Letters in Applied Microbiology 1991, 12, 1&19
ADONIS 0266825491oooO5J
Isolation and characterization of a cryptic plasmid from mesophilic aeromonads : potential as a cloning vector I . F . CONNERTON, J . S . K A U R ,S. ROGERS& R . W . A . P A R KDepartmentof Microbiology, University of Reading, London Road, Reading RG1 5AQ, U K Received I4 September I990 and accepted 14 September 1990 Paper number: CRH/022 C O N N E R T OIN. F, . , K A U R ,J.S., ROGERS,S . & P A R K R , . W . A . 1991. Isolation and characterizationof a cryptic plasmid from mesophilic aeromonads:potential as a cloning vector. Letters in Applied Microbiology 12, 1619. A 2.6 kb covalently closed circular plasmid has been isolated from clinical and environmental isolates of Aeromonas sobria and A. hydrophila. The possibility that the plasmid carries genetic determinantsthat mediate resistance to a variety of antimicrobial agents has been eliminated. The plasmid is stable at approximately 20-25 copies per chromosome equivalent which, together with its relatively small size and the presence of unique restrictionsites, makes it a good candidate for development as a cloning vector.
Species of the genus Aeromonas are ubiquitous in aquatic environments. The mesophilic aeromonads are potential pathogens of mammals and lower vertebrates including amphibia, reptiles and fish (Janda et al. 1984; Dooley & Trust 1988). Recent attention has focussed on the species Aeromonas sobria and A. hydrophila, as they are persistently implicated as causative agents of clinical bacteraemia (Harris et al. 1985; Janda & Brendan 1987) and gastroenteritis (Holmberg & Farmer 1984). It is likely that these species are water-borne and foodborne pathogens. In addition Aeromonas species have been shown to be good indicators of the trophic state of aquatic environments (Rippey & Cabelli 1980). As a result of this interest a series of studies has been initiated on the genetics and molecular biology of species of the genus. These studies range from the directed investigation of pathogenic factors such as enterotoxins (Chakraborty et al. 1984) and the cytotoxic haemolysins (aerolysin) (Chakraborty et al. 1986; Howard & Buckley 1986), to more fundamental genetic aspects which may provide enabling technology (Resnick & Nelson 1988). All of these studies have relied upon Escherichia coli to express functional Aeromonas genes. This has been
necessary since most routine cloning vectors utilize the ColEl origin of replication which does not function in Aeromonas. Although transcription so far has proved faithful, E. coli does not always process the protein products eficiently (Howard & Buckley 1986). To help facilitate further studies we have investigated a small stable plasmid native to Aeromonas with a view to developing an Aeromonas-based vector system.
Materials and Methods STRAINS
Mesophilic Aeromonas spp., isolated from faeces, food or water using alkaline peptone water enrichment followed by Aeromonas selective medium (Difco), were classified according to Popoff & Veron (1976). These assignments were later verified by DNA/DNA hybridization (Rogers et al. 1990). Cultures were stored frozen at -70°C in 15% glycerol with Nutrient Broth (Oxoid No. 2). Routine subcultures were made on Nutrient Agar (Oxoid, 37"C, 24 h). For isolation of the plasmids, strains were grown to late exponential phase in Nutrient Broth (Oxoid No. 2) at 37°C.
Cloning vector plasmid in Aeromonas I S O L A T I O N OF P L A S M I D D N A
Routine small-scale plasmid preparations were performed according to Holmes & Quigley (1981). Larger preparations of closed circular DNAs were carried out by the alkaline-lysis method of Birnboim & Doly (1979) and purified further by caesium chloride/ethidium bromide density gradient centrifugation. A N A L Y S I S OF P L A S M I D D N 4
Plasmid DNA was analysed electrophoretically on 0.9% flat-bed agarose gels with 50 mmol/l Tris/borate 1 mmol/l EDTA (pH 8.3) as running buffer, before visualization under U.V.light with ethidium bromide (0.5 mg/l). Restriction digests were performed following the manufacturer’s instructions (BRL) and analysed on similar gels. The plasmid copy number was estimated by U.V. fluorescence spectrophotometry of ethidium bromide-stained gels containing total cell lysates as described by Eckhardt (1978). PLASMID CURING
Acridine orange or ethidium bromide dyes were used as curing agents (Trevors 1986). After incubation, those cells demonstrating visible growth at the highest dye concentration were transferred to dye-free medium and plasmid preparations performed. Cured variants that displayed normal growth characteristics were verified by colony hybridization against plasmid DNA (Grunstein & Hogness 1975). S E N S I TI V I TI ES TO 4 N T I MI C R O B I A L A G EN T S
A range of compounds was tested at various concentrations on filter paper discs to screen for resistance phenotypes associated with the plasmid. Agents used were : chloramphenicol, colistin sulphate, erythromycin, gentamycin, kanamycin, nalidixic acid, nitrofurantoin, streptomycin, tetracycline and vancomycin (Sigma). It should be noted that many strains of Aeromonas are inherently resistant to ampicillin and amoxycillin. Results and Discussion
As part of our work on plasmid profiling and replicon typing in the mesophilic aeromonads, we have observed a relatively small sized
17
plasmid on several occasions. A single plasmid of similar size was isolated from five independent strains of Aerornonas (3 of A. sobria and 2 of A. hydrophila) out of some 122 clinical and environmental isolates. One of the A. sobria isolates was selected as a prototype strain and a large-scale plasmid preparation performed by caesium chloride/ethidium bromide buoyant density gradient centrifugation. The resulting plasmid was called pAER027. Colony hybridization with the purified plasmid DNA confirmed that the plasmids isolated from alternative strains were related with respect to their DNA sequences, and restriction digests confirmed that these plasmids had a similar molecular organization to that of pAER027. The restriction endonuclease cleavage patterns for 17 enzymes were examined by agarose gel electrophoresis. Plasmid pAER027 had cleavage sites for six of the enzymes (Table I). Analysis of the results showed that the plasmid is a circle of 2.6 kb. Single, double and triple digests were performed to construct a physical map for the enzymes ApaI, BylII, EcoRI, MluI, PouIl and SstI (Fig. 1). The restriction enzymes BamHI, Ben, ClaI, DraI, HindIII, NdeI, PstI, SalI, ScaI, XbaI and XhoI did not cut the plasmid. The relatively small size of this plasmid together with its unique restriction sites (especially those adjacent to each other) and, just as important, the absence of a number of common sites which may be introduced as unique, make the plasmid amenable for genetic manipulation. Yields of plasmid DNA confirmed our earlier suspicion that the molecule was present at a higher copy number than many other plasmids observed in our initial survey. Fluorescence monitoring of total cell DNA separated on
Table 1. Numbers and sizes of fragments generated by endonucleases with different restriction digestions of the 2.6 kb plasmid pAER027 from Aeromonas sohria
Fragment Restriction enzyme
Number
ApaI BglII EcoRI MluI PVUII SstI
3 1
1 1
1 1
Size (kb) 1.5
+ 0.95 + 0.15 2.60 2.60 2.60 2.60 2.60
Z. F. Connerton et al.
18
EcoRI 0.00
MIuI 2.55
I
Financial support from the Ministry of Agriculture, Fisheries and Food for Susan Rogers is gratefully acknowledged.
Refereaces
ApaI 2.05 pAER027
SSlI 1-25
Fig. 1. The restriction map of pAER027 from Aeromonas sobria.
