Las Bacterias

Revista de Biología Marina y Oceanografía 35(2): 121-125, diciembre de 2000

ARTICULOS

Probing transfer of an IncP replicon to natural marine bacteria*

Ensayo de transferencia de un replicón IncP a bacterias marinas naturales

James Robeson1 and Ana María Skarmeta1

1Instituto de Biología, Universidad Católica de Valparaíso. Avenida Brasil 2950, Valparaíso, Chile

jrobeson@ucv.cl

ABSTRACT

An IncP plasmid probe (pUCV2), coding for Cm-r, Km-r and bearing Ap (am) and Tc (am) resistance determinants was constructed by transposition of Tn9 (Cm-r) onto plasmid pLM2 for an efficient selection of potential recipients among natural marine bacteria. Using a Dap- E. coli donor, transmission of pUCV2 to marine bacteria was tested. pUCV2 is transferred to about 4-8% of natural, marine bacterial cells capable of forming colonies on a low nutrient, marine agar medium. The following bacterial genera, commonly found in the marine environment, could be detected when twenty of the transconjugant colonies obtained were identified: Vibrio, Pseudomonas and Aeromonas.

Key words: Horizontal transfer, IncP probe, marine bacteria

RESUMEN

Una sonda plasmidial IncP (pUCV2), que codifica para Cm-r, Km-r y que tiene determinantes de resistencia Ap (am) y Tc (am) fue construida por transposición de Tn9 (Cm-r) al plásmido pLM2, para una eficiente selección de potenciales receptores entre bacterias marinas naturales. Usando un donador E. coli Dap-, se ensayó la transmisión de pUCV2 a bacterias marinas. pUCV2 es transferido a cerca del 4 - 8% de las células bacterianas marinas naturales, capaces de formar colonias en un medio de agar marino con bajos niveles de nutrientes. Los siguientes géneros bacterianos, comúnmente encontrados en el ambiente marino, pudieron ser detectados cuando veinte de las colonias transconjugantes obtenidas fueron identificadas: Vibrio, Pseudomonas y Aeromonas.

Palabras clave: Transferencia horizontal, sonda IncP, bacterias marinas

INTRODUCTION

The carriage of plasmids by antibiotic-resistant marine bacteria was described in a pioneering study by Sizemore & Colwell (1977). However, only recently more attention has been devoted to the nature of such plasmids and their potential for horizontal transfer. In this vein, resistance plasmids have been described for bacteria in the marine air-water interface (Hermansson et al. 1987), in marine sediments (Sobecky et al. 1997, 1998) and other marine habitats (Dahlberg et al. 1997). This seems relevant to the emergence of antibiotic-resistant marine bacteria in aquafarming as its has been revealed by studies of Sandaa et al. (1992), who found transferable drug resistance among bacteria from fishfarm sediments and showed dispersal of a promiscuous plasmid from Aeromonas salmonicida to bacteria in marine sediments (Sandaa & Enger 1994).

Besides plasmids in indigenous marine bacteria, those carried by allochthonous bacteria that enter the marine environment are also considered a potential source of genetic variation for marine bacteria, in the event that heterologous transmission takes place and the allochthonous plasmids are stabilized in the marine bacterial recipients. This situation was perceived in an early study by Patt et al. (1972), who showed transfer of Escherichia coli plasmids to marine bacteria. In this regard, promiscuous plasmids in Gram negative bacteria, such as the IncP replicon RP4 (Smith & Thomas 1989) are prime candidates for such a role because they are capable of transfer to different genera of Proteobacteria (Hodgson 1989), a dominant group among marine bacteria (González & Moran 1997).

Studies pertaining transfer of IncP plasmids to marine bacteria are overall very scarce. Goodman et al. (1993) have shown transfer of RP1 between E. coli and strains of the marine Vibrio S14 under starvation conditions in artificial seawater. Furthermore, Sorensen (1993) demonstrated transfer of RP4 from E. coli to different marine bacterial isolates in filter crosses and in sterile seawater and to indigenous marine bacteria using an auxotrophic donor strain to select transconjugants on selective minimal media. In addition, Sandaa (1993) used Vibrio sp. S141, containing RP4, as a genetic donor in experiments to detect plasmid transfer and maintenance in marine sediments with marine bacterial isolates acting as genetic recipients. In this investigation, no transfer was detected to the marine recipients, in spite of the fact that the plasmid was maintained for more than 67 days in the released host.

Within the latter context, in the study we hereby report we aimed at developing a selection based strategy for the facile investigation of the dispersal of IncP replicons from an E. coli donor to a wide range of natural marine bacteria, using a genetically tagged plasmid probe.

