Complete nucleotide sequence of a Selenomonas ruminantium plasmid and definition of a region necessary for its replication in Escherichia coli

PLASMID

28,

123-129

Complete

(1992)

Nucleotide Sequence of a Selenomonas ruminantium and Definition of a Region Necessary for Its Replication in Escherichia co/i

Plasmid

G. T. ATTWOOD* AND J. D. BROOKER’ Department ofAnimal Sciences, Waite Agricultural Research Institute, Glen Osmond, South Australia 5064, Australia. and *Department of Animal Science& University o/Illinois, Urbana, Illinois 61801 Received January 2 I, 1992; revised April I 3, 1992 A plasmid from Selenomonas ruminantium subspecies lactilytica has been subcloned in Escherichiu coli K-l 2 and completely sequenced. Three open reading frames (ORFs) of 909, 801, and 549 bp were identified and the complete sequence was analyzed by comparison with DNA and protein databases. No significant deoxynucleotide or amino acid sequence homology with other published genes or proteins was detected. The plasmid was shown to replicate independently in E. coli K-12 by a DNA polymerase I-dependent mechanism and deletion analysis defined the DNA sequence responsible for this phenotype. o 1992 Academic press. inc.

The development of recombinant DNA systems for species of the anaerobic ruminal bacterium Selenomonas has progressed slowly due to lack of detailed information on the genetics of these organisms and of any native plasmids that may serve as possible transformation vectors. Selenomonas ruminantium has been reported to contain a number of cryptic plasmids (Martin and Dean, 1989) ranging in size from 2.5 kb to greater than 50 kb. These may be present singly or in combination (Zhang et al., 199 1). A number of cryptic plasmids from other ruminal bacteria have been described (Hazlewood and Teather, 1988; Mann et al., 1986; Thomson et al., 1992) but there are no reports of any plasmid DNA sequence analysis. In this work, we report the entire nucleotide sequence of the 3.7-kb cryptic plasmid pJDB23 from S. ruminantium subsp. lactilytica and analyze its genetic organisation. A derivative of this plasmid, pJDB23 1, containing the Tn9 chloramphenicol acetyltransferase (CAT) gene but lacking any pUC 19 sequence, expressed chloramphenico1 resistance (Cmr) and replicated autono’ To whom correspondence

should be addressed.

mously in Escherichia coli K-12 in a DNA polymerase I-dependent fashion as do ColEl-type plasmids. This plasmid may form the basis of a SelenomonasjE. co/i K- 12 shuttle vector. MATERIALS

AND

METHODS

Bacterial strains and plasmids. Bacterial plasmids used in this work and derived from pJDB23 are listed in Table 1. E. coli plasmids were pUCl9 and pHC79. The E. coli K- 12 strains used were HB 10 1 and UB 1636polA,, (J. I. Rood, Monash University, Australia). DNA sequencing. Subclones of pJDB23I were obtained by cloning PstI/XmnIJmnI, AvaI,AluI, and RsaI fragments of pJDB231 DNA separately into the appropriate sites in pUC19. Recombinant clones were verified by Southern analysis of isolated DNA using a radiolabeled pJDB23 probe and by replica plating onto LB containing ampicillin (Ap), 50 pg/ml, or Cm, 25 &ml. Nested deletions were generated using the Double Stranded Nested Deletion Kit supplied by Pharmacia (Pharmacia LKB Biotechnology, Australia). Plasmid DNA from deletion clones was sequenced (Sanger et al., 1977) using the Amersham multiwell microtiter plate se123

0147-619X/92

$5.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form rescrvcd.