agarose gels and stained with ethidium bromide established copy numbers of between 20 and 25 per chromosomal equivalent. Re-introduction of recombinant versions of this plasmid carrying additional aeromonas genes may well cause expression over and above that of chromosomal counterparts, as a consequence of the increased copy number. None of the five isolates possessing the plasmid could be ascribed phenotypic traits that could be associated with its possession. The cured version of the prototype A. sobria strain displayed similar growth characteristics and responded similarly to all the antimicrobial agents tested. Since we have been unable to assign any phenotypic character to the plasmid it may be considered as cryptic. To facilitate initial selection of the plasmid in Aeromonas, a genetic marker must be introduced onto it. Our initial experiments suggest that at least the unique EcoRI site will tolerate the insertion of DNA. The tetracycline resistance gene derived from vectors based on E. coli would seem a good candidate as many strains of Aeromonas are sensitive to this antibiotic. It should be possible to design a selectable cloning vector based upon the cryptic replicon. However, direct transformation into Aeromonas is likely to be difficult, and electroporation may prove a useful alternative.
BIRNBOIM,H.C. & D ~ L YJ. , 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. NucIeic Acids Research 7, 15131523. CHAKRABORTY, T., MO"EGRO, M.A., SANYAL, S.C., HELMUTH,R., BULLING,E. & TIMMIS, K.N. 1984 Cloning of enterotoxin gene from A e r o m o m hydrophila provides conciusive evidence of production of a cyctotoxic enterotoxin. Infection and Immunity 46, 435-441. -n, T., HUHLE, B, BERG8AUER, H. & GOEBEL, W. 1986 Cloning, expression, and mapping of the A e r o m o ~ hydrophh s aerolysin gene determinant in EsEherichia eofi K-12. Journal of Bacterwlogy 167,36&374. DOOLEY, J.S.G. & TRUST,TJ. 1988 Surface protein components of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein Journal of Bacteriology 170,49%506. ECKHARDT, T . 1978 A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1,584-588. GRUNSTEIN, M. & HOGNESS, D. 1975 Colony hybridization: a method for the isolation of cloned DNAs that contain a spccific gene. Proceedings of the National Academy of Sciences, U S A 72,3961-3965. I-Luws, R.L., FAINSTEIN,V., ELTING,L.E., HOPFER, R.L. & BODm, G.D. 1985 Bacteremia caused by Aeromonas species in hospitalizad cancer patients. Review o f l ~ e c t i o u Diseases s 7,314-320. HOLMBERG, S.D. & FARMEX, 111, J.J. 1984 Aeromonas hydrophila and Plesiomom shigelloides as causes of intestinal infections. Review of Infectious Diseoses 6, 633439. HOLMES,D.S. & QUIGLEY, M. 1981 A rapid boiling method for the preparation of bacterial plasrnids. Analytical Biochemistry 114, 193-197. HOWARD. S.P. & BUCKLEY,J.T. 1986 Molecular cloning and expression in Escherichia coli of the structural gene for hemolytic toxin aerolysin from Aeromonas hydrophila. Molecular and General Genetics 204,289-295. JANDA,J.M. & BRENDAN, R. 1987 Importance of Aeromonas sobria in Aeromonas bacteremia. Journal of Infectious Diseases 115,589-591. JANDA,J.M., ~ I T A N O M. , & BOTTONE, E.J. 1984 Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum. Journal of Clinical Microbiology 19,4447. Popom, M. & VERON,M. 1976 A taxonomic study of the Aeromonas hydrophila-Aeromonas punctata group. Journal of General Microbiology 94, 11-22. &NICK, D. & NELSON,D.R. 1988 Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant. Journal of Bacteriology 170,48-55.
Cloning vector plasmid in Aeromonas CABELLI,V.J. 1980 Occurrence of Amomonas hydrophila in limnetic environments: relationship of the organism to trophic state. Microbial Ecology 6,45554. ROGERS,S., M O F F A ~M.F., . CONNERTON, LF. & PARK, R.W.A. 1990 Plasmid profiling and D N A P N A hybridization for distinguishing between mesophilic ~ P P E Y , S.R. &
19
Aeromonas bacteria. In Genetic Manipulation: Techniques and Applications ed. Grange, J.M., Fox, A. & Morgan, N.L. Society for Applied Bacteriology Technical Series no. 28; in press. TREVORS, J.T. 1986 Plasmid curing in bacteria. FEMS Microbiology Reviews 32,14%157.
