Role of the Pseudomonas aeruginosa PlcH Tat signal peptide in protein secretion, transcription, and cross-species Tat secretion system compatibility

The secretion of PlcH and its homolog PlcN of Pseudomonas aeruginosa through the inner membrane depends upon a functional twin arginine translocase (Tat) system and a Tat signal sequence. Conserved twin arginine (Arg) residues within the Tat signal sequence consensus motif (S/TRRxFLK) are considered essential for the secretion of Tat substrates, but some exceptions (e.g., Lys and Arg) to the twin Arg residues in this motif have been noted. The roles of all three Arg residues within the PlcH RRRTFLK consensus motif were examined. Data are presented which indicate that Arg-9 and Arg-10 are essential for PlcH secretion across the inner membrane, but the mutation of Arg-8 (e.g., to Ala or Ser) had no observable effect on the localization of PlcH. In the signal sequence of PlcH and in all of its homologs in other bacteria, there are basic amino acid residues (Arg, Lys, and Gln) immediately adjacent to the signal peptidase cleavage site (Ala-X-Ala) that are not seen in Sec-dependent signal sequences. The mutation of these basic residues to Ala caused slightly decreased levels of extracellular PlcH, but normal localization was still observed. Deletion of the entire Tat signal sequence of PlcH not only resulted in the absence of detectable extracellular PlcH activity and protein but also caused a substantial decrease in the detectable level of plcH mRNA. Finally, data are presented which indicate that P. aeruginosa PlcH exhibits cross-species compatibility with the Escherichia coli Tat secretion machinery, but only when the E. coli Tat machinery is expressed in a P. aeruginosa host.     

A novel class of microbial phosphocholine-specific phospholipases C

In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.     

Purification,characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa. PlcH is a multifunctional enzyme

Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of approximately 75 kDa and one of 30-35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.     

Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces two secreted phospholipase C (PLC) enzymes. The expression of both PLCs is regulated by Pi. One of the PLCs is hemolytic, and one is nonhemolytic. Low-stringency hybridization studies suggested that the genes encoding these two PLCs shared DNA homology. This information was used to clone plcN, the gene encoding the 77-kilodalton nonhemolytic PLC, PLC-N. A fragment of plcN was used to mutate the chromosomal copy of plcN by the generation of a gene interruption mutation. This mutant produces 55% less total PLC activity than the wild type, confirming the successful cloning of plcN. plcN was sequenced and encodes a protein which is 40% identical to the hemolytic PLC (PLC-H). The majority of the homology lies within the NH2 two-thirds of the proteins, while the remaining third of the amino acid sequence of the two proteins shows very little homology. Both PLCs hydrolyze phosphatidylcholine; however, each enzyme has a distinct substrate specificity. PLC-H hydrolyzes sphingomyelin in addition to phosphatidylcholine, whereas PLC-N is active on phosphatidylserine as well as phosphatidylcholine. These studies suggest structure-function relationships between PLC activity and hemolysis.     

Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms

Pseudomonas aeruginosa forms diverse matrix-enclosed surface-associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air-liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface-associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild-type P. aeruginosa PA14 produces a cellulase-sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild-type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose-rich matrix material required for the formation of biofilms by P. aeruginosa PA14.     

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg(-1) protein which was more than a recombinant P. pastoris GS115 (552 U mg(-1) protein) or KM71H (539 U mg(-1) protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg(-1) protein by P. pastoris GS115, 1176 U mg(-1) protein by P. pastoris KM71H and 1522 U mg(-1) protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 degrees C) than the wild-type PLC from B. cereus . Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co(2+) and Mn(2+) etc., also influenced the activity of the recombinant PLCs.     

Proteolytic Pathways of Activation and Degradation of a Bacterial Phospholipase C during Intracellular Infection by Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment. Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC). PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytogenes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway. Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity. Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner. Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1–positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases. Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A₁, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.     

