Department of Genetics1 and Department of Biochemistry2, University of Leicester, Leicester LE1 7RH, UK
Author for correspondence: Brian Wilkins. Tel: +44 116 252 3432. Fax: +44 116 252 5101. e-mail: bmw1{at}le.ac.uk
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ABSTRACT |
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Keywords: single strand promoters, IncI1 plasmid, bacterial conjugation, plasmid early genes, plasmid leading region
The EMBL accession number for the sequence reported in this paper is AJ238399.
a Present address: Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.
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INTRODUCTION |
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ColIb oriT flanks one end of the I1 transfer region and is orientated such that the transfer genes are the last to enter the recipient cell (Howland & Wilkins, 1988 ). The first portion of the plasmid to be transferred is called the leading region. This sector of ColIb carries some inessential genes that aid establishment of the entrant DNA in the new host cell. These genes are: ssb, encoding a ssDNA-binding protein of the type required for bacterial DNA replication (Howland et al., 1989
); psiB, determining a protein that inhibits induction of the bacterial SOS stress response in conjugation (Jones et al., 1992
); and ardA, which functions specifically in the conjugatively active recipient cell to alleviate type I restriction of the immigrant plasmid (Read et al., 1992
; Althorpe et al., 1999
). Highly conserved homologues of these genes are found in different combinations in the leading regions of enterobacterial plasmids of other Inc groups (Golub et al., 1988
; Chilley & Wilkins, 1995
). Measurement of transcript levels and the accumulation of gene products indicate that ardA, psiB and ssb genes function as early genes that are expressed rapidly and transiently in the conjugatively infected cell (Bagdasarian et al., 1992
; Jones et al., 1992
; Althorpe et al., 1999
). This mode of gene expression is called zygotic induction.
We report here tests indicating that zygotic induction is regulated by a process which is independent of a trans-acting repressor but dependent on the transcriptional orientation of the inducible gene on the T-strand. To identify motifs responsible for this mode of gene expression, the nucleotide sequence of 11·7 kb of the ColIb leading region was determined. The sequence contains three dispersed repeats indicative of single strand promoters. We propose that these signals initiate rapid transcription of genes on the incoming T-strand to facilitate establishment of the incoming plasmid in the new host cell.
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METHODS |
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ß-Galactosidase assays.
Assays were performed as described previously (Jones et al., 1992 ). Strain MC4110 carries a lac deletion; MC4100N is a derivative resistant to nalidixic acid.
Nucleotide sequencing and sequence analysis.
Substrates for sequencing reactions were pLG290 and subclones of pBR328-based recombinants containing the S3 (pLG2012) and S4 (pCRS4) fragments of ColIbdrd-1 (Rees et al., 1987 ). Subclones were generated by cleaving pLG2012 and pCRS4 with BglII, ClaI, PstI and SalI in single and double digests followed by ligation of fragments into vector pIC19H (Marsh et al., 1984
). Escherichia coli strain DH5
(recA1; Raleigh et al., 1989
) was used as host. Both DNA strands were sequenced. The sequence at a cloning junction was confirmed using a larger recombinant and primers designed to generate sequence data in one direction over the restriction site. The sequence at the junction of S3 and S4 was confirmed by sequencing the equivalent portion of E1 (see Fig. 3
). Oligonucleotides for sequencing primers were generated using a Perkin Elmer Biosystems (PE Biosystems) 394 synthesizer. Sequencing reactions used standard dye terminator kits from PE Biosystems and were analysed with a PE Biosystems 377 automated DNA sequencer. Both machines were operated by the PNACL service of Leicester University. Sequence data were analysed using the Wisconsin package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin, USA. The complete sequence was compiled using the GEL set of programs. FASTA and BLAST searches were carried out through the European Bioinformatics Institute site on the web (http://www.ebi.ac.uk). The entire sequence of ColIb-P9 has recently been made available as EMBL accession number AB021078.
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RESULTS AND DISCUSSION |
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The repressor hypothesis was tested by measuring the effect that carriage of pRR8 exerts on zygotic induction of a psiB::lacZ operon fusion transferred on a ColIb plasmid (Fig. 1a). Transfer of the lacZ fusion resulted in a burst of ß-galactosidase accumulation shown previously to occur in the transconjugant cell (Jones et al., 1992
). In such a conjugation, transfer is initiated asynchronously in the cell population typically to give a burst of transconjugant production between 7 and 20 min by which time about 60% of the input recipient cells have acquired the ColIbdrd plasmid. Subsequently, the rate of increase of transconjugant production diminishes. Within this framework, transfer of a single ColIb molecule is thought to take less than 3 min and to be associated with an even briefer burst of psiB transcription. Whereas psiB transcripts are unstable, ß-galactosidase is a metabolically stable protein (Althorpe et al., 1999
).
