Screening of genes involved in biosurfactant production using a Mini-Tn5 Insertion Library of Pseudomonas extremaustralis

PAULA M. TRIBELLI*; NANCY I. LOPEZ; LAURA J. RAIGER IUSTMAN

Mar del Plata

Congreso; ? VII Congreso Argentino de Microbiologia General SAMIGE; 2012

SAMIGE

Random insertion libraries provide invaluable advantages in the study of bacterial genomes and in the analysis of metabolic pathways. Pseudomonas extremaustralis is an Antarctic bacterium with high stress resistance in association with the accumulation of large amounts of polyhydroxybutyrate. Recently, the complete sequence of its genome was obtained. Previous reports of our group described that this strain was capable to synthesize biosurfactants of lipopeptide nature. Biosurfactants have high relevance in the biotechnology field, because they could be applied in several industries like petrochemical, pharmaceutical, food, etc. The finding of new biosurfactants and the study of their synthesis pathways are of great interest, because it can contribute to develop products for new applications and to reduce production costs. A mini Tn5 library was constructed in P. extremaustralis to detect biosurfactant production?s related genes. Transposon mutagenesis was performed by conjugation, using Escherichia coli S17-1λ Pir carrying the plasmid pUTmini-Tn5. Conjugation process was carried out for 6 hours to avoid redundant clones. The resulting clone collection was screened for the detection of mutants affected in biosurfactant production in blood agar plates supplemented with 2% glucose. Plates were incubated for 36 h at 30°C until hemolysis development. Two clones, H and N, showed different hemolytic activity respect the wild type (WT) strain, and were selected for further assays. H showed a higher hemolytic activity than the WT strain, while N showed no hemolytic halo. To corroborate if the differences observed in the hemolytic activity correspond to a change in the surfactant production, a drop-collapsed assay was performed. Clone H?s contact angle was 17% smaller than the WT, showing an increased surfactant production in this mutant. No difference was observed in mutant N. A TLC assay developed with ninhydrin and Iodine armosphera showed that, while mutant H presented the same spots pattern than the WT, in mutant N some spots were missing. To identify the insertion site in the genome sequence, a PCR based strategy was used. For H clone a PCR fragment of 1200 bp was obtained. Sequence analysis indicated that the transposon insertion site was in the polyhydroxybutyrate biosynthesis cluster, specifically in the phaR gene, coding for a putative activator. The results suggest that the mutation in a regulator involved in PHB metabolism, increased surfactant production perhaps by redirecting the carbon flux to this metabolism.