Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities

Marisa A. Cook1, A. Mark Osborn2, Juli Bettandorff1 and Patricia A. Sobecky1

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Six functional replication origins (repGA14, repGA33, repGA70, repSD41, repSD164 and repSD172), obtained from endogenously isolated, broad-host-range (BHR) marine plasmids ranging in size from 5 to 60 kb, were used to determine plasmid occurrence in three coastal marine sediment sites (in California, Georgia and South Carolina, USA). The plasmid-specific replicons were isolated from plasmid-bearing marine sediment bacteria belonging to the {alpha} and {gamma} subclasses of the Proteobacteria. The plasmid sources of the endogenous replicons were considered to be cryptic due to a lack of identifiable phenotypic traits. The putative Rep proteins from a number of these replicons showed similarity to replicons of two recognized families: RCR group III (repSD164) and the FIA family of theta group A (repSD41, repSD121, repGA33 and repGA14). Plasmids isolated from marine bacteria belonging to the genera Pseudoalteromonas, Shewanella and Vibrio cultivated from geographically different coastal sites exhibited homology to two of the marine plasmid replicons, repSD41 and repGA70, obtained from a Vibrio sp. The repGA33 plasmid origin, obtained from a Shewanella sp. isolated from coastal Georgia, was detected in 7% of the Georgia marine sediment Shewanella sp. isolates. Microbial community DNA extracted from marine sediments was also screened for the presence of the plasmid replication sequences. Community DNA samples amplified by PCR yielded a positive signal for the repSD172 and repGA14 replication sequences. The replication origin of BHR plasmid RK2 (IncP) was also detected in marine Vibrio sp. and microbial community DNA extracted from the three coastal sites. These findings provide molecular evidence that marine sediment bacteria harbour an untapped population of BHR plasmids.

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
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INTRODUCTION
METHODS
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DISCUSSION
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Plasmids are mobile genetic elements that can range in size from several hundred base pairs to several thousand kilobases (Kado, 1998 ). While small plasmids (i.e. <1kb) lack ORFs that code for proteins, they do contain an essential region, the origin of replication (oriV), which contains the genes and loci involved in replication and its control. The basic replicon of a plasmid consists of (i) a cis-acting region which supports autonomous replication, (ii) cop and inc gene(s) involved in the control of replication initiation, and often (iii) rep gene(s) encoding a protein involved in replication control (Couturier et al., 1988 ; del Solar et al., 1998 ). Plasmids containing similar or related replication systems are considered incompatible if they cannot coexist in a host cell (Datta, 1979 ; Novick, 1987 ). The typical clustering of plasmid replication genes has facilitated isolation and characterization of incompatibility and replication (inc/rep) loci suitable for use as DNA probes to type plasmids isolated from bacteria of clinical and animal origin (Chaslus-Dancla et al., 1991 ; Couturier et al., 1988 ; Davey et al., 1984 ). However, these inc/rep replicon probes, derived from plasmids found predominantly in members of the Enterobacteriaceae, are not suitable for classifying plasmids from aquatic and terrestrial environments (Dahlberg et al., 1997 ; Kobayashi & Bailey, 1994 ; Osborn et al., 2000b ; Sobecky et al., 1997 ; van Elsas et al., 1998 ).

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 (Dr{oslash}nen 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 {alpha} and {gamma} subclasses of the Proteobacteria. Plasmid-containing marine bacteria and microbial community DNA obtained from marine sediments were subjected to DNA–DNA 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
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INTRODUCTION
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DISCUSSION
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Marine sediment sampling and bacterial isolation.
In this study, three coastal locales in the USA were investigated. Charleston Harbor, South Carolina, and San Diego Bay, California, represent sites with industrial activity, and Sapelo Island, Georgia, a barrier island located along the southeastern coast of the USA, represents a habitat with minimal human disturbances and little to no industrial activity. Surface sediment samples (0–12 cm) were collected with a Shipek grab sampler in the Cooper River branch of Charleston Harbor during August 1998 and December 1998. Sediments were aseptically removed, placed in sterile tubes and processed immediatelBy. Sediment samples (0–15 cm) were collected with a Van Veen grab sampler in the Paleta Creek and Shelter Island boat basin located in San Diego Bay during January 1999 and November 1999. Sediment samples were kept at 4 °C until processing (36–48 h). Sediment samples were collected from a large tidal creek (Factory Creek) on the western side of Sapelo Island during January 1998 and January 1999. Sediment samples were collected at low tides using a hand coring device (5–10 cm in length) and processed immediately. At the time of sample collection, water temperatures ranged from 15 °C (winter months) to 28 °C (summer months) and salinity ranged from 17 to 30{per thousand}.

