phnE and glpT genes enhance utilization of organophosphates in Escherichia coli K-12

• Published in: Applied and environmental microbiology 1998-07, Vol.64 (7), p.2601-2608
• Main Authors: Elashvili, I. (US Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD.), DeFrank, J.J, Culotta, V.C
• Format: Article
• Language: English
• Subjects: Anion Transport Proteins - genetics; Bacteria; BIODEGRADACION; BIODEGRADATION; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; COMPOSE ORGANOPHOSPHORE; COMPUESTO ORGANICO DEL FOSFORO; ESCHERICHIA COLI; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Fundamental and applied biological sciences. Psychology; Genes; Genes, Bacterial - genetics; Genetics; Isoflurophate - metabolism; Membrane Transport Proteins - genetics; METABOLISM; METABOLISME; METABOLISMO; MICROBIAL DEGRADATION; Microbiology; Mission oriented research; MODEL COMPOUNDS; MODELE; MODELOS; Organophosphates - metabolism; ORGANOPHOSPHORUS COMPOUNDS; Organophosphorus Compounds - metabolism; PESTICIDE; PESTICIDES; Physiology and Biotechnology; PLAGUICIDAS
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.64.7.2601-2608.1998

Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (less than or equal to mM) of diisopropyl fluorophosphate and its hydrolysis product, diisoprapyl phosphate (DIPP), as sole phosphorus sources. Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate. A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation. These genes were identified as phnE and glpT. The original OPU mutation represented phnE gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type E. coli K-12 phnE gene that has been shown previously to result in phnE activation. In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the glpT gene was not the result of a mutation. PCR clones of glpT from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid. This suggests that the OPU phenotype associated with the glpT gene is the result of amplification and overproduction of the glpT gene product. Both the active phnE and multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnE gene could confer the ability to break down a chromogenic substrate, S-bromo-4 chloro-3-indoxyl phosphate-p-toluidine (X-Pi). This result indicates that in E. coli, X-Pi is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems