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Plasmid incidence in the subgroups of two bacterial communities
ARTICLE IN PRESS Microbiological Research 163 (2008) 350—353
www.elsevier.de/micres
SHORT COMMUNICATION
Plasmid incidence in the subgroups of two bacterial communities Frauke Beilsteina, Anja Battermannb, Brigitte Dreiseikelmanna, a
Department of Microbiology/Genetechnology, Faculty of Biology, University of Bielefeld, Universitaetsstr. 25, 33615 Bielefeld, Germany b Department of Transfusion Medicine, MH Hannover, Medical Park, Feodor-Lynen Str. 21, 30625 Hannover, Germany Received 24 February 2006; received in revised form 23 June 2006; accepted 23 June 2006
KEYWORDS Bacterial community; Plasmid incidence
Summary The plasmid incidence of two bacterial communities from soil and freshwater was determined by endogenous plasmid isolation. The overall plasmid incidence for the communities was about 10%, while the frequency of plasmid-containing members in different subgroups ranged from 0% to 100%. Both communities included a minor population where all members contained several plasmids. & 2006 Elsevier GmbH. All rights reserved.
Introduction It is generally accepted that horizontal gene transfer by conjugation promotes the evolution and adaptation of individual bacteria and of the microbial communities in natural systems. Laboratory experiments have shown that conjugation of broad-host-range plasmids is possible between distantly related bacteria and even eukaryotes (Dro ¨ge et al., 1999; Davison, 1999). A lot of studies about conjugal plasmids and conjugation in natural habitats support these findings, but the extent of conjugation events and the involvement of different groups of a community are not known because Corresponding author. Tel.: +49 5211066907; fax: 49 5211066015. E-mail address: [email protected] (B. Dreiseikelmann).
of the complexity of a bacterial community and of the biotic and abiotic parameters of the environment. Nowadays cultivation dependent methods were mostly replaced by fingerprint-techniques which allow to study a broader spectrum. Nevertheless, cultivation dependent isolation methods and endogenous plasmid isolation are necessary to get an idea about the identity of indigenous partners involved in horizontal gene transfer. Data about the genetic potential for conjugation are mainly restricted to the determination of the incidence and abundance of conjugal and mobilizable plasmids in total communities or in a special preselected group (e.g. Campbell et al., 1995; Cook et al., 2001). We wanted to determine the plasmid incidence in different groups of a bacterial community which could give an idea whether special groups are more or less involved in conjugational gene transfer.
0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2006.06.015
ARTICLE IN PRESS Plasmid incidence in the subgroups of two bacterial communities
Materials and methods ARDRA and 16S rDNA sequencing ARDRA experiments were done as described previously (Disque´-Kochem et al., 2001) with PCR products amplified from cells resuspended in PCR mixtures and with standard 16S rDNA primers. The PCR products were digested with HinfI, HpaII and HaeIII, respectively. After electrophoresis in a 2.5% agarose gel the DNA fragments patterns were compared and length determination was done with the help of a 100 bp DNA ladder. For 16S rDNA sequencing the PCR products were inserted into pUC18 and sequenced with standard primers (primers M13-FP: TGTAAAACGACGGCCAGT, M13RP: CAGGAAACAGCTATGACC).
Analysis of total cell protein Total cell protein was prepared according to Lanka and Barth (1981) and subjected to SDS-polyacrylamide-gel electrophoresis (17.5%; Laemmli, 1970).
Identification of plasmids Isolation of plasmids was done according to Ramos-Gonzalez et al. (1991). This alkaline isolation method allowed the detection of plasmids with molecular masses up to about 200 kb. All strains were screened three times for plasmids.
