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

Summary P seudomonas putida   DOT ‐ T 1 E ‐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 ‐ T 1 E ‐18 on solid L uria– B ertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT ‐ T 1 E ‐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 G ram‐positive bacteria, G ram‐negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against B acillus , L ysinibacillus and R hodococcus 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 G ram‐positive bacteria and showed an interesting antimicrobial effect against P seudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin.