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A consensus Porphyromonas gingivalis promoter sequence
FEMS Microbiology Letters 186 (2000) 133^138
www.fems-microbiology.org
A consensus Porphyromonas gingivalis promoter sequence Christine A. Jackson, Brigitte Ho¡mann, Nada Slakeski, Steven Cleal, Anne J. Hendtlass, Eric C. Reynolds * Biochemistry and Molecular Biology Unit, The School of Dental Science, The University of Melbourne, 711 Elizabeth St., Melbourne, Vic. 3000, Australia Received 29 November 1999; received in revised form 8 March 2000 ; accepted 11 March 2000
Abstract We have determined the transcription start points (tsp) for recently identified Porphyromonas gingivalis W50 genes, kgp, rgpA, rgpB (formerly designated prtK, prtR, and prtRII respectively), fetB and the mcmAB operon. Alignment of the DNA upstream of these tsp and those from the literature has enabled us to identify consensus sequences that may represent a P. gingivalis promoter. There is a potential 310 hexamer sequence, 5P-TATATT-3P centred on average at 310/11 nt which is repeated at 319/20 nt and an upstream consensus, 5P-CAGAT(A/G)-3P which is centred at 339/40 nt. ß 2000 Published by Elsevier Science B.V. All rights reserved. Keywords : RNA polymerase; Sigma-70; Transcription start point; Promoter ; Expression ; Porphyromonas gingivalis
1. Introduction Porphyromonas gingivalis is a Gram-negative, anaerobic, coccobacillary pathogen implicated in the onset and progression of adult periodontitis [1]. Many P. gingivalis genes have been sequenced, including those encoding proteinases [2^4], and ¢mbriae [5,6]. Recently, the complete genome of P. gingivalis strain W83 has been determined (www.tigr.org) and although amino acid sequences can be deduced using this approach, little information can be gathered regarding the regulatory elements of these genes, and how protein expression is controlled. Transcription start points (tsp) have been reported for the P. gingivalis genes, prtT, which encodes a cysteine proteinase [7], hemR, a haemin-regulated gene [8], the groESL operon [9], the proteinase tpr [10], sodA the superoxide dismutase gene [11] and the ¢mbrillin gene ¢mA [12]. Promoter-like sequences were subsequently designated for prtT, groESL and tpr using the Escherichia coli sigma-70 (c70 ) consensus binding sequence, although E. coli is only distantly related to the Porphyromonas genus [13]. The groESL operon also appears to exhibit a homologue of a sigma-32 heat shock promoter, which produces a heat inducible, shorter, tran-
script [9]. The promoter of ¢mA has been subjected to sequence analysis by Xie and Lamont [12], which involved the construction of site-speci¢c and deletion mutants. The results of this study suggested that two sequences, 5P-TATGAC-3P centred at 320/21 and 5P-TTGTTG-3P centred at 343/44 comprised the promoter element. Further, Xie and Lamont [12] showed that AT-rich upstream sequences were required for full expression of the ¢mA gene. In an approach to enhance our understanding of gene regulation in P. gingivalis we have determined the tsp for a number of recently identi¢ed P. gingivalis genes. This information combined with the published tsp data has enabled us to identify a putative P. gingivalis promoter consensus sequence. 2. Materials and methods 2.1. Bacterial strains and culture conditions E. coli strain JM109 was host for plasmid clones used for DNA sequencing and was grown using standard conditions [14]. P. gingivalis W50 was grown as described previously [15]. Cells were harvested at a density of approximately 3U108 cells ml31 . 2.2. Genes, plasmids and DNA sequencing
* Corresponding author. Tel. : +61 (3) 93410270; Fax: +61 (3) 93410236; E-mail: [email protected]
Genes for which tsp were determined were the mcmAB
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Table 1 Oligonucleotides used in primer extension and sequencing reactions Gene
Oligonucleotide sequencea
Rangeb
mcmAB fetB kgp rgpA rgpB
5P-CCGGAGGGAATTCGGAAAAGAGC-3P 5P-CCATAAGTCCCAAAGTCGC-3P 5P-CCAAAAGGGACGCCGCGATCAGCA-3P 5P-GCAAAGAGCAATCGAAACAAACTTGTTCAAG-3P 5P-GAATGCTACGATCGAAACGATCCTGC-3P
+21^+43 +19^+37 +17^+40 +9^+39 +17^+42
a b
The oligonucleotides are complementary to the coding strand. +1 is `A' of the methionine start codon on the coding strand.