Results Isolation and analysis of two bacterial communities About 1000 members of the bacterial community present in an agricultural soil sample (Fuessinger Au, Kiel, Germany) and near 600 isolates of a second community from three freshwater ponds (Meierteich, Oetkerparteich, Schlosshofteich, Bielefeld, Germany) were isolated. For this purpose, suitable dilutions of a soil suspension (in 50 mM potassium phosphate buffer, pH 7.5), respectively, a freshwater sample were spread onto solid rich medium (TBY; 10 g tryptone, 5 g yeast extract and 0.5 g NaCl per litre, pH7.5) and incubated at 26 1C. Single colonies were purified by streaking onto at least three consecutive agar plates. After a macroscopic and microscopic grouping, all isolates of the soil community were taxonomically grouped by ARDRA analysis with HinfI, HpaII and HaeIII,
351
respectively. A collection of about 600 members of the freshwater community was analyzed by comparing the total protein patterns in a SDSPAGE. 16S rDNA sequence analysis of some members of the main groups of both communities allowed affiliation at least to a genus. Further analyses were restricted to the Gramnegative isolates. Nearly half of the members from the soil community (510 isolates) were Gramnegative, while 298 of 600 freshwater isolates fitted into this group. As expected the Pseudomonas strains represented the major group in both communities, 70.8% of all soil isolates and 46% of the screened freshwater community members affiliated with this genus (Fig. 1). The remaining minor groups of the soil community represented the following six genera: Stenotrophomonas, Xanthomonas, Alcaligenes, Paracoccus, Agrobacterium and Enterobacter. Among the Gram-negative strains of the freshwater habitat, the most abundant group beside the Pseudomonads were the Aeromonads with 20% of the community members. The minor groups which all amounted to less than 10% of the whole community belonged to the genera Flavobacterium, Hydrogenophaga, Escherichia, Shigella, Brevundimonas, Flexibacter and Chromobacter
Plasmid incidence in the communities Plasmids between 1 and 200 kb in size were visualized by agarose gel electrophoresis after an alkaline isolation (Ramos-Gonzalez et al., 1991). Forty-five plasmid containing isolates (plasmid incidence 8.8%) were detected among the members of the soil community while 30 freshwater strains (plasmid incidence 10%) harboured plasmids (Fig. 1). The latter result is comparable to published data about the plasmid incidence of 9.4% determined by endogenous isolation for another freshwater habitat (Burton et al., 1982). In the different subgroups the plasmid incidence ranged from 0% to 100%, also among strains with nearly the same abundance. In both communities a small group attracted attention because all members were plasmid-containing and each isolate contained more than one plasmid (Fig. 2). Both groups seemed to be real populations because they included only one species. The multiplasmid containing strains isolated from soil belonged to a Paracoccus species closely related to Paracoccus aminophilus, those isolated from water were classified as Escherichia coli. All members of both populations contained several plasmids, some of them identical in
ARTICLE IN PRESS 352
F. Beilstein et al. 100%
100% A
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Figure 1. Abundance and plasmid incidence of different groups of a bacterial freshwater (A), respectively, soil (B) community. The major groups of the freshwater community (A) consisted of the genera (1) Pseudomonas (140/8), (2) Aeromonas (73/10) (3) Flavobacterium (25/0), (4) Hydrogenophaga (14/0), (5) E. coli (12/12), (6) Shigella (12/0), (7) Brevundimonas (9/0), (8) Flexibacter (8/0), (9) Chromobacterium (5/0). The most abundant groups of the soil community (B) were represented by (1) Pseudomonas (361/21), (2) Stenotrophomonas (51/1), (3) Xanthomonas (34/3), (4) Alcaligenes (25/2), (5) Paracoccus (14/14), (6) Agrobacterium (13/2), (7) Enterobacter (12/2). Number of isolates/ number of plasmid-containing members in parantheses. Black columns represent the abundance of a genus, grey columns the incidence of plasmids.
Figure 2. Agarose gel electrophoresis of plasmids from multiplasmid populations. Plasmid DNA was isolated according to the method of Ramos-Gonzalez et al. (1991). (A) Plasmids from freshwater E. coli isolates. For size estimation plasmids of known molecular weights were loaded on the gel. Lane M, l DNA EcoRI/HindIII, Plasmid DNA: Lane 1, F plasmid (100 kb); lane 2, SAL (80 kb); lane 3, RP4 (54 kb); plasmid DNA from isolates: lane 4, M15; lane 5, M26; lane 6, M11; lane 7, M25; lane 8, M27; lane 9, M52; lane 10, M78; lane 11, M101; lane 12, O221; lane 13, M2; lane 14, M4; lane 15, M7. (B) Plasmid DNA from Paracoccus spp. soil isolates. Lane M, l DNA EcoRI/HindIII, plasmid DNA isolated from: Lane 1, Bi1590; lane 2, Bi1663; lane 3, Bi1664; lane 4, Bi1058; lane 5, Bi1094; lane 6, Bi1099; lane 7, Bi1802; lane 8, Bi161; lane 9, Bi 165; lane 10, Bi237; lane 11, Bi271; lane 12, Ol11; lane 13, Ol18; lane 14, Ol24; lane 15, Bi144.
different isolates, but the whole plasmid pattern differed in most isolates indicating that they are not siblings but independent isolates. For example, most of the Paracoccus isolates contained a 23 kb plasmid as well as several smaller or larger ones (Battermann et al., 2003). In the E. coli strains up
to 15 different plasmids were identified, some plasmids seemed to occur in several isolates. Approximately 45% of the E. coli plasmids were smaller than 15 kb suggesting that they are not conjugative. Preliminary experiments with separated plasmids revealed that at least some of the
ARTICLE IN PRESS Plasmid incidence in the subgroups of two bacterial communities large plasmids were conjugative and most of the smaller ones were mobilizable.