ADONIS 0266825491oooO5J
Isolation and characterization of a cryptic plasmid from mesophilic aeromonads : potential as a cloning vector I . F . CONNERTON, J . S . K A U R ,S. ROGERS& R . W . A . P A R KDepartmentof Microbiology, University of Reading, London Road, Reading RG1 5AQ, U K Received I4 September I990 and accepted 14 September 1990 Paper number: CRH/022 C O N N E R T OIN. F, . , K A U R ,J.S., ROGERS,S . & P A R K R , . W . A . 1991. Isolation and characterizationof a cryptic plasmid from mesophilic aeromonads:potential as a cloning vector. Letters in Applied Microbiology 12, 1619. A 2.6 kb covalently closed circular plasmid has been isolated from clinical and environmental isolates of Aeromonas sobria and A. hydrophila. The possibility that the plasmid carries genetic determinantsthat mediate resistance to a variety of antimicrobial agents has been eliminated. The plasmid is stable at approximately 20-25 copies per chromosome equivalent which, together with its relatively small size and the presence of unique restrictionsites, makes it a good candidate for development as a cloning vector.
Species of the genus Aeromonas are ubiquitous in aquatic environments. The mesophilic aeromonads are potential pathogens of mammals and lower vertebrates including amphibia, reptiles and fish (Janda et al. 1984; Dooley & Trust 1988). Recent attention has focussed on the species Aeromonas sobria and A. hydrophila, as they are persistently implicated as causative agents of clinical bacteraemia (Harris et al. 1985; Janda & Brendan 1987) and gastroenteritis (Holmberg & Farmer 1984). It is likely that these species are water-borne and foodborne pathogens. In addition Aeromonas species have been shown to be good indicators of the trophic state of aquatic environments (Rippey & Cabelli 1980). As a result of this interest a series of studies has been initiated on the genetics and molecular biology of species of the genus. These studies range from the directed investigation of pathogenic factors such as enterotoxins (Chakraborty et al. 1984) and the cytotoxic haemolysins (aerolysin) (Chakraborty et al. 1986; Howard & Buckley 1986), to more fundamental genetic aspects which may provide enabling technology (Resnick & Nelson 1988). All of these studies have relied upon Escherichia coli to express functional Aeromonas genes. This has been
necessary since most routine cloning vectors utilize the ColEl origin of replication which does not function in Aeromonas. Although transcription so far has proved faithful, E. coli does not always process the protein products eficiently (Howard & Buckley 1986). To help facilitate further studies we have investigated a small stable plasmid native to Aeromonas with a view to developing an Aeromonas-based vector system.
Materials and Methods STRAINS
Mesophilic Aeromonas spp., isolated from faeces, food or water using alkaline peptone water enrichment followed by Aeromonas selective medium (Difco), were classified according to Popoff & Veron (1976). These assignments were later verified by DNA/DNA hybridization (Rogers et al. 1990). Cultures were stored frozen at -70°C in 15% glycerol with Nutrient Broth (Oxoid No. 2). Routine subcultures were made on Nutrient Agar (Oxoid, 37"C, 24 h). For isolation of the plasmids, strains were grown to late exponential phase in Nutrient Broth (Oxoid No. 2) at 37°C.