operon, which encodes methylmalonyl-CoA mutase [16], fetB (formerly designated pga30, GenBank accession number AF195649), which encodes an outer membrane haemebinding protein [17] and three proteinase-encoding genes, kgp (formerly prtK) [4], rgpA (formerly prtR) [2], and rgpB (formerly prtRII) [3]. Plasmids containing the genes under study were prepared using the method of Li and Schweizer [18]. DNA sequencing was performed using standard methods. The oligonucleotides used in the primer extension and sequencing reactions are listed in Table 1. 2.3. Preparation of total RNA and primer extension reactions Total P. gingivalis RNA was isolated as described previously [2]. Primer extension was performed using 10 Wg of total RNA and the AMV reverse transcriptase primer extension system (Promega Corporation, Madison, WI, USA), as per instructions. 2.4. Sequence alignment analysis The nucleotide sequences upstream of the determined tsp were aligned using the Pileup program of the University of Wisconsin Genetics Computing Group, accessed from The Australian National Genomic Information Service. Gap penalties used were 5.0 and gap extension penalties 0.3. To improve a region of local homology between the 5P section of the sequences small gaps were introduced as appropriate. 3. Results and discussion 3.1. The promoter consensus Primer extension reactions were performed to determine the tsp of mcmAB, fetB, kgp, rgpA and rgpB (Fig. 1). The tsp for mcmAB was nt-64 (the translation start codon methionine being +1), while the others were further upstream, the fetB tsp was nt 3267, the kgp tsp was nt 3170, the rgpA tsp was nt 3109, and for rgpB transcription was determined to begin at nt 3131. The tsp of kgp at nt 3170 supports the proposal of Simpson et al. [19] that
an insertion of IS1126 at nt 3185 is within the promoter region of the kgp gene. Transcription initiation well upstream of the translation initiation sites has been observed with other P. gingivalis genes, for prtT the tsp is at nt 3510, for tpr it is nt 3215, for hemR nt 3240 and for sodA it is nt 3315. The tsp of ¢mA is reported at the moderate distance of nt 341, the tsp associated with the sigma-32 promoter-like region of groESL is at nt 334 (and possibly nt 339) with the more distal groESL tsp at nt 369 and 370. It would appear, therefore, for some P. gingivalis genes that transcription is initiated well upstream of the translation start site, a situation which has also been observed in other members of the Bacteroidaceae [20,21]. This should be considered when cloning P. gingivalis genes, especially if the aim is gene expression from endogenous promoters. The DNA sequences upstream of the tsp of the above genes (using the more distal groESL tsp) were aligned (Fig. 1). Nucleotides that occurred at least four times (44% frequency) in any position were considered to be of potential signi¢cance. Clusters of conserved sequence became apparent, two of which coincided with the location of the ¢mA promoter sequences recently reported by Xie and Lamont [12]. The ¢rst consensus sequence, 5P-TATATT-3P centred at 319/20, which will hereafter be designated P3P, has nt frequencies of 5/10, 8/10, 6/10, 6/10, 7/10 and 5/10, respectively. The other consensus sequence 5P-CAGAT(A/G)-3P centred at 339/40, hereafter designated P5P, has nt frequencies of 6/10, 6/10, 6/10, 7/10, 5/10 and 4/10 respectively. The average spacing between these clusters is 14 nt, with a range of 12^17 nt. Of note is a cluster similar to P3P centred at nt 310/11 (Fig. 2). This sequence 5P-TATATT-3P (P3Q) occurs with nt frequencies of 9/10, 5/10, 5/10, 4/10, 6/10 and 6/10, respectively. Further, 3 nt upstream of both P3P and P3Q `G' occurs with high frequency, whilst 3 nt downstream of each, `C' frequently occurs. Interestingly with rgpA and rgpB the sequence around P3Q, 5P-TGNNTATATTTGC-3P is identical, yet upstream of this the sequences have no more than random similarity. These genes encode cysteine proteinases that have a high degree of amino acid sequence similarity and may have evolved following a gene duplication event [3,22]. Although the conservation of this sequence may be an evolutionary remnant, the sub-sequence 5P-TTGC-3P, or a close derivative thereof, is found in association with many of the other P3Q elements (Fig. 2). A consensus sequence for Bacteroides fragilis promoters has recently been reported [20]. This sequence 5P-TAXXTTTG-3P occurs within 15 nt upstream of the +1 site, and bears striking similarity to, or indeed matches exactly, many of the sequences at P3Q (Fig. 2). This consensus sequence is also present upstream of the tsp of Prevotella spp. [21], which suggests that the Bacteroidaceae may have a common RNA polymerase (RNAP) promoter recognition sequence. A role for the P3Q element in P. gingivalis promoter func-
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Fig. 1. Primer extension analysis of the tsp of the P. gingivalis genes rgpA, rgpB, fetB and the mcmAB operon. [35 S]dCTP-labeled DNA sequencing ladders and 32 P-labeled primer extension cDNAs were electrophoresed in parallel using 8% denaturing polyacrylamide gels. Both the cDNA and sequencing ladders were synthesised using the same oligonucleotide primer (Table 1). The tsp is indicated by an arrow.