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 1–14 d at 30 °C. With the exception of the 2216E medium, artificial seawater was used in all the media and 1·7–2·0% 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 519–536; primer 2, positions 907–926; and primer 3, positions 1392–1406 (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 228–1295 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 (Jukes–Cantor 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{alpha} 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 ).


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Table 1. Bacterial strains and plasmids used in this study

 
Southern hybridizations.
DNA fragments containing the inc/rep regions of clinical plasmids were digested as previously described (Sobecky et al., 1997 ). Plasmids pTM41, pTM164, pTM172, pBJ014 and pBJ03 were digested with PstI, and pUBL2420 was digested with HaeII (Table 1). DNA fragments containing the marine plasmid replication region were excised from the gel and eluted with a QIAquick gel extraction kit (Qiagen). The isolated DNA fragments to be used as probes were labelled by random priming with either [{alpha}-32P]dATP [6000 Ci mmol-1 (220 TBq mmol-1); NEN Dupont] or with digoxigenin-11-dUTP and the Boehringer Mannheim random-primed DNA labelling system according to the manufacturers’ instructions. Following electrophoresis of plasmid DNA on 0·7% LE agarose (FMC), gels were denatured, neutralized and blotted onto nylon membranes (Schleicher & Schuell) according to the manufacturer’s recommendations except that blotting was routinely carried out overnight to ensure complete DNA transfer. Membranes were then rinsed in 2xSSPE (1xSSPE is 0·18 M NaCl, 10 mM NaH2PO4 pH 7·7, 1 mM EDTA), UV-cross-linked and stored until hybridization. The membranes were then washed in prewarmed (42 °C) 2xSSPE, placed in hybridization bottles (Hybaid Instruments) and pre-hybridized in 30 ml hybridization solution consisting of 50% (v/v) deionized formamide, 6xSSPE, 5xDenhardt’s solution, 1% SDS and 100 µg salmon sperm DNA ml-1 for 4–8 h at 37–42 °C and 7 r.p.m. Radiolabelled probes were added at approximately 2x106 c.p.m. ml-1 and incubated for 16 h at 37–42 °C and 7 r.p.m. Unbound probe was removed by washing membranes twice in 2xSSPE/0·1% SDS for 15 min at 65 °C, and twice in 1xSSPE/0·1% SDS for 30 min at 65 °C in a Hybaid oven at 10 r.p.m. The final wash step was in 0·1xSSPE at 65 °C. Membranes were then exposed to BioMax X-ray film (Kodak) at -70 °C with an intensifying screen. Hybridizations conducted with DIG-labelled DNA fragments were done as previously described (Reyes et al., 1999 ). The stringency of the hybridization conditions used in all assays detected greater than 75% sequence homology. When necessary to reprobe membranes with a different environmental plasmid replication probe, bound labelled probe was removed according to the manufacturer’s recommendations (Schleicher & Schuell).

DNA extraction and purification from marine sediments.
Total DNA (chromosomal and plasmid DNA) was extracted from sediment samples (1–5 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 5–10 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.


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Table 2. Sequences of PCR primers used for replicon amplification in this study

 
Nucleotide sequence accession numbers.
The nucleotide sequences of plasmid replication origins repGA14 and repGA33 have been deposited in the GenBank database under accession numbers AF250848 and AF250849, respectively. The 16S rRNA gene sequences obtained in this study have been deposited in the GenBank database under accession numbers AF249334AF249338 and AF284226AF284230.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmid incidence in marine sediment microbial communities from different locations
The endogenous plasmid isolation method, which requires culturability of the host, was used to determine plasmid incidence in the heterotrophic bacterial populations from three different coastal environments. The percentage of culturable hosts in which plasmids were detected varied between sites and in some cases between sampling times (Fig. 1).



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Fig. 1. Percentage of plasmid-containing marine sediment bacterial isolates from three coastal marine environments. The total number of isolates screened for the presence of plasmids is indicated above each bar.

 
There were significant differences in plasmid incidence between sites as bacterial isolates obtained from Charleston Harbor and San Diego Bay sediments were more likely to contain plasmids than were isolates obtained from Sapelo Island sediments (G-squared values of 24·94 and 53·05, respectively, and P values of <0·0001). Within sites, there was a significantly higher incidence of plasmids occurring in the culturable microbial community from San Diego Bay during November 1999 (Fig. 1; G-squared value of 41·73 and P value of <0·0001) relative to isolates screened in January 1999. Similarly, a significantly higher plasmid incidence was detected in isolates from Charleston Harbor sediments obtained during December 1998 relative to isolates screened in August 1998 (Fig. 1; G-squared value of 6·26 and P value of <0·05). In contrast, there was no significant difference in plasmid incidence in isolates cultivated from Sapelo Island sediments from the two different sample dates (Fig. 1).

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 35–100 kb size ranges (Fig. 2). A significant increase in Charleston Harbor and San Diego Bay bacterial isolates containing plasmids in the 35–100 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).



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Fig. 2. Distribution of plasmid sizes for plasmid-containing marine bacteria. Plasmid size for plasmid-containing isolates from (a) Charleston Harbor, (b) San Diego Bay and (c) Sapelo Island was estimated by comparison with known plasmid size standards.

 
Specificity of marine plasmid replicon probes
Replication-proficient loci (replicons) of endogenously isolated plasmids have been obtained from Gram-negative marine heterotrophs belonging to the {alpha} and {gamma}-3 subclasses of Proteobacteria cultivated previously from coastal California (repSD41, repSD121, repSD164 and repSD172; Sobecky et al., 1998 ) and Georgia (repGA14, repGA33 and repGA70; this study). These marine plasmid rep origins are considered to be BHR in nature due to their propagation in E. coli and distantly related marine bacteria (Sobecky et al., 1998 ). The replicons obtained from the marine plasmids were initially screened for homology to the 14 broad- and narrow-host-range inc/rep probes presently available (Couturier et al., 1988 ). No homology or cross-reactivity to these clinical inc/rep probes was observed (Table 3). Cross-reactivity between the marine plasmid replicons was observed between repSD41 and repSD121 as previously reported (Sobecky et al., 1998 ) and between repSD41 and repGA70, a replicon isolated from Vibrio sp. 70 obtained during this study (Table 3). Thus, the collection of replicons represents five BHR marine plasmid types with several members of one group (repGA70, repSD41 and repSD121) represented by the small (7–8 kb) cryptic plasmids occurring in marine Vibrio sp. (Table 3). It is also of importance to note that there was a lack of hybridization between the marine replicon probes and chromosomal DNA, indicating that the plasmids were not integrated into their host chromosome (data not shown).


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Table 3. Replication-proficient (replicon) fragments derived from plasmids isolated from marine bacteria used as probes in this study

 
Plasmid typing of marine sediment microbial communities
The collection of marine plasmid replicons was used to determine the prevalence and distribution of the plasmid types in marine sediment bacterial communities from three different geographical locations (Table 4). Southern blot analyses of endogenous plasmids (Fig. 1) and microbial community DNA extracted from marine sediments subjected to PCR amplification with the replicon-specific primers (Table 2) was conducted. RepSD121 was omitted from these analyses due to its extensive sequence homology to repSD41 (Sobecky et al., 1998 ).


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Table 4. Occurrence of endogenous marine plasmids and PCR products from sediment microbial community DNA exhibiting homology to plasmid replicons by Southern hybridization

 
Screening of the endogenous plasmid populations with the marine replicon probes indicated a limited occurrence of the marine plasmid types in culturable hosts (Table 4). For example, a marine Shewanella sp. isolated from Sapelo Island sediment contained a plasmid that exhibited homology to repSD41 and repGA70 (Table 4; Fig. 3). The plasmid was approximately 5 kb (data not shown). Screening with the repGA70 DNA probe revealed homology to a 50 kb plasmid present in only two different culturable isolates (Vibrio sp. strain 01 and Pseudoalteromonas sp. strain 05; Fig. 3) obtained from San Diego Bay sediments collected in November 1999 (Table 4). Both isolates also contained an additional plasmid of approximately 40 kb (data not shown). Although repSD41 exhibits cross-reactivity to repGA70 (Table 3), repSD41 did not hybridize to either of these large plasmids, indicating that either the plasmid source of the repGA70 replicon probe contains a second and functional replication origin or that repSD41 is more distantly related to these large plasmids. The repGA33 DNA probe, derived from a 5 kb plasmid isolated from a Shewanella sp. (Table 3) hybridized to a 5 kb plasmid present in 7% of the plasmid-containing isolates cultivated from Sapelo Island in January 1998 (data not shown). Cellular fatty acid methyl ester analyses indicated that the isolates belonged to the genus Shewanella (data not shown). Lastly, three plasmid-containing Vibrio sp. strains isolated from San Diego Bay in November 1999 (Table 4) exhibited homology to the 750 bp IncP replicon probe (Fig. 4a, b). Lowering the stringency of hybridization (i.e. <75% homology) did not reveal any additional endogenous plasmids with homology to any of the other replicon probes (data not shown).



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Fig. 3. Phylogenetic relationship among marine bacterial isolates containing plasmids with homology to replicon probes repSD41 and repGA70. The tree is unrooted and was constructed with 1000 bases of aligned 16S rRNA gene sequences using Bacillus subtilis as the outgroup. The bootstrap values (100 replicates) are indicated at the nodes. The bar indicates Jukes–Cantor distances. Isolates denoted in bold are the host sources of the plasmid replicons: Vibrio sp. 41, source of repSD41; Vibrio sp. 70, source of repGA70. An asterisk indicates marine isolates containing a plasmid exhibiting homology to repGA70. Those marine isolates containing a plasmid exhibiting homology to repGA70 and repSD41 are underlined.

 


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Fig. 4. Agarose gel electrophoresis and corresponding Southern blot analysis of plasmid-containing strains of E. coli and marine Vibrio sp. using the IncP replicon probe. (a) Ethidium-bromide-stained 0·7% agarose gel of plasmid DNA isolated by the modified Keiser method (Sobecky et al., 1997 ). Lanes: 1, Vibrio sp. strain 5; 2, Vibrio sp. strain 259; 3, Vibrio sp. strain 373; 4, positive control, E. coli HB101 containing the 60 kb IncP plasmid RK2. All three plasmid-containing Vibrio sp. were isolated from San Diego Bay marine sediments. The expected position of the chromosomal DNA band (Chr) is indicated. The expected position of the plasmid RK2 is indicated by the arrow. (b) Corresponding Southern blot analysis using the 750 bp IncP probe. Hybridization signals were obtained from plasmid DNA isolated from Vibrio sp. strain 5 (lane 1), Vibrio sp. strain 259 (lane 2), Vibrio sp. strain 373 (lane 3), and E. coli HB101 containing RK2 (lane 4). The strongest signals observed for the plasmid DNA corresponded to positions of plasmids visible by gel electrophoresis (a, lanes 2–4). The additional signals in lanes 3 and 4 above the asterisk may be due to plasmid DNA with a different superhelical density or a different (low-copy-number) plasmid not readily visible by gel electrophoresis that also exhibits homology to the IncP probe. The weaker signal indicated by the arrow in lane 1 may be due to a nicked form of the plasmid, plasmid integration into the host chromosome or a different plasmid with less homology to IncP not readily visible by the assay conditions used in this study.

