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Sequence analysis and characterization of sulfonamide resistance plasmid pRF-1 from Salmonella enterica serovar Choleraesuis
Plasmid 52 (2004) 218–224 www.elsevier.com/locate/yplas
Short communication
Sequence analysis and characterization of sulfonamide resistance plasmid pRF-1 from Salmonella enterica serovar Choleraesuis Takeshi Haneda, Nobuhiko Okada*, Tsuyoshi Miki, Hirofumi Danbara Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan Received 16 April 2004, revised 8 July 2004 Available online 13 October 2004 Communicated by D. Figurski
Abstract The nucleotide sequence of a small plasmid, designated pRF-1, isolated from Salmonella enterica serovar Choleraesuis, was determined. We identified seven open reading frames (ORFs) encoded by 6066 nucleotides with a total G + C content of 53.6%. Analysis of the complete nucleotide sequence revealed a replicon of pRF-1 to have high similarity to the p15A origin of replication, with a possible cer-like region. ORF1, which is composed of 816 nucleotides, shows a high degree of similarity to dihydropteroate synthetase encoded by the sulII gene from plasmids in several enteropathogenic bacteria, which functions as the sulfonamide resistance determinant. In fact, Salmonella and Escherichia coli strains carrying pRF-1 were found to show strong resistance to sulfathiazole, suggesting that orf1 is a functional gene. Four of seven ORFs were found to encode putative proteins of unknown function. 2004 Elsevier Inc. All rights reserved. Keywords: Salmonella enterica serovar Choleraesuis; Drug-resistance plasmid; Nucleotide sequence
Salmonellosis is an important public health problem throughout the world (Cohen et al., 1987; Cohen and Tauxe, 1986; Mead et al., 1999). Salmonella enterica is a Gram-negative and facultative intracellular bacterium that causes
*
Corresponding author. Fax: +81 3 3444 4831. E-mail address: [email protected] (N. Okada).
a broad spectrum of diseases such as gastroenteritis and bacteremia. Among more than 2000 Salmonella serovars, S. enterica serovar Choleraesuis is an important swine pathogen and a cause of serious systemic infections, including typhoid disease, and pneumonia. Although S. enterica serovar Choleraesuis is highly host-adapted, the bacterium usually causes systemic infection such as bacteremia in humans (Blaser and Feldman,
0147-619X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2004.07.002
Short communication / Plasmid 52 (2004) 218–224
1981; Cohen et al., 1987). In general, the primary approach to the treatment and control of Salmonella infections is the use of antimicrobial agents. However, bacteria are becoming increasingly resistant to multiple antibiotics in many areas of the world (Lee et al., 1994; Salmon et al., 1995). In a number of bacteria, resistance is acquired through natural mutations or resistance determinants located on plasmids, transposons, and constitute cassettes in integrons (Mabilat et al., 1992; Poirel et al., 1999; Smith and Lewin, 1993). Virulent strains of S. enterica serovar Choleraesuis commonly possess a 50-kb plasmid containing several virulence-associated genes (Haneda et al., 2001), and sometimes one or more small cryptic plasmids (Kawahara et al., 1988). We report herein the complete nucleotide sequence of the 6.1-kb plasmid pRF-1 from S. enterica serovar Choleraeusis strain RF-1 (Kawahara et al., 1988), which was isolated from swine in Japan, and reveal the presence of the antibiotic-resistance determinant gene for sulfonamide. The 6.1-kb plasmid pRF-1 was isolated from S. enterica serovar Choleraesuis strain 31N-1, an isogenic derivative of RF-1 cured of a 50-kb large virulence plasmid (Kawahara et al., 1988). The plasmid pRF-1 was obtained by the method as described previously (Kado and Liu, 1981). The nucleotide sequence of pRF-1 was determined on both strands by cloning suitable overlapping restriction fragments into plasmid pBluescript II SK(+) (Stratagene, Heidelberg, German). Purified pBluescript II SK(+) templates were sequenced using cycle-sequencing reactions with Texas red-labeled forward and reverse primers (Hitachi HighTechnologies, Tokyo, Japan) with SQ5500E sequencer (Hitachi High-Technologies), as described previously (Haneda et al., 2001). For the sequence analysis, open reading frames (ORFs) were initially identified using GENETYX-Mac (version 11.2.3) software (Software Development, Tokyo, Japan) and a BLAST database of putative genes. For subsequent analysis, each ORF was compared to the current nonredundant protein database of the National Center for Biotechnology Information by using BLAST software through the Internet. The sequence of pRF-1 is deposited in DDBJ under Accession No. AB076707.
