phnE and glpT Genes Enhance Utilization of Organophosphates in Escherichia coli K-12

Abstract

Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (≤1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl 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 asphnE 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. coliK-12 phnE gene that has been shown previously to result inphnE 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 theglpT gene is the result of amplification and overproduction of the glpT gene product. Both the active phnEand multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnEgene could confer the ability to break down a chromogenic substrate, 5-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.

Microbial degradation of organophosphorus compounds

“…The major problem associated with whole cell bioreactors is mass transport limitation of substrate across the cell membrane where OPH resides (Mulchandani et al, 1999a). Uptake of organophosphorus compounds as a rate limiting factor has been reported by several groups (Hung & Liao, 1996;Elashvili et al, 1998). This barrier of substrate transport can be overcome by treating cells with permeabilizing agents such as EDTA and DMSO.…”

Simultaneous degradation of organophosphorus pesticides and p‐nitrophenol by a genetically engineered Moraxella sp. with surface‐expressed organophosphorus hydrolase

“…for in situ degradation is advantageous because they are known to survive in contaminated environments for an extended period of time (Tresse et al, 1998). Because the transport of these pesticides across the cell membrane has been shown to be restrictive (Elashvili et al, 1998;Hung and Liao, 1996), one solution is to target OPH directly on the surface of Moraxella sp. (Richins et al, 1997).…”

The Abc of Phosphonate Breakdown: A Mechanism for Bacterial Survival

“…The resulting 5 0deoxyadenosyl radical can then abstract a hydrogen atom and generate a radical on a substrate, or in the case of glycyl radical enzymes, directly on the backbone of a conserved glycine residue in the enzyme itself. Based on careful deuterium exchange [18] PhnD ABC transport system for phosphonate, periplasmic binding protein 3QK6 [46] PhnE ABC transport system for phosphonate, membrane-spanning protein N/A [47] PhnF Transcriptional regulator 2FA1 [48] PhnG Purine ribonucleoside triphosphate phosphonylase component, component of PhnGHIJK 4XB6 [26] PhnH Purine ribonucleoside triphosphate phosphonylase component, component PhnGHIJK 2FSU 4XB6 [26,31] PhnI Purine ribonucleoside triphosphate phosphonylase component, component of PhnGHIJK 4XB6 [26,49] PhnJ C À P lyase, component of PhnGHIJK 4XB6 [26] PhnK Component of PhnGHIJK, accessory protein, nucleotide binding protein of an ABC transport system.…”