 
Screening microbial community DNA extracted from marine sediments indicated a similar limited occurrence of marine plasmid types as observed with the endogenous plasmid populations (Table 4). For example, the repSD172 DNA probe derived from a 30 kb plasmid (Table 3) was obtained from the marine Vibrio sp. strain 172 previously cultivated from a different coastal California site (Mission Bay; Sobecky et al., 1997 ). A positive signal from microbial community DNA extracted from San Diego Bay sediments collected in January 1999 was obtained (Table 4). The PCR product obtained with the repSD172 primers was confirmed by Southern hybridization (data not shown). The repGA14 DNA probe was initially obtained from an approximately 60 kb plasmid present in the culturable Vibrio sp. strain 14 isolated from Georgia sediments (Table 3). PCR products obtained with repGA14-specific primers were detected in community DNA extracted from San Diego Bay in November 1999 (Table 4). The PCR product obtained with the repGA14 primers was also confirmed by Southern hybridization (data not shown). Interestingly, PCR products obtained with IncP-specific primers were detected in microbial community DNA extracted from Sapelo Island in January 1998 and January 1999 (Table 4). In addition, PCR products amplified with IncP primers were also detected in community DNA extracted from Charleston Harbor in December 1998 (Table 4). Southern hybridization of the PCR products with a DNA probe internal to the IncP replicon confirmed that these amplicons were related to the IncP{alpha} replicon (data not shown). In contrast, no PCR products were obtained for repGA33, repSD41 or repSD164 from microbial community DNA extracted from the marine sediments (Table 4). Since the sensitivity of detection of target sequences amplified from plasmid replication regions with the PCR conditions used in this study has been previously determined to be approximately 10–100 cells (g wet wt sediment)-1 (Sobecky et al., 1997 ), these replicons are either present below our detection limits or are not found in these coastal environments.

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).


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Table 5. Similarities between putative marine plasmid Rep proteins and previously characterized Rep proteins

 


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Fig. 5. Alignment of the repSD164 putative Rep protein from pTM164 with Rep proteins belonging to the RCR group III replicon family with common motifs 1 and 2 (as described by Ilyina & Koonin, 1992 ) and the refined motif 3 containing the putative active site (as described by Seery et al., 1993 ). Amino acids shown in bold are those conserved across most RCR group III Rep proteins, where u indicates a bulky hydrophobic residue (F, I, L, M, V) and underlining represents almost complete conservation of residues across this group.

 
Replicons repSD41, repSD121, repGA33 and repGA14 have putative ORFs that show similarity to Rep proteins from the FIA family of theta group A replicons (Table 5; Bruand et al., 1993 ; Osborn et al., 2000a ). Putative Rep proteins from repSD41 and repSD121 show greatest similarity to each other (Table 5) as well as to Rep proteins from R6K in E. coli, pCFC1 in Riemerella anatipestifer and pMD136 from Pediococcus pentosaceus (Table 5). Translation of the GA33 DNA sequence identified one ORF (ORF1) that shows highest similarity to the Rep proteins of pL6.5 (TOL plasmid) from Pseudomonas fluorescens and pYC from Yersinia pestis (Table 5). ORF1 shows only limited similarity over relatively short regions to the putative Rep proteins from repSD41 and repSD121 (data not shown). Additionally, the GA33 ORF would appear to be truncated when compared against other Rep proteins from the theta group A FIA family, suggesting that this is one of the more divergent members of this replicon family.

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmid-encoded genes represent a considerable pool of mobile DNA that can contribute to the genetic adaptation of microbial communities. Advantageous traits such as heavy metal resistance genes have been reported to be encoded on marine plasmids obtained by exogenous isolation methods (Dahlberg et al., 1997 ). However, many plasmids obtained by endogenous isolation techniques occurring in marine sediment bacterial populations are considered to be cryptic due to a lack of readily identifiable phenotypic traits (Sobecky et al., 1997 ). The use of molecular tools such as DNA probes specific for plasmid backbone genes (i.e. replicon probes) provides an opportunity to gain insights into the contribution of mobilizable and conjugative cryptic plasmids and their genes in structuring microbial communities in natural habitats.

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{oslash}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 {gamma}-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 {gamma}-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 {gamma}-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 {gamma}-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
 
This work was supported by Office of Naval Research grant N00014-98-1-0078 and NOAA Office of Sea grant RR100-289/2000607. We thank Drs M. Montgomery and S. Apitz for providing sampling assistance and A. Toukdarian for helpful suggestions. The collaboration between P.A.S. and A.M.O. benefited from the support of the EU concerted action BIO4-CT-0099 on ‘Mobile Genetic Elements’ Contribution to Bacterial Adaptability and Diversity (MECBAD)’.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 5 January 2001; revised 22 March 2001; accepted 27 April 2001.