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Fig. 2. Alignment of the DNA sequences upstream of the tsp of several P. gingivalis genes. Putative promoter recognition sequences are single-underlined, with the proposed ¢mA promoter boxes [12] double-underlined. The tsp are in lower case. The TTGC-like sequences (see text) are in bold.
tion may also be indicated by the increase in activity observed with the ¢mA promoter following mutation of nt 39 to 311 TAA to GCC [12]. The P3P and P3Q boxes have a 5/6 match with the E. coli c70 -10 consensus sequence (5P-TATAAT-3P) and indeed have the highly conserved TAXXXT motif [23,24]. Therefore it would be anticipated that these sequences may be recognised by c70 in E. coli. The P5P box however, is less similar to the c70 -35 box consensus sequence 5P-TTGACA-3P, so c70 recognition of the P5P hexamer is less likely. Also the spacer sequences between the P5P and P3P hexamers were generally smaller than the optimum 17 nt spacing observed with E. coli c70 promoters. These factors may have a bearing on the observed poor expression of P. gingivalis genes in E. coli from endogenous promoters. The mRNAs of two genes which are expressed in E. coli, tpr and ¢mA, have each been shown to be initiated from a site other than that used in P. gingivalis [10,12]. Functional di¡erences between the E. coli and P. gingivalis RNAP were suggested in an in vitro study reported by Klimpel and Clark [25], which demonstrated that the
Table 2 Percentage A+T upstream of tsp Gene
A+T (%) nt 31 to 350
A+T (%) nt 31 to 3100
hemR tpr groESL mcmAB kgp rgpA rgpB prtT ¢mA fetB
56 62 66 70 68 70 72 64 58 42
53 65 60 61 66 64 69 62 68 42
P. gingivalis RNAP could not initiate transcription from E. coli and bacteriophage T7 DNA promoters. E. coli RNAP however could initiate transcription using P. gingivalis DNA, but with an e¤ciency of only 25% of that when using E. coli DNA as template [25]. Similarly the B. fragilis RNAP also fails to initiate transcription from E. coli promoters. E. coli promoter: :cat (chloramphenicol acetyl transferase gene) fusions are non-functional in B. fragilis, whereas in E. coli, B. fragilis promoter : :cat fusions do function, albeit very poorly [26]. The P. gingivalis consensus promoter sequence determined here is a further indicator that the P. gingivalis RNAP may di¡er from that of E. coli. 3.2. Analysis of the 5P DNA sequences The majority of the nucleotide sequences upstream of the tsp are A+T-rich (Table 2). With the exception of hemR and fetB, there is 58^72% A+T over nt 31 to 350 in a genome that is only 52^54% A+T overall [27]. Over 100 nt, the percentage A+T is still high at 60^69%. DNA rich in A+T has a tendency to bends and groove distortions, particularly where there are polyadenine (A4ÿ6 ) runs [28,29]. The architecture of the DNA within a promoter region may in£uence the e¤ciency of promoter recognition by the core polymerase, the polymerase holoenzyme and accessory proteins. DNA structural predictions [30,31] reveal a number of bends (up to 90³), as well as distortions to the DNA grooves, in these upstream sequences. This suggests that the DNA in the region of P. gingivalis promoters could have secondary structural features that may be relevant to gene expression. The 340 to 360 region in several E. coli promoters has now been recognised as a site to which the K subunit of RNA polymerase (RpoA) binds and has been designated
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the UP element [32,33]. Binding of RpoA to the UP-element enhances recognition of the core promoter sequences by the core polymerase which in turn enhances the rate of transcription. The A+T-rich nature of some of the P. gingivalis sequences in these 340 to 360 regions suggests there is potential for UP-element type regulation. This proposal is supported by the results of the ¢mA promoter studies, in which deletion of the DNA between 345 and 363 reduced activity of the promoter, as did deletion of A+T-rich sequences between 390 and 3240 [12]. Our understanding of P. gingivalis gene regulation is just beginning, but the consensus promoter sequences determined here give a starting point from which to identify potential promoter sequences of other P. gingivalis genes.
