School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, GA 30332-0230, USA1
Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK2
Author for correspondence: Patricia A. Sobecky. Tel: +1 404 894 5819. Fax: +1 404 894 0519. e-mail: patricia.sobecky{at}biology.gatech.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: extrachromasomal elements, marine sediment, replication origins, DNA extraction
Abbreviations: BHR, broad-host-range
The GenBank accession numbers for the 16S rRNA sequences determined in this work are AF249334AF249338 and AF284226AF284230.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ability of plasmids to replicate in different bacterial hosts and to either self-transfer or be mobilized by transfer-proficient plasmids ensures plasmid survival and provides a pool of mobile DNA which contributes to the genetic adaptation of microbial communities. Such plasmid-mediated adaptation has been documented by increased frequencies (2- to 10-fold) of catabolic plasmids in polluted marine and freshwater ecosystems (Burton et al., 1982 ; Hada & Sizemore, 1981
; Ogunseitan et al., 1987
). However, to better understand the role of plasmids in promoting the horizontal transfer of ecologically advantageous genes within marine microbial communities, knowledge of the molecular diversity of replicons, host range and transfer capabilities of marine plasmids is required.
Previous efforts to assess plasmid diversity have often relied on RFLP patterns for grouping and characterizing naturally occurring plasmid populations from bacterial assemblages in freshwater and terrestrial environments (Drnen et al., 1998
, 1999
; Lilley et al., 1996
; Pickup, 1989
). However, in response to changing environmental conditions or when attempting to colonize new niches, plasmids can sequester additional novel genes (Szpirer et al., 1999
), thereby altering their RFLP pattern yet retaining the basic replicon. Numerous studies have documented bacterial plasmid incidence in microbial communities occurring in marine sediments and estuarine and pelagic ecosystems (Belliveau et al., 1991
; Hermansson et al., 1987
; Kobori et al., 1984
; Reyes et al., 1999
; Sizemore & Colwell 1977
; Sobecky et al., 1997
). However, few studies have attempted to assess plasmid diversity and distribution in marine environments by molecular typing due to a lack of inc/rep DNA probes derived from plasmids found in marine bacterial populations (Dahlberg et al., 1997
; Sobecky et al., 1998
).
Previously, we developed a strategy for the isolation of plasmid oriVs from marine bacteria (Sobecky et al., 1998 ). In this study we describe a collection of inc/rep probes derived from endogenously isolated, cryptic marine plasmids, their relationship to known replicon groups and their use in assessing plasmid distribution and ecology in marine sediment microbial communities. To date, replicons have been obtained from broad-host-range (BHR) plasmids obtained from marine bacteria isolated from coastal California (Sobecky et al., 1998
), Georgia and South Carolina (this study), USA, sediments. The replicon probes were derived from plasmids ranging in size from approximately 5 to 60 kb isolated from hosts belonging to
and
subclasses of the Proteobacteria. Plasmid-containing marine bacteria and microbial community DNA obtained from marine sediments were subjected to DNADNA hybridization and PCR analyses using the endogenous marine replicon probes. In addition, the relationship between the marine plasmid replicons and those from previously characterized plasmids was investigated by comparative analysis of putative Rep protein sequences.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Prior to plating, samples collected from each of the three sites were homogenized. Replicate samples (1 g wet wt) were serially diluted in artificial sea water (Sobecky et al., 1997 ) and spread onto the following solid media: (i) Difco 2216E medium; (ii) YTSS (Sobecky et al., 1997
); and (iii) TSS (0·1% tryptone; Sobecky et al., 1997
) and incubated for 114 d at 30 °C. With the exception of the 2216E medium, artificial seawater was used in all the media and 1·72·0% agar was added when necessary. One hundred to five hundred colonies from each medium were restreaked at least twice on the same medium to ensure purity. Sodium ion requirements were determined by the method of Baumann et al. (1972)
. Only those isolates with a sodium ion growth requirement were considered to be of marine origin.
Identification of plasmid-bearing marine sediment isolates.
