Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs

Abstract

Background: Lysophosphatidic acid acyltransferase (LPAAT) is a phospholipid biosynthesis enzyme that introduces a particular set of fatty acids at the sn-2 position of phospholipids. Many bacteria have multiple LPAAT paralogs, and these enzymes are considered to have different fatty acid selectivities and to produce diverse phospholipids with distinct fatty acid compositions. This feature is advantageous for controlling the physicochemical properties of lipid membranes to maintain membrane integrity in response to the environment. However, it remains unclear how LPAAT paralogs are functionally differentiated and biologically significant.

Results: To better understand the division of roles of the LPAAT paralogs, we analyzed the functions of two LPAAT paralogs, PlsC4 and PlsC5, from the psychrotrophic bacterium Shewanella livingstonensis Ac10. As for their enzymatic function, lipid analysis of plsC4- and plsC5-inactivated mutants revealed that PlsC4 prefers iso-tridecanoic acid (C₁₂-chain length, methyl-branched), whereas PlsC5 prefers palmitoleic acid (C₁₆-chain length, monounsaturated). Regarding the physiological role, we found that plsC4, not plsC5, contributes to tolerance to cold stress. Using bioinformatics analysis, we demonstrated that orthologs of PlsC4/PlsC5 and their close relatives, constituting a new clade of LPAATs, are present in many γ-proteobacteria. We also found that LPAATs of this clade are phylogenetically distant from principal LPAATs, such as PlsC1 of S. livingstonensis Ac10, which are universally conserved among bacteria, suggesting the presence of functionally differentiated LPAATs in these bacteria.

Conclusions: PlsC4 and PlsC5, which are LPAAT paralogs of S. livingstonensis Ac10, play different roles in phospholipid production and bacterial physiology. An enzyme belonging to PlsC4/PlsC5 subfamilies and their close relatives are present, in addition to principal LPAATs, in many γ-proteobacteria, suggesting that the division of roles is more common than previously thought. Thus, both principal LPAATs and PlsC4/PlsC5-related enzymes should be considered to decipher the metabolism and physiology of bacterial cell membranes.

Keywords: Fatty acid composition; Lysophosphatidic acid acyltransferase; Phospholipid; PlsC; YihG.

Fig. 1

Schematic illustration of the LPAAT reaction (A) and genomic organization of plsC4, plsC5, and flanking genes (B). R, hydrocarbon chain; CoA, coenzyme A; ACP, acyl carrier protein

Fig. 2

Construction of plsC4^inact. and plsC5^inact. mutants. A The wild-type (upper) and mutated (lower) nucleotide and amino acid sequences of catalytic site-coding regions. The small characters indicate the mutated nucleotides. A SalI site in the wild-type plsC5, of which loss indicates the successful mutation, is shaded. Raw sequencing data for the mutated sequences are shown at the bottom. B The growth curves of parent (black), plsC4^inact. (blue), and plsC5^inact. cells (red). Solid and broken lines indicate growth profiles at 18 °C and 4 °C, respectively

Fig. 3

ESI-MS analysis of phospholipids. A Relative abundances of PE and PG species in the parent cells harboring pJRD-Cm^R (closed black bar) and plsC4^inact. cells harboring pJRD-Cm^R (open blue bar) and pJRD_PlsC4^His (closed blue bar). B Relative abundances of PE and PG species in the parent cells harboring pJRD-Cm^R (closed black bar) and plsC5^inact. cells harboring pJRD-Cm^R (open red bar) and pJRD_PlsC5^His (closed red bar). The data were obtained from three independent experiments

Fig. 4

GC-MS analysis of sn-2 fatty acids. A Relative abundances of each fatty acid at sn-2 position in the parent cells harboring pJRD-Cm^R (closed black bar) and plsC4^inact. cells harboring pJRD-Cm^R (open blue bar) and pJRD_PlsC4^His (closed blue bar). B Relative abundances of each fatty acid at the sn-2 position in the parent cells harboring pJRD-Cm^R (closed black bar) and plsC5^inact. cells harboring pJRD-Cm^R (open red bar) and pJRD_PlsC5^His (closed red bar). The data were obtained from three independent experiments

Fig. 5

Phylogenetic analysis of PlsC4 and PlsC5. PlsC4 and PlsC5 homologs and well-characterized PlsCs from a broad range of γ-proteobacteria were grouped by a neighbor-joining method using MEGA X software. Symbols attached to proteins from S. livingstonensis Ac10 (asterisk), E. coli MG1655 (dagger), and P. fluorescens F113 (sharp). Bootstrap values greater than 50% are indicated for each node