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In a parallel experiment (Fig. 1b), the psiB::lacZ fusion was transferred on an oriT-recombinant plasmid (pRR2-12) which was mobilized by a non-transmissible ColIb plasmid (pLG2062) in the donor. The latter plasmid determines all of the transfer functions but is immobile due to deletion of the nic site. Specific activity of ß-galactosidase was found to increase linearly in both the plasmid-free and pRR8-containing recipient cells to give approximately threefold higher values than those attained in Fig. 1(a)
. The increases are attributed to enhanced transfer of the psiB::lacZ recombinant plasmid (pRR2-12) rather than to its higher copy number relative to ColIb. The favoured explanation is that multiple rounds of pRR2-12 transfer occur when the plasmid is mobilized by a non-transmissible ColIb plasmid, possibly because the conjugation cycle is not terminated until the recipient has acquired a copy of the conjugative plasmid. This interpretation is suggested by data in Fig. 2
, showing that when pRR2-12 was mobilized by a transmissible ColIb plasmid, the amount of zygotic induction per transconjugant cell was similar to that determined when the psiB::lacZ fusion was located on ColIb itself (Fig. 1a
). Taken together, the results in Fig. 1
contradict the repressor hypothesis by showing that a recipient cell carrying a pre-existing copy of ColIb can support one or more cycles of zygotic induction.
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Inversion of the S4 fragment disrupts the downstream orf5 gene (Fig. 3), raising the hypothesis that zygotic induction requires carriage of a functional orf5 gene in cis. This possibility can be rejected because the lacZkanamycin resistance cassette used to construct the psiB::lacZ operon fusion contains a bidirectionally active transcriptional terminator downstream of the lacZ component (Kokotek & Lotz, 1989
). Since orf5 is thought to be part of the same transcriptional unit as psiB (see next section), the terminator should prevent orf5 transcription on both pRR2-12 and pRR2-14. Another possible explanation of the orientation-dependent induction of psiB is that inversion of S4 uncouples the gene from a promoter outside the fragment. If so, the hypothetical promoter would be at least 7·7 kb upstream of psiB (Fig. 3
). A more intriguing possibility is that psiB can only undergo zygotic induction when orientated in a specific direction relative to oriT.
Orientation of ORFs in the ColIb leading region
To establish whether all ORFs in the leading region have the same orientation, the sequence of the first 11712 nt in the ColIb leading region was determined. The sequence starts at the nic site which was located previously by sequence alignment with known features of the R64 oriT region (Althorpe et al., 1999 ). Ten ORFs were identified (Table 2
; Fig. 3
): three correspond to ardA, psiB and ssb, while a fourth is a homologue of the psiA gene of F-like plasmids (Loh et al., 1990
). Six other candidate genes (orf16) were identified on the basis of a codon preference bias similar to that in known leading region genes and a separation of at least 4 nt between the initiator codon and the putative ShineDalgarno sequence. Importantly, all 10 ORFs have the same transcriptional orientation.
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Orf6 is predicted to be a DNA-binding protein since it contains a helixturnhelix (residues 186205) motif of the AraC family of regulatory proteins which is indicative of sequence-specific DNA-binding proteins. The protein also contains a potential ATP/GTP binding-site motif A (P-loop; residues 178185). Apparently F-like plasmids also carry an orf6 homologue, as indicated by strong sequence similarity of orf6 to the sequence upstream of psiB on F and R6-5 (Dutreix et al., 1988 ). Thus it is proposed that psiA-psiB-orf6-ssb comprise a conserved module on ColIb and F-like plasmids. The module is inferred to be expressed as a single transcriptional unit on both plasmids, as indicated by activation of the PsiB phenotype by insertions in the upstream ssb gene (Dutreix et al., 1988
; Jones et al., 1992
) and the overlap between the start codon of psiA and the stop codon of psiB (Loh et al., 1990
; see also Table 2
).
Three copies of a putative single strand promoter
The leading region is remarkable in containing three repeats of a 328 bp element homologous to a novel promoter in ssDNA called Frpo (Masai & Arai, 1997 ). The repeats (ssi1, ssi2 and ssi3; Table 2
, Fig. 3
) are located in the same orientation approximately 55 nt upstream of orf1, orf4 and ssb; they share
80% sequence identity and display
59% identity to the central 150 nt portion of Frpo shown in Fig. 4
.