The modified Keiser method used to determine the presence of plasmids in marine bacterial isolates has been described previously (Sobecky et al., 1997 ). Escherichia coli strains containing pRR15 (4·8 kb), pRR10 (5·0 kb) and pRR54 (8·3 kb) (Roberts et al., 1990
), R6K (38 kb) (Stalker et al., 1979
), RK2 (60 kb) (Pansegrau et al., 1994
), pRK290 (20 kb) (Ditta et al., 1980
) and Rhizobium meliloti 102F34 containing three plasmids of 100, 150 and 220 kb (G. Ditta, personal communication) were used as sources of known plasmid size standards to approximate sizes of the marine plasmids. Plasmid frequency data were analysed for statistical significance using Statview (release 4.1). Data were subjected to the G-squared test for significance. Significance was accepted at a P value of 0·05. The entire 16S rRNA gene was amplified from genomic DNA as previously described (Sobecky et al., 1998
) by using primers fD1 and rD1 (Weisburg et al., 1991
) to obtain the identities of selected marine hosts containing plasmids. Amplified products were electrophoresed in 1·0% agarose gels, purified with a Qiagen PCR purification kit and partial insert sequences were obtained by using three reverse primers corresponding to the following positions in the E. coli sequence: primer 1, positions 519536; primer 2, positions 907926; and primer 3, positions 13921406 (Lane et al., 1985
). The rRNA gene sequence of each of the marine isolates was compared by database searches carried out through NCBI using BLAST 2.0 (Altschul et al., 1997
). 16S rRNA sample sequences, supplemented with 16S rRNA sequences from GenBank (region spanning positions 2281295 of the E. coli numbering system, were aligned using CLUSTAL W on BioNavigator.com provided by eBioinformatics. Phylogenetic trees were constructed and a bootstrap analysis was performed by using the evolutionary distances (JukesCantor distances) and the neighbour-joining method (Saitou & Nei, 1987
).
Large-scale isolation of plasmid DNA from marine isolates.
The isolation of plasmid DNA from marine bacteria has been described previously (Sobecky et al., 1998 ). Briefly, isolates were grown overnight in 1 litre of either TSS or 0·5xYTSS (Sobecky et al., 1997
) at 30 °C and cells were harvested by centrifugation (6000 g, 10 min). The cell pellets were resuspended in 100 ml solution 1 (10 mg lysozyme ml-1, 50 mM Tris/HCl pH 8·0, 10 mM EDTA, 100 µg RNaseA ml-1). This was followed by addition of 100 ml solution 2 (200 mM NaOH, 1% SDS) and incubation for 5 min at room temperature, and then addition of 100 ml solution 3 (3 M potassium acetate pH 5·5) and incubation on ice for 30 min. Samples were centrifuged (20000 g, 30 min), each supernatant was extracted with 0·5 vols phenol/chloroform (1:1) and the phases were separated by centrifugation (10000 g, 10 min). The aqueous layer was then precipitated with 0·8 vol. 2-propanol at room temperature and immediately centrifuged (20000 g, 30 min). Plasmid DNA was subsequently purified by caesium chloride gradient centrifugation (Maniatis et al., 1982
).
Cloning and sequencing of plasmid replication origins from marine bacteria.
The cloning and sequencing of marine plasmid replication origins have been described previously (Sobecky et al., 1998 ). Mixtures of marine plasmid origins ligated to the Tn903 npt gene were transformed into E. coli strains DH5
and C2110 (Table 1
). Plasmids pBJ033 and pBJ014 containing repGA33 and repGA14 origins, respectively, were generated by inserting the replication-proficient fragments into pBR325 (Table 1
). The plasmids were purified as previously described (Sobecky et al., 1998
) and the nucleotide sequences of the cloned replication origins were determined by using the vector primer sequences 5'-ATTGTTGCCGGGAAGCTAGAGTAAGTAGTT-3' and 5'-AATGAAGCCATACCAAACGACGAGCGTGAC-3' and a model ABI 377 automated DNA sequencer (Perkin-Elmer Applied Biosystems). Both strands of repGA14 and repGA33 were sequenced. Initial DNA sequence analysis and alignment were performed by the pairwise correlation method using BioNavigator.com provided by eBioinformatics. Complete replicon sequences were then analysed using the GCG suite of programs (version 8, Genetics Computer Group, Madison, WI, USA). DNA and protein sequences were compared with the EMBL DNA and protein databases using FASTA 3. Alignments of protein sequences were constructed using CLUSTAL W (Thompson et al., 1994
).
|
DNA extraction and purification from marine sediments.
