Identification of Genes and Proteins Necessary for Catabolism of Acyclic Terpenes and Leucine/Isovalerate in Pseudomonas aeruginosa

Abstract

ABSTRACT Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters ( atuABCDEFGH and liuRABCDE ) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads ( P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens . This result concurred with the finding that P. fluorescens , but not P. putida , could grow on acyclic terpenes (citronellol and citronellate), while both species were able to utilize leucine and isovalerate. A regulatory gene, atuR , was identified upstream of atuABCDEFGH and negatively regulated expression of the atu gene cluster.