124

ATTWOOD AND BROOKER TABLE I

DERIVATION ANDPHENOTYPE OF~JDB~~-BASED PLASMIDS Plasmid

Derivation

Antibiotic resistance

pJDB23 pJDB24 pJDB24 1 pJDB23 I pJDB232

S. ruminantium 3.7-kb cryptic plasmid pJDB23 cloned into the PstI site of pUC 19 CAT gene from Tn9, cloned into the Hind111site of pJDB24 pJDB24 1 with the pUC 19 sequencesremoved using PstI 2.3 PstI/PvuII fragment of pJDB23 1 ligated with the 6.3-kb PstI/PvuII fragment of pHC79 Ba/3 1 deletion of PstI-digested pJDB23 I

AP Ap Cm Cm Tc

pJDB1 pJDB 13 pJDB 17 pJDB18 pJDB602

Cm I

quencing system and 7 M urea/8% polyacrylamide gel electrophoresis. Regions of pJDB23 not covered by nested deletions were sequenced using pJDB23 1 subclones with custommade primers for sequencing outward from both ends of the CAT gene (Alton and Vapnek, 1979). DNA sequence was entered on a Macintosh computer using the MacMolly DNA analysis program. Nuclease Bal 31 deletions. Deletions of PstI- and AvaI-digested pJDB23 1 and PvuIIdigested pJDB232 were generated by digestion with nuclease Ba131 (Boehringer-Mannheim). Reactions containing 5 pg of each linearized plasmid and 3 units of nuclease Ba131were incubated at 30°C and aliquots were removed at regular intervals. Half of each aliquot was analyzed by agarosegel electrophoresis and reaction mixtures containing appropriately spaced deletions were treated with T4 DNA polymerase to provide blunt ends (Maniatis et al., 1982). Deletion fragments were ligated and used to transform E. coli HB 101. Deletion clones from pJDB23 1 were selected on plates containing 25 pg/ml Cm and those from pJDB232 on plates containing 12 pg/ml tetracycline (Tc). RESULTS

striction sites mapped experimentally. The sequence was computer analyzed and shown to contain three large open reading frames (ORFs) with coding probabilities of 0.88 or greater (Table 2). The coding sequence for each ORF is preceded by a predicted ribosome binding site (RBS) (based on the E. coli consensus sequence) and a predicted promoter sequence. Downstream of the coding region for each ORF there is an A + T-rich sequence containing an inverted repeat that may act as a transcription terminator. The predicted promoter for ORF 2 overlaps the control region of ORF 1 but lies on the opposite strand. The predicted amino acid sequence for ORF 3 indicates an unusually high (14%) proportion of lysine residues. The pJDB23 sequencewas compared with sequences present in the GenBank and EMBL DNA databasesbut no significant homology was detected. The derived amino acid sequence from each ORF was also compared with the protein sequenceslisted in the Swissprot Protein Database but a simple analysis of the normalized alignment score (NAS) (Doolittle, 1986) of each sequenceoverlap indicated that none of the 40 best matches for any of the amino acid sequenceswere significant.

Sequencingof pJDB23

Localization of the pJDB231 ori

The nucleotide sequence of pJDB23 (3759 bp) (Fig. 1) confirms the presence of all re-

To determine whether pJDB23 could replicate autonomously in E. coli K-12, the 4.5-

SEQUENCE OF A Selenomonas PLASMID

125

126

ATTWOOD AND BROOKER TABLE 2 PREDICTED PROTEIN-ENCODING ORFs FROM pJDB23 SEQUENCE

ORF

(bp)

Location (nucleotide)

Predicted protein size (daltons)

AC (termination) (kcal)

I

909 801 549

3114-2203 3652-196 1126-1674

35,518 30,567 22,292

-4.6 -5.4 -3.8, -5.2

Size

2 3

a AC values of putative termination hairpin loops determined by computer analysis.

kb plasmid pJDB231 (Table 1) was constructed and electroporated into E. coli K- 12. Transformation frequency was at least 5 X lo5 CFUs/pg of DNA. Restriction mapping and Southern analysis confirmed the absence of pUC 19 sequence in pJDB23 1 and suggested that the plasmid could replicate in E. coli K- 12 independently of the pUC 19 ori sequence. To determine whether pJDB23 1 replication in E. coli K-12 was mediated by DNA polymerase I, pJDB231 was electroporated into E. coli K-12 strain UB 1636polA,, and screened for growth on plates containing Cm at the permissive (30°C) and nonpermissive (42°C) temperatures. Plasmids pJDB24 1 and pUC 19 and a DNA polymerase I-independent derivative of R6K, pcos2EMBL (Poustka et al., 1984), were controls. All transformants grew at 30°C but only transformants carrying pcos2EMBL grew at 42°C. Plasmid pJDB231 was therefore dependent upon DNA polymerase I for replication in E. coli K-12, strain UB 1636polA,, . ColEl and related plasmids are the only plasmids previously reported to require DNA polymerase I for plasmid replication. Therefore we defined the region of pJDB231 involved in replication in E. coli K-l 2 and compared it with ColEI-type sequences.Eighteen clones were obtained from Ba131 digestions of &I-linearized pJDB231 and one clone from Ba131-digested, AvaI-linearized pJDB231. DNA from the four largest deletion clones of &I-digested pJDB231 (pJDB1, 13, 17 and 18) was digested with XmnI + AvaI. Restriction fragments were

consistent with deletions of 0.9, 1.05, 1.3, and 1.5 kb as shown in Fig. 2. The single transformant obtained after Ba131 digestion of AvaI-linearized pJDB23 1 contained fulllength plasmid DNA (4.5 kb) that comigrated with AvaI-linearized pJDB23 1 (result not shown). Since the selective CAT marker gene is located between the two XmnI sites, these results suggest that the sequence encompassing the region between the Hind111 site and the AvaI site is necessary for pJDB23 1 replication in E. coli K- 12. When the 2.3-kb PstI/PvuII fragment of pJDB231 was ligated with the 6.3-kb PstI/ PvuII fragment of pHC79 containing a tetracycline resistance (Tc’) gene, the hybrid plasmid (pJDB232) replicated and expressedTc’ in E. coli. Because the pHC79-derived fragment did not contain the pBR322 ori (Hohn and Collins, 1980) the replication function must be located on the 2.3-kb PstI/PvuII pJDB231 fragment. To localize the ori further, Ba131 deletions of PvuII-linearized pJDB232 were constructed. The largest deletion plasmid (pJDB602) had lost approximately 1 kb of pJDB23 sequence(Fig. 2). Together with the previous data, the pJDB231 ori can therefore be located to within a 1-kb region which overlaps the AvaI and adjacent XmnI sites. SequenceAnalysis of ori Examination of the DNA sequence spanning the 1-kb region defined by deletion analysis revealed a number of possible secondary structures. The region contained most of

127

SEQUENCE OF A Selenomonas PLASMID

Pst I

Xmn I

Hind III

Xhn I

riind III

Ala I

tist I

b

rl 2.2 kb Xmn I fragment cannot replicate independantly

Legend 0 m

pJDB23 CAT gene

_

ORFS

Deletion

clones

-

Remaining

1,1,,1,‘,

Deleted

DNA DNA

FIG. 2. Localization of the pJDB23 origin of replication. Exonuclease Bu131 deletion plasmids pJDB 1, 13, 17, 18,and 602 are mapped in relation to pJDB23 1. The region of pJDB23 necessaryfor replication in E. coli, as defined by Bal3 1 deletions and pJDB23 1 subcloning, is indicated by the shaded box.

dated and a l.O-kb region of the plasmid responsible for replication in E. coli K-12 has been defined. The pJDB23 DNA sequence appears unique as it does not show significant homology with DNA sequenceslisted in the GenBank or EMBL databases. However, DNA sequences around the replication origins of many plasmids have structural characteristics which may not be obvious in a database search. Although three ORFs were identified, their functions are unknown. Most of ORF 1 and approximately 230 bp of associated upstream sequence were required for redication of pJDB23 1 in E. coli K- 12. DISCUSSION Nevertheless, apart from the inserted CAT sequence, we could not demonstrate expresIn this paper, the complete nucleotide se- sion of any pJDB23 l-encoded proteins in E. quence of plasmid pJDB23 has been eluci- co/i K- 12 minicells (data not shown). ORF 3 ORF 1, an 80-bp direct repeat and two 7-bp and one 9-bp inverted repeats, The direct repeat contained A + T-rich and G + C-rich regions as well as indirectly repeated and palindrome structures, and overlapped with the putative ORF 2 promoter. The direct repeat was completely contained within the control region predicted for ORF 1 (Fig. 1). This region may be involved in the formation of a regulatory transcript with the last inverted repeat and sequenceof five T’s acting as a transcriptional terminator.