Total DNA (chromosomal and plasmid DNA) was extracted from sediment samples (15 g) by the method of Tsai & Olson (1991) , modified to include two phenol/chloroform (1:1) extractions rather than one phenol and one phenol/chloroform extraction. The crude DNA extract was purified to remove humic acids and other co-extracted contaminants by the method of Tebbe & Vahjen (1993)
with ion-exchange columns (Qiagen-Tip 500).
PCR amplification.
The sequences of the primers used for detection of the plasmid replication regions are listed in Table 2. Primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Aliquots of marine sediment community DNA (1 µl, corresponding to the DNA recovered from approximately 510 mg sediment) were PCR-amplified in reaction mixtures containing (as final concentrations) 1xPCR buffer (Stratagene), 1·5 mM MgCl2, 200 µM of each deoxynucleoside triphosphate, 1 pmol of each forward and reverse primer, 0·025 ng T4 gene 32 protein (Boehringer Mannheim) and 0·025 U Taq 2000 (Stratagene) µl-1. The addition of T4 gene 32 protein has been shown to improve PCR amplification by binding and stabilizing single-stranded DNA (Tebbe & Vahjen, 1993
). Reaction mixtures were incubated in a model 2400 GeneAmp thermal cycler (Perkin Elmer) for 2 min at 95 °C, 2 min at the annealing temperature recommended for the respective primer sets (Table 2
) and 1 min at 72 °C, followed by a final extension for 5 min at 72 °C. Amplified products were analysed on 1·0% agarose gels run in TBE buffer, stained with ethidium bromide and UV-illuminated.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Plasmid sizes were approximated by comparison to known plasmid size standards for all plasmid-containing isolates. A bimodal pattern was observed with highest percentage detected in the <15 kb and 35100 kb size ranges (Fig. 2). A significant increase in Charleston Harbor and San Diego Bay bacterial isolates containing plasmids in the 35100 kb size range was observed during December 1998 (Charleston) and November 1999 (San Diego) samplings (Fig. 2a
, b
); G-squared values of 13·78 and 6·73 and P values of 0·0002 and 0·01, respectively). While plasmid-containing isolates obtained from San Diego Bay bacterial populations in November 1999 lacked plasmids greater than 100 kb (Fig. 2b
), the majority of isolates contained three or more plasmids (data not shown). Although bacteria from Sapelo Island sediments had a significantly lower plasmid incidence (Fig. 1
), a similar bimodal pattern was observed (Fig. 2c
). Although plasmids were readily detected in marine bacteria (Fig. 1
), the localization of plasmid genes encoding traits such as antibiotic and heavy metal resistance could not be confirmed (data not shown).
|
|
|
|
|
Assignment of marine plasmid replicons to replicon families
The relationship of the marine plasmid replicons to known plasmid families was determined by sequence comparison. Translation analysis revealed a number of potential ORFs with similarities to previously characterized Rep proteins (Table 5). An ORF in repSD164 (Table 5
) shows similarity to other outliers within the RCR group III (pC194-like) replicon family (Novick, 1989
), specifically, to Rep proteins from pAP1 in Arcanobacterium pyogenes and pIJ101 from Streptomyces lividans (Table 5
). Alignment of the putative repSD164 Rep protein with those from other members of this family reveal conservation of two of the three motifs (Fig. 5
) identified by Ilyina & Koonin (1992)
. Significantly, the repSD164 replicon lacks the His-Hydr-His motif 2 common to the majority of RCR Rep proteins (Fig. 5
).
|
|
The repGA14 replicon contains a partial ORF (ORF2; Table 5) at the extreme 3' end of the cloned replicon sequence that shows similarity to the Rep proteins of pCFC1 and pCFC2 from R. anatipestifer (Table 5
). However ORF2 appears to be a truncated homologue as it is relatively short when compared to the carboxyl termini of pCFC1 (54 aa longer) and pCFC2 (117 aa longer) Rep proteins. It is, however, likely that the gene encoding this putative ORF may extend upstream of the cloned region in the parental plasmid, i.e. the region encoding the amino terminus. It is possible that this ORF is in fact not involved in replication and represents part of a vestigial replicon. The only other significantly sized ORF in the GA14 cloned replicon shows similarity to resolvases from members of the Tn21 family of transposons. Although repGA14 may not encode a Rep protein, no similarity was observed between the repGA14 DNA sequence and any of the plasmid replicons in which a Rep protein is not encoded (i.e. ColE1 family). Lastly, two potential ORFs were identified in the repSD172 replicon sequence (Table 5
). Interestingly, FASTA analysis of both ORFs shows similarity to a hypothetical protein (ORF1) from pYV in Yersinia enterocolitica that is itself located between other recognized replication genes. Moreover, the repSD172 ORF1 also shares limited similarity with the RepA protein from the IncL/M replicon of the IncFIC-related theta-replicating plasmid pMU407 (Table 5
).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Numerous studies have, however, clearly demonstrated that the set of replicon probes derived from plasmids found in members of the Enterobacteriaceae (Couturier et al., 1988 ) has not been suitable for classifying plasmids from marine, freshwater and terrestrial habitats (Dahlberg et al., 1997
; Dr
nen et al., 1998
, 1999
; Kobayashi & Bailey 1994
; Osborn et al., 2000b
; Smit et al., 1998
; Sobecky et al., 1997
; van Elsas et al., 1998
). The development and validation of replicon probes specific for plasmids from environmental sources, such as marine sediments, would greatly facilitate genetic and biochemical studies designed to characterize plasmid replication systems and the distribution and transfer of ecologically relevant genes in microbial communities.
In the present study, plasmid replicons were used as DNA probes to identify the presence of BHR marine plasmid groups and a clinical BHR group (IncP) in endogenous plasmid populations and sediment microbial community DNA obtained from geographically separated marine locales. To our knowledge, this is the first report of plasmid groups detected by replicon-specific DNA probes derived from plasmid-bearing marine bacteria. The detection of plasmids present in phylogenetically distinct members of the -Proteobacteria that exhibited homology to repSD41 and repGA70 provides evidence for the occurrence of a common plasmid type(s) in marine sediment microbial communities. Although the source of the repSD41 origin, a 7 kb marine plasmid, appears to lack an identifiable phenotype, it has a predicted copy number of 15 per host chromosome and can be mobilized at a high frequency (i.e. 10-1) by a conjugative plasmid (Powers et al., 2000
). Taken together, such attributes are likely to contribute to its maintenance and dissemination in the microbial community. Interestingly, the repGA70 origin, derived from an 8 kb cryptic plasmid, hybridized to 50 kb plasmid(s) present in two phylogenetically distinct members of the
-Proteobacteria. These findings suggest this cryptic BHR marine plasmid is important in capturing and disseminating beneficial genes throughout the marine bacterial community. The 50 kb plasmid sequence is presently being determined. On the basis of hybridization experiments that show homology between the repSD41 and repGA70 replicons it is likely that both the GA70 replicon and the related replicons from the two 50 kb plasmids will belong to the theta A group FIA replicon family. The repSD172, obtained from a 30 kb plasmid present in a Californian marine Vibrio sp. (Sobecky et al., 1998
) was detectable only in community DNA from a coastal Californian site, albeit in a different location than that from which the host was isolated. The long-term persistence of a replicon within a bacterial population has also been reported for bacteria within a sugar beet phytosphere (Viegas et al., 1997
). These findings would suggest that the traits encoded on such plasmids are advantageous for hosts occurring in either localized niches or specific environmental conditions that vary temporally or spatially (Eberhard, 1990
).
