T A T A A A T C T 420 S , ^ ~ ^ ^ A A A ^
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721 T A A T A T A T C A T G G C T A T T A A A A A T A C T A A A G C T A G A A A T T T T G G A T T T T T A T T A T A T C C T 780 M A I K N T K A R N F G F L L Y P start RepA 781 GACTCAATTCCTPatkTGATTGGAAAGAAAIb~TTAGAGAGTTTGGGCGTATCTATGGCTGTC 840 D S I P N D W K E K L E S L G V S M A V 841 A G T C C T T T A C A C G A T A T G G A C G A ~ G A T A A A G A T A C A T G G A A T A G T A G T G A T G T T S P L H D M D E K K D K D T W N S $ D
900 V
901 ATACGAAATGGAAAGCACTATAAAAAACCACACTATCACGTTATATATATTGCACGAJUKT 960 I R N G K H Y K K P H Y H V I Y I A R N 961 C C T G T A A C A A T A C 4 ~ G C G T T A G G A A C A A G A T T A A G C G A A A A T T G G G G A A T A G T T C A G T T P V T I E S V R N K I K R K L G N S S V
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1021 G C T C A T G S T G A G A T A C T T G A T T A T A T C A A A G G T T C A T A T G A A T A T T T G A C T C A T G A A T C A 1080 A H V E I L D Y I K G S Y E Y L T H E S 1081 /~AGGACGCTATTGCTAAGAAT/~%ACATATATACGACCCAAAAGATATTTTGAACATTAAT 1140 K D A I A K N K H I Y D P K D I L N I N 1141 GATTTTC@,TATTGACCGATATAffAACACTTGATGAAAGCCAAAAAAGAGAATTGAAGAAT 1200 D F D I D R Y I T L D E 3 Q K R E L K N 1201 TTACTTT~ ~ G A T A T A G T G G A T G A C T A T A A T T T G G T A A A T A C C A A A G A T T T A A T G G C T T T T 1260 L L L D ~ V D D Y N L V N T K D L M A F 1261 A T T C G ~c'' .'~%GGGGAGCGGAGTTTGGAATTTTAAATACGAATGATGTAAAAGATATTGTT 1320 i R L R G A E F G I L N T N D V K D I V 1321 TCaACAAACTCTAGCGCCTTTAGATTATGGTTTGAGGGCJkATTATCAGTGTGGATATAGA 1380 S T N S S A F R L W F E G N Y Q C G Y R 1381 GCAAGTTATGCAAAGGTTCTTGATGCTGAAACGGGGGAAATAAAATGACAAACAAACdkAA 1440 A S Y A K V L D A E T G E $ K * 1441 A A G A G T T A T T T G C T ~ T G A G G A A T T A A A A A A A G A A A T T A A G G A C T T A ~ G A G C G T A
1500
1501 T T G A A A G A T A C A G A G A A A T G G A A G T T G A A T T A A G T A C A A C A A T A G A T T T A T T G A G A G G A G 1560
Taq I 1561 G G A T T A T T G A A T A A A T A A A A G C C C C C C T G A C G A A A G T L ~ 1600
Fig. I. Nucleotide sequence of the Lactobacillus portion (Taql-Taql-Taql) of pPSC22. The deduced amino acid sequences of the potenti;fl OP Fs are shown. Double-underlined and asterisks indicate potential promoters and ribosome binding sites. A possible dso site lined) and iterons (inverted letter v) are indicated. The possit~e area coding for the CtRNA is by a dotted line. These sequence data were assigned accession no. X95843 in the EMBL data
regions (undermarked library.
622
P.S. COCCONCELLI ET AL.
functions of these plasmids are nearly identical (96% sequence homology).
pPSC22 double-stranded origin of replication The essential region containing the elements required for in cis initiation of the leading strand synthesis (dso) was identified within the 1,600-bp fragment of pPSC22. The location of the dso in pPSC22, previously identified in the 520-kb Taql fragment through complementation experiments (Cocconcelli et al., 1991), was confirmed by deleting with exonuclease III and transforming into L. reuteri DSM20016 and E. coil HB101. Deletions resulted in the plasrnid no longer being functional in lactobacilli and in the Gram-negative host. By sequence comparison, the two regions, bind and nic, composing the dso of the pLS1 family, were found in the 520-bp TaqI fragment of pPSC22. The nic site was identified as a region of dyad symmetry (positions 243-317) potentially capable of forming a stem-loop structure (AG° = -22 kcal/mol) containing, on the side of the stem, the possible site of DNA nicking TACTACG\A as described for pLSI (de la Campa et al., 1990). Downstream from the palindromic structures of the nic site, iterated sequences, potentially forming the bind locus involved in the binding of the initiation protein (de la Campa et al., 1990; del Solar et al., 1993a), were present. The three iterons (TCGCCAACG) found in the sequences of the dso of pPSC22 showed a high degree of homology with iterated sequences of the pLS1 family, as reported by del Solar et al. (1993a). In addition, the motif GTTT found in pLS1, pWVOI, pFX2 and pKMK1 was located in the direct repeats of the bind site of pPSC22.