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The complete nucleotide sequence of pRF-1 revealed a size of 6066 bp, slightly smaller than that estimated from earlier electrophoresis analysis (Kawahara et al., 1988). pRF-1 has a total G + C content of 53.6% in the range typical for Salmonella genomic DNA (McClelland et al., 2001). The unique EcoRI site was arbitrarily considered as base 1. Nucleotides 6025–4193 containing ORF1 to ORF5 and plasmid replication region on pRF-1 have 99% identity to the DNA regions of p9123 from Escherichia coli, and pKKTET7, pSS4, and pSSTAV from Shigella sonnei (Fig. 1 and Table 1). Interestingly, the remaining portion of pRF-1 is also identical to the chromosomal region from Shigella flexneri strain 2457T (Fig. 1 and Table 1). A putative origin of replication (p15A ori) of pRF-1 was found to be located between positions 1077 and 1931, and to contain a sequence capable of controlling the initiation of DNA replication and the plasmid copy number. This region is predicted to express a putative antisense RNA molecule (RNA I, positions 1250–1145), which controls the initiation of plasmid DNA replication, and primer RNA (RNA II, positions 1145– 1699). The nucleotide sequences of putative promoters for the genes encoding RNA I and RNA II of pRF-1 are highly homology to the respective promoters of p15A-derived plasmid. For plasmid copy number assays, plasmids isolated by alkaline lysis from 1 · 1011 bacterial cells were digested with appropriate restriction endonuclease to produce linear DNA, followed by agarose gel electrophoresis. A densitometric analysis of the amount of plasmid DNA was performed using image analysis program (Image J) with a control containing a known amount of plasmid DNA separated on the same agarose gel. The average number of plasmid monomers per cell was then calculated. The copy number of pRF-1 in S. entreica serovar Choleraesuis was approximately 50 copies per bacterial cell, which showed to be lower than that of pACYC184 (a p15A-derived replicon), numbering 100 copies per bacterial cell, in S. entreica serovar Choleraesuis. It appeared that plasmid number of pACYC184 was higher than that reported in E. coli, probably due to differences in nature of the host, and interactions between the
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Short communication / Plasmid 52 (2004) 218–224
Fig. 1. Genetic organization of plasmid pRF-1. (A) A circular map of pRF-1 with selected restriction sites. Seven open reading frames are shown, as indicated with arrows. The noncoding regions containing the putative ori region and the cer-like region are indicated with open boxes. (B) A linear map of pRF-1 with the corresponding regions of other plasmids or chromosome from enteropathogenic bacteria (see also Table 1). The nucleotide positions in the pRF-1 sequence and the corresponding positions in each plasmid or chromosome are indicated. The positions of putative genes are shown above the plasmid.
plasmid-encoded elements and the host transcription and replication machineries. The 145-bp segment (positions 2752–2896) showed 100% identity with the cer (ColE1 resolution)-like recombination region of plasmid p9123 from E. coli, and pKKTET7, pSS4, and pSSTAV from S. sonnei (Table 1). The cer region plays a role in plasmid stability through plasmid monomerization (Leung et al., 1985; Summers and Sherratt, 1984). Although the cer region is responsible for the stability of the ColE1-type plasmid, the function of cer-like region in pRF-1 remains unknown. Com-
puter analysis revealed that pRF-1 contains seven putative ORFs of more than 50 amino acids that are preceded by putative Shine-Dalgarno sequences (Table 1) and two possible promoters are identified upstream of ORF1 and ORF5. Of the seven putative ORFs, four (ORF2 to ORF5) encode putative proteins of unknown function. To further analyze the genetic feature of pRF-1, the replication of pRF-1 in E. coli strain JC411 carrying a temperature-sensitive polA214ts mutation (obtained from NIG collection, National Institute of Genetics, Mishima, Japan) was determined.
Table 1 ORFs and noncoding element of pRF-1 Position (bp)a
Size (aa)
Homologue found by blast
Plasmid or chromosome/host
% Identity (aa)
Accession No.
