The phylogeny of marine and freshwater caulobacters reflects their habitat.

Caulobacter is a distinctive genus of prosthecate bacteria. Because caulobacters adhere to surfaces and are found in diverse locales, their role in oligotrophic environments and bacterial biofilm communities is of interest. The phylogenetic relationships of a group of marine and freshwater caulobacters were examined in part to address whether the taxonomic grouping of these bacteria (based primarily on morphological characters) was consistent with 16S rRNA sequence divergence. The caulobacters examined (9 marine and 11 freshwater species or strains) were affiliated with the alpha proteobacteria. They made up a diverse yet, with the possible exception of a strain of Caulobacter subvibrioides, coherent assemblage. The diversity was most apparent in a comparison of freshwater and marine isolates; an early divergence within the main caulobacter lineage generally corresponded to strains isolated from freshwater and marine habitats. The marine caulobacter assemblage was not exclusive; it also embraced strains of marine hyphomonads and Rhodobacter capsulatus. We hypothesize that these genera are derived from more ancestral caulobacters. Overall, the data are consistent with the interpretation that all of the caulobacters examined, with the possible exception of C. subvibrioides, are ancestrally related, albeit anciently, and that most often division by terrestrial and marine habitats corresponds to an early evolutionary divergence within the genus.     

Characterization of the Adhesive Holdfast of Marine and Freshwater Caulobacters

Caulobacters are prosthecate (stalked) bacteria that elaborate an attachment organelle called a holdfast at the tip of the cellular stalk. We examined the binding of lectins to the holdfasts of 16 marine Caulobacter strains and 10 freshwater species or strains by using a panel of fluorescein-conjugated lectins and fluorescence microscopy. The holdfasts of all the marine isolates bound to only wheat germ agglutinin (WGA) and other lectins that bind N-acetylglucosamine (GlcNac) residues. The freshwater caulobacters showed more variability in holdfast composition. Some bound only to WGA and comparable lectins as the marine strains did. Others bound additional or other lectins, and some did not bind to the lectins tested. The binding of WGA appeared to involve the regions of the holdfast involved with adhesion; a holdfast bound to WGA was significantly less adhesive to glass. Competition experiments with WGA-binding holdfasts and oligomers of GlcNac demonstrated that trimers of GlcNac (the preferred substrate for WGA binding) were more effective than dimers or monomers in preventing WGA binding to holdfasts, suggesting that stretches of contiguous GlcNac residues occur in the WGA-binding holdfasts. In addition, differences between freshwater and marine holdfasts in the strength of WGA binding were noted. The effect of a number of proteolytic and glycolytic enzymes on holdfast integrity was examined; the proteases had no effect for all caulobacters. None of the glycolytic enzymes had an effect on marine caulobacter holdfasts, but chitinase and lysozyme (both attack oligomers of GlcNac) disrupted the holdfasts of those freshwater caulobacters that bound WGA. Despite some similarity to chitin, holdfasts did not bind Calcofluor and no measurable effects on holdfast production were detectable after cell growth in the presence of diflubenzuron or polyoxin D, inhibitors of chitin synthesis in other systems. Finally, the holdfasts of all caulobacters bound to colloidal gold particles, without regard to the coating used to stabilize the gold particles. This binding was stronger or more specific than WGA binding; treatment with colloidal gold particles prevented WGA binding, but the reverse was not the case.     

Transformation of freshwater and marine caulobacters by electroporation.

We performed plasmid electrotransformation of Caulobacter crescentus strains and obtained up to 3 x 10(8) transformants per micrograms of pKT230. The presence and integrity of the paracrystalline protein surface (S) layer influenced electroporation; caulobacters lacking the S layer were electrotransformed 10 times more efficiently than caulobacters possessing the S layers. A procedure yielding 1,500 transformants per micrograms of pKT230 was developed for a marine caulobacter. Electroporation was used in combination with several genetic techniques, including introduction of ligation mixtures, suicide transposon mutagenesis, gene replacement, and plasmid electrotransfer from Escherichia coli to caulobacters.     

Bacterial Plasmids in Antarctic Natural Microbial Assemblages

Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica.     

Plasmids isolated from marine sediment microbial communities contain replication and incompatibility regions unrelated to those of known plasmid groups.

Two hundred ninety-seven bacteria carrying plasmids that range in size from 5 to 250 kb were identified from more than 1,000 aerobic heterotrophic bacteria isolated from coastal California marine sediments. While some isolates contained numerous (three to five) small (5- to 10-kb) plasmids, the majority of the natural isolates typically contained one large (40- to 100-kb) plasmid. By the method of plasmid isolation used in this study, the frequency of plasmid incidence ranged from 24 to 28% depending on the samples examined. Diversity of the plasmids occurring in the marine sediment bacterial populations was examined at the molecular level by hybridization with 14 different DNA probes specific for the incompatibility and replication (inc/rep) regions of a number of well-characterized plasmid incompatibility groups (repB/O, FIA, FII, FIB, HI1, HI2, I1, L/M, X, N, P, Q, W, and U). Interestingly, we found no DNA homology between the plasmids isolated from the culturable bacterial population of marine sediments and the replicon probes specific for numerous incompatibility groups developed by Couturier et al. (M. F. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Our findings suggest that plasmids in marine sediment microbial communities contain novel, as-yet-uncharacterized, incompatibility and replication regions and that the present replicon typing system, based primarily on plasmids derived from clinical isolates, may not be representative of the plasmid diversity occurring in some marine environments. Since the vast majority of marine bacteria are not culturable under laboratory conditions, we also screened microbial community DNA for the presence of broad- and narrow-host-range plasmid replication sequences. Although the replication origin of the conjugally promiscuous broad-host-range plasmid RK2 (incP) was not detectable in any of the plasmid-containing culturable marine isolates, DNA extracted from the microbial community and amplified by PCR yielded a positive signal for RK2 oriV replication sequences. The strength of the signal suggests the presence of a low level of the incP replicon within the marine microbial community. In contrast, replication sequences specific for the narrow-host-range plasmid F were not detectable in DNA extracted from marine sediment microbial communities. With the possible exception of mercuric chloride, phenotypic analysis of the 297 plasmid-bearing isolates did not demonstrate a correlation between plasmid content and antibiotic or heavy metal resistance traits.     

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Frequency of antibiotic and heavy metal resistance, pigmentation, and plasmids in bacteria of the marine air-water interface

A field investigation of marine coastal waters revealed that the frequency of pigmented bacteria and the occurrence of bacterial antibiotic resistance were higher at the air-water interface than in the bulk water. The differences in the frequency of pigmented colonies at the surface and in the bulk-water samples could not be explained by the degree of cell surface hydrophobicity or by bacterial adhesion to air-water interfaces. Pigmented strains exhibited a higher degree of multiple drug resistance than did nonpigmented strains. However, the frequency of multiple drug resistance in nonpigmented strains was also substantial. An average of 91% of all strains were resistant to more than one antibiotic, and 21% of the bacteria isolated were resistant to five of the eight antibiotics tested. High numbers of plasmid-carrying strains were found among selected surface isolates, but the presence of detectable plasmids could not be correlated with either pigmentation or multiple drug resistance. Furthermore, selected surface isolates were significantly more resistant to mercury than were bulk-water bacteria. The higher frequency of pigmented, antibiotic-resistant, and mercury resistant strains at the air-water interface than in the bulk water are discussed in terms of various forms of selective pressure and genetic exchange at the surface.     

Mercury and methylmercury detoxification potential by sponge-associated bacteria

Ionic and organic forms of mercury (Hg) are powerful cytotoxic and neurotoxic agents in both humans and wild life. The aim of this study was to analyze the resistance profile and potential detoxification of inorganic and organic forms of Hg of bacteria isolated from marine sponges on the coast of Rio de Janeiro, Brazil. Out of the 1,236 colony forming units associated with eleven species of marine sponges, 100 morphologically different bacterial strains were analyzed in this study. Of these, 21 strains were resistant to Hg, 14 of which were classified as highly resistant because they grew despite exposure to 100 µM HgCl₂. Fifteen resistant strains reduced Hg and presented merA in their genomes. The remaining six strains produced biosurfactants, suggesting that they may tolerate Hg by sequestration. Eleven strains grew in the presence of methylmercury. Our results suggest a potential for mercury detoxification by marine sponge-associated resistant bacteria, either through reduction or sequestration, as well as the possibility of bioremediation of toxic waste containing mercury.     

Frequency of antibiotic and heavy metal resistance, pigmentation, and plasmids in bacteria of the marine air-water interface.

A field investigation of marine coastal waters revealed that the frequency of pigmented bacteria and the occurrence of bacterial antibiotic resistance were higher at the air-water interface than in the bulk water. The differences in the frequency of pigmented colonies at the surface and in the bulk-water samples could not be explained by the degree of cell surface hydrophobicity or by bacterial adhesion to air-water interfaces. Pigmented strains exhibited a higher degree of multiple drug resistance than did nonpigmented strains. However, the frequency of multiple drug resistance in nonpigmented strains was also substantial. An average of 91% of all strains were resistant to more than one antibiotic, and 21% of the bacteria isolated were resistant to five of the eight antibiotics tested. High numbers of plasmid-carrying strains were found among selected surface isolates, but the presence of detectable plasmids could not be correlated with either pigmentation or multiple drug resistance. Furthermore, selected surface isolates were significantly more resistant to mercury than were bulk-water bacteria. The higher frequency of pigmented, antibiotic-resistant, and mercury resistant strains at the air-water interface than in the bulk water are discussed in terms of various forms of selective pressure and genetic exchange at the surface.     

The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L. lactis

Abstract Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm^t-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 × 10³ transformants per μg DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 × 10² transformants per μg pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.     

Construction and Properties of Cloning Vectors Based on a 7.2-kb Rhizobium meliloti Cryptic Plasmid

Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were constructed by extension of the host range of a 7.2-kb Rhizobium meliloti cryptic plasmid (pRm1132f) with the ColE1-based plasmids, pBR322, pACYC177, pACYC 184, pSUP301, or pHC179; mobilization was facilitated by introduction of the ori T region from pRK2, a broad-host-range plasmid. The vector plasmids transferred readily into a wide range of gram-negative bacteria and had relatively low copy number in R. meliloti; two constructs, pFD1001 and pFD1212, were completely stable in R. meliloti isolated from nodules of alfalfa (Medicago sativa). A representative of the vector constructs (pFD1001) could be maintained in R. meliloti in the presence of the broad-host-range shuttle plasmid pRK290. These two vector plasmids could be introduced into R. meliloti, either simultaneously or singly when pRK290 was the resident plasmid; however, entry of pRK290 was blocked when pFD1001 was the resident plasmid. The cloning vectors constructed in this study should prove to be useful for the genetic manipulation of Rhizobium.     

