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

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