[10]
[11]
[12]
[13] [14]
[15]
[16]
Acknowledgements This work was supported by an International Association for Dental Research John A. Gray Fellowship. We wish to thank John Davies for DNA structural predictions.
References
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[19]
[1] Ha¡ajee, A.D., Cugini, M.A., Tanner, A., Pollack, R.P., Smith, C., Kent Jr., R.L. and Socransky, S.S. (1998) Subgingival microbiota in healthy, well-maintained elder and periodontitis subjects. J. Clin. Periodontol. 25, 346^353. [2] Slakeski, N., Cleal, S.M. and Reynolds, E.C. (1996) Characterization of a Porphyromonas gingivalis gene prtR that encodes an argininespeci¢c thiol proteinase and multiple adhesins. Biochem. Biophys. Res. Commun. 224, 605^610. [3] Slakeski, N., Bhogal, P.S., O'Brien-Simpson, N. and Reynolds, E.C. (1998) Characterization of a second cell-associated Arg-speci¢c cysteine proteinase of Porphyromonas gingivalis and identi¢cation of an adhesin binding motif involved in association of the prtR and prtK proteinases and adhesins into large complexes. Microbiology 144, 1583^1592. [4] Slakeski, N., Cleal, S.M., Bhogal, P.S. and Reynolds, E.C. (1999) Characterization of a Porphyromonas gingivalis gene prtK that encodes a lysine-speci¢c cysteine proteinase and three sequence-related adhesins. Oral Microbiol. Immunol. 14, 92^97. [5] Dickinson, D.P., Kubiniec, M.A., Yoshimura, F. and Genco, R.J. (1988) Molecular cloning and sequencing of the gene encoding the ¢mbrial subunit protein of Bacteroides gingivalis. J. Bacteriol. 170, 1658^1665. [6] Yoshimura, F. and Takahashi, Y. (1991) Cloning of virulence factors of periodontopathic bacteria. In: Periodontal Disease: Pathogens and Host Immune Responses (Hamada, S., Holt, S.C. and McGhee, J.R., Eds.), pp. 155^165. Quintessence, Tokyo. [7] Madden, T.E., Clark, V.L. and Kuramitsu, H.K. (1995) Revised sequence of the Porphyromonas gingivalis PrtT cysteine protease/ hemagglutinin gene: homology with streptococcal pyrogenic exotoxin B/streptococcal proteinase. Infect. Immun. 63, 238^247. [8] Karunakaran, T., Madden, T. and Kuramitsu, H. (1997) Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis. J. Bacteriol. 179, 1898^1908. [9] Hotokezaka, H., Ohara, N., Hayashida, H., Matsumoto, S., Matsuo, T., Naito, M., Kobayashi, K. and Yamada, T. (1997) Transcriptional
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analysis of the groESL operon from Porphyromonas gingivalis. Oral Microbiol. Immunol. 12, 236^239. Lu, B. and McBride, B.C. (1998) Expression of the tpr protease gene of Porphyromonas gingivalis is regulated by peptide nutrients. Infect. Immun. 66, 5147^5156. Lynch, M.C. and Kuramitsu, H.K. (1999) Role of superoxide dismutase activity in the physiology of Porphyromonas gingivalis. Infect. Immun. 67, 3367^3375. Xie, H. and Lamont, R.J. (1999) Promoter architecture of the Porphyromonas gingivalis ¢mbrillin gene. Infect. Immun. 67, 3227^ 3253. Woese, C.R. (1987) Bacterial Evolution. Microbiol. Rev. 51, 221^271. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. McKee, A.S., McDermid, A.S., Baskerville, A., Dowsett, A.B., Ellwood, D.C. and Marsh, P.D. (1986) E¡ect of Hemin on the Physiology and Virulence of Bacteroides gingivalis W50. Infect. Immun. 