Molecular analysis of a sphingomyelinase C gene from Leptospira interrogans serovar hardjo

Abstract

A thermolabile hemolysin from Leptospira interrogans serovar hardjo, strain Sponselee, was shown to specifically degrade sphingomyelin. Nucleotide sequence determination revealed that sphingomyelinase activity was encoded by an open reading frame of 1,668 nucleotides. Although a putative signal sequence could be identified, no evidence for protein export in either L. interrogans or Escherichia coli was obtained. The apparent molecular mass of the expression product in E. coli minicells was 41.2 kilodaltons, whereas open reading frame 1 encoded a protein of 63,268 daltons. The observed difference may be explained by processing at the carboxy-terminal part of the hemolysin in E. coli. A high degree of similarity on the DNA and protein levels with Staphylococcus aureus ,-hemolysin and sphingomyelinase C from three Bacillus cereus strains was observed. The presence of various sphingomyelinase genes within the L. interrogans species is demonstrated.

Plasmodium falciparum Phospholipase C Hydrolyzing Sphingomyelin and Lysocholinephospholipids Is a Possible Target for Malaria Chemotherapy

“…Fatty acid-free BSA was used as lipid-free BSA (6). Egg lysoPtdCho was from Avanti Polar Lipids, Inc., and 1-hexadecyl-2-acetyl-sn -glycerol from Biomol Research Laboratories Inc. [ Choline-methyl - 14 (18)(19)(20)(21)(22)(23)(24)(25)(26)(27) Ci/ mmol) were from Amersham Pharmacia Biotech, and [ palmitoyl -1-14 C]lysoPtdCho l-1-monopalmitoyl (55 mCi/mmol) from NEN Life Science Products. Scyphostatin was generously provided from Sankyo Co. and d, l -threo-1-phenyl-2-hexadecanoidylamino-3-morphiolino-1-propanol (PPMP) was from Matreya, Inc. Scyphostatin and PPMP were dissolved in DMSO at 10 mM and stored at -20 Њ C until use.…”

“…Some bacteria produce extracellular Mg 2 ϩ -dependent neutral SMases (18) that are presumably implicated in degradation of environmental SM for nutritional use or for infection to vertebrate hosts. Several cDNAs encoding acid and neutral SMases (18,(20)(21)(22)(23) and genes for bacterial SMase (24)(25)(26) have been identified so far. There has been no report, however, of a plasmodial SMase gene.…”

Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni

“…Examination of codon usage within ORF10 and ORF58 showed a greater similarity with the gene for sphingomyelinase C of Leptospira borgpetersenii serovar hardjo (Segers et al, 1990) and the trpE and trpG genes of Leptospira biflexa (Yelton and Peng, 1989) than with E coli (Ikemura and Ozeki. 1982), particularly for the amino acids glutamine, arginine.…”

Presence of putative sphingomyelinase genes among members of the family Leptospiraceae

“…Saprophytic members of the family Leptospiraceae now include the species Leptospira biflexa, Leptospira meyeri, Leptospira wolbachii, and Leptospira parva (19). Previously, a sphingomyelinase gene (sphA) from a serovar hardjo strain, which was provisionally classified in the species L. borgpetersenii (15,17), was characterized at the molecular level and shown to be homologous to sphingomyelinase genes from gram-positive bacteria (14). As shown by hybridization under stringent conditions, this sphA gene is present in a limited number of pathogenic leptospires only (4,15).…”