Discussion Multiplasmid strains were first described for clinical isolates (Laufs and Kleinmann, 1978) but in the meantime various environmental strains with a high incidence of different plasmids were isolated (e.g. Coplin et al., 1981; Toranzo et al., 1983). A high number of plasmids with different sizes, copy number, and genetic equipment in a single bacterium could possibly increase its fitness (Kado, 1998) by allowing adaptation to special environmental conditions or exploitation of new ecological niches. These advantages may exceed the disadvantage of high-energy costs for plasmid replication and maintenance. A high plasmid pool represents diverse opportunities for horizontal gene transfer and thus further evolution of the population. But does it influence significantly the whole community? If conjugational gene transfer occurs with high frequency between all members of the community, the plasmid incidence of the total community should be comparable to that of the subgroups assuming a stable maintenance of the plasmids. There are several possible explanations for the significant differences in the plasmid incidence of subgroups. One hypothesis may be that some groups are more active in horizontal gene transfer – especially in intraspecies transfer – than others. Successful interspecies transfer seems to be very unusual. The high plasmid content may indicate that, for unknown reasons, these strains are more potent receptors and/or allow establishment and stable maintenance for a lot of plasmids more frequently than others. On the other hand, differences in the plasmid incidence of various populations could be based on colonization of the habitat at different times, so that dissemination of the plasmids is still going on. The multiplasmid strains may have acqired the high number of plasmids in an ecological niche where high concentrations of this strain allowed high frequencies of conjugation, e.g. the E. coli population in the intestine of mammals. Further studies are necessary to elucidate these open questions.
353
References Battermann, A., Disse-Kro ¨mker, C., Dreiseikelmann, B., 2003. A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus. Microbiology 149, 3587–3593. Burton, N.F., Day, M.J., Bull, A.T., 1982. Distribution of bacterial plasmids in clean and polluted sites in a south Wales river. Appl. Environ. Microbiol. 44, 1026–1029. Campbell, J.I.A., Jacobsen, C.S., Sørensen, J., 1995. Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils. FEMS Microbiol. Ecol. 18, 51–62. Cook, M.A., Osborn, A.M., Bettandorff, J., Sobecky, P.A., 2001. Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities. Microbiologica 147, 2089–2101. Coplin, D.L., Rowan, R.G., Chrisholm, D.A., Whitmoyer, R.E., 1981. Characterization of plasmids in Erwinia stewartii. Appl. Environ. Microbiol. 42, 599–604. Davison, J., 1999. Genetic exchange between bacteria in the environment. Plasmid 42, 73–91. Disque´-Kochem, C., Battermann, A., Stra ¨tz, M., Dreiseikelmann, B., 2001. Screening for trbB- and traG-like sequences by PCR for the detection of conjugative plasmids in bacterial soil isolates. Microbiol. Res. 156, 159–168. Dro ¨ge, M., Pu ¨hler, A., Selbitschka, W., 1999. Horizontal gene transfer among bacteria in terrestrial and aquatic habitats as assessed by microcosm and field studies. Biol. Fertil. Soils 29, 221–245. Kado, C.I., 1998. Origin and evolution of plasmids. Antonie van Leeuwenhoek 73, 117–126. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lanka, E., Barth, P.T., 1981. Plasmid RP4 specifies a deoxyribonucleic acid primase involved in its conjugal transfer and maintenance. J. Bacteriol. 148, 769–781. Laufs, R., Kleinmann, F., 1978. Antibiotic resistance factors and other plasmids in bacterial isolates from hospitalized patients. Zentralblatt Bakteriol. 240, 503–516. Ramos-Gonzalez, M.I., Duque, E., Ramos, J.L., 1991. Conjugal transfer of recombinant DNA in cultures and in soil: host range of Pseudomonas putida TOL plasmids. Appl. Environ. Microbiol. 57, 3020–3027. Toranzo, A.E., Barja, J.L., Colwell, R.R., Hetrick, F.M., 1983. Characterisation of plasmids in bacterial fish pathogens. Infection Immunity 39, 184–192.