Isolation of Broad-Host-Range Replicons from Marine Sediment Bacteria

Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties. Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P. A. Sobecky, T. J. Mincer, M. C. Chang, and D. R. Helinski, Appl. Environ. Microbiol. 63:888–895, 1997). In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates. 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the α and γ subclasses of the class Proteobacteria. Three of the marine sediment isolates are related to the γ-3 subclass organisms Vibrio splendidus and Vibrio fischeri, while the fourth isolate may be related to Roseobacter litoralis. Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria. In addition to replication in Escherichia coli DH5α and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the genera Achromobacter, Pseudomonas, Serratia, and Vibrio. While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other. The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates. Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences. In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.  The maintenance and horizontal transfer of extrachromosomal elements provide one mechanism by which microbial communities can rapidly adapt to changes in environmental conditions. This adaptation can be in the form of plasmid rearrangements and duplications (18, 40), a change in the plasmid copy number (40, 54), or lateral or horizontal movement of plasmids within bacterial populations. An example demonstrating the importance of plasmid-mediated genetic adaptation in natural microbial communities, likely caused by lateral transfer, is the increased frequencies (2- to 10-fold) of catabolic plasmids reported in bacterial isolates obtained from polluted marine and freshwater environments compared to isolates from nonpolluted or less impacted ecosystems (8, 23, 43). Plasmids also play a major role in promoting the widespread distribution of antibiotic resistance genes attributed to the intense and increased use of antibiotics (42).The ability of plasmids to self-transfer or to be mobilized by transfer-proficient plasmids and the ability to replicate in different bacterial hosts are key factors in the spread of plasmid-encoded genes within microbial communities. Plasmids which are considered to have broad host ranges in nature have the potential to significantly affect the microbial community structure and function due to their ability to replicate and be maintained in members of distantly related genera. Thus, to better understand gene flux in natural systems and hence the potential role of plasmids in promoting horizontal transfer within microbial communities, knowledge of the distribution, diversity, and host ranges of naturally occurring plasmids is necessary.At present, most indigenous plasmids from marine and freshwater systems have been only partially characterized with respect to host range, replication mechanisms, incompatibility groups, and conjugal abilities. Plasmids containing similar or related replication systems are considered incompatible if they cannot coexist in a host cell (12, 41). This trait has facilitated the grouping of plasmids from gram-negative bacteria, mainly members of the family Enterobacteriaceae, into more than 30 different incompatibility groups (3). While molecularly based plasmid classification or replicon typing by using DNA sequences of replication origins and incompatibility loci of well-characterized plasmids has been useful in classifying plasmids from bacterial isolates of medical importance (9, 10, 14), plasmids from various marine microbial communities, including sediments, biofilms, bulk water, and the marine air-water interface, have been recently shown to contain incompatibility and replication regions unrelated to those currently defined (11, 53).The present study was undertaken to characterize, at the molecular level, the replication and incompatibility loci of naturally occurring plasmids isolated from gram-negative marine heterotrophs for use as replicon probes to classify and type, at the molecular level, plasmids present in bacterial populations of marine sediments. Replication origins were obtained from plasmids ranging in size from 6 to 30 kb isolated from culturable bacteria of coastal California marine sediments (53). Phylogenetic analysis indicated that the plasmids were initially isolated from bacteria belonging to the α and γ-3 subclasses of the class Proteobacteria. Although a sequence and hybridization analysis of the replication origins from the marine plasmids confirmed the lack of homology with previously described plasmids, the replication regions contained features commonly found in previously characterized plasmid replication origins. The replication origins of the naturally occurring plasmids appear to have a broad host range, as indicated by their ability to replicate in members of diverse gram-negative marine genera. In addition to molecular characterization of the indigenous plasmids, the persistence of the replicons in marine sediment bacterial populations was determined by PCR amplification of microbial community DNA extracted on different dates and examined for the presence of homologous plasmid replication sequences.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment.

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Conjugal transfer of R68.45 and FP5 between Pseudomonas aeruginosa strains in a freshwater environment

Recent concern over the release of genetically engineered organisms has resulted in a need for information about the potential for gene transfer in the environment. In this study, the conjugal transfer in Pseudomonas aeruginosa of the plasmids R68.45 and FP5 was demonstrated in the freshwater environment of Fort Loudoun Resevoir, Knoxville, Tenn. When genetically well defined plasmid donor and recipient strains were introduced into test chambers suspended in Fort Loudoun Lake, transfer of both plasmids was observed. Conjugation occurred in both the presence and absence of the natural microbial community. The number of transconjugants recovered was lower when the natural community was present. Transfer of the broad-host-range plasmid R68.45 to organisms other than the introduced recipient was not observed in these chambers but was observed in laboratory simulations when an organism isolated from lakewater was used as the recipient strain. Although the plasmids transferred in laboratory studies were genetically and physically stable, a significant number of transconjugants recovered from the field trials contained deletions and other genetic rearrangements, suggesting that factors which increase gene instability are operating in the environment. The potential for conjugal transfer of genetic material must be considered in evaluating the release of any genetically engineered microorganism into a freshwater environment.     

Conjugal transfer of R68.45 and FP5 between Pseudomonas aeruginosa strains in a freshwater environment.

Recent concern over the release of genetically engineered organisms has resulted in a need for information about the potential for gene transfer in the environment. In this study, the conjugal transfer in Pseudomonas aeruginosa of the plasmids R68.45 and FP5 was demonstrated in the freshwater environment of Fort Loudoun Resevoir, Knoxville, Tenn. When genetically well defined plasmid donor and recipient strains were introduced into test chambers suspended in Fort Loudoun Lake, transfer of both plasmids was observed. Conjugation occurred in both the presence and absence of the natural microbial community. The number of transconjugants recovered was lower when the natural community was present. Transfer of the broad-host-range plasmid R68.45 to organisms other than the introduced recipient was not observed in these chambers but was observed in laboratory simulations when an organism isolated from lakewater was used as the recipient strain. Although the plasmids transferred in laboratory studies were genetically and physically stable, a significant number of transconjugants recovered from the field trials contained deletions and other genetic rearrangements, suggesting that factors which increase gene instability are operating in the environment. The potential for conjugal transfer of genetic material must be considered in evaluating the release of any genetically engineered microorganism into a freshwater environment.     

The use of cluster analysis to help determine the function of plasmids in marine Vibrio species

A phenotypic matrix was established for 512 marine Vibrio species previously surveyed for plasmid incidence and characteristics. The organisms were subjected to a variety of tests to determined phenetic groups and plasmid functions. Both physiologic and plasmid function tests were used to cluster the strains by numerical taxonomy. The resulting phenotypic clusters showed an uneven distribution of plasmids. Some clusters contained isolates with no plasmids and other clusters contained isolates with a high incidence of plasmids. Traits correlating with plasmid occurrence in clusters included bioluminescence and antibiotic and heavy metal resistance. These characteristics were assumed to be either plasmid-mediated or plasmid-associated traits. Cluster analysis was found to be a successful method for relating the presence of plasmids with specific traits.     

Plasmid-borne mercury resistance in aquatic bacteria

Abstract Bacteria isolated from the River Mersey were analysed for their tolerance to mercury (HgCl₂). About 40% of the population was tolerant to mercury and in 13 of 52 mercury-tolerant isolates tested the mercury resistance (Hg^®) was transferred to Escherichia coli in conjugal matings. These 13 isolates represented a range of gram-negative genera and in each case mercury resistance was coded by a conjugative plasmid. These plasmids (75 kb to > 250 kb in size) all expressed mercury resistance of the narrow spectrum variety, volatilised HgCl₂ to elemental Hg° vapour and showed some degree of temperature sensitivity of transfer. None expressed resistance to nine different antibiotics. These 13 Hg^R plasmids were classified by restriction mapping into three distinct groups typified by pMER11, pMER327 and pMER610. The eight pMER610 group plasmids are identical and belong to the IncHI-2 group. Two of the four pMER327 group plasmids are closely related while the other two contain some common restriction fragments. pMER11 is quite distinct from the other groups. These results imply that within this aquatic environment plasmids play an important role in the response of bacteria to contaminating mercury and that there is widespread plasmid transfer and considerable genetic rearrangement.     

Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Demonstration of tra+ plasmid activity in bacteria indigenous to the phyllosphere of sugar beet; gene transfer to a recombinant pseudomonad

Abstract The presence of transfer proficient plasmids in bacteria isolated from the leaves of sugar beet ( Beta vulgaris L.) was studied. Of 435 bacteria sampled 79 (18%) contained plasmids. Pseudomonads (30%), Erwinia (12%) and Klebsiella (9%) were the largest populations sampled of which 22%, 33% and 29%, respectively, contained plasmids. The ability of these plasmids to self-transfer or mediate the mobilization of the tra⁻ mob⁺ broad host range IncQ plasmid R300B was determined. R300B was maintained in 61/79 natural plasmid containing isolates, the Gram positive isolates could not support R300B. Pseudomonas aureofaciens SBW25, isolated from sugar beet leaves, was chromosomally marked with a tetracycline resistance gene and used as a recipient (SBW25ETc). Five isolates of Erwinia herbicola and one of Erwinia salicis containing natural plasmids were able to mobilize R300B into the recombinant, SBW25ETc. These mobolizing ( tra⁺ ) plasmids were not maintained in transconjugant SBW25 cells. Analysis of the fragment patterns of Pst I digested plasmid DNA demonstrated that four (pSB139, pSB140, pSB142, pSB146; 110 kb) were identical, one (pSB153; 65 kb) was common to a subset of fragments in these four and another (pSB169; 100 kb) was unique. Other natural isolates were able to transfer copper resistance ( Erwinia rhapontici , 2 strains) or mercury resistance ( Pseudomonas fluorescens SBW340) to a rifampicin resistant recipient Pseudomonas putida UWC1 but not to SBW25ETc. These self-transferable plasmids were not able to mobilize R300B. These data demonstrate that the phyllosphere supports indigenous microbial populations which have the capacity to transfer genetic material between bacteria of different genera.