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Journal of Bacteriology, August 1999, p. 4680-4685, Vol. 181, No. 15
Department of Biological Sciences, Louisiana
State University, Baton Rouge, Louisiana 70803
Received 29 March 1999/Accepted 28 May 1999
A database search revealed extensive sequence similarity between
Streptomyces lividans plasmid pIJ101 and
Streptomyces plasmid pSB24.2, which is a deletion
derivative of Streptomyces cyanogenus plasmid pSB24.1. The
high degree of relatedness between the two plasmids allowed the
construction of a genetic map of pSB24.2, consisting of putative
transfer and replication loci. Two pSB24.2 loci, namely, the
cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of
complementing the pIJ101 clt and korB
functions, respectively, a result that is consistent with the notion
that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if
not identical, conjugation systems.
The conjugative plasmid pIJ101 is
one of the most extensively studied of the Streptomyces
extrachromosomal elements. Originally purified from Streptomyces
lividans ISP5434 (14), pIJ101 (Fig. 1A) is an 8,830-bp (11),
circular, high-copy-number plasmid which possesses a broad host range
among actinomycete bacteria (14). To produce copies of
itself, pIJ101 uses rolling-circle replication (RCR) (6), a
process that involves the 456-amino-acid Rep protein encoded by the
rep gene of pIJ101 (11), a double-strand origin
(DSO) of replication where Rep-mediated single-stranded nicking and
initiation of first-strand synthesis occurs, and a likely single-strand
origin (SSO) termed sti, where second-strand synthesis is
normally initiated (4). Certain motifs present within its
Rep protein, along with conserved sequences present at the site of Rep
nicking, have allowed for the classification of pIJ101 as a member of
the pIJ101-pJV1 family of RCR plasmids (20), which is
comprised mostly of Streptomyces replicons (13). The DSO of pIJ101, which has been mapped (3) to a 517-bp
region that includes the likely nicking site (20) as well as
other sequences required for plasmid replication and copy number
control (3), is located between the rep gene and
orf56, a small open reading frame (ORF) of undetermined
function (11). The sti locus of pIJ101
(4) includes sequences that may form an extensive secondary
structure and may contain consensus SSO hexanucleotide base positions
(28).
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Complementation of Conjugation Functions of Streptomyces
lividans Plasmid pIJ101 by the Related Streptomyces
Plasmid pSB24.2
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FIG. 1.
Physical and genetic maps of pIJ101 (A) and pSB24.2 (B).
Filled arrows represent ORFs which were determined from the published
pIJ101 (11) or pSB24.2 (2) sequence or from the
pSB24.2 sequence reported here. Genetic functions were determined for
either pIJ101 genes (12, 14) or loci (solid bars with no
arrows) (4, 17) as described previously. Small pIJ101 ORFs
of undetermined genetic function (i.e., orf56,
orf66, orf79, and orf85) are
indicated. Certain pSB24.2 genetic functions (i.e., korB and
clt) were determined here; other genetic functions (i.e.,
DSO and SSO) or small ORFs of unknown function (i.e., orf56)
were assigned based on their similarity to corresponding pIJ101
sequences or in the case of the rep gene on the similarity
of the deduced Rep protein to Rep proteins of other
Streptomyces plasmids (9, 13).
The efficient transfer of pIJ101 is dependent on both the tra gene (12, 14), which encodes a trans-acting membrane protein with an as yet undetermined function (16), and a cis-acting transfer locus (clt), which appears to represent a site or sequence that participates in some unknown manner in the transfer event (17). The clt locus was originally subcloned on a 142-bp fragment of pIJ101 that spans the 3' end of the korB gene, although sequences essential for clt function may be present only downstream of the korB ORF (17). Although its exact sequence determinants remain to be elucidated, clt does not appear to resemble cis-acting transfer sequences found on other bacterial plasmids (27), including the only other Streptomyces plasmid clt function so far identified (19).
