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Journal of Bacteriology, January 2002, p. 540-546, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.540-546.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Cloning and Expression of Mn²⁺-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp. Strain TK4

Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received 13 August 2001/ Accepted 25 October 2001

We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn²⁺. The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.

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