52, 349^355. Jackson, C.A., Kirszbaum, L., Dashper, S. and Reynolds, E.C. (1995) Cloning, expression and sequence analysis of the genes encoding the heterodimeric methylmalonyl-CoA mutase of Porphyromonas gingivalis W50. Gene 167, 127^132. Hendtlass, A.J. (1999) A 30 kDa antigenic heme-binding protein of Porphyromonas gingivalis. PhD thesis, The School of Dental Science, The University of Melbourne, Melbourne. Li, M. and Schweizer, H.P. (1993) Resolution of common DNA sequencing ambiguities of GC-rich DNA templates by terminal deoxynucleotidal transferase without dGTP analogues. Focus 15, 19^ 20. Simpson, W., Wang, C.-Y., Mikolajczyk-Pawlinska, J., Potempa, J., Travis, J., Bond, V.C. and Genco, C.A. (1999) Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis. Infect. Immun. 67, 5012^ 5020. Bayley, D.P. and Smith, C.J. (1998) In:. Abstr. Annu. Meet. ASM, p. 293. Atlanta, GA. Manch-Citron, J.N., Dey, A., Schneider, R. and Nguyen, N.Y. (1999) The translational hop junction and the 5P transcriptional start site for the Prevotella loescheii adhesin encoded by plaA. Curr. Microbiol. 38, 22^26. Nakayama, K. (1997) Domain-speci¢c rearrangement between the two Arg-gingipain-encoding genes in Porphyromonas gingivalis : possible involvement of non-reciprocal recombination. Microbiol. Immunol. 41, 185^196. Rosenberg, M. and Court, D. (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Ann. Rev. Genet. 13, 319^353. Hawley, D.K. and McClure, W.R. (1983) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11, 2237^2255. Klimpel, K.W. and Clark, V.L. (1990) The RNA polymerase of Porphyromonas gingivalis and Fusobacterium nucleatum are unrelated to the RNA polymerase of Escherichia coli. J. Dent. Res. 69, 1567^ 1572. Smith, C.J., Rogers, M.B. and McKee, M.L. (1992) Heterologous gene expression in Bacteroides fragilis. Plasmid 27, 141^154. Shah, H.N. and Collins, M.D. (1988) Proposal for reclassi¢cation of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus Porphyromonas. Int. J. Syst. Bacteriol. 38, 128^131. Koo, H.-S., Wu, H.-M. and Crothers, D.M. (1986) DNA bending at adenine thymine tracts. Nature 320, 501^506. Trifonov, E.N. (1991) DNA in pro¢le. Trends Biochem. Sci. 16, 467^ 470. Cacchione, S., Cerone, P., De Santis, P. and Savino, M. (1995) Superstructural features of the upstream regulatory regions of two b
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pea rbcS genes and nucleosomes positioning: theoretical prediction and experimental evaluation. Biophys. Chem. 53, 267^281. [31] De Santis, P., Fua, M., Anselmi, C. and Bocchinfuso, G. (1996) Sequence dependent circularization of DNAs : a physical model to predict the DNA sequence dependent propensity to circularization and its changes in the presence of protein-induced bending. J. Phys. Chem. 100, 9968^9976. [32] Landini, P. and Volkert, M.R. (1995) RNA polymerase K subunit
binding site in positively controlled promoters: a new model for RNA polymerase-promoter interaction and transcriptional activation in the Escherichia coli ada and aidB genes. EMBO J. 14, 4329^ 4335. [33] Ross, W., Aiyar, S.E., Salomon, J. and Gourse, R.L. (1998) Escherichia coli promoters with UP elements of di¡erent strengths: modular structure of bacterial promoters. J. Bacteriol. 180, 5375^5383.