128

ATTWOOD AND BROOKRR

was interrupted by insertion of the Tn9 CAT gene. Plasmids fall into two general categories with regard to their mode of replication control. One group uses an inhibitor-target mechanism in which a small countertranscript RNA molecule binds with a complementary RNA transcript that acts either as a primer for replication initiation (Co1 El and related plasmids) (Tomizawa and Masukata, 1987) or as a mRNA molecule for a Rep protein [Inc FII plasmids (Light and Molin, 1982) and the staphylococcal plasmids related to pT181 (Kumar and Novick, 1985)]. The second group of plasmids are regulated by an iteron (direct repeat)-binding mechanism (Scott, 1984). The iteron-binding replicons have a series of 19- to 22-bp direct repeats located near the ori, some of which overlap the promoter region of a gene encoding a 29- to 38-kDa Rep protein (Couturier et al., 1988). These plasmids also have A + Tand G + C-rich regions located next to these direct repeats (Smith and Thomas, 1985). The DNA sequencelocated within the 1.Okb region of pJDB23 has characteristics similar to those of the iteron-binding type plasmids. However, this type of plasmid replication is difficult to reconcile with the finding that pJDB23 1 replicates in E. coli K- 12 using host-encoded DNA polymerase I, characteristic of ColEl and related plasmids that use an RNA preprimer to hybridize with template DNA near the ori (Itoh and Tomizawa, 1980). The RNA primer also forms a displacement loop downstream of the ori to allow proteins involved in lagging strand synthesis to assemble on the exposed single strand. If similar events occur during pJDB23 1 replication in E. coli K- 12, this is most probably carried out by pJDB23’s own priming mechanism. The 230 bp upstream of ORF 1 contains a potential promoter sequenceand an ORF for a small transcript that could act as a primer for pJDB23 replication in E. coli K- 12 similar to the 175-bp transcript described in RK2 (Stalker et al., 1981). The second stem loop in the predicted secondary structure of this

proposed transcript resembles the originproximal stem loop X of the ColEl RNA II primer molecule (Masukata and Tomizawa, 1986), with seven A’s near the baseof its stem similar to the five A’s in RNA II which form the target sequence for RNase H cleavage at the ori (Kues and Stahl, 1989). The proposed pJDB23 small transcript may therefore be recognized by DNA polymerase I as a primer to initiate replication in E. coli. It seemslikely that pJDB23 1 replication in E. coli is an opportunistic event and does not represent the normal situation in Selenomonas.The lack of expression or the interruption of pJDB23encoded replication proteins in E. coli has probably forced pJDB231 into an alternate mode of replication. Clarification of the authentic mode of pJDB23 replication was not possible because transformation of Selenomenuswith pJDB23 1 was not achieved. This could be due to the presence of a restriction barrier, poor DNA uptake through the cell wall, or lack of expression of the CAT selection marker. The characterization of the major ORFs encoded by plasmid pJDB23, including sequencesinvolved in replication in E. coli, has not defined plasmid function. Nevertheless, it represents a significant step toward achieving gene manipulation in Selenomonas since it allows the functionally important regions of the plasmid to be identified and forms the basis of future plasmid manipulations to construct an E. coZi/SeZenomenus shuttle vector. ACKNOWLEDGMENTS This work was supported by a grant from the Australian Meat Research Corporation. We acknowledge the excellent technical assistancefrom Mrs. D. K. Lum and helpful comments from Dr. J. A. Hackett and Dr. A. Thomson in proofreading the manuscript.

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