The detection of a significantly (1·6- to 3-fold) higher plasmid incidence in two industrialized coastal sites relative to a marine ecosystem with limited human activity supports previous findings that plasmid frequency increases in response to environmental disturbances (Baya et al., 1986 ; Burton et al., 1982
; Glassman & McNicol, 1981
; Hada & Sizemore, 1981
; Reyes et al., 1999
). A bimodal distribution of plasmid sizes was observed for each of the three marine sites, presumably reflecting the overall strategies of the plasmid populations, with the smaller size peak representing mobilizable plasmids and the larger size range peak representing self-transmissible conjugative plasmids (Eberhard, 1990
). While culturable marine bacterial populations often do not adequately reflect the diversity of bacteria in marine microbial communities, previous phylogenetic analyses of surface marine sediments have reported that, of the major lineages of Bacteria detected, members of the
-Proteobacteria were predominant amongst amplified environmental clones (Gray & Herwig, 1996
; Urakawa et al., 1999
). Although efforts in this study focused on endogenously isolated replicons (i.e. plasmids from culturable hosts), plasmid-bearing
-proteobacterial hosts appear to be important in promoting dissemination of plasmids within marine sediment bacterial populations. Although attempts to characterize the exact nature of the traits encoded by these endogenously isolated marine plasmids have been limited to selectable traits (e.g. antibiotic and heavy metal resistances; Sobecky et al., 1997
), no obvious phenotypic markers could be detected. These findings indicate that the BHR plasmids are cryptic, at least with respect to these resistance genes. However, these plasmids are likely to encode ecologically advantageous traits or host fitness determinants that promote their dispersal and persistence in marine bacterial populations. Sequencing of a number of marine plasmids obtained from this study is currently under way to determine the nature of the marine plasmid-encoded traits.
Alternatively, it has been suggested that the promiscuous nature of BHR plasmids ensures their presence in microbial communities (Guiney, 1993 ) and that this plasmid pool, while possibly a small fraction of the total plasmid population, is important in mediating a response to changing environmental conditions. More recently, it has been suggested that some cryptic plasmids may serve as a reservoir of DNA-redesigning-associated functions (Hoflack et al., 1999
) providing bacteria with the capability to adapt to environmental fluctuations, perhaps with limited alteration to the host chromosome. It is noteworthy that, with the exceptions of the BHR origins repGA33 and repSD41 and IncP plasmids, marine plasmids were not detected either more than once at a site or at a high frequency in the culturable microbial community. In contrast, several studies have previously indicated widespread dissemination of the clinical IncP plasmid RK2 (Gotz et al., 1996
; Smalla et al., 2000
; Sobecky et al., 1997
). Results from our comparative analysis of the putative Rep proteins in this study, together with those from previously characterized plasmid replicons, indicates that both RCR- and theta-replicating plasmids are to be found in marine isolates. Specifically, some of these replicons are related, albeit distantly, to replicons from RCR group III and the theta group A (FIA) family. However, these replicons are clearly highly divergent from the well-characterized replicons from plasmids isolated primarily from clinical sources, which underlines the need to develop environmentally relevant molecular tools for studies of plasmid ecology.
A number of limitations to the approach employed in this study exist and should be noted. PCR-based detection of plasmid types from community DNA will not provide information on the nature of the bacterial host. As marine plasmid replicons were selected for their ability to replicate in E. coli hosts, many other replicon types (i.e. narrow host range) will be excluded. Expanding the range of hosts used in the capture of replication origins should, however, allow for the isolation of additional plasmid replicons. In addition, the marine replicon probes used in this study were initially obtained from culturable hosts. However, in many cases only a minor fraction of aquatic and terrestrial bacterial populations can be obtained using cultivation techniques. The use of exogenous plasmid techniques and subsequent isolation of plasmid replicons may provide a means to access plasmid populations occurring in the nonculturable bacterial populations in the microbial community. Continued efforts and techniques for plasmid typing and replicon isolation will facilitate studies to characterize the role of plasmids in microbial communities from diverse habitats.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Athanasopoulus, V., Praszkier, J. & Pittard, A. J. (1995). The replication of an IncL/M plasmid is subject to antisense control. J Bacteriol 177, 4730-4741.[Abstract]
Baumann, L., Baumann, P., Mandel, M. & Allen, R. D. (1972). Taxonomy of aerobic marine eubacteria. J Bacteriol 110, 402-409.[Medline]
Baya, A. M., Brayton, P. R., Brown, V. L., Grimes, D. J., Russek-Cohen, E. & Colwell, R. R. (1986). Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples. Appl Environ Microbiol 51, 1285-1292.[Medline]
Belliveau, B. H., Starodub, M. E. & Trevors, J. T. (1991). Occurrence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment. Can J Microbiol 37, 513-520.[Medline]
Bethesda Research Laboratories (1986). BRL pUC host: E. coli DH5TM competent cells. Bethesda Res Lab Focus 8, 9.