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A consensus Porphyromonas gingivalis promoter sequence Christine A. Jackson, Brigitte Ho¡mann, Nada Slakeski, Steven Cleal, Anne J. Hendtlass, Eric C. Reynolds * Biochemistry and Molecular Biology Unit, The School of Dental Science, The University of Melbourne, 711 Elizabeth St., Melbourne, Vic. 3000, Australia Received 29 November 1999; received in revised form 8 March 2000 ; accepted 11 March 2000
Abstract We have determined the transcription start points (tsp) for recently identified Porphyromonas gingivalis W50 genes, kgp, rgpA, rgpB (formerly designated prtK, prtR, and prtRII respectively), fetB and the mcmAB operon. Alignment of the DNA upstream of these tsp and those from the literature has enabled us to identify consensus sequences that may represent a P. gingivalis promoter. There is a potential 310 hexamer sequence, 5P-TATATT-3P centred on average at 310/11 nt which is repeated at 319/20 nt and an upstream consensus, 5P-CAGAT(A/G)-3P which is centred at 339/40 nt. ß 2000 Published by Elsevier Science B.V. All rights reserved. Keywords : RNA polymerase; Sigma-70; Transcription start point; Promoter ; Expression ; Porphyromonas gingivalis
1. Introduction Porphyromonas gingivalis is a Gram-negative, anaerobic, coccobacillary pathogen implicated in the onset and progression of adult periodontitis [1]. Many P. gingivalis genes have been sequenced, including those encoding proteinases [2^4], and ¢mbriae [5,6]. Recently, the complete genome of P. gingivalis strain W83 has been determined (www.tigr.org) and although amino acid sequences can be deduced using this approach, little information can be gathered regarding the regulatory elements of these genes, and how protein expression is controlled. Transcription start points (tsp) have been reported for the P. gingivalis genes, prtT, which encodes a cysteine proteinase [7], hemR, a haemin-regulated gene [8], the groESL operon [9], the proteinase tpr [10], sodA the superoxide dismutase gene [11] and the ¢mbrillin gene ¢mA [12]. Promoter-like sequences were subsequently designated for prtT, groESL and tpr using the Escherichia coli sigma-70 (c70 ) consensus binding sequence, although E. coli is only distantly related to the Porphyromonas genus [13]. The groESL operon also appears to exhibit a homologue of a sigma-32 heat shock promoter, which produces a heat inducible, shorter, tran-
script [9]. The promoter of ¢mA has been subjected to sequence analysis by Xie and Lamont [12], which involved the construction of site-speci¢c and deletion mutants. The results of this study suggested that two sequences, 5P-TATGAC-3P centred at 320/21 and 5P-TTGTTG-3P centred at 343/44 comprised the promoter element. Further, Xie and Lamont [12] showed that AT-rich upstream sequences were required for full expression of the ¢mA gene. In an approach to enhance our understanding of gene regulation in P. gingivalis we have determined the tsp for a number of recently identi¢ed P. gingivalis genes. This information combined with the published tsp data has enabled us to identify a putative P. gingivalis promoter consensus sequence. 2. Materials and methods 2.1. Bacterial strains and culture conditions E. coli strain JM109 was host for plasmid clones used for DNA sequencing and was grown using standard conditions [14]. P. gingivalis W50 was grown as described previously [15]. Cells were harvested at a density of approximately 3U108 cells ml31 . 2.2. Genes, plasmids and DNA sequencing
* Corresponding author. Tel. : +61 (3) 93410270; Fax: +61 (3) 93410236; E-mail: [email protected]
Genes for which tsp were determined were the mcmAB
0378-1097 / 00 / $20.00 ß 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 1 3 1 - 2
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Table 1 Oligonucleotides used in primer extension and sequencing reactions Gene
Oligonucleotide sequencea
Rangeb
mcmAB fetB kgp rgpA rgpB
5P-CCGGAGGGAATTCGGAAAAGAGC-3P 5P-CCATAAGTCCCAAAGTCGC-3P 5P-CCAAAAGGGACGCCGCGATCAGCA-3P 5P-GCAAAGAGCAATCGAAACAAACTTGTTCAAG-3P 5P-GAATGCTACGATCGAAACGATCCTGC-3P
+21^+43 +19^+37 +17^+40 +9^+39 +17^+42
a b
The oligonucleotides are complementary to the coding strand. +1 is `A' of the methionine start codon on the coding strand.