ORF1
6062–811 (comp)
271
Dihydropteroate synthase (sulII)
ORF2
1999–2148
49
Hypothetical protein
ORF3
2354–2602
82
Hypothetical protein
ORF4
2914–3402 (comp)
162
Hypothetical protein
ORF5
3572–3926
129
Hypothetical protein
ORF6
4005–5498
497
Putative transposase
ORF7
5674–6018 (comp)
114
Phosphoglucosamine mutase (glmM)
p9123/E. coli pKKTET7/S. sonnei pSS4/S. sonnei pHCM1/S. enterica Chromosome/S. flexneri pSS4/S. sonnei pSSTAV/S. sonnei p9123/E. coli pSSTAV/S. sonnei p9123/E. coli pKKTET7/S. sonnei pSS4/S. sonnei pSSTAV/S. sonnei p9123/E. coli pSS4/S. sonnei pSSTAV/S. sonnei Chromosome/S. flexneri Chromosome/Vibrio cholerae Chromosome/S. flexneri
99 99 99 99 99 100 100 65 (truncated) 65 (truncated) 98 98 98 98 98 98 98 100 100 100 (truncated)
NP_957531 AAM21663 AAN06711 NP_569414 NP_838053 AAN06711 AAM22226 NP_957537 AAM22225 NP_957536 AAM21662 AAN06710 AAM22224 NP_957535 AAN06709 AAM22223 NP_838055 AAK64580 NP_838054
p9123/E. coli pKKTET7/S. sonnei pSS4/S. sonnei p9123/E. coli pKKTET7/S. sonnei pSS4/S. sonnei pSSTAV/S. sonnei
% Identity (nt) 98 98 98 100 100 100 100
AY360321 AF497970 AF534183 AY360321 AF497970 AF534183 AF502943
Noncoding
a
1077–1931
p15A ori
2752–2896
cer-like region
Short communication / Plasmid 52 (2004) 218–224
ORF element
‘‘comp’’ denotes a gene encoded on the minus strand.
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The number of JC411 transformants for the pRF-1 was decreased when selected at 42 C, a temperature that abolishes the DNA polymerase I function, necessary for the replication of ColE1 plasmids, as compared to selection at 30 C, suggesting that pRF-1 requires DNA polymerase I for its replication. Next, we tested the ability of the pRF-1 to coexistent in the same bacterial cells with plasmid pACYC184 (a p15A-derived replicon). We found that E. coli recA strain DH5a carrying pACYC184 can be transformed with pRF-1 and maintained under antibiotic pressure. Furthermore, pRF-1 was maintained with pACYC184 after growth for 60 generations in the absence of selection, indicating that pRF-1 is compatible with pACYC184. In addition, the conjugal transfer properties of pRF-1 was determined. E. coli S17.1, which contains the chromosomally integrate transfer genes of a broad-host-range conjugal IncP-type plasmid RP4, were transformed with pRF-1, and used a donor cells. Using the filter mating procedures, donor cells were mated with S. enterica serovar Choleraesuis 2N-3 (RifR), a pRF-1 cured-derivative of RF-1 (Kawahara et al., 1988), as a recipient strain. Transconjugants were selected on medium containing rifampicin and sulfathiazole. Consistent with the lack of a predicted oriT locus in pRF-1, no transconjugants were obtained (frequency <1.0 · 108) (data not shown). ORF1 (nucleotides 6062–811), with 271 amino acids, is highly homologous to dihydropteroate synthase encoded by the sulII gene, the sulfonamide resistance determinant, from E. coli (p9123) S. enterica serovar Typhi (plasmid pHCM1), S. sonnei (plasmids pKKTET7 and pSS4), and S. flexneri strain 2457T (chromosome). To test the enzymatic activity of the gene-product of orf1 in pRF-1, the minimal inhibitory concentrations (MIC) of sulfonamide for S. enterica serovar Choleraesuis wild-type RF-1 and 2N-3, a pRF-1cured derivative of RF-1, were compared. The MICs were determined by an agar dilution method (Jones et al., 1985) using Mu¨ller-Hinton agar for Salmonella and E. coli. Bacterial suspensions cultured overnight in broth were inoculated at a final concentration of 5 · 105 cfu/ml. Each inoculated agar plate was incubated at 37 C for 24 h, and the lowest drug concentration at which
growth was completely suppressed was expressed as the MIC. While strain RF-1 showed strong resistance (>400 lg/ml) of sulfathiazole (4-aminoN-2-thiazolylbenzensulfonamide, Sigma, MI, USA), the resistance of strain 2N-3 was somewhat weaker (50 lg/ml). Similarly, the MIC of sulfathiazole for E. coli strain DH5a with and without pRF-1 were >400 and 6 lg/ml, respectively. To further confirm that orf1 is responsible for sulfonamide resistance, orf1 was deleted by inverse PCR with primers, Dorf1-FW (5 0 -CGGAATTCATC GAGCATGAGTCTCATACGG-3 0 ) and Dorf1RV (5 0 -TGGCGGCGCTGAAAGAAACCGCA AGAATTC-3 0 ), using pRF-1 DNA as template. PCR product was digested with EcoRI and ligated with EcoRI-digested XKm2 fragment (Perez-Casal et al., 1991), a kanamycin resistance cassette. The resulting plasmid was transformed to S. enterica serovar Choleraesuis strain 2N-3 and E. coli DH5a. The MIC of sulfathiazole for these transformants was lower than that of transformants carrying intact pRF-1 (data not shown). Thus, ORF1 functions as the sulfonamide resistance determinant. ORF6 and ORF7 were found to be 100% identical to the chromosomal region from S. flexneri strain 2457T. ORF6 could encode a 497-amino-acid