Billington, S. J., Jost, B. H. & Songer, J. G. (1998). The Arcanobacterium (Actinomyces) pyogenes plasmid pAP1 is a member of the pIJ101/pJV1 family of rolling circle replication plasmids. J Bacteriol 180, 3233-3236.[Abstract]
Bruand, C., Le Chatelier, E., Ehrlich, S. D. & Janniere, L. (1993). A fourth class of theta-replicating plasmids: the pAMß1 family from Gram-positive bacteria. Proc Natl Acad Sci USA 90, 11668-11672.[Abstract]
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.[Medline]
Carter, P., Bedouelle, H. & Winter, G. (1985). Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res 13, 4431-4443.[Abstract]
Chang, C. F., Hung, P. E. & Chang, Y. F. (1998). Molecular characterization of a plasmid isolated from Riemerella anatipestifer. Avian Pathol 27, 339-345.
Chaslus-Dancla, E., Pohl, P., Meurisse, M., Marin, M. & Lafont, L. P. (1991). High genetic homology between plasmids of human and animal origins conferring resistance to the aminoglycosides gentamicin and apramycin. Antimicrob Agents Chemother 35, 590-593.[Medline]
Couturier, M., Bex, F., Bergquist, P. L. & Maas, W. K. (1988). Identification and classification of bacterial plasmids. Microbiol Rev 52, 375-395.
Dahlberg, C., Linberg, C., Torsvik, V. L. & Hermansson, M. (1997). Conjugative plasmids isolated from bacteria in marine environments show various degrees of homology to each other and are not closely related to well-characterized plasmids. Appl Environ Microbiol 63, 4692-4697.[Abstract]
Datta, N. (1979). Plasmid classification: incompatibility grouping. In Plasmids of Medical, Environmental and Commercial Importance , pp. 3-12. Edited by K. Timmis & A. Puhler. Amsterdam:Elsevier/North Holland.
Davey, R. B., Bird, P. I., Nikoletti, S. M., Praszkier, J. & Pittard, J. (1984). The use of mini-Gal plasmids for rapid incompatibility grouping of conjugative R plasmids. Plasmid 11, 234-242.[Medline]
Ditta, G., Stanfield, S., Corbin, D. & Helinski, D. R. (1980). Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene of Rhizobium meliloti. Proc Natl Acad Sci USA 77, 7347-7351.[Abstract]
Dong, X. Q., Lindler, L. E. & Chu, M. C. (2000). Complete DNA sequence and analysis of an emerging cryptic plasmid isolated from Yersinia pestis. Plasmid 43, 144-148.[Medline]
Drnen, A. K., Torsvik, V., Goks
yr, J. & Top, E. M. (1998). Effect of mercury addition on plasmid incidence and gene mobilizing capacity in bulk soil. FEMS Microbiol Ecol 27, 381-394.
Drnen, A. K., Torsvik, V. & Top, E. M. (1999). Comparison of the plasmid types obtained by two distantly related recipients in biparental exogenous plasmid isolations from soil. FEMS Microbiol Lett 176, 105-110.
Eberhard, W. G. (1990). Evolution in bacteria and levels of selection. Q Rev Biol 65, 3-22.[Medline]
van Elsas, J. D., McSpadden Gardener, B. B., Wolters, A. C. & Smit, E. (1998). Isolation, characterization, and transfer of cryptic gene-mobilizing plasmids in the wheat rhizosphere. Appl Environ Microbiol 67, 880-889.
Glassman, D. L. & McNicol, L. A. (1981). Plasmid frequency in natural populations of estuarine microorganisms. Plasmid 5, 231.
Gotz, A., Pukall, R., Smit, E., Tietze, E., Prager, R., Tschape, H., van Elsas, J. D. & Smalla, K. (1996). Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl Environ Microbiol 62, 2621-2628.[Abstract]
Gray, J. P. & Herwig, R. P. (1996). Phylogenetic analysis of the bacterial communities in marine sediments. Appl Environ Microbiol 62, 4049-4059.[Abstract]
Guiney, D. G. (1993). Broad host range conjugative and mobilizable plasmids in Gram-negative bacteria. In Bacterial Conjugation , pp. 75-103. Edited by D. Clewell. New York:Plenum.
