Manganese Is Essential for PlcP Metallophosphoesterase Activity Involved in Lipid Remodeling in Abundant Marine Heterotrophic Bacteria

Abstract

In vast areas of the ocean, microbes must adapt to the availability of scarce nutrients, and a key strategy for reducing the cellular phosphorus (P) quota is to remodel membranes by replacing phospholipids with non-P surrogate lipids. A metallophosphoesterase, PlcP, is essential for lipid remodeling in cosmopolitan marine bacteria of the Roseobacter (e.g., Phaeobacter sp. strain MED193) and SAR11 (e.g., Pelagibacter sp. strain HTCC7211) clades, and transcription of plcP is known to be induced by P limitation. In order to better understand PlcP-mediated lipid remodeling, we sought to characterize PlcP for its metal ion requirement and to determine its selectivity for native bacterial phospholipids. Here, we report the occurrence of a highly conserved binuclear ion center in PlcPs from MED193 and HTCC7211 and show that manganese is the preferred metal for metallophosphoesterase activity. PlcP displayed high activity towards the major bacterial phospholipids, e.g., phosphatidylglycerol but also phosphatidic acid, a key intermediate in phospholipid biosynthesis. In contrast, phosphatidylserine and phosphatidylinositol, both of which are rare lipids in bacteria, are not preferred substrates. These data suggest that PlcP undertakes a generic lipid remodeling role during the cellular response of marine bacteria to P deficiency and that manganese availability may play a key role in regulating the lipid remodeling process.IMPORTANCE Membrane lipids form the structural basis of all cells. In the marine environment, it is well established that phosphorus availability significantly affects lipid composition in cosmopolitan marine bacteria, whereby non-phosphorus-containing lipids are used to replace phospholipids in response to phosphorus stress. Central to this lipid remodeling pathway is a newly identified phospholipase C-type metallophosphoesterase (PlcP). However, little is known about how PlcP activity is regulated. Here, we determined the role of metal ions in regulating PlcP activity and compared PlcP substrate specificities in PlcP enzymes from two model marine bacteria from the marine Roseobacter clade and the SAR11 clade. Our data provide new insights into the regulation of lipid remodeling in these marine bacteria.

Keywords: PlcP; Roseobacter; SAR11; lipid remodeling.

FIG 1

Multiple-sequence alignment and functional domain analyses of PlcP proteins. PLC₁₉₃, PlcP of Phaeobacter sp. strain MED193; PLC₇₂₁₁, PlcP of Pelagibacter sp. strain HTCC7211; PlcP_Sm, PlcP of Sinorhizobium meliloti (10). (A) Multiple-sequence alignment of PlcP and closely related LpxH enzymes. The 6 conserved motifs are highlighted in gray. The conserved histidine residue in PlcP (H82) is highlighted in green. (B) Neighbor-joining phylogenetic analysis between members of the metallophosphoesterase family (PFam 00149) including proteins closely related to PlcP: PaLpxH, LpxH from Pseudomonas aeruginosa (14); HiLpx, LpxH from Haemophilus influenzae (13). LpxH displays pyrophosphatase activity and acts on UDP-2,3-diacylglucosamine to produce lipid X, a key precursor for the formation of lipid A in lipopolysaccharide biosynthesis. More-distantly related members of the metallophosphoesterase family include the following: MJ0936, which represents a group of novel phosphodiesterases that do not degrade phosphomonoesters (26); Mre11/SbcD, which are bacterial and archaeal DNA phosphodiesterases involved in DNA repair (27, 28); Dbr1, which is a group of phosphodiester nucleases that act on RNA (29); CpdA, CpdB, and cAMP phosphodiesterases, which are cyclic nucleotide phosphodiesterases; ApaH, which represents a group of enzymes with pyrophosphatase and protein phosphatase activities (30); YfcE, which represents a group of small metallophosphoesterases showing phosphodiesterase activity (31); sphingomyelinase, which is a group of hydrolases responsible for breaking down sphingomyelin to phosphocholine and ceramide (32). Numbers indicate bootstrap values (only values of >50 are shown).

FIG 2

Homology modeling showing the predicted structure of PlcP₁₉₃ and the metal-binding pocket. The signature arginine residue in LpxH (Arg81) is replaced by a histidine residue in PlcP (His82).

FIG 3

PlcP displays manganese-dependent phosphomonoesterase and phosphodiesterase activities. (A) Overexpression and purification of PlcP from Phaeobacter sp. strain MED193 and Pelagibacter sp. strain HTCC7211. M, protein molecular weight marker. Lane 1, cell-free supernatant induced with isopropyl β-d-1-thiogalactopyranoside (IPTG); lane 2, cell-free supernatant without IPTG induction; lane 3, purified PlcP protein (molecular weight estimated to be ∼27 kDa). (B) PlcP activity assays in the presence of various divalent metal ions (1 mM). Values are means ± standard deviations from three replicated measurements. (C) PlcP activity assays using p-nitrophenylphosphorylcholine (NPPC) or p-nitrophenylphosphate (PNPP). Values are means ± standard deviations from three replicated measurements.

FIG 4

Homology modeling prediction of the metal coordination center in the PlcP₁₉₃ enzyme (A) and the site-directed mutants of His82 to Ala82 (B), Arg82 (C), and Asn82 (D). Specific activities of site-directed mutants of PlcP₁₉₃ are measured using p-nitrophenylphosphorylcholine (NPPC) as the substrate (E). Values are means ± standard deviations from three replicated measurements.

FIG 5

Specific activity of the degradation of phospholipids by PlcP₁₉₃ and PlcP₇₂₁₁. Activity was measured by quantifying the formation of the common product diacylglycerol (DAG) in these reactions. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PA, phosphatidic acid; PI, phosphatidylinositol; PS, phosphatidylserine. Values are means ± standard deviations from three replicated measurements.

FIG 6

Schematic overview of the PlcP-mediated lipid remodeling pathway and its regulation in representative marine bacteria. The major lipids in Phaeobacter sp. strain MED193 and Pelagibacter sp. strain HTCC7211 under P-replete conditions are two phospholipids (highlighted in gray), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Under P stress, the two-component signal transduction system PhoBR is activated and the phosphorylated PhoB activates not only the expression of the high-affinity ABC transporter for phosphate (PstSABC) but also the transcription of the plcP gene. A conserved phoB binding site in the plcP promoter has previously been identified in these bacteria (8). The purified PlcP protein requires manganese for activity (Fig. 3). Manganese is likely transported into the cell through either the SitABCD or the MntX transporter system, both of which are present in these marine heterotrophic bacteria (4). Active PlcP can convert PE, PG, or its biosynthesis precursor phosphatidic acid (PA) to diacylglycerol (DAG), which serves as the building block for the biosynthesis of alternative P-free surrogate lipids (highlighted in blue), including diacylglyceryl trimethylhomoserine (DGTS) and the glycolipids monoglycosyl diacylglycerol (MGDG) and glucuronic acid diacylglycerol (GADG) (7, 8).