putative transposase. ORF7, which could encode a 114-amino-acid protein, showed high homology to the phosphoglucosamine mutase (GlmM), which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell-wall precursor UDP-N-acetylglucosamine (MenginLecreulx and van Heijenoort, 1996). Because repeated passages of strain RF-1 without selective pressure in the laboratory did not result in a loss of the plasmid from the bacteria, the stability of pRF-1 was tested according to a previously described method (Takamatsu et al., 2001) and compared with that of various conventional plasmid vectors commonly used for Salmonella, including pMW118 (Nippon Gene, Tokyo, Japan), pACYC184 (New England Biolabs, MA, USA), pBR322 (Bolivar et al., 1977), and pBluescript II SK(+). As shown in Fig. 2, pRF-1, pMW118, and pBR322 in S. enterica serovar Choleraesuis strain 2N-3 were stably maintained for
Short communication / Plasmid 52 (2004) 218–224
Fig. 2. Stability of various plasmids in S. enterica serovar Choleraesuis in the absence of antibiotic resistance selection.
approximately 100 generations of growth without selective pressure. The proportion of cells carrying pRF-1 in the culture of strain 2N-3 was slightly higher than that of pMW118 and pBR322. In contrast, pACYC184 in strain 2N-3 was lost gradually under nonselective conditions, and after approximately 100 generations the proportions of cells carrying pACYC184 were reduced to 14%. In addition, pBluescript II SK(+) carried by strain 2N-3 showed to be lost at markedly high rate under nonselective conditions. Thus, the smaller size of pRF-1 and its high stability in the S. enterica serovar Choleraesuis host would be advantageous for use of the plasmid as a cloning and/or expression vector for S. enterica serovar Choleraesuis. Construction of such a vector is currently under way.
Acknowledgments We thank Miki Mizuhara and Mariko Sugimoto for their technical assistance. This work was supported in part by a Grant-in-aid for Exploratory Research (15659105) and by a 21st Century COE Program Grant from the Japanese Ministry of Education, Culture, Sports, Sciences, and Technology (MEXT).
References Blaser, M.J., Feldman, R.A., 1981. From the centers for disease control. Salmonella bacteremia: reports to the centers for disease control, 1968–1979. J. Infect. Dis. 143, 743–746.
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Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95–113. Cohen, J.I., Bartlett, J.A., Corey, G.R., 1987. Extra-intestinal manifestations of Salmonella infections. Medicine (Baltimore) 66, 349–388. Cohen, M.L., Tauxe, R.V., 1986. Drug-resistant Salmonella in the United States: an epidemiologic perspective. Science 234, 964–969. Haneda, T., Okada, N., Nakazawa, N., Kawakami, T., Danbara, H., 2001. Complete DNA sequence and comparative analysis of the 50-kilobase virulence plasmid of Salmonella enterica serovar Choleraesuis. Infect. Immun. 69, 2612–2620. Jones, R.N., Barry, A.L., Gavan, T.L., Washington II, J.A., 1985. Susceptibility Tests: Microdilution and Macrodilution Broth Procedures. American Society for Microbiology, Washington, DC. Kado, C.I., Liu, S.T., 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145, 1365–1373. Kawahara, K., Haraguchi, Y., Tsuchimoto, M., Terakado, N., Danbara, H., 1988. Evidence of correlation between 50kilobase plasmid of Salmonella choleraesuis and its virulence. Microb. Pathog. 4, 155–163. Lee, L.A., Puhr, N.D., Maloney, E.K., Bean, N.H., Tauxe, R.V., 1994. Increase in antimicrobial-resistant Salmonella infections in the United States, 1989–1990. J. Infect. Dis. 170, 128–134. Leung, D.W., Chen, E., Cachianes, G., Goeddel, D.V., 1985. Nucleotide sequence of the partition function of Escherichia coli plasmid ColE1. DNA 4, 351–355. Mabilat, C., Lourencao-Vital, J., Goussard, S., Courvalin, P., 1992. A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM3. Mol. Gen. Genet. 235, 113–121. McClelland, M., Sanderson, K.E., Spieth, J., Clifton, S.W., Latreille, P., Courtney, L., Porwollik, S., Ali, J., Dante, M., Du, F., Hou, S., Layman, D., Leonard, S., Nguyen, C., Scott, K., Holmes, A., Grewal, N., Mulvaney, E., Ryan, E., Sun, H., Florea, L., Miller, W., Stoneking, T., Nhan, M., Waterston, R., Wilson, R.K., 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413, 852–856. Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.M., Tauxe, R.V., 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5, 607–625. Mengin-Lecreulx, D., van Heijenoort, J., 1996. Characterization of the essential gene glmM encoding phosphoglucosamine mutase in Escherichia coli. J. Biol. Chem. 271, 32–39. Perez-Casal, J., Caparon, M.G., Scott, J.R., 1991. Mry, a transacting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of twocomponent regulatory systems. J. Bacteriol. 173, 2617–2624.