The korB gene encodes a repressor protein that binds within its own promoter region as well as that of kilB (21, 22), a gene that is involved in, although not absolutely required for, pIJ101 transfer (12). The ability of the KorB protein to repress or override kilB is essential, since the unregulated expression of the kilB gene on pIJ101 is lethal in Streptomyces (12). Like kilB, the genes spdA and spdB contribute to plasmid transfer in an as yet undetermined ancillary manner (11, 12), while similar to korB control, the pIJ101 korA gene codes for a repressor (21) that appears to negatively regulate the expression of both korA and a transcript that includes tra, spdA, and spdB (8).
pSB24.1 is a conjugative plasmid isolated from Streptomyces cyanogenus (2), a species notable for its production of the antitumor agent landomycin A (26). Upon introduction of pSB24.1 into S. lividans, a stable, transfer-defective, deletion derivative designated pSB24.2 forms (Fig. 1B), which has a reported size of 3,706 bp (2). The deduced Rep protein of pSB24.2 (9) and potential site of Rep-mediated nicking (5) show homology to those of the other members of the pIJ101-pJV1 family of RCR plasmids (13). Here we show that a database search for sequences showing homology to the pIJ101 clt region (and thus plasmids that may share the same transfer mechanism as pIJ101) subsequently revealed extensive similarity between both transfer and replication loci of pIJ101 and corresponding previously unidentified loci on pSB24.2. We further show that the clt and korB functions encoded by pSB24.2 can complement their pIJ101 counterparts. These results support the hypothesis that pIJ101 and pSB24.1 share the same conjugation system and that pIJ101-like conjugative plasmids may be dispersed throughout at least a portion of the Streptomyces genus.
pIJ101 and pSB24.2 show extensive sequence similarity. By using the BLASTN algorithm (1) and the 142-bp clt+ region of plasmid pIJ101 (Fig. 2) as a query sequence, a database search revealed only one other highly similar sequence, which belonged to Streptomyces plasmid pSB24.2. Subsequent BLASTN alignment of the entire pIJ101 and pSB24.2 sequences revealed extensive similarity between them (i.e., approximately 77% of the pSB24.2 sequence aligned to pIJ101 with 89% identity and 1% gaps by this method) and allowed the identification of putative transfer and replication loci on pSB24.2 (Fig. 1B) as detailed below. To resolve discrepancies arising through probable errors, both strands of the korB-clt region of pSB24.2 were sequenced by using relevant primers and an ABI Prism dye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Inc., Foster City, Calif.), followed by analysis of reactions on a model 310 genetic analyzer (PE Applied Biosystems, Inc.).
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10 but not a completely conserved possible 35 promoter
determinant. Also within this region of pSB24.2 are inverted repeat
sequences that are identical to those found in the pIJ101
korB promoter which were shown previously to be important
for the autoregulation of korB gene expression (25). Downstream of the korB ORFs on both
plasmids, there is complete conservation of sequences in the immediate
vicinity of the MluI restriction site, a location at which
the insertion of DNA into pIJ101 was previously shown to eliminate
clt function (17).
The alignment of the two plasmids' sequences also revealed that
pSB24.2 contains an additional transfer-related sequence: in this case,
a partial copy of a putative kilB ORF (Fig. 1B). The partial
kilB ORF of pSB24.2 aligns with the 147-codon
kilB ORF of pIJ101 (11) beginning one base prior
to codon 42, extends to the end of the ORF, and is 95 and 75%
identical at the respective nucleotide and deduced amino acid levels to
the corresponding portion of the pIJ101 kilB gene (data not shown).
