Organophosphate Hydrolase in Brevundimonas diminuta Is Targeted to the Periplasmic Face of the Inner Membrane by the Twin Arginine Translocation Pathway

Abstract

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in B. diminuta. The localization of OPH to the periplasmic face of the inner membrane in B. diminuta was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.Bacterial organophosphate hydrolases (OPH), also known as phosphotriesterases, have been shown to hydrolyze a structurally diverse group of phosphotriesters used as insecticides and chemical warfare agents (26,37). The genetic information required to encode these dimeric metalloenzymes is highly conserved and often located on plasmids known as organophosphate-degrading (opd) plasmids (27). Among the opd plasmids, pPDL2 (40 kb), isolated from Flavobacterium sp. strain ATCC27551, and pCMS1 (66 kb), isolated from Brevundimonas diminuta (formerly Pseudomonas diminuta), are well characterized (27). In these two indigenous plasmids, a 7-kb region that includes the 1.5-kb organophosphate-degrading (opd) gene is highly conserved and has the features of a complex transposon (38).OPH has been crystallized from a number of sources and has been shown to be a dimeric metalloenzyme with zinc at its catalytic center (1, 2, 28). In Flavobacterium and B. diminuta, the protein has been shown to be membrane associated, and a 29-amino-acid-long signal peptide found in its precursor form has been deduced to be responsible for membrane targeting (24,25,36). A similar signal sequence is also encoded in opd genes identified in Agrobacterium radiobacter (15) and Sphingomonas sp. strain JK1 (GenBank accession no. ACD85809). While the conservation of a signal peptide in this group of organophosphate hydrolases has been recognized for some time, its biological role and its precise involvement in the membrane localization of OPH have not been investigated. In this study, we expressed OPH with a C-terminal His tag in opd-negative mutants of B. diminuta and established a system to differentiate and localize precursor and mature ...

Covalent immobilization of recombinant organophosphorus hydrolase on spores ofBacillus subtilis

“…OPH signal peptide has a twin arginine translocation (Tat) motif, which is involved in the transportation of the folded protein across the bacterial inner membrane (Gorla et al . ). The complete opd gene along with its native signal peptide was codon optimized and used for functional secretion of OPH into the extracellular medium of E. coli .…”

Organophosphate Hydrolase Is a Lipoprotein and Interacts with Pi-specific Transport System to Facilitate Growth of Brevundimonas diminuta Using OP Insecticide as Source of Phosphate

“…Twin-arginine signal peptides serve to target proteins to the twin-arginine protein transport (Tat) pathway, which translocates folded proteins across the bacterial cytoplasmic membrane (14). Proteinase K treatment confirmed that OPH is exported to the periplasmic side of the inner membrane in Brevundimonas diminuta and dependence on the Tat pathway was demonstrated because substitution of the invariant arginine residues of the Tat signal peptide affected both processing and localization of OPH (15). However, the mechanism by which OPH is anchored to the inner membrane and the physiological role of OPH are currently unclear.…”

“…In bacteria these usually have either a single transmembrane domain at the C terminus (35) or an N-terminal non-cleaved twin-arginine signal anchor sequence (37). However, bioinformatic analysis supported by structural studies show that OPH has no C-terminal hydrophobic helical domain (38) and the N-terminal signal peptide of OPH is cleaved off during biosynthesis and therefore cannot serve as a signal anchor (15).…”

“…It has previously been demonstrated that OPH is associated with the inner membrane of B. diminuta (15). To probe the nature of the interaction of OPH with the membrane, we first determined whether the protein could be released from membrane fractions following washing under alkaline conditions using sodium carbonate (Fig.…”

“…Aliquots of OPH antisera (15) were passed through the OPH-coupled Sepharose column at a flow rate of 0.5 ml per min. The flow-through was collected carefully and reloaded onto the column to ensure complete binding of OPH-specific antibodies to the column.…”