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Poirel, L., Naas, T., Guibert, M., Chaibi, E.B., Labia, R., Nordmann, P., 1999. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene. Antimicrob. Agents Chemother. 43, 573–581. Salmon, S.A., Watts, J.L., Case, C.A., Hoffman, L.J., Wegener, R.J., Yancey Jr., R.J., 1995. Comparison of MICs of ceftiofur and other antimicrobial agents against bacterial pathogens of swine from the United States, Canada, and Denmark. J. Clin. Microbiol. 33, 2435–2444.
Smith, J.T., Lewin, C.S., 1993. Mechanisms of antimicrobial resistance and implications for epidemiology. Vet. Microbiol. 35, 233–242. Summers, D.K., Sherratt, D.J., 1984. Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability. Cell 36, 1097–1103. Takamatsu, D., Osaki, M., Sekizaki, T., 2001. Construction and characterization of Streptococcus suis–Escherichia coli shuttle cloning vectors. Plasmid 45, 101–113.
Short communication
Sequence analysis and characterization of sulfonamide resistance plasmid pRF-1 from Salmonella enterica serovar Choleraesuis Takeshi Haneda, Nobuhiko Okada*, Tsuyoshi Miki, Hirofumi Danbara Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan Received 16 April 2004, revised 8 July 2004 Available online 13 October 2004 Communicated by D. Figurski
Abstract The nucleotide sequence of a small plasmid, designated pRF-1, isolated from Salmonella enterica serovar Choleraesuis, was determined. We identified seven open reading frames (ORFs) encoded by 6066 nucleotides with a total G + C content of 53.6%. Analysis of the complete nucleotide sequence revealed a replicon of pRF-1 to have high similarity to the p15A origin of replication, with a possible cer-like region. ORF1, which is composed of 816 nucleotides, shows a high degree of similarity to dihydropteroate synthetase encoded by the sulII gene from plasmids in several enteropathogenic bacteria, which functions as the sulfonamide resistance determinant. In fact, Salmonella and Escherichia coli strains carrying pRF-1 were found to show strong resistance to sulfathiazole, suggesting that orf1 is a functional gene. Four of seven ORFs were found to encode putative proteins of unknown function. 2004 Elsevier Inc. All rights reserved. Keywords: Salmonella enterica serovar Choleraesuis; Drug-resistance plasmid; Nucleotide sequence
Salmonellosis is an important public health problem throughout the world (Cohen et al., 1987; Cohen and Tauxe, 1986; Mead et al., 1999). Salmonella enterica is a Gram-negative and facultative intracellular bacterium that causes
*
Corresponding author. Fax: +81 3 3444 4831. E-mail address: [email protected] (N. Okada).