Cloning vector plasmid in Aeromonas I S O L A T I O N OF P L A S M I D D N A
Routine small-scale plasmid preparations were performed according to Holmes & Quigley (1981). Larger preparations of closed circular DNAs were carried out by the alkaline-lysis method of Birnboim & Doly (1979) and purified further by caesium chloride/ethidium bromide density gradient centrifugation. A N A L Y S I S OF P L A S M I D D N 4
Plasmid DNA was analysed electrophoretically on 0.9% flat-bed agarose gels with 50 mmol/l Tris/borate 1 mmol/l EDTA (pH 8.3) as running buffer, before visualization under U.V.light with ethidium bromide (0.5 mg/l). Restriction digests were performed following the manufacturer’s instructions (BRL) and analysed on similar gels. The plasmid copy number was estimated by U.V. fluorescence spectrophotometry of ethidium bromide-stained gels containing total cell lysates as described by Eckhardt (1978). PLASMID CURING
Acridine orange or ethidium bromide dyes were used as curing agents (Trevors 1986). After incubation, those cells demonstrating visible growth at the highest dye concentration were transferred to dye-free medium and plasmid preparations performed. Cured variants that displayed normal growth characteristics were verified by colony hybridization against plasmid DNA (Grunstein & Hogness 1975). S E N S I TI V I TI ES TO 4 N T I MI C R O B I A L A G EN T S
A range of compounds was tested at various concentrations on filter paper discs to screen for resistance phenotypes associated with the plasmid. Agents used were : chloramphenicol, colistin sulphate, erythromycin, gentamycin, kanamycin, nalidixic acid, nitrofurantoin, streptomycin, tetracycline and vancomycin (Sigma). It should be noted that many strains of Aeromonas are inherently resistant to ampicillin and amoxycillin. Results and Discussion
As part of our work on plasmid profiling and replicon typing in the mesophilic aeromonads, we have observed a relatively small sized
17
plasmid on several occasions. A single plasmid of similar size was isolated from five independent strains of Aerornonas (3 of A. sobria and 2 of A. hydrophila) out of some 122 clinical and environmental isolates. One of the A. sobria isolates was selected as a prototype strain and a large-scale plasmid preparation performed by caesium chloride/ethidium bromide buoyant density gradient centrifugation. The resulting plasmid was called pAER027. Colony hybridization with the purified plasmid DNA confirmed that the plasmids isolated from alternative strains were related with respect to their DNA sequences, and restriction digests confirmed that these plasmids had a similar molecular organization to that of pAER027. The restriction endonuclease cleavage patterns for 17 enzymes were examined by agarose gel electrophoresis. Plasmid pAER027 had cleavage sites for six of the enzymes (Table I). Analysis of the results showed that the plasmid is a circle of 2.6 kb. Single, double and triple digests were performed to construct a physical map for the enzymes ApaI, BylII, EcoRI, MluI, PouIl and SstI (Fig. 1). The restriction enzymes BamHI, Ben, ClaI, DraI, HindIII, NdeI, PstI, SalI, ScaI, XbaI and XhoI did not cut the plasmid. The relatively small size of this plasmid together with its unique restriction sites (especially those adjacent to each other) and, just as important, the absence of a number of common sites which may be introduced as unique, make the plasmid amenable for genetic manipulation. Yields of plasmid DNA confirmed our earlier suspicion that the molecule was present at a higher copy number than many other plasmids observed in our initial survey. Fluorescence monitoring of total cell DNA separated on
Table 1. Numbers and sizes of fragments generated by endonucleases with different restriction digestions of the 2.6 kb plasmid pAER027 from Aeromonas sohria
Fragment Restriction enzyme
Number
ApaI BglII EcoRI MluI PVUII SstI
3 1
1 1
1 1
Size (kb) 1.5
+ 0.95 + 0.15 2.60 2.60 2.60 2.60 2.60
Z. F. Connerton et al.
18
EcoRI 0.00
MIuI 2.55
I
Financial support from the Ministry of Agriculture, Fisheries and Food for Susan Rogers is gratefully acknowledged.
Refereaces
ApaI 2.05 pAER027
SSlI 1-25
Fig. 1. The restriction map of pAER027 from Aeromonas sobria.
agarose gels and stained with ethidium bromide established copy numbers of between 20 and 25 per chromosomal equivalent. Re-introduction of recombinant versions of this plasmid carrying additional aeromonas genes may well cause expression over and above that of chromosomal counterparts, as a consequence of the increased copy number. None of the five isolates possessing the plasmid could be ascribed phenotypic traits that could be associated with its possession. The cured version of the prototype A. sobria strain displayed similar growth characteristics and responded similarly to all the antimicrobial agents tested. Since we have been unable to assign any phenotypic character to the plasmid it may be considered as cryptic. To facilitate initial selection of the plasmid in Aeromonas, a genetic marker must be introduced onto it. Our initial experiments suggest that at least the unique EcoRI site will tolerate the insertion of DNA. The tetracycline resistance gene derived from vectors based on E. coli would seem a good candidate as many strains of Aeromonas are sensitive to this antibiotic. It should be possible to design a selectable cloning vector based upon the cryptic replicon. However, direct transformation into Aeromonas is likely to be difficult, and electroporation may prove a useful alternative.