Hada, H. S. & Sizemore, R. K. (1981). Incidence of plasmids in marine Vibrio spp. isolated from an oil field in the northwestern Gulf of Mexico. Appl Environ Microbiol 41, 199-202.
Hermansson, M., Jones, G. W. & Kjelleberg, S. (1987). Frequency of antibiotic and heavy metal resistance, pigmentation and plasmids in bacteria of the marine air-water interface. Appl Environ Microbiol 53, 2338-2342.[Medline]
Hoffmann, B., Strauch, E., Gewinner, C., Nattermann, H. & Appel, B. (1998). Characterization of plasmid regions of foodborne Yersinia enterocolitica biogroup 1A strains hybridizing to the Yersinia enterocolitica virulence plasmid. Syst Appl Microbiol 21, 201-211.[Medline]
Hoflack, L., Wilcks, A., Andrup, L. & Mahillon, J. (1999). Functional insights into pGI2, a cryptic rolling-circle replicating plasmid from Bacillus thuringiensis. Microbiology 145, 1519-1530.[Abstract]
Ilyina, T. V. & Koonin, E. V. (1992). Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res 20, 3279-3285.[Abstract]
Kado, C. I. (1998). Origin and evolution of plasmids. Antonie Leeuwenhoek 73, 117-126.
Kahn, M., Ow, D., Sauer, R., Rabinowitz, A. & Calendar, R. (1980). Genetic analysis of bacteriophage P4 using P4-plasmid ColE1 hybrids. Mol Gen Genet 177, 399-412.[Medline]
Kantor, A., Montville, T. J., Mett, A. & Shapira, R. (1997). Molecular characterization of the replicon of the Pediococcus pentosaceus 43200 pediocin A plasmid pMD136. FEMS Microbiol Lett 151, 237-244.[Medline]
Kendall, K. J. & Cohen, S. N. (1988). Complete nucleotide sequence of the Streptomyces lividans plasmid pIJ101 and correlation of the sequence with genetic properties. J Bacteriol 170, 4634-4651.[Medline]
Kobayashi, N. & Bailey, M. J. (1994). Plasmids isolated from the sugar beet phyllosphere show little or no homology to molecular probes currently available for plasmid typing. Microbiology 140, 289-296.[Abstract]
Kobori, H., Sullivan, C. W. & Shizuya, H. (1984). Bacterial plasmids in Antarctic natural assemblages. Appl Environ Microbiol 48, 515-518.
Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace, N. R. (1985). Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 82, 6955-6959.[Abstract]
Lilley, A. K., Bailey, M. J., Day, M. J. & Fry, J. C. (1996). Diversity of mercury resistance plasmids obtained by exogenous isolation from the bacteria of sugar beet in three successive years. FEMS Microbiol Ecol 20, 211-227.
Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Novick, R. P. (1987). Plasmid incompatibility. Microbiol Rev 51, 381-395.
Novick, R. P. (1989). Staphylococcal plasmids and their replication. Annu Rev Microbiol 43, 537-565.[Medline]
Ogunseitan, O. A., Tedford, E. T., Pacia, D., Sirotkin, K. M. & Sayler, G. S. (1987). Distribution of plasmids in groundwater bacteria. J Ind Microbiol 1, 311-317.
Osborn, M., Bron, S., Firth, N. & 11 other authors (2000a). The evolution of bacterial plasmids. In The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread, pp. 301361. Edited by C. M. Thomas. Amsterdam: Harwood Academic.
Osborn, A. M., Pickup, R. W. & Saunders, J. R. (2000b). Development and application of molecular tools in the study of IncN-related plasmids from lakewater sediments. FEMS Microbiol Lett 186, 203-208.[Medline]
Pansegrau, W., Lanka, E., Barth, P. T. & 7 other authors (1994). Complete nucleotide sequence of Birmingham IncP plasmids. Compilation and comparative analysis. J Mol Biol 239, 623663.[Medline]
Pickup, R. W. (1989). Related plasmids found in an English Lake District stream. Microb Ecol 18, 211-220.