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The ColIb ssi elements have predicted secondary structures similar to that of Frpo (Fig. 4). The structure shown for Frpo is based on sensitivity to a single-strand-specific nuclease (Masai & Arai, 1997
); a thermodynamically favoured variant predicted by the MFOLD program (Mathews et al., 1999
) is shown as Frpo* (Fig. 4
). The secondary structures for ssi1, 2 and 3 are similar thermodynamically favoured predictions of the program. We therefore postulate that ssi1, 2 and 3 are also promoters active in ssDNA. The putative -10 regions contain multiple mismatches. As shown with synthetic heteroduplexes, mismatches in the -10 region may aid promoter function by facilitating melting of the duplex and formation of the open complex necessary for transcription. Strand bias exists in that E. coli RNA polymerase
70 primarily recognizes bases in the non-template strand of the -10 region, with -7T, -11A and -12T being the three most conserved residues (Roberts & Roberts, 1996
). Two of these three bases are present in the non-template strand of the putative -10 region of Frpo and ColIb ssi2 and ssi3.
Promoter strength is also affected by the spacing between the -35 and -10 hexamers. While a separation of 17 bp in duplex DNA is optimal, changes of 3 bp can be tolerated albeit with some loss of function possibly as a result of DNA deformation induced by the holoenzyme (Dombroski et al., 1996 ). The spacing between the putative -35 and -10 regions of the ssi elements and Frpo ranges from 18 to 20 nt. Such elongated spacings might be required to compensate for the mispaired nucleotides within them.
Frpo and the ColIb ssi elements are orientated in the respective leading regions such that the promoter structure is formed in the T-strand. This feature leads to the appealing model that zygotic induction of leading region genes is due to the activity of Frpo-like sequences in directing transcription of the transferring plasmid strand. The model predicts that a psiB inversion will be non-inducible, as observed in Fig. 2, because the T-strand will contain the antisense strand of the gene. Termination of transcription may occur immediately downstream of orf1 and orf2 at sequences t1 and t2 (Table 2
, Fig. 3
), which have the characteristics of
-independent transcription terminators (Platt, 1986
). Termination at t1 will prevent transcription across oriT and potential interference with reactions involved in the termination of a round of DNA transfer at the nic region and circularization of the transferring plasmid strand (see Lanka & Wilkins, 1995
).
The ssi promoters will be silenced by synthesis of the complementary DNA strand. There is no need to invoke a role for ColIb ssi elements in initiating complementary strand synthesis, since this mode of DNA synthesis is normally initiated by RNA primers made by a dedicated plasmid-encoded DNA primase. This enzyme is transmitted selectively from the donor to the recipient cell during bacterial conjugation (Rees & Wilkins, 1989 ).
Transcription of the incoming T-strand has biological appeal. It allows the ArdA antirestriction protein to accumulate in the newly infected cell before the T-strand is converted into duplex DNA, the substrate of type-I restriction enzymes. It allows rapid expression of PsiB as a function that prevents induction of the bacterial SOS response by the presumptive trigger of transferring ssDNA and it gives elevated levels of SSB protein, which may prevent drainage of the cellular counterpart and potentiate proteinDNA interactions peculiar to conjugation. The mechanism will also cause a burst of Orf5 transposase production in the new cell, which is expected to initiate transposition of the IS element and allow its spread to the genomes of bacteria outside the host range of ColIb. Finally, the concept that the plasmid leading region contains early genes that are expressed transiently in the conjugatively infected cell to promote establishment of the incoming genetic element puts plasmids on a mechanistic parallel with bacterial viruses which are known to have evolved a variety of strategies for phasing gene expression during cellular infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bagdasarian, M., Bailone, A., Angulo, J. F., Scholz, P., Bagdasarian, M. & Devoret, R. (1992). PsiB, an anti-SOS protein, is transiently expressed by the F sex factor during its transmission to an Escherichia coli K-12 recipient. Mol Microbiol 6, 885-893.[Medline]
Bancroft, I. & Wolk, C. P. (1989). Characterization of an insertion sequence (IS891) of novel structure from the cyanobacterium Anabaena sp. strain M-131. J Bacteriol 17, 5949-5954.
Billington, S. J., Sinistaj, M., Cheetham, B. F., Ayres, A., Moses, E. K., Katz, M. E. & Rood, J. I. (1996). Identification of a native Dichelobacter nodosus plasmid and implications for the evolution of the vap region. Gene 172, 111-116.[Medline]
Chilley, P. M. & Wilkins, B. M. (1995). Distribution of the ardA family of antirestriction genes on conjugative plasmids. Microbiology 141, 2157-2164.[Abstract]
Dombroski, J., Johnson, B. D., Lonetto, M. & Gross, C. A. (1996). The sigma subunit of Escherichia coli RNA polymerase senses promoter spacing. Proc Natl Acad Sci USA 93, 8858-8862.