www.elsevier.de/micres
SHORT COMMUNICATION
Plasmid incidence in the subgroups of two bacterial communities Frauke Beilsteina, Anja Battermannb, Brigitte Dreiseikelmanna, a
Department of Microbiology/Genetechnology, Faculty of Biology, University of Bielefeld, Universitaetsstr. 25, 33615 Bielefeld, Germany b Department of Transfusion Medicine, MH Hannover, Medical Park, Feodor-Lynen Str. 21, 30625 Hannover, Germany Received 24 February 2006; received in revised form 23 June 2006; accepted 23 June 2006
KEYWORDS Bacterial community; Plasmid incidence
Summary The plasmid incidence of two bacterial communities from soil and freshwater was determined by endogenous plasmid isolation. The overall plasmid incidence for the communities was about 10%, while the frequency of plasmid-containing members in different subgroups ranged from 0% to 100%. Both communities included a minor population where all members contained several plasmids. & 2006 Elsevier GmbH. All rights reserved.
Introduction It is generally accepted that horizontal gene transfer by conjugation promotes the evolution and adaptation of individual bacteria and of the microbial communities in natural systems. Laboratory experiments have shown that conjugation of broad-host-range plasmids is possible between distantly related bacteria and even eukaryotes (Dro ¨ge et al., 1999; Davison, 1999). A lot of studies about conjugal plasmids and conjugation in natural habitats support these findings, but the extent of conjugation events and the involvement of different groups of a community are not known because Corresponding author. Tel.: +49 5211066907; fax: 49 5211066015. E-mail address: [email protected] (B. Dreiseikelmann).
of the complexity of a bacterial community and of the biotic and abiotic parameters of the environment. Nowadays cultivation dependent methods were mostly replaced by fingerprint-techniques which allow to study a broader spectrum. Nevertheless, cultivation dependent isolation methods and endogenous plasmid isolation are necessary to get an idea about the identity of indigenous partners involved in horizontal gene transfer. Data about the genetic potential for conjugation are mainly restricted to the determination of the incidence and abundance of conjugal and mobilizable plasmids in total communities or in a special preselected group (e.g. Campbell et al., 1995; Cook et al., 2001). We wanted to determine the plasmid incidence in different groups of a bacterial community which could give an idea whether special groups are more or less involved in conjugational gene transfer.
0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2006.06.015
ARTICLE IN PRESS Plasmid incidence in the subgroups of two bacterial communities
Materials and methods ARDRA and 16S rDNA sequencing ARDRA experiments were done as described previously (Disque´-Kochem et al., 2001) with PCR products amplified from cells resuspended in PCR mixtures and with standard 16S rDNA primers. The PCR products were digested with HinfI, HpaII and HaeIII, respectively. After electrophoresis in a 2.5% agarose gel the DNA fragments patterns were compared and length determination was done with the help of a 100 bp DNA ladder. For 16S rDNA sequencing the PCR products were inserted into pUC18 and sequenced with standard primers (primers M13-FP: TGTAAAACGACGGCCAGT, M13RP: CAGGAAACAGCTATGACC).
Analysis of total cell protein Total cell protein was prepared according to Lanka and Barth (1981) and subjected to SDS-polyacrylamide-gel electrophoresis (17.5%; Laemmli, 1970).
Identification of plasmids Isolation of plasmids was done according to Ramos-Gonzalez et al. (1991). This alkaline isolation method allowed the detection of plasmids with molecular masses up to about 200 kb. All strains were screened three times for plasmids.