Using a molecular model and kinetic experiments in the presence of divalent cations to study the active site and catalysis of Pseudomonas aeruginosa phosphorylcholine phosphatase

Phosphorylcholine phosphatase (PchP) of Pseudomonas aeruginosa, a product of the PA5292 gene, catalyzes the hydrolysis of phosphocholine to choline and inorganic phosphate (Pi). Phosphocholine is produced after hemolytic phospholipase C (PlcH) acts upon phosphatidylcholine or sphingomyelin. Therefore, PlcH and PchP are involved in the pathogenesis of P. aeruginosa. PchP belongs to the HAD superfamily as it contains three conserved sequences motifs. In mature PchP, the motifs I, II, and III are (31)DMDNT(35), (166)S, and (261)GDTPDSD(267), respectively. Kinetic characterization of wild-type and mutated proteins, obtained by site-directed mutagenesis, in addition to a molecular model of PchP helped us to understand the contribution of key residues in the conserved motifs I, II and III that are involved in the catalysis of p-nitrophenylphosphate processing after the addition of Mg(2+), Zn(2+) or Cu(2+) (these are activators of PchP activity). Our results are explained by invoking the concept of chemical hardness and softness introduced by Pearson in 1963 and its extension that "hard acids prefer to coordinate to hard bases and soft acids to soft bases" [Parr and Pearson, J. Am. Chem. Soc., 105, 7512-7516 (1983)].     

The latest phospholipase C, PLCeta, is implicated in neuronal function

Members of the phosphoinositide-specific phospholipase C (PLC) family have key roles in cell signalling. In response to many extracellular stimuli, such as hormones, neurotransmitters, antigens and growth factors, PLCs catalyse the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)], thereby generating two well-established second messengers, inositol (1,4,5)-trisphosphate and diacylglycerol. Eleven PLC isozymes encoded by different genes have been identified in mammals and, on the basis of their structure and sequence relationships, have been classified into five families designated PLCbeta (1-4), PLCgamma (1 and 2), PLCdelta (1, 3 and 4), PLCepsilon (1) and PLCzeta (1). All PLCs contain the catalytic X and Y domain, in addition to other regulatory domains including the C2 domain and the EF-hand domain. In 2005, four groups independently identified an entirely new family of PLCs--eta1 and eta2--using data mining of mammalian genomes. The properties of the PLCeta enzyme suggest that it might act as a Ca(2+) sensor, in particular, functioning during formation and maintenance of the neuronal network in the postnatal brain.     

Prediction of missing enzyme genes in a bacterial metabolic network. Reconstruction of the lysine-degradation pathway of Pseudomonas aeruginosa

The metabolic network is an important biological network which consists of enzymes and chemical compounds. However, a large number of metabolic pathways remains unknown, and most organism-specific metabolic pathways contain many missing enzymes. We present a novel method to identify the genes coding for missing enzymes using available genomic and chemical information from bacterial genomes. The proposed method consists of two steps: (a) estimation of the functional association between the genes with respect to chromosomal proximity and evolutionary association, using supervised network inference; and (b) selection of gene candidates for missing enzymes based on the original candidate score and the chemical reaction information encoded in the EC number. We applied the proposed methods to infer the metabolic network for the bacteria Pseudomonas aeruginosa from two genomic datasets: gene position and phylogenetic profiles. Next, we predicted several missing enzyme genes to reconstruct the lysine-degradation pathway in P. aeruginosa using EC number information. As a result, we identified PA0266 as a putative 5-aminovalerate aminotransferase (EC 2.6.1.48) and PA0265 as a putative glutarate semialdehyde dehydrogenase (EC 1.2.1.20). To verify our prediction, we conducted biochemical assays and examined the activity of the products of the predicted genes, PA0265 and PA0266, in a coupled reaction. We observed that the predicted gene products catalyzed the expected reactions; no activity was seen when both gene products were omitted from the reaction.     