some-binding site (RBS) (GGAG) 8 bp upstream from the translation initiation codon ATG. Ten bp upstream from the ATG of ORb-E, another possible RBS (AAGG) was located. One putative promoter structure (ATrACA-17nucleotides-TATAAA) was present 23 bp upstream from the ATG of ORF1. The putative product of ORFI (copA gene), a peptide containing 53 amino acids, showed a high degree of homology with the Cop transcription repressor proteins of the RC plasmids of the pLS 1 family. The predicted secondary structures showed an o~-helix-turn-ot-helix composed of two helixes from residues 16-27 and between residues 34-52, and the turn motif present in the spacing region (28-33) between the helixes. The 26.8-kDa peptide (RepA), coded by ORF2, showed strong homologies with the replication initiation proteins (Rep) of the pLS 1 family. Five conserved motifs, R-I to R-V, as described by del Solar et al. (1993b), and two conserved H residues were found in the amino acid sequence of RepA.
Possible regulative mechanism of pPSC22 replication in the sequence of the replication region of pPSC22, the two elements involved in the control of replication and copy number present in the pLSl family were found: the CopA protein, as described above, and a small antisense RNA that overlapped the translation initiation signal of the repA gene (positions 750-654). The area coding for the countertranscribed RNA (CtRNA) is shown in figure 1. A p u t a l ~ nromoter from position 750 to position 721 (-35 region TITAGT-17-bp spacing, -10 region TATATT) was fouod. Downt, Zre,tm from the promoter region, inverted repeats were present, forming a possible transcription terminator.
Plasmid replication proteins Downstream from the dso, computer-assisted analysis revealed the presence of two open reading frames, ORFI (positions 504-662) and ORF2 (positions 730-I425), potentially capable of encoding polypeptides of 53 and 234 amino acids, respectively. ORFI was preceded by a typical ribo-
DISCUSSION In the study described here, the nucleotide sequence of the 1600-bp replication region of the Lactobacillus plasmid vector pPSC22 was determined and analysed. On the basis of sequence
THE REPLICATION REGION OF LACTOBACILLUS VECTOR pPSC22
analysis of the leading strand initiation and control region, the Lactobacillus vector pPSC22 was demonstrated to belong to the recently described pLS1 family (del Solar et al., 1993a) of highly interrelated plasmids. The genetic elements involved in the initiation of replication and in the copy number control of the p L S I family, the loci b i n d and nic of dso (del Solar et al., 1993a), the repA gene, the copA gene, coding for the Cop repressor protein (del Solar and Espinosa 1992), and a CtRNA complementary to the cop-rep m R N A (Novick, 1989), were found in the sequence of pPSC22. Comparison of the sequence data of the Lactobacillus portion of pPSC22 with the replication regions of the l a c t o c o c c a l p l a s m i d s p W V O I (Leenhouts et al., 1991) and pFX2 (Xu et aL, 1991), a vector derived from the plasmid pDI25 (Xu et aL, 1990), demonstrated that the replication functions of these plasmids are nearly identical ( 9 6 % sequence h o m o l o g y ) . The presence, in the highly interrelated family of plasmids that replicate via an RC mechanism, of common casseres of genetic information was already reported (Projan and Novick, 1e~88). The high degree of homology among replication regions of plasmids of different molecular mass and isolated from different bacterial hosts (pPSC22 was derived from a 7-kb cryptic plasmid of L. planmnm~, p W V O l was a native 2,2-kb plasrnid of Lactococcus lactis subsp, creraoris, and pFX2 was obtained from p D I 2 5 , a 5,5-kb plasmid of L. lactis subsp, lactis) is an example of blocks of genetic information spread among microorganisms.
Acknowledgements This work was supported by the National Research Council of Italy (CNR), Special Project RAISA, Subproject N. 4 Paper N.
Analyse g~nttique de la r~gion de rtplication du vecteur plasmidique pPSC22 de LactobacUlus Nous avons dttermin6 la stquence et I'organisation gtnttique de la rtgion de rtplication, de
623
1.600 bp, du vect,~ur plasmidique pPSC22 de /_x+ctoba¢'illt+s, un plasmide dtriv6 d'un plasmide cryptique de 7 kb de I. plantarum utilisd pour le clonage des g~nes h&drelogues chez divers lactobacilleg. L'analyse s6quentielle r~v~le la prtsence d'une oridine de rtplication <
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