title: Identification and characterization of the AgmR regulator of Pseudomonas putida: role in alcohol utilization.
abstract: Two-phase partitioning bioreactors (TPPBs) comprise an aqueous phase containing all non-carbon nutrients necessary for microbial growth and a solvent phase containing high concentrations of inhibitory or toxic substrates that partition at sub-inhibitory levels to the aqueous phase in response to cellular demand. This work aimed at eliminating the growth of Pseudomonas putida ATCC 11172 on medium-chain-length (C8-C12) aliphatic alcohols, hence enabling their use as xenobiotic delivery solvents within two-phase partitioning bioreactors. Experiments resulted in the isolation of a mini-Tn5 mutant unable to utilize these alcohols. The mutation, which also eliminated growth on glycerol and ethanol, was identified to be within a homologue of the P aeruginosa agmR gene, which encodes a response regulator. Enzyme analysis of the agmR::Tn5Km mutant cell extracts revealed a 10-fold decrease in pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase activity. A knockout in a gene (exaA) encoding a PQQ-linked alcohol dehydrogenase slowed but did not eliminate growth on medium-chain-length alcohols or ethanol, suggesting metabolic redundancy within P. putida ATCC 11172. Analysis of P. putida KT2440 genome sequence data indicated the presence of two PQQ-linked alcohol dehydrogenase-encoding genes. The successful elimination of alcohol utilization in the agmR mutant indicates control by AgmR on multiple pathways and presents a useful strain for biotechnological applications requiring alcohol non-utilizing microbial catalysts.
authors.affiliation: Department of Chemical Engineering, Queen's University, Kingston, Ontario, Canada.
authors.forename: H A
authors.initials: HA
authors.lastname: Vrionis
authors.forename: A J
authors.initials: AJ
authors.lastname: Daugulis
authors.forename: A M
authors.initials: AM
authors.lastname: Kropinski
mesh_terms: D000426:Alcohol Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D000438:Alcohols / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019149:Bioreactors; D015870:Gene Expression; D005798:Genes, Bacterial; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators
chemical_list: D000438:Alcohols; D001426:Bacterial Proteins; D015534:Trans-Activators; C071444:AgmR protein, Pseudomonas aeruginosa; D000426:Alcohol Dehydrogenase