Characterization of the twin-arginine translocase secretion system of Mycobacterium smegmatis

The twin-arginine translocation (TAT) system secretes fully folded proteins that contain a twin-arginine motif within their signal sequence across the cytoplasmic membrane in bacteria. Using a green fluorescent protein fused with a TAT signal sequence, we demonstrated that Mycobacterium smegmatis contains a TAT system. By inactivating individual genes, we showed that three genes (tatA, tatB, and tatC) are required for a functional TAT system in M. smegmatis. The tat mutants exhibited a decreased growth rate and altered colony morphology compared to the parent strain. Comparison of the secreted proteins of the deltatatC and parent strain by two-dimensional polyacrylamide gel electrophoresis revealed an alteration in the secretion of at least five proteins, and one of the major TAT-dependent secreted proteins was identified as beta-lactamase (BlaS). The genome of M. smegmatis was analyzed with the TATFIND program, and 49 putative TAT substrates were identified, including the succinate transporter DctP. Because disruption of the TAT secretion system has a direct effect on the physiology of M. smegmatis and homologs of the TAT proteins are also present in the genome of Mycobacterium tuberculosis, the TAT secretion system or its substrates may be good candidates for drug or vaccine development.     

Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins

We describe here a new mutant of Pseudomonas aeruginosa PAO, strain D10C (genotype plcB), which produces phospholipase C and alkaline phosphatase constitutively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracellular proteins produced by this mutant in high- and low-Pi media revealed that the mutation resulted in a marked deficiency of one major Pi-regulated protein of 41,000 molecular weight and constitutive synthesis of all other major extracellular Pi-regulated proteins. Furthermore, the plcB mutant was deficient in phosphate transport. A plcA mutation, which also led to a loss of the 41,000-molecular-weight protein, was similarly deficient in Pi transport. The genetic loci, plcA and plcB, located at 22 to 23 min on the PAO chromosome, were indistinguishable by conjugational and transductional mapping, and may therefore be in the same gene or in a cluster of genes which regulate the synthesis of Pi-repressible proteins.     

Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development

An analysis of the Pseudomonas aeruginosa genomic sequence revealed three gene clusters, PA1381-1393, PA2231-2240, and PA3552-3558, in addition to the alginate biosynthesis gene cluster, which appeared to encode functions for exopolysaccharide (EPS) biosynthesis. Recent evidence indicates that alginate is not a significant component of the extracellular matrix in biofilms of the sequenced P. aeruginosa strain PAO1. We hypothesized that at least one of the three potential EPS gene clusters revealed by genomic sequencing is an important component of P. aeruginosa PAO1 biofilms. Thus, we constructed mutants with chromosomal insertions in PA1383, PA2231, and PA3552. The mutant with a PA2231 defect formed thin unstructured abnormal biofilms. The PA3552 mutant formed structured biofilms that appeared different from those formed by the parent, and the PA1383 mutant formed structured biofilms that were indistinguishable from those formed by the parent. Consistent with a previous report, we found that polysaccharides were one component of the extracellular matrix, which also contained DNA. We suggest that the genes that were inactivated in our PA2231 mutant are required for the production of an EPS, which, although it may be a minor constituent of the matrix, is critical for the formation of P. aeruginosa PAO1 biofilms.     

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.     

Cloning and heterologous expression of a gene encoding an alkane-induced extracellular protein involved in alkane assimilation from Pseudomonas aeruginosa.

Pseudomonas aeruginosa PG201 produces a 16-kDa extracellular protein in media containing n-hexadecane as a carbon source but not in media containing glycerol or glucose. This protein was purified, and the N-terminal amino acid sequence was determined. The amino acid composition of the protein was found to be very similar to that of the so-called protein-like activator for n-alkane oxidation (PA) from P. aeruginosa S7B1. This extracellular protein was previously characterized (K. Hisatsuka, T. Nakahara, Y. Minoda, and K. Yamada, Agric. Biol. Chem. 41:445-450, 1977) and found to stimulate the growth of P. aeruginosa on n-hexadecane and to possess emulsifying activity. To study the role(s) of the PA protein and to make it accessible for possible future applications, we have cloned the PA-encoding (pra) gene and determined its nucleotide sequence. This analysis revealed a protein-coding region of 162 amino acids, with the first 25 residues being reminiscent of those of a typical bacterial signal sequence. The pra gene was inactivated by insertional mutagenesis, and the resulting strain was found to lack extracellular PA protein and to be retarded in its growth in n-hexadecane-containing media. These results are consistent with the growth stimulatory role of the PA protein. The pra gene was expressed in Escherichia coli, and substantial amounts of the recombinant protein were found in the extracellular growth medium. The recombinant protein was purified by metal chelate affinity chromatography. The ability to produce secreted PA protein by E. coli provides a simple and safe means to analyze its function(s) in alkane assimilation in the future.     

Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.

While searching for a phospholipase C (PLC) specific for phosphatidylcholine in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold. The secreted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol. The active enzyme is a monomer of approximately 40 kDa. As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for activity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2. The compound D609, described to selectively inhibit phosphatidylcholine-specific PLCs, caused half-inhibition of the P. fluorescens enzyme at approximately 420 microM, while 50-fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens. Partial peptide sequences obtained from the pure P. fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P. fluorescens gene library prepared from our laboratory isolate. It contains an ORF of 1155 nucleotides encoding the PLC. There is no significant sequence homology to other PLCs, suggesting that the P. fluorescens enzyme represents a distinct subclass of bacterial PLCs. The protein lacks cysteine residues and consequently contains no disulfide bonds. Interestingly, P. fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant coding sequence.     

Phospholipase C: Diverse functions in plant biotic stress resistance and fungal pathogenicity

Phospholipase C (PLC) generates various second messenger molecules and mediates phospholipid hydrolysis. In recent years, the important roles of plant and fungal PLC in disease resistance and pathogenicity, respectively, have been determined. However, the roles of PLC in plants and fungi are unintegrated and relevant literature is disorganized. This makes it difficult for researchers to implement PLC-based strategies to improve disease resistance in plants. In this comprehensive review, we summarize the structure, classification, and phylogeny of the PLCs involved in plant biotic stress resistance and fungal pathogenicity. PLCs can be divided into two groups, nonspecific PLC (NPC) and phosphatidylinositol-specific PLC (PI-PLC), which present marked differences in phylogenetic evolution. The products of PLC genes in fungi play significant roles in physiological activity and pathogenesis, whereas those encoded by plant PLC genes mediate the immune response to fungi. This review provides a perspective for the future control of plant fungal diseases.     

Phospholipase C families: Common themes and versatility in physiology and pathology

Phosphoinositide-specific phospholipase Cs (PLCs) are expressed in all mammalian cells and play critical roles in signal transduction. To obtain a comprehensive understanding of these enzymes in physiology and pathology, a detailed structural, biochemical, cell biological and genetic information is required. In this review, we cover all these aspects to summarize current knowledge of the entire superfamily. The families of PLCs have expanded from 13 enzymes to 16 with the identification of the atypical PLCs in the human genome. Recent structural insights highlight the common themes that cover not only the substrate catalysis but also the mechanisms of activation. This involves the release of autoinhibitory interactions that, in the absence of stimulation, maintain classical PLC enzymes in their inactive forms. Studies of individual PLCs provide a rich repertoire of PLC function in different physiologies. Furthermore, the genetic studies discovered numerous mutated and rare variants of PLC enzymes and their link to human disease development, greatly expanding our understanding of their roles in diverse pathologies. Notably, substantial evidence now supports involvement of different PLC isoforms in the development of specific cancer types, immune disorders and neurodegeneration. These advances will stimulate the generation of new drugs that target PLC enzymes, and will therefore open new possibilities for treatment of a number of diseases where current therapies remain ineffective.     

Critical evaluation of p-nitrophenylphosphorylcholine (p-NPPC) as artificial substrate for the detection of phospholipase C*

Phospholipase C (PLC) activity secreted by bacteria as a virulence factor is commonly detected by use of the artificial substrate p-nitrophenylphosphorylcholine (p-NPPC). We examined several commercially available enzymes (phosphodiesterases, phosphomonoesterases, phospholipase A, lipase, protease) for their hydrolytic activity towards p-NPPC and compared these results with those of PLC tests using phospholipid substrates. Our data indicate that, in addition to PLC, several other enzymes which can affect phosphate esters are able to hydrolyze p-NPPC. We therefore suggest to use lipid substrates for correct characterization of bacterial PLCs, especially when whole bacteria or crude enzyme preparations are investigated.