Journal of Microbiology

Research Support, U.S. Gov't, Non-P.H.S.

Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance§: Ananya Barman , Ranjan Tamuli; J. Microbiol. 2015;53(4):226-235. Published online January 31, 2015; DOI: https://doi.org/10.1007/s12275-015-4465-1

Abstract: Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca2+/H+ exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca2+/H+ exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca2+ concentration ([Ca2+]c) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)- survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca2+]c, carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

Research Support, Non-U.S. Gov't

NOTE] The Activity of Phosphoinositide-Specific Phospholipase C Is Required for Vegetative Growth and Cell Wall Regeneration in Coprinopsis cinerea: Young Taek Oh , Chun-Seob Ahn , Kyung-Jin Lee , Jeong-Geun Kim , Hyeon-Su Ro , Jae Won Kim , Chang-Won Lee; J. Microbiol. 2012;50(4):689-692. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2004-x

Abstract: Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.

Isolation and Characterization of Fatty Acid Derivatives from an Actinomycetes and Examination of the Effects on Activities of Phospholipase C and Protein Kinase C: Ko, Hack Ryong , Kim, Bo Yeon , Lee, Hyun Sun , Kang, Dae Ook , Ryu, Sung Ho , Suh, Pann Ghill , Mheen, Tae Ick , Ahnm Jong Seog; J. Microbiol. 1998;36(4):316-321.

Abstract: In our screening to search inhibitors of phosphoinositide(PI)-specific phospholipase C (PI-PLC), two inhibitors, MT965-A and-B were isolated from a culture broth of an actinomycetes. MT965-A and-B were identified as fatty acid deribatives, 14-methylpentadecanoic acid and 16-methyllinoleic acid methyl ester, respectively, based on the spectral data including NMR and MS. Both inhibitors directly inhibited not only in vitro PLCγ1 activity but also the platelet-derived growth factor(PDGF)-induced inositol phosphates(IPt) formation in NIH 3T3γ1 cells ocerexpressing PLCγ1. However, the inhibitors enhanced in vitro protein kinase C (PKC) activity. On examination of the effects of various fatty acids(FAs) on activities of PLC, PKC, and PDGF-induced IPt formation, the unsaturated FAs(UFAs) showed the same activities like the inhibitors, but the saturated FAs(SFAs) did not show similar activities. It was inferred that the chain length, degree of unsaturation, methyl esterification, branching with a methyl group, and cis-configuration were important for their activity.

Bioluminescent Assay of Phospholipase C Using A Luminescent Marine Mutant Bacterium Vibrio harveyi M-17: Ki Woong Cho , SangJun Mo , Hyi-Seung Lee , Jung-Rae Rho , Jongheon Shin; J. Microbiol. 2000;38(3):150-155.

Abstract: A bioluminescent assay method for detecting the activity of phospholipase C (PLC; phosphatidyl choline cholinephosphohydrolase, EC 3.1.4.3) was developed using bioluminescent marine bacteria. Phospholipase C from Bacillus cereus and sn-1,2-dimyristoyl phosphatidyl choline (DMPC) as a substrate were used in the demonstration, and the produced sn-1,2-dimyristoyl glycerol was further hydrolyzed with lipase from Candida cylidracea. The hydrolyzed myristic acid was quantified using a dark mutant of Vibrio harveyi (designated as M-17). The in vivo light intensity of which was stimulated specifically up to one thousand fold in the presence of myristic acid. The rates of the hydrolysis of the DMPC substrate by the phospholipase measured by the luminescence method were linear with time and the amount of enzyme added. Activity measurement conditions (at 25 C, pH 6.5, 10 min fixed time assay) were established to detect as little as 0.1 mUnit of phospholipase C and 5 nM of myristic acid production.

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