Efflux pump-deficient mutants as a platform to search for microbes that produce antibiotics

Microb Biotechnol. 2015 Jul;8(4):716-25. doi: 10.1111/1751-7915.12295. Epub 2015 Jun 8.

Abstract

Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria-Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / isolation & purification
  • Anti-Bacterial Agents / metabolism*
  • Antibiosis*
  • Bacillus
  • Bacteria / isolation & purification
  • Bacteria / metabolism*
  • Biological Transport, Active
  • Drug Synergism
  • Environmental Microbiology*
  • Fungi
  • Humans
  • Membrane Transport Proteins / deficiency*
  • Microbial Sensitivity Tests
  • Pseudomonas / drug effects*
  • Pseudomonas / genetics
  • Pseudomonas / growth & development

Substances

  • Anti-Bacterial Agents
  • Membrane Transport Proteins