While sequences related to pIJ101 and other Streptomyces
plasmids were previously identified for both the deduced Rep protein of
pSB24.2 (9, 13), as well as for its putative site of
single-stranded nicking (5), our analysis here identifies
additional potential replication determinants (i.e., DSO and SSO
sequences) present within the published pSB24.2 nucleotide sequence
(2). The 480-bp region immediately 5' to the pSB24.2
rep gene (designated DSO in Fig. 1B) aligned with 79%
identity and 7% gaps (data not shown) to the corresponding 517-bp
region of pIJ101 shown previously to contain all essential
cis-acting sequences necessary for plasmid replication and
copy number control (3). Included within this region of
pSB24.2 is a stretch of 224 bp that is 100% identical to pIJ101 and
includes the putative nicking site at the end proximal to the
rep gene. The extended similarity between the pIJ101 DSO and
the corresponding region of pSB24.2 may indicate that essential replication sequences, besides the site of Rep-mediated nicking, have
remained conserved between the two plasmids.
Between its putative DSO region and partial kilB gene,
pSB24.2 also contains a copy of orf56 (Fig. 1B) that is 99%
identical at the nucleotide level and 96% identical at the deduced
amino acid level (data not shown) to orf56 of pIJ101
(11). The significance, however, of this sequence
conservation for orf56, an ORF that has been shown to be
dispensable for pIJ101 replication (29) and copy number
regulation (3), remains unknown.
While DSO regions within families of RCR plasmids display some sequence
similarity, SSO sequences are typically not well conserved, although
they generally show the potential for an extensive secondary structure
(13). In pIJ101, the SSO locus has been termed
sti and appears to confer a strong incompatibility phenotype
on pIJ101, such that Sti
plasmids cannot coexist with
Sti+ plasmids (4). The sti locus
(Fig. 1A) has been mapped to a point within roughly 200 bp
(4) upstream of the korB promoter (22). A 485-bp region of pSB24.2 located upstream of its
korB gene was found to align with 90% identity and 1% gaps
(data not shown) to a 491-bp region of pIJ101 that contains the pIJ101
sti locus and extends from the korB promoter to a
point within the small overlapping ORFs of unknown function
(11), orf79 and orf85 (Fig. 1A).
Within this region of pIJ101 exists a previously identified (28) potential stem-and-loop motif that contains five of six bases of the consensus hexanucleotide sequence 5' TAGCGT 3'
(Fig. 3A), which is present in many
SSOs (13). A nearly identical motif including the same
hexanucleotide sequence was also found within the corresponding region
of pSB24.2 (Fig. 3B). Because of the close similarity between the
sequences located upstream of korB on the two plasmids, we
have tentatively identified this region of pSB24.2 as the SSO (Fig.
1B); however, the exact determinants required for the efficient
conversion of single-strand plasmid intermediates into double-stranded
molecules as well as their potential to elicit strong incompatibility
effects similar to the pIJ101 sti locus remain to be
determined.
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Possible physical derivation of pSB24.2 from the parental plasmid pSB24.1. The genetic organization and nucleotide sequence of plasmid pSB24.2 thus strongly resemble the corresponding region of pIJ101 that extends from the clt locus clockwise to 3' sequences within the kilB gene (Fig. 1A). It therefore seems reasonable to speculate that the parental plasmid pSB24.1 may be organized similarly to pIJ101 throughout and that upon passage through S. lividans, pSB24.1, for reasons unknown, undergoes a spontaneous deletion of sequences (including its transfer-essential tra gene) that correspond to the portion of pIJ101 extending from within kilB clockwise to just beyond the korA gene (Fig. 1A); such a deletion would thereby result in the formation of the transfer-defective pSB24.2 derivative (Fig. 1B).
pSB24.2 encodes a functional clt locus that complements
the pIJ101 clt locus.
Our interest in conjugation in
Streptomyces prompted us to examine whether pSB24.2 encodes
a functional clt locus, and if so, whether this pSB24.2
locus can complement the clt function of the closely related
pIJ101 plasmid. Such complementation could potentially allow for the
subsequent identification of conserved functional sequences that are
important for clt activity. To test these possibilities, we
used a previously described mating system (16, 17) involving
an S. lividans donor strain (TK23.42), which contains
chromosomally integrated copies of the pIJ101 tra and
korA genes, and an S. lividans recipient strain
(TK23) containing pHYG1 (12), a transfer-defective,
hygromycin-resistant pIJ101 derivative. TK23.42 was transformed with
pSB24.202 (Table 1), a
thiostrepton-resistant derivative of pSB24.2, and plasmid-containing spores of this strain were mixed with TK23(pHYG1) recipient spores, plated on nonselective MY (malt extract-yeast extract) agar
(17), and then quantified following mating for donor,
recipient, and transconjugant cell types as described previously
(17).