a broad spectrum of diseases such as gastroenteritis and bacteremia. Among more than 2000 Salmonella serovars, S. enterica serovar Choleraesuis is an important swine pathogen and a cause of serious systemic infections, including typhoid disease, and pneumonia. Although S. enterica serovar Choleraesuis is highly host-adapted, the bacterium usually causes systemic infection such as bacteremia in humans (Blaser and Feldman,
0147-619X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2004.07.002
Short communication / Plasmid 52 (2004) 218–224
1981; Cohen et al., 1987). In general, the primary approach to the treatment and control of Salmonella infections is the use of antimicrobial agents. However, bacteria are becoming increasingly resistant to multiple antibiotics in many areas of the world (Lee et al., 1994; Salmon et al., 1995). In a number of bacteria, resistance is acquired through natural mutations or resistance determinants located on plasmids, transposons, and constitute cassettes in integrons (Mabilat et al., 1992; Poirel et al., 1999; Smith and Lewin, 1993). Virulent strains of S. enterica serovar Choleraesuis commonly possess a 50-kb plasmid containing several virulence-associated genes (Haneda et al., 2001), and sometimes one or more small cryptic plasmids (Kawahara et al., 1988). We report herein the complete nucleotide sequence of the 6.1-kb plasmid pRF-1 from S. enterica serovar Choleraeusis strain RF-1 (Kawahara et al., 1988), which was isolated from swine in Japan, and reveal the presence of the antibiotic-resistance determinant gene for sulfonamide. The 6.1-kb plasmid pRF-1 was isolated from S. enterica serovar Choleraesuis strain 31N-1, an isogenic derivative of RF-1 cured of a 50-kb large virulence plasmid (Kawahara et al., 1988). The plasmid pRF-1 was obtained by the method as described previously (Kado and Liu, 1981). The nucleotide sequence of pRF-1 was determined on both strands by cloning suitable overlapping restriction fragments into plasmid pBluescript II SK(+) (Stratagene, Heidelberg, German). Purified pBluescript II SK(+) templates were sequenced using cycle-sequencing reactions with Texas red-labeled forward and reverse primers (Hitachi HighTechnologies, Tokyo, Japan) with SQ5500E sequencer (Hitachi High-Technologies), as described previously (Haneda et al., 2001). For the sequence analysis, open reading frames (ORFs) were initially identified using GENETYX-Mac (version 11.2.3) software (Software Development, Tokyo, Japan) and a BLAST database of putative genes. For subsequent analysis, each ORF was compared to the current nonredundant protein database of the National Center for Biotechnology Information by using BLAST software through the Internet. The sequence of pRF-1 is deposited in DDBJ under Accession No. AB076707.
219
The complete nucleotide sequence of pRF-1 revealed a size of 6066 bp, slightly smaller than that estimated from earlier electrophoresis analysis (Kawahara et al., 1988). pRF-1 has a total G + C content of 53.6% in the range typical for Salmonella genomic DNA (McClelland et al., 2001). The unique EcoRI site was arbitrarily considered as base 1. Nucleotides 6025–4193 containing ORF1 to ORF5 and plasmid replication region on pRF-1 have 99% identity to the DNA regions of p9123 from Escherichia coli, and pKKTET7, pSS4, and pSSTAV from Shigella sonnei (Fig. 1 and Table 1). Interestingly, the remaining portion of pRF-1 is also identical to the chromosomal region from Shigella flexneri strain 2457T (Fig. 1 and Table 1). A putative origin of replication (p15A ori) of pRF-1 was found to be located between positions 1077 and 1931, and to contain a sequence capable of controlling the initiation of DNA replication and the plasmid copy number. This region is predicted to express a putative antisense RNA molecule (RNA I, positions 1250–1145), which controls the initiation of plasmid DNA replication, and primer RNA (RNA II, positions 1145– 1699). The nucleotide sequences of putative promoters for the genes encoding RNA I and RNA II of pRF-1 are highly homology to the respective promoters of p15A-derived plasmid. For plasmid copy number assays, plasmids isolated by alkaline lysis from 1 · 1011 bacterial cells were digested with appropriate restriction endonuclease to produce linear DNA, followed by agarose gel electrophoresis. A densitometric analysis of the amount of plasmid DNA was performed using image analysis program (Image J) with a control containing a known amount of plasmid DNA separated on the same agarose gel. The average number of plasmid monomers per cell was then calculated. The copy number of pRF-1 in S. entreica serovar Choleraesuis was approximately 50 copies per bacterial cell, which showed to be lower than that of pACYC184 (a p15A-derived replicon), numbering 100 copies per bacterial cell, in S. entreica serovar Choleraesuis. It appeared that plasmid number of pACYC184 was higher than that reported in E. coli, probably due to differences in nature of the host, and interactions between the
220
Short communication / Plasmid 52 (2004) 218–224
Fig. 1. Genetic organization of plasmid pRF-1. (A) A circular map of pRF-1 with selected restriction sites. Seven open reading frames are shown, as indicated with arrows. The noncoding regions containing the putative ori region and the cer-like region are indicated with open boxes. (B) A linear map of pRF-1 with the corresponding regions of other plasmids or chromosome from enteropathogenic bacteria (see also Table 1). The nucleotide positions in the pRF-1 sequence and the corresponding positions in each plasmid or chromosome are indicated. The positions of putative genes are shown above the plasmid.