operon, which encodes methylmalonyl-CoA mutase [16], fetB (formerly designated pga30, GenBank accession number AF195649), which encodes an outer membrane haemebinding protein [17] and three proteinase-encoding genes, kgp (formerly prtK) [4], rgpA (formerly prtR) [2], and rgpB (formerly prtRII) [3]. Plasmids containing the genes under study were prepared using the method of Li and Schweizer [18]. DNA sequencing was performed using standard methods. The oligonucleotides used in the primer extension and sequencing reactions are listed in Table 1. 2.3. Preparation of total RNA and primer extension reactions Total P. gingivalis RNA was isolated as described previously [2]. Primer extension was performed using 10 Wg of total RNA and the AMV reverse transcriptase primer extension system (Promega Corporation, Madison, WI, USA), as per instructions. 2.4. Sequence alignment analysis The nucleotide sequences upstream of the determined tsp were aligned using the Pileup program of the University of Wisconsin Genetics Computing Group, accessed from The Australian National Genomic Information Service. Gap penalties used were 5.0 and gap extension penalties 0.3. To improve a region of local homology between the 5P section of the sequences small gaps were introduced as appropriate. 3. Results and discussion 3.1. The promoter consensus Primer extension reactions were performed to determine the tsp of mcmAB, fetB, kgp, rgpA and rgpB (Fig. 1). The tsp for mcmAB was nt-64 (the translation start codon methionine being +1), while the others were further upstream, the fetB tsp was nt 3267, the kgp tsp was nt 3170, the rgpA tsp was nt 3109, and for rgpB transcription was determined to begin at nt 3131. The tsp of kgp at nt 3170 supports the proposal of Simpson et al. [19] that
an insertion of IS1126 at nt 3185 is within the promoter region of the kgp gene. Transcription initiation well upstream of the translation initiation sites has been observed with other P. gingivalis genes, for prtT the tsp is at nt 3510, for tpr it is nt 3215, for hemR nt 3240 and for sodA it is nt 3315. The tsp of ¢mA is reported at the moderate distance of nt 341, the tsp associated with the sigma-32 promoter-like region of groESL is at nt 334 (and possibly nt 339) with the more distal groESL tsp at nt 369 and 370. It would appear, therefore, for some P. gingivalis genes that transcription is initiated well upstream of the translation start site, a situation which has also been observed in other members of the Bacteroidaceae [20,21]. This should be considered when cloning P. gingivalis genes, especially if the aim is gene expression from endogenous promoters. The DNA sequences upstream of the tsp of the above genes (using the more distal groESL tsp) were aligned (Fig. 1). Nucleotides that occurred at least four times (44% frequency) in any position were considered to be of potential signi¢cance. Clusters of conserved sequence became apparent, two of which coincided with the location of the ¢mA promoter sequences recently reported by Xie and Lamont [12]. The ¢rst consensus sequence, 5P-TATATT-3P centred at 319/20, which will hereafter be designated P3P, has nt frequencies of 5/10, 8/10, 6/10, 6/10, 7/10 and 5/10, respectively. The other consensus sequence 5P-CAGAT(A/G)-3P centred at 339/40, hereafter designated P5P, has nt frequencies of 6/10, 6/10, 6/10, 7/10, 5/10 and 4/10 respectively. The average spacing between these clusters is 14 nt, with a range of 12^17 nt. Of note is a cluster similar to P3P centred at nt 310/11 (Fig. 2). This sequence 5P-TATATT-3P (P3Q) occurs with nt frequencies of 9/10, 5/10, 5/10, 4/10, 6/10 and 6/10, respectively. Further, 3 nt upstream of both P3P and P3Q `G' occurs with high frequency, whilst 3 nt downstream of each, `C' frequently occurs. Interestingly with rgpA and rgpB the sequence around P3Q, 5P-TGNNTATATTTGC-3P is identical, yet upstream of this the sequences have no more than random similarity. These genes encode cysteine proteinases that have a high degree of amino acid sequence similarity and may have evolved following a gene duplication event [3,22]. Although the conservation of this sequence may be an evolutionary remnant, the sub-sequence 5P-TTGC-3P, or a close derivative thereof, is found in association with many of the other P3Q elements (Fig. 2). A consensus sequence for Bacteroides fragilis promoters has recently been reported [20]. This sequence 5P-TAXXTTTG-3P occurs within 15 nt upstream of the +1 site, and bears striking similarity to, or indeed matches exactly, many of the sequences at P3Q (Fig. 2). This consensus sequence is also present upstream of the tsp of Prevotella spp. [21], which suggests that the Bacteroidaceae may have a common RNA polymerase (RNAP) promoter recognition sequence. A role for the P3Q element in P. gingivalis promoter func-
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135
Fig. 1. Primer extension analysis of the tsp of the P. gingivalis genes rgpA, rgpB, fetB and the mcmAB operon. [35 S]dCTP-labeled DNA sequencing ladders and 32 P-labeled primer extension cDNAs were electrophoresed in parallel using 8% denaturing polyacrylamide gels. Both the cDNA and sequencing ladders were synthesised using the same oligonucleotide primer (Table 1). The tsp is indicated by an arrow.
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Fig. 2. Alignment of the DNA sequences upstream of the tsp of several P. gingivalis genes. Putative promoter recognition sequences are single-underlined, with the proposed ¢mA promoter boxes [12] double-underlined. The tsp are in lower case. The TTGC-like sequences (see text) are in bold.
tion may also be indicated by the increase in activity observed with the ¢mA promoter following mutation of nt 39 to 311 TAA to GCC [12]. The P3P and P3Q boxes have a 5/6 match with the E. coli c70 -10 consensus sequence (5P-TATAAT-3P) and indeed have the highly conserved TAXXXT motif [23,24]. Therefore it would be anticipated that these sequences may be recognised by c70 in E. coli. The P5P box however, is less similar to the c70 -35 box consensus sequence 5P-TTGACA-3P, so c70 recognition of the P5P hexamer is less likely. Also the spacer sequences between the P5P and P3P hexamers were generally smaller than the optimum 17 nt spacing observed with E. coli c70 promoters. These factors may have a bearing on the observed poor expression of P. gingivalis genes in E. coli from endogenous promoters. The mRNAs of two genes which are expressed in E. coli, tpr and ¢mA, have each been shown to be initiated from a site other than that used in P. gingivalis [10,12]. Functional di¡erences between the E. coli and P. gingivalis RNAP were suggested in an in vitro study reported by Klimpel and Clark [25], which demonstrated that the
Table 2 Percentage A+T upstream of tsp Gene
A+T (%) nt 31 to 350
A+T (%) nt 31 to 3100
hemR tpr groESL mcmAB kgp rgpA rgpB prtT ¢mA fetB
56 62 66 70 68 70 72 64 58 42
53 65 60 61 66 64 69 62 68 42
P. gingivalis RNAP could not initiate transcription from E. coli and bacteriophage T7 DNA promoters. E. coli RNAP however could initiate transcription using P. gingivalis DNA, but with an e¤ciency of only 25% of that when using E. coli DNA as template [25]. Similarly the B. fragilis RNAP also fails to initiate transcription from E. coli promoters. E. coli promoter: :cat (chloramphenicol acetyl transferase gene) fusions are non-functional in B. fragilis, whereas in E. coli, B. fragilis promoter : :cat fusions do function, albeit very poorly [26]. The P. gingivalis consensus promoter sequence determined here is a further indicator that the P. gingivalis RNAP may di¡er from that of E. coli. 3.2. Analysis of the 5P DNA sequences The majority of the nucleotide sequences upstream of the tsp are A+T-rich (Table 2). With the exception of hemR and fetB, there is 58^72% A+T over nt 31 to 350 in a genome that is only 52^54% A+T overall [27]. Over 100 nt, the percentage A+T is still high at 60^69%. DNA rich in A+T has a tendency to bends and groove distortions, particularly where there are polyadenine (A4ÿ6 ) runs [28,29]. The architecture of the DNA within a promoter region may in£uence the e¤ciency of promoter recognition by the core polymerase, the polymerase holoenzyme and accessory proteins. DNA structural predictions [30,31] reveal a number of bends (up to 90³), as well as distortions to the DNA grooves, in these upstream sequences. This suggests that the DNA in the region of P. gingivalis promoters could have secondary structural features that may be relevant to gene expression. The 340 to 360 region in several E. coli promoters has now been recognised as a site to which the K subunit of RNA polymerase (RpoA) binds and has been designated
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the UP element [32,33]. Binding of RpoA to the UP-element enhances recognition of the core promoter sequences by the core polymerase which in turn enhances the rate of transcription. The A+T-rich nature of some of the P. gingivalis sequences in these 340 to 360 regions suggests there is potential for UP-element type regulation. This proposal is supported by the results of the ¢mA promoter studies, in which deletion of the DNA between 345 and 363 reduced activity of the promoter, as did deletion of A+T-rich sequences between 390 and 3240 [12]. Our understanding of P. gingivalis gene regulation is just beginning, but the consensus promoter sequences determined here give a starting point from which to identify potential promoter sequences of other P. gingivalis genes.
[10]
[11]
[12]
[13] [14]
[15]
[16]
Acknowledgements This work was supported by an International Association for Dental Research John A. Gray Fellowship. We wish to thank John Davies for DNA structural predictions.
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