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The pSB24.2 korB gene can complement the pIJ101 korB function. We also examined whether the pSB24.2 korB gene could functionally substitute for its pIJ101 counterpart to override the lethal effects of unregulated kilB gene expression in Streptomyces. When 100 ng of plasmid pGSP317, a pHYG1 derivative containing the unregulated pIJ101 kilB gene, was used to transform S. lividans TK23 according to the method of Hopwood et al. (7), a relatively low number of slow-growing transformants were observed after 4 days (i.e., an average of 2.8 × 102 transformants with a diameter of 1 mm or less was obtained in three independent assays). These transformants eventually grew to normal size and were therefore likely to be equivalent to similar transformants obtained previously (12) in which incoming plasmids carrying the unregulated kilB gene had subsequently deleted their kilB sequences. When TK23 was transformed with an equimolar amount of plasmid pGSP322, which is a derivative of pGSP317 containing the pSB24.2 korB gene, an average of 1.0 × 103 faster-growing transformants (i.e., with a diameter of greater than or equal to 2 mm after 4 days) was obtained in three independent assays; such numbers and growth phenotypes were similar to the results seen (i.e., an average of 1.8 × 103 faster-growing transformants) when TK23 was transformed with an equimolar amount of plasmid pGSP321, a pGSP317 derivative containing the pIJ101 korB gene.
Results here demonstrating that two important conjugation functions (i.e., clt and korB) have been conserved between plasmids pIJ101 and pSB24.2 are consistent with the hypothesis that pIJ101 and the parental plasmid pSB24.1 most likely encode the same conjugation system. Given the high degree of overall sequence relatedness between pIJ101 and pSB24.2, plasmids pIJ101 and pSB24.1 (along with its deletion derivative pSB24.2) may also encode highly similar, if not identical, replication systems. The possibility, however, that pIJ101 and pSB24.1 instead represent separate replicons possessing the same conjugation system is intriguing, as we are unaware of any example from other bacterial conjugation systems in which specific cis-acting transfer sequences (e.g., oriT sequences) (27) encoded by distinct replicons are functionally interchangeable. The hosts of pIJ101 and pSB24.1 (i.e., S. lividans and S. cyanogenus, respectively) have 99.8% similarity in their 16S rRNA gene sequences, indicating that they are closely related species (18). Our results are therefore consistent with the notion that conjugative pIJ101-like plasmids may be dispersed throughout at least a portion of the Streptomyces genus. Such dispersal has the potential to be even more widespread, however, given the extremely high, albeit limited, degree of relatedness between pIJ101 and pSN22, two plasmids that are derived from species (i.e., S. lividans and S. nigrifaciens, respectively) that have only 95.7% homology in their 16S rRNA genes and thus fall into different phylogenetic clusters (18).Nucleotide sequence accession number. The revised pSB24.2 korB-clt sequence determined in this study has been deposited in the GenBank database under accession no. AF133709.
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ACKNOWLEDGMENTS |
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We are very grateful to Alexei Sorokin and Sergey Sineoky, for their assistance in obtaining plasmid pSB24.2, and to Fred A. Rainey for assistance with nucleotide sequencing. We thank Naomi Ward-Rainey, John Battista, and Matthew Ducote for critical reading of the manuscript.
This work was supported by grant MCB-9604879 from the National Science Foundation (to G.S.P.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biological Sciences, Louisiana State University, 508 Life Sciences Building, Baton Rouge, LA 70803. Phone: (225) 388-2798. Fax: (225) 388-2597. E-mail: gpettis{at}unix1.sncc.lsu.edu.
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Journal of Bacteriology, August 1999, p. 4680-4685, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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