Powers, L. A., Mallonee, J. & Sobecky, P. A. (2000). Nucleotide sequence and characterization of a cryptic plasmid from a marine Vibrio sp. Plasmid 43, 99-102.[Medline]
Prentki, P., Karch, F., Iida, S. & Meyer, J. (1981). The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication. Gene 14, 289-299.[Medline]
Reyes, N., Frischer, M. E. & Sobecky, P. A. (1999). Characterization of mercury resistance mechanisms in marine sediment microbial communities. FEMS Microbiol Ecol 30, 273-284.[Medline]
Roberts, R. C., Burioni, R. & Helinski, D. R. (1990). Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2. J Bacteriol 172, 6204-6216.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.[Abstract]
Seery, L. T., Nolan, N. C., Sharp, P. M. & Devine, K. M. (1993). Comparative analysis of the pC194 group of rolling circle plasmids. Plasmid 30, 185-196.[Medline]
Sizemore, R. K. & Colwell, R. R. (1977). Plasmids carried by antibiotic-resistant marine bacteria. Antimicrob Agents Chemother 12, 373-382.[Medline]
Smalla, K., Krogerrecklenfort, E., Heuer, H. & 21 other authors (2000). PCR-based detection of mobile genetic elements in total community DNA. Microbiology 146, 12561257.
Smit, E., Wolters, A. & van Elsas, J. D. (1998). Self-transmissible mercury resistance plasmids with gene-mobilizing capacity in soil bacterial populations: influence of wheat roots and mercury addition. Appl Environ Microbiol 64, 1210-1219.
Sobecky, P. A., Mincer, T. J., Chang, M. C. & Helinski, D. R. (1997). Plasmids isolated from marine sediment microbial communities contain replication and incompatibility regions unrelated to those of known plasmid groups. Appl Environ Microbiol 63, 888-895.[Abstract]
Sobecky, P. A., Mincer, T. J., Chang, M. C., Toukdarian, A. & Helinski, D. R. (1998). Isolation of broad-host-range replicons from marine sediment bacteria. Appl Environ Microbiol 64, 2822-2830.
del Solar, G., Giraldo, R., Ruiz-Echevarria, M. J., Espinosa, M. & Diaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev 62, 434-464.
Stalker, D. M., Kolter, R. & Helinski, D. R. (1979). Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K. Proc Natl Acad Sci USA 76, 1150-1154.[Abstract]
Stalker, D. M., Kolter, R. & Helinski, D. R. (1982). Plasmid R6K DNA replication. I. Complete nucleotide sequence of an autonomously replicating segment. J Mol Biol 161, 33-43.[Medline]
Szpirer, C., Top, E., Courturier, M. & Mergeay, M. (1999). Retrotransfer or gene capture: a feature of conjugative plasmids, with ecological and evolutionary significance. Microbiology 145, 3321-3329.
Tebbe, C. C. & Vahjen, W. (1993). Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast. Appl Environ Microbiol 59, 2657-2665.[Abstract]
Thomas, C. M., Meyer, R. & Helinski, D. R. (1980). Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance. J Bacteriol 141, 213-222.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]
Tsai, Y.-L. & Olsen, B. H. (1991). Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol 57, 1070-1074.[Medline]
Urakawa, H., Kita-Tsukamoto, K. & Ohwada, K. (1999). Microbial diversity in marine sediments from Sagami Bay and Tokyo Bay, Japan, as determined by 16S rRNA gene analysis. Microbiology 145, 3305-3315.
Viegas, C. A., Lilley, A. K., Bruce, K. & Bailey, M. J. (1997). Description of a novel plasmid replication origin from a genetically distinct family of conjugative plasmids associated with phytosphere microflora. FEMS Microbiol Lett 149, 121-127.[Medline]
Vieria, J. & Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268.[Medline]
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697-703.[Medline]
Weng, S., Lin, W., Chang, Y. & Chang, C. (1999). Identification of a virulence-associated protein homolog gene and ISRa1 in a plasmid of Riemerella anatipestifer. FEMS Microbiol Lett 179, 11-19.[Medline]
Received 5 January 2001;
revised 22 March 2001;
accepted 27 April 2001.