Department of Physics, East Carolina University, Greenville, NC 27858-4353, USA.
J Appl Microbiol. 2012 Oct;113(4):824-36. doi: 10.1111/j.1365-2672.2012.05387.x. Epub 2012 Aug 2.
To analyse the effect of wet heat treatment on nutrient and non-nutrient germination of individual spores of Clostridium perfringens.
Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat-treated spores of Cl. perfringens with various germinants. When incubated in water at 90-100°C for 10-30 min, more than 90% of spores were inactivated but 50-80% retained their Ca(2+) -dipicolinic acid (CaDPA). The wet heat-treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640-1680 cm(-1) (amide I) and 1230-1340 cm(-1) (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat-treated spores that retained CaDPA germinated with KCl or l-asparagine, but wet heat treatment increased values of T(lag) , ΔT(release) and ΔT(lys) , during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl. perfringens spores lacking the essential cortex-lytic enzyme (CLE), SleC, exhibited longer T(lag) and ΔT(release) values during KCl germination than wild-type spores and germinated poorly with CaDPA. Wet heat-treated wild-type spores germinating with CaDPA or dodecylamine exhibited increased T(lag) , ΔT(release) and ΔT(lys) values, as did wet heat-treated sleC spores germinating with dodecylamine.
(i) Some proteins important in Cl. perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat.
This study provides information on the germination of individual Cl. perfringens spores and improves the understanding of effects of wet heat treatment on spores.
分析湿热处理对梭状芽胞杆菌单个孢子营养和非营养萌发的影响。
使用拉曼光谱和微分干涉对比（DIC）显微镜监测未经处理和湿热处理的梭状芽胞杆菌单个孢子在不同萌发剂中的动态萌发。在 90-100°C 的水中孵育 10-30 分钟后，超过 90%的孢子失活，但 50-80%的孢子保留其钙（Ca 2+ ）-二吡啶羧酸（CaDPA）。失去 CaDPA 的湿热处理孢子表现出广泛的蛋白质变性，如拉曼光谱的 1640-1680 cm(-1)（酰胺 I）和 1230-1340 cm(-1)（酰胺 III）区域所示，而保留 CaDPA 的孢子则表现出部分蛋白质变性。保留 CaDPA 的湿热处理孢子用 KCl 或 L-天冬酰胺萌发，但湿热处理增加了 T(lag)、ΔT(release)和ΔT(lys)的值，在此期间，孢子在与萌发剂混合后开始释放大部分 CaDPA，释放超过 90%的 CaDPA，并由于皮层水解而完成其 DIC 强度的降低。缺乏必需的皮层裂解酶（CLE）SleC 的未经处理的梭状芽胞杆菌孢子在 KCl 萌发期间表现出更长的 T(lag)和ΔT(release)值，比野生型孢子萌发差，并且用 CaDPA 萌发不良。用 CaDPA 或十二胺萌发的湿热处理野生型孢子和用十二胺萌发的湿热处理 sleC 孢子表现出增加的 T(lag)、ΔT(release)和ΔT(lys)值。
（i）湿热处理会破坏一些对梭状芽胞杆菌孢子萌发很重要的蛋白质；（ii）CLE SleC 或激活 SleC 的丝氨酸蛋白酶 CspB 可能是被湿热处理破坏的萌发蛋白；（iii）CaDPA 释放过程可能受到湿热处理的破坏。
本研究提供了关于单个梭状芽胞杆菌孢子萌发的信息，并提高了对湿热处理对孢子影响的理解。
