Advertisement

New methods for the isolation and characterization of biofilm-persistent mutants in Pseudomonas putida

Authors

  • Aroa López-Sánchez,

    1. Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, Sevilla, Spain
    2. Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Sevilla, Spain
    Search for more papers by this author
  • Alicia Jiménez-Fernández,

    1. Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, Sevilla, Spain
    2. Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Sevilla, Spain
    Search for more papers by this author
  • Patricia Calero,

    1. Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, Sevilla, Spain
    2. Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Sevilla, Spain
    Search for more papers by this author
  • Laura D. Gallego,

    1. Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, Sevilla, Spain
    2. Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Sevilla, Spain
    Search for more papers by this author
  • Fernando Govantes

    Corresponding author
    1. Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Sevilla, Spain
    • Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, Sevilla, Spain
    Search for more papers by this author

For correspondence. E-mail fgovrom@upo.es; Tel. (+34) 954977877; Fax (+34) 954349376.

Summary

Here we describe two new methods for the genetic characterization of bacterial biofilm development. First, we have designed a microtitre dish-based approach for high-throughput screening of Pseudomonas putida mutants showing increased biofilm under dispersal conditions. Using this method, nine such biofilm-persistent mutants, bearing transposon insertions in four loci: lapG, bifA, mvaB and dksA, were isolated. Second, we have developed a serial dilution-based scheme to monitor biofilm development and dispersal in microtitre dish wells in a simple, time-efficient and reproducible manner. Using this method, we showed that (i) mutants in bifA and dksA do not undergo starvation-induced biofilm dispersal in LB or minimal medium, (ii) a mvaB mutant does not disperse the biofilm in LB, but shows a normal dispersal response in minimal medium, and (iii) unlike the lapG mutant, the bifA, mvaB and dksA mutants do not show an increase in biofilm production. The procedures shown here are useful tools for the identification of previously uncharacterized biofilm-related genes and considerably simplify the characterization of biofilm growth phenotypes.

Ancillary