Results Isolation and analysis of two bacterial communities About 1000 members of the bacterial community present in an agricultural soil sample (Fuessinger Au, Kiel, Germany) and near 600 isolates of a second community from three freshwater ponds (Meierteich, Oetkerparteich, Schlosshofteich, Bielefeld, Germany) were isolated. For this purpose, suitable dilutions of a soil suspension (in 50 mM potassium phosphate buffer, pH 7.5), respectively, a freshwater sample were spread onto solid rich medium (TBY; 10 g tryptone, 5 g yeast extract and 0.5 g NaCl per litre, pH7.5) and incubated at 26 1C. Single colonies were purified by streaking onto at least three consecutive agar plates. After a macroscopic and microscopic grouping, all isolates of the soil community were taxonomically grouped by ARDRA analysis with HinfI, HpaII and HaeIII,
351
respectively. A collection of about 600 members of the freshwater community was analyzed by comparing the total protein patterns in a SDSPAGE. 16S rDNA sequence analysis of some members of the main groups of both communities allowed affiliation at least to a genus. Further analyses were restricted to the Gramnegative isolates. Nearly half of the members from the soil community (510 isolates) were Gramnegative, while 298 of 600 freshwater isolates fitted into this group. As expected the Pseudomonas strains represented the major group in both communities, 70.8% of all soil isolates and 46% of the screened freshwater community members affiliated with this genus (Fig. 1). The remaining minor groups of the soil community represented the following six genera: Stenotrophomonas, Xanthomonas, Alcaligenes, Paracoccus, Agrobacterium and Enterobacter. Among the Gram-negative strains of the freshwater habitat, the most abundant group beside the Pseudomonads were the Aeromonads with 20% of the community members. The minor groups which all amounted to less than 10% of the whole community belonged to the genera Flavobacterium, Hydrogenophaga, Escherichia, Shigella, Brevundimonas, Flexibacter and Chromobacter
Plasmid incidence in the communities Plasmids between 1 and 200 kb in size were visualized by agarose gel electrophoresis after an alkaline isolation (Ramos-Gonzalez et al., 1991). Forty-five plasmid containing isolates (plasmid incidence 8.8%) were detected among the members of the soil community while 30 freshwater strains (plasmid incidence 10%) harboured plasmids (Fig. 1). The latter result is comparable to published data about the plasmid incidence of 9.4% determined by endogenous isolation for another freshwater habitat (Burton et al., 1982). In the different subgroups the plasmid incidence ranged from 0% to 100%, also among strains with nearly the same abundance. In both communities a small group attracted attention because all members were plasmid-containing and each isolate contained more than one plasmid (Fig. 2). Both groups seemed to be real populations because they included only one species. The multiplasmid containing strains isolated from soil belonged to a Paracoccus species closely related to Paracoccus aminophilus, those isolated from water were classified as Escherichia coli. All members of both populations contained several plasmids, some of them identical in
ARTICLE IN PRESS 352
F. Beilstein et al. 100%
100% A
B
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Figure 1. Abundance and plasmid incidence of different groups of a bacterial freshwater (A), respectively, soil (B) community. The major groups of the freshwater community (A) consisted of the genera (1) Pseudomonas (140/8), (2) Aeromonas (73/10) (3) Flavobacterium (25/0), (4) Hydrogenophaga (14/0), (5) E. coli (12/12), (6) Shigella (12/0), (7) Brevundimonas (9/0), (8) Flexibacter (8/0), (9) Chromobacterium (5/0). The most abundant groups of the soil community (B) were represented by (1) Pseudomonas (361/21), (2) Stenotrophomonas (51/1), (3) Xanthomonas (34/3), (4) Alcaligenes (25/2), (5) Paracoccus (14/14), (6) Agrobacterium (13/2), (7) Enterobacter (12/2). Number of isolates/ number of plasmid-containing members in parantheses. Black columns represent the abundance of a genus, grey columns the incidence of plasmids.
Figure 2. Agarose gel electrophoresis of plasmids from multiplasmid populations. Plasmid DNA was isolated according to the method of Ramos-Gonzalez et al. (1991). (A) Plasmids from freshwater E. coli isolates. For size estimation plasmids of known molecular weights were loaded on the gel. Lane M, l DNA EcoRI/HindIII, Plasmid DNA: Lane 1, F plasmid (100 kb); lane 2, SAL (80 kb); lane 3, RP4 (54 kb); plasmid DNA from isolates: lane 4, M15; lane 5, M26; lane 6, M11; lane 7, M25; lane 8, M27; lane 9, M52; lane 10, M78; lane 11, M101; lane 12, O221; lane 13, M2; lane 14, M4; lane 15, M7. (B) Plasmid DNA from Paracoccus spp. soil isolates. Lane M, l DNA EcoRI/HindIII, plasmid DNA isolated from: Lane 1, Bi1590; lane 2, Bi1663; lane 3, Bi1664; lane 4, Bi1058; lane 5, Bi1094; lane 6, Bi1099; lane 7, Bi1802; lane 8, Bi161; lane 9, Bi 165; lane 10, Bi237; lane 11, Bi271; lane 12, Ol11; lane 13, Ol18; lane 14, Ol24; lane 15, Bi144.
different isolates, but the whole plasmid pattern differed in most isolates indicating that they are not siblings but independent isolates. For example, most of the Paracoccus isolates contained a 23 kb plasmid as well as several smaller or larger ones (Battermann et al., 2003). In the E. coli strains up
to 15 different plasmids were identified, some plasmids seemed to occur in several isolates. Approximately 45% of the E. coli plasmids were smaller than 15 kb suggesting that they are not conjugative. Preliminary experiments with separated plasmids revealed that at least some of the
ARTICLE IN PRESS Plasmid incidence in the subgroups of two bacterial communities large plasmids were conjugative and most of the smaller ones were mobilizable.