Dutreix, M., Bäckman, A., Célérier, J., Bagdasarian, M. M., Sommer, S., Bailone, A., Devoret, R. & Bagdasarian, M. (1988). Identification of psiB genes of plasmids F and R6-5. Molecular basis for psiB enhanced expression in plasmid R6-5. Nucleic Acids Res 16, 10669-10679.[Abstract]
Furuya, N. & Komano, T. (1991). Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids. J Bacteriol 173, 6612-6617.[Medline]
Furuya, N. & Komano, T. (1994). Surface exclusion gene of IncI1 plasmid R64: nucleotide sequence and analysis of deletion mutants. Plasmid 32, 80-84.[Medline]
Golub, E. I., Bailone, A. & Devoret, R. (1988). A gene encoding an SOS inhibitor is present in different conjugative plasmids. J Bacteriol 170, 4392-4394.[Medline]
Howland, C. J. & Wilkins, B. M. (1988). Direction of conjugative transfer of IncI1 plasmid ColIb-P9. J Bacteriol 170, 4958-4959.[Medline]
Howland, C. J., Rees, C. E. D., Barth, P. T. & Wilkins, B. M. (1989). The ssb gene of plasmid ColIb-P9. J Bacteriol 171, 2466-2473.[Medline]
Jones, L., Barth, P. T. & Wilkins, B. M. (1992). Zygotic induction of plasmid ssb and psiB genes following conjugative transfer of IncI1 plasmid ColIb-P9. Mol Microbiol 6, 605-613.[Medline]
Kim, S.-R. & Komano, T. (1997). The plasmid R64 thin pilus identified as a type IV pilus. J Bacteriol 179, 3594-3603.[Abstract]
Kokotek, W. & Lotz, W. (1989). Construction of a lacZkanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. Gene 84, 467-471.[Medline]
Lanka, E. & Wilkins, B. M. (1995). DNA processing reactions in bacterial conjugation. Annu Rev Biochem 64, 141-169.[Medline]
Loh, S., Cram, D. & Skurray, R. (1989). Nucleotide sequence of the leading region adjacent to the origin of transfer on plasmid F and its conservation among conjugative plasmids. Mol Gen Genet 219, 177-186.[Medline]
Loh, S., Skurray, R., Célérier, J., Bagdasarian, M., Bailone, A. & Devoret, R. (1990). Nucleotide sequence of the psiA (plasmid SOS inhibition) gene located on the leading region of plasmids F and R6-5. Nucleic Acids Res 18, 4597.[Medline]
Marsh, J. L., Erfle, M. & Wykes, E. J. (1984). The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32, 481-485.[Medline]
Masai, H. & Arai, K. (1997). Frpo: a novel single-stranded DNA promoter for transcription and for primer RNA synthesis of DNA replication. Cell 89, 897-907.[Medline]
Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288, 911-940.[Medline]
Nomura, N., Masai, H., Inuzuka, M. & 7 other authors (1991). Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids. Gene 108, 1522.[Medline]
Platt, T. (1986). Transcript termination and the regulation of gene expression. Annu Rev Biochem 55, 339-372.[Medline]
Raleigh, E. A., Lech, K. & Brent, R. (1989). Selected topics from classical bacterial genetics. In Current Protocols in Molecular Biology, Vol. I, Unit 1.4. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Wiley.
Read, T. D., Thomas, T. & Wilkins, B. M. (1992). Evasion of type I and type II restriction systems by IncI1 plasmid ColIb-P9 during transfer by bacterial conjugation. Mol Microbiol 6, 1933-1941.[Medline]
Rees, C. E. D. & Wilkins, B. M. (1989). Transfer of tra proteins into the recipient cell during bacterial conjugation mediated by plasmid ColIb-P9. J Bacteriol 171, 3152-3157.[Medline]
Rees, C. E. D., Bradley, D. E. & Wilkins, B. M. (1987). Organization and regulation of the conjugation genes of IncI1 plasmid ColIb-P9. Plasmid 18, 223-236.[Medline]
Roberts, C. W. & Roberts, J. W. (1996). Base-specific recognition of the nontemplate strand of promoter DNA by E. coli RNA polymerase. Cell 86, 495-501.[Medline]
Roscoe, R. A. (1996). Regulation of leading region genes on IncI1 plasmid ColIb-P9. PhD thesis. University of Leicester, Leicester, UK.
Vapnek, D., Lipman, M. B. & Rupp, W. D. (1971). Physical properties and mechanism of transfer of R factors in Escherichia coli. J Bacteriol 108, 508-514.[Medline]
Wilkins, B. & Lanka, E. (1993). DNA processing and replication during plasmid transfer between Gram-negative bacteria. In Bacterial Conjugation, pp. 105-136. Edited by D. B. Clewell. New York: Plenum.
Received 2 June 1999;
revised 28 July 1999;
accepted 29 July 1999.