MATERIALS AND METHODS

Bacteriological techniques

The main bacterial strains and plasmids used in this work are listed in Table 1. Additional bacterial strains are described in Tables 2 and 3. E. coli strains were routinely grown at 37º C, unless otherwise indicated, in L medium (Robeson & Skarmeta, 1998). Marine bacteria were grown at 25º C in a marine medium prepared in 75% seawater that contained in g/l: Bactopeptone, 5 and Yeast extract, 1. For plates, agar was added at 1.5% (MA). Low Nutrient (LN) medium was as MA, but with Bactopeptone and Yeast Extract 100 fold less concentrated. Mc Conkey agar base supplemented with 1% sugar was used as indicator medium.

Table 1. Bacterial strains and plasmids used in this study

Tabla 1. Cepas bacterianas y plásmidos utilizados en éste estudio

Table 2. Transmission of pUCV2 to culture collection marine bacteria

Tabla 2. Transmisión de pUCV2 a bacterias marinas de colección

Table 3. Transfer of pUCV2 to natural marine bacteria in LN marine agar plates

Tabla 3. Transferencia de pUCV2 hacia bacterias marinas naturales en placas de agar marino LN

Antibiotics were used in the following concentrations in µg/ml: Ampicillin (Ap), 50; Chloramphenicol (Cm), 20; Kanamycin (Km), 50; Nalidixic acid (Nal), 100; Tetracycline (Tc), 25 and Streptomycin (Sm), 200. Diaminopimelic acid (Dap) was used at 50 µg/ml. All media were from Difco (Detroit, Mi.) and supplements from Sigma (St. Louis, Mo.).

Characterization of marine bacterial isolates by biochemical tests and physiological features was according to Smibert & Krieg (1981) and by the API 20B identification system (Bio Mérieux, Montalieu, France).

Genetical techniques

Conjugative spin matings between donor and recipient strains in Eppendorf centrifuge tubes were as described (Robeson & Skarmeta, 1998). Surface matings between E. coli strains were performed spreading 2x109 cells of each donor and recipient on the surface of L agar plates, which were incubated for 6 h at 37º C prior to selection of recombinants.

Transfer of the plasmid probe pUCV2 (see below) from E. coli VAL1 (Table 1) to natural marine bacteria was detected by a plate transfer assay (Robeson et al., 1990) that consisted in spreading about 1x107 washed E. coli donor cells on the surface of an LN plate, which was then seeded with 50 µl of seawater to allow development of marine bacterial colonies. All potential recipient colonies could then be tested for acquisition of plasmid markers in MA supplemented with Cm and Km.

For the construction of pUCV2, E. coli VAL2, containing the plasmid pLM2 was spin-mated with E. coli VAL53 that contains Tn9 in its chromosome. One of the VAL53 (pLM2) transconjugants obtained was then surface-mated with E. coli c2605. The bacterial cell mixture was then replica-plated onto McConkey-galactose agar containing Sm, Cm and Km to select E. coli c2605 (pLM2::Tn9 = pUCV2) exconjugants.

RESULTS

To study transfer of IncP replicons to natural marine bacteria our first aim was to construct a suitable IncP plasmid probe that could be genetically recognizable and would allow easy selection of transconjugants. In plating non-polluted seawater samples in the presence of various antibiotics we found that the Cm-Km combination was particularly effective in eliminating all background growth of marine bacteria in MA medium. Therefore, we decided to derive an IncP probe containing Cm and Km resistance determinants. As a starting base we used plasmid pLM2, an IncP derivative that has amber mutated Ap and Tc resistance genes, which are only expressed in amber supressor (sup E) bacterial strains (Mindich et al., 1976).

We transposed Tn9 onto pLM2 and selected exconjugants of E. coli c2605 as Gal- Sm-r colonies that became simultaneously resistant to Cm and Km. These exconjugants were Ap and Tc sensitive due the fact that E. coli c2605 is not an amber supressor strain (Su0). We finally worked with a plasmid probe designated pUCV2, which conferred the expected phenotypes to sup E and Su0 E. coli strains ; it rendered both E. coli strains c2605 and c1849 resistant to Cm and Km but only c1849 expressed, in addition, Ap and Tc resistance. Furthermore, pUCV2 DNA could be detected as a single plasmid band with increased molecular mass in relation to pLM2 DNA, upon comparison of both molecules in a 0.5% agarose gel (data not shown). This difference in mass is expected due to insertion of

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