plasmid-encoded elements and the host transcription and replication machineries. The 145-bp segment (positions 2752–2896) showed 100% identity with the cer (ColE1 resolution)-like recombination region of plasmid p9123 from E. coli, and pKKTET7, pSS4, and pSSTAV from S. sonnei (Table 1). The cer region plays a role in plasmid stability through plasmid monomerization (Leung et al., 1985; Summers and Sherratt, 1984). Although the cer region is responsible for the stability of the ColE1-type plasmid, the function of cer-like region in pRF-1 remains unknown. Com-
puter analysis revealed that pRF-1 contains seven putative ORFs of more than 50 amino acids that are preceded by putative Shine-Dalgarno sequences (Table 1) and two possible promoters are identified upstream of ORF1 and ORF5. Of the seven putative ORFs, four (ORF2 to ORF5) encode putative proteins of unknown function. To further analyze the genetic feature of pRF-1, the replication of pRF-1 in E. coli strain JC411 carrying a temperature-sensitive polA214ts mutation (obtained from NIG collection, National Institute of Genetics, Mishima, Japan) was determined.
Table 1 ORFs and noncoding element of pRF-1 Position (bp)a
Size (aa)
Homologue found by blast
Plasmid or chromosome/host
% Identity (aa)
Accession No.
ORF1
6062–811 (comp)
271
Dihydropteroate synthase (sulII)
ORF2
1999–2148
49
Hypothetical protein
ORF3
2354–2602
82
Hypothetical protein
ORF4
2914–3402 (comp)
162
Hypothetical protein
ORF5
3572–3926
129
Hypothetical protein
ORF6
4005–5498
497
Putative transposase
ORF7
5674–6018 (comp)
114
Phosphoglucosamine mutase (glmM)
p9123/E. coli pKKTET7/S. sonnei pSS4/S. sonnei pHCM1/S. enterica Chromosome/S. flexneri pSS4/S. sonnei pSSTAV/S. sonnei p9123/E. coli pSSTAV/S. sonnei p9123/E. coli pKKTET7/S. sonnei pSS4/S. sonnei pSSTAV/S. sonnei p9123/E. coli pSS4/S. sonnei pSSTAV/S. sonnei Chromosome/S. flexneri Chromosome/Vibrio cholerae Chromosome/S. flexneri
99 99 99 99 99 100 100 65 (truncated) 65 (truncated) 98 98 98 98 98 98 98 100 100 100 (truncated)
NP_957531 AAM21663 AAN06711 NP_569414 NP_838053 AAN06711 AAM22226 NP_957537 AAM22225 NP_957536 AAM21662 AAN06710 AAM22224 NP_957535 AAN06709 AAM22223 NP_838055 AAK64580 NP_838054
p9123/E. coli pKKTET7/S. sonnei pSS4/S. sonnei p9123/E. coli pKKTET7/S. sonnei pSS4/S. sonnei pSSTAV/S. sonnei
% Identity (nt) 98 98 98 100 100 100 100
AY360321 AF497970 AF534183 AY360321 AF497970 AF534183 AF502943
Noncoding
a
1077–1931
p15A ori
2752–2896
cer-like region
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ORF element
‘‘comp’’ denotes a gene encoded on the minus strand.