Discussion Multiplasmid strains were first described for clinical isolates (Laufs and Kleinmann, 1978) but in the meantime various environmental strains with a high incidence of different plasmids were isolated (e.g. Coplin et al., 1981; Toranzo et al., 1983). A high number of plasmids with different sizes, copy number, and genetic equipment in a single bacterium could possibly increase its fitness (Kado, 1998) by allowing adaptation to special environmental conditions or exploitation of new ecological niches. These advantages may exceed the disadvantage of high-energy costs for plasmid replication and maintenance. A high plasmid pool represents diverse opportunities for horizontal gene transfer and thus further evolution of the population. But does it influence significantly the whole community? If conjugational gene transfer occurs with high frequency between all members of the community, the plasmid incidence of the total community should be comparable to that of the subgroups assuming a stable maintenance of the plasmids. There are several possible explanations for the significant differences in the plasmid incidence of subgroups. One hypothesis may be that some groups are more active in horizontal gene transfer – especially in intraspecies transfer – than others. Successful interspecies transfer seems to be very unusual. The high plasmid content may indicate that, for unknown reasons, these strains are more potent receptors and/or allow establishment and stable maintenance for a lot of plasmids more frequently than others. On the other hand, differences in the plasmid incidence of various populations could be based on colonization of the habitat at different times, so that dissemination of the plasmids is still going on. The multiplasmid strains may have acqired the high number of plasmids in an ecological niche where high concentrations of this strain allowed high frequencies of conjugation, e.g. the E. coli population in the intestine of mammals. Further studies are necessary to elucidate these open questions.
353
References Battermann, A., Disse-Kro ¨mker, C., Dreiseikelmann, B., 2003. A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus. Microbiology 149, 3587–3593. Burton, N.F., Day, M.J., Bull, A.T., 1982. Distribution of bacterial plasmids in clean and polluted sites in a south Wales river. Appl. Environ. Microbiol. 44, 1026–1029. Campbell, J.I.A., Jacobsen, C.S., Sørensen, J., 1995. Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils. FEMS Microbiol. Ecol. 18, 51–62. Cook, M.A., Osborn, A.M., Bettandorff, J., Sobecky, P.A., 2001. Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities. Microbiologica 147, 2089–2101. Coplin, D.L., Rowan, R.G., Chrisholm, D.A., Whitmoyer, R.E., 1981. Characterization of plasmids in Erwinia stewartii. Appl. Environ. Microbiol. 42, 599–604. Davison, J., 1999. Genetic exchange between bacteria in the environment. Plasmid 42, 73–91. Disque´-Kochem, C., Battermann, A., Stra ¨tz, M., Dreiseikelmann, B., 2001. Screening for trbB- and traG-like sequences by PCR for the detection of conjugative plasmids in bacterial soil isolates. Microbiol. Res. 156, 159–168. Dro ¨ge, M., Pu ¨hler, A., Selbitschka, W., 1999. Horizontal gene transfer among bacteria in terrestrial and aquatic habitats as assessed by microcosm and field studies. Biol. Fertil. Soils 29, 221–245. Kado, C.I., 1998. Origin and evolution of plasmids. Antonie van Leeuwenhoek 73, 117–126. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lanka, E., Barth, P.T., 1981. Plasmid RP4 specifies a deoxyribonucleic acid primase involved in its conjugal transfer and maintenance. J. Bacteriol. 148, 769–781. Laufs, R., Kleinmann, F., 1978. Antibiotic resistance factors and other plasmids in bacterial isolates from hospitalized patients. Zentralblatt Bakteriol. 240, 503–516. Ramos-Gonzalez, M.I., Duque, E., Ramos, J.L., 1991. Conjugal transfer of recombinant DNA in cultures and in soil: host range of Pseudomonas putida TOL plasmids. Appl. Environ. Microbiol. 57, 3020–3027. Toranzo, A.E., Barja, J.L., Colwell, R.R., Hetrick, F.M., 1983. Characterisation of plasmids in bacterial fish pathogens. Infection Immunity 39, 184–192.