BIRNBOIM,H.C. & D ~ L YJ. , 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. NucIeic Acids Research 7, 15131523. CHAKRABORTY, T., MO"EGRO, M.A., SANYAL, S.C., HELMUTH,R., BULLING,E. & TIMMIS, K.N. 1984 Cloning of enterotoxin gene from A e r o m o m hydrophila provides conciusive evidence of production of a cyctotoxic enterotoxin. Infection and Immunity 46, 435-441. -n, T., HUHLE, B, BERG8AUER, H. & GOEBEL, W. 1986 Cloning, expression, and mapping of the A e r o m o ~ hydrophh s aerolysin gene determinant in EsEherichia eofi K-12. Journal of Bacterwlogy 167,36&374. DOOLEY, J.S.G. & TRUST,TJ. 1988 Surface protein components of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein Journal of Bacteriology 170,49%506. ECKHARDT, T . 1978 A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1,584-588. GRUNSTEIN, M. & HOGNESS, D. 1975 Colony hybridization: a method for the isolation of cloned DNAs that contain a spccific gene. Proceedings of the National Academy of Sciences, U S A 72,3961-3965. I-Luws, R.L., FAINSTEIN,V., ELTING,L.E., HOPFER, R.L. & BODm, G.D. 1985 Bacteremia caused by Aeromonas species in hospitalizad cancer patients. Review o f l ~ e c t i o u Diseases s 7,314-320. HOLMBERG, S.D. & FARMEX, 111, J.J. 1984 Aeromonas hydrophila and Plesiomom shigelloides as causes of intestinal infections. Review of Infectious Diseoses 6, 633439. HOLMES,D.S. & QUIGLEY, M. 1981 A rapid boiling method for the preparation of bacterial plasrnids. Analytical Biochemistry 114, 193-197. HOWARD. S.P. & BUCKLEY,J.T. 1986 Molecular cloning and expression in Escherichia coli of the structural gene for hemolytic toxin aerolysin from Aeromonas hydrophila. Molecular and General Genetics 204,289-295. JANDA,J.M. & BRENDAN, R. 1987 Importance of Aeromonas sobria in Aeromonas bacteremia. Journal of Infectious Diseases 115,589-591. JANDA,J.M., ~ I T A N O M. , & BOTTONE, E.J. 1984 Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum. Journal of Clinical Microbiology 19,4447. Popom, M. & VERON,M. 1976 A taxonomic study of the Aeromonas hydrophila-Aeromonas punctata group. Journal of General Microbiology 94, 11-22. &NICK, D. & NELSON,D.R. 1988 Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant. Journal of Bacteriology 170,48-55.
Cloning vector plasmid in Aeromonas CABELLI,V.J. 1980 Occurrence of Amomonas hydrophila in limnetic environments: relationship of the organism to trophic state. Microbial Ecology 6,45554. ROGERS,S., M O F F A ~M.F., . CONNERTON, LF. & PARK, R.W.A. 1990 Plasmid profiling and D N A P N A hybridization for distinguishing between mesophilic ~ P P E Y , S.R. &
19
Aeromonas bacteria. In Genetic Manipulation: Techniques and Applications ed. Grange, J.M., Fox, A. & Morgan, N.L. Society for Applied Bacteriology Technical Series no. 28; in press. TREVORS, J.T. 1986 Plasmid curing in bacteria. FEMS Microbiology Reviews 32,14%157.
