Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate

Abstract

Escherichia coli outer membrane protein A (OmpA) is a well-established model for the study of membrane assembly. Previous studies have shown that the essential sequence for outer membrane localization, known as the sorting signal, is contained in a segment of the eighth beta-strand, residues 163-171. Sequential digestion of OmpA, purified from outer membranes or inclusion bodies with cyanogen bromide and Staphylococcus aureus GluC, yielded peptides 162-174(LSLGVSYRFGQGE). Western blot and chemical assays indicated that the peptide was covalently modified by oligo-(R)-3-hydroxybutyrate (cOHB), a flexible, amphipathic oligoester. MALDI/MS was consistent with modification of peptides 162-174 by up to ten R-3-hydroxybutyrate (HB) residues. Western blot analysis of mutants of the peptide, using anti-OHB IgG, indicated that cOHB modification was not inhibited by the single mutations S163G, S167G, Y168F, R169N or R169D; however, cOHB was not detected on peptides containing the double mutations S163G:S167G S163G:V166G, L162G:S167G, and L164G:S167G. MALDI/MS/MS of double mutant S163G:S167G confirmed the absence of cOHB-modification. The results suggest that cOHB may be attached to one or both serines, and point to the importance of the flanking hydrophobic residues. Modification by cOHB may play a role in outer membrane targeting and assembly of OmpA.

New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3‐hydroxybutyrate)

“…Covalent modification of proteins by cPHB, designated as PHBylation, can have important functions on the physiological properties of proteins. One of the first identified PHBylated proteins is OmpA of E. coli (Xian et al, 2007;Negoda et al, 2010b,c). PHBylation of specific serine residues was important for correct integration and orientation of OmpA in the outer membrane.…”

The Role of Short-Chain Conjugated Poly-(R)-3-Hydroxybutyrate (cPHB) in Protein Folding

“…, heating in dilute acid or refluxing in methanol:chloroform mixtures [14]. cPHB-peptides may also be recognized by MALDI MS [26] (see Figure 6 below).…”

Involvement of the AtoSCDAEB regulon in the high molecular weight poly-(R)-3-hydroxybutyrate biosynthesis in phaCAB+ Escherichia coli

“…AtoSC signaling also regulates a number of indispensable E. coli processes, including motility and chemotaxis regulation (Theodorou et al, 2011c), as well as the biosynthesis and intracellular distribution regulation of the complexed short-chain poly-(R)-3-hydroxybutyrate (cPHB) cPHB, is an inherent E. coli macromolecule of low molecular weight, synthesized by native E. coli enzymes and is considered as a member of the biodegradable high-molecular weight PHAs family (Reusch et al, 2002). However, only many physiological roles have been attributed to E. coli cPHB, such as Ca 2 þ homeostasis through the non-proteinaceous complexes of PHB-polyphosphate-Ca 2 þ , acting as voltage-gated Ca 2 þ channels, competence for genetic transformation, protection of the complexed proteins from proteolysis, participation in DNA organization, and modification of the sorting signal of the OmpA protein (Reusch et al, 2002;Xian et al, 2007). Its regulation by AtoSC TCS is achieved through its direct effects on atoDAEB operon (Theodorou et al, 2006) as well as according to recent studies through its involvement in fatty acids metabolism (Theodorou et al, 2011a).…”