Rhizobia are Gram-negative soil bacteria able to establish nitrogenfixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithinecontaining lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse-chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria.nimal cells have access to relatively abundant sources of phosphorus for the formation of biomolecules such as membrane phospholipids and nucleic acids. The characteristic lipid composition for a particular animal cell membrane is thought to result from a steady state between formation and turnover of the lipids. In contrast, plants and many environmental microbes often live in environments where available phosphorus is a growth-limiting factor. The strategies employed by organisms to deal with phosphorus limitation include: (i) increased solubilization of phosphorus-containing compounds; (ii) more efficient uptake into cells; and (iii) less phosphorus use when synthesizing their biomolecules (1). The replacement of phospholipids by galacto-and sulfolipids in plant membranes constitutes an important adaptive process for growth on phosphate-limited soils. In Arabidopsis thaliana, several phospholipases D and C (2-5) are induced under phosphate-limiting conditions, and they degrade membrane phospholipids to phosphatidic acid or diacylglycerol (DAG), respectively. DAG then serves as the initial substrate for the formation of galacto-and sulfolipids, which lack phosphorus.In some bacteria, the membrane phospholipids are partially replaced during phosphate limitation by phosphoru...

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.