221
222
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The number of JC411 transformants for the pRF-1 was decreased when selected at 42 C, a temperature that abolishes the DNA polymerase I function, necessary for the replication of ColE1 plasmids, as compared to selection at 30 C, suggesting that pRF-1 requires DNA polymerase I for its replication. Next, we tested the ability of the pRF-1 to coexistent in the same bacterial cells with plasmid pACYC184 (a p15A-derived replicon). We found that E. coli recA strain DH5a carrying pACYC184 can be transformed with pRF-1 and maintained under antibiotic pressure. Furthermore, pRF-1 was maintained with pACYC184 after growth for 60 generations in the absence of selection, indicating that pRF-1 is compatible with pACYC184. In addition, the conjugal transfer properties of pRF-1 was determined. E. coli S17.1, which contains the chromosomally integrate transfer genes of a broad-host-range conjugal IncP-type plasmid RP4, were transformed with pRF-1, and used a donor cells. Using the filter mating procedures, donor cells were mated with S. enterica serovar Choleraesuis 2N-3 (RifR), a pRF-1 cured-derivative of RF-1 (Kawahara et al., 1988), as a recipient strain. Transconjugants were selected on medium containing rifampicin and sulfathiazole. Consistent with the lack of a predicted oriT locus in pRF-1, no transconjugants were obtained (frequency <1.0 · 108) (data not shown). ORF1 (nucleotides 6062–811), with 271 amino acids, is highly homologous to dihydropteroate synthase encoded by the sulII gene, the sulfonamide resistance determinant, from E. coli (p9123) S. enterica serovar Typhi (plasmid pHCM1), S. sonnei (plasmids pKKTET7 and pSS4), and S. flexneri strain 2457T (chromosome). To test the enzymatic activity of the gene-product of orf1 in pRF-1, the minimal inhibitory concentrations (MIC) of sulfonamide for S. enterica serovar Choleraesuis wild-type RF-1 and 2N-3, a pRF-1cured derivative of RF-1, were compared. The MICs were determined by an agar dilution method (Jones et al., 1985) using Mu¨ller-Hinton agar for Salmonella and E. coli. Bacterial suspensions cultured overnight in broth were inoculated at a final concentration of 5 · 105 cfu/ml. Each inoculated agar plate was incubated at 37 C for 24 h, and the lowest drug concentration at which
growth was completely suppressed was expressed as the MIC. While strain RF-1 showed strong resistance (>400 lg/ml) of sulfathiazole (4-aminoN-2-thiazolylbenzensulfonamide, Sigma, MI, USA), the resistance of strain 2N-3 was somewhat weaker (50 lg/ml). Similarly, the MIC of sulfathiazole for E. coli strain DH5a with and without pRF-1 were >400 and 6 lg/ml, respectively. To further confirm that orf1 is responsible for sulfonamide resistance, orf1 was deleted by inverse PCR with primers, Dorf1-FW (5 0 -CGGAATTCATC GAGCATGAGTCTCATACGG-3 0 ) and Dorf1RV (5 0 -TGGCGGCGCTGAAAGAAACCGCA AGAATTC-3 0 ), using pRF-1 DNA as template. PCR product was digested with EcoRI and ligated with EcoRI-digested XKm2 fragment (Perez-Casal et al., 1991), a kanamycin resistance cassette. The resulting plasmid was transformed to S. enterica serovar Choleraesuis strain 2N-3 and E. coli DH5a. The MIC of sulfathiazole for these transformants was lower than that of transformants carrying intact pRF-1 (data not shown). Thus, ORF1 functions as the sulfonamide resistance determinant. ORF6 and ORF7 were found to be 100% identical to the chromosomal region from S. flexneri strain 2457T. ORF6 could encode a 497-amino-acid putative transposase. ORF7, which could encode a 114-amino-acid protein, showed high homology to the phosphoglucosamine mutase (GlmM), which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell-wall precursor UDP-N-acetylglucosamine (MenginLecreulx and van Heijenoort, 1996). Because repeated passages of strain RF-1 without selective pressure in the laboratory did not result in a loss of the plasmid from the bacteria, the stability of pRF-1 was tested according to a previously described method (Takamatsu et al., 2001) and compared with that of various conventional plasmid vectors commonly used for Salmonella, including pMW118 (Nippon Gene, Tokyo, Japan), pACYC184 (New England Biolabs, MA, USA), pBR322 (Bolivar et al., 1977), and pBluescript II SK(+). As shown in Fig. 2, pRF-1, pMW118, and pBR322 in S. enterica serovar Choleraesuis strain 2N-3 were stably maintained for
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Fig. 2. Stability of various plasmids in S. enterica serovar Choleraesuis in the absence of antibiotic resistance selection.
approximately 100 generations of growth without selective pressure. The proportion of cells carrying pRF-1 in the culture of strain 2N-3 was slightly higher than that of pMW118 and pBR322. In contrast, pACYC184 in strain 2N-3 was lost gradually under nonselective conditions, and after approximately 100 generations the proportions of cells carrying pACYC184 were reduced to 14%. In addition, pBluescript II SK(+) carried by strain 2N-3 showed to be lost at markedly high rate under nonselective conditions. Thus, the smaller size of pRF-1 and its high stability in the S. enterica serovar Choleraesuis host would be advantageous for use of the plasmid as a cloning and/or expression vector for S. enterica serovar Choleraesuis. Construction of such a vector is currently under way.
Acknowledgments We thank Miki Mizuhara and Mariko Sugimoto for their technical assistance. This work was supported in part by a Grant-in-aid for Exploratory Research (15659105) and by a 21st Century COE Program Grant from the Japanese Ministry of Education, Culture, Sports, Sciences, and Technology (MEXT).
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