Keevil C W, Major N C, Davies D B, Robinson A
Pathogenic Microbes Research Laboratory, PHLS Centre for Applied Microbiology and Research, Salisbury, UK.
J Gen Microbiol. 1986 Dec;132(12):3289-302. doi: 10.1099/00221287-132-12-3289.
Piliated Neisseria gonorrhoeae forming small, transparent colonies (P+O-) on clear typing agar have been grown in prolonged continuous culture to ascertain how different growth environments might affect gonococcal physiology and the expression of virulence determinants. Virulence of the penicillin-sensitive P9-2 and the penicillin-resistant KW1 strains was assessed by their ability to survive in polypropylene chambers implanted into the flanks of guinea pigs. Initial continuous culture experiments in the defined medium of Manchee et al. (FEMS Microbiology Letters 7, 115-118, 1980) indicated that growth was actually cystine-limited, rather than the anticipated glucose-limited. Surprisingly, cysteine was not completely metabolized and ammonium salts remained in excess. The molar growth yield on glucose (YGlc) was 65 g dry wt mol-1 and 45% of the glucose carbon metabolized was converted to biomass. Gonococci, whilst retaining the P+O- phenotype for over 100 generations of growth, did not survive in the subcutaneous chambers when inoculated at a variety of doses. When the cystine and glucose concentrations were increased and decreased respectively, growth became glucose-limited, the YGlc increased to 108 g mol-1 for strain KW1 and 75% of the metabolized glucose carbon was converted to biomass. After 17 generations of growth, however, only 2% of the gonococci retained the P+O- phenotype and P-O- bacteria predominated. Nevertheless, these bacteria were virulent in the chamber model, as was strain P9-2, which also retained only 2% of the P+O- phenotype during glucose-limited continuous culture. By contrast, the P+O- phenotype was retained during prolonged cystine- or oxygen-limited growth but only the latter was virulent. SDS-PAGE of membrane extracts confirmed that opaque colonies (O+) selected from the glucose-limited cultures contained a heat-modifiable protein (protein II) whereas transparent colony types lacked such proteins. The initial phenotype of virulent gonococci recovered from the subcutaneous chambers was P+O- but opaque variants dominated after several days. A 40 kDa outer-membrane protein was apparently induced during oxygen-limited continuous culture whereas a 44 kDa protein was absent during cystine-limited growth.
在透明分型琼脂上形成小的透明菌落（P+O-）的有菌毛淋病奈瑟菌，已在长期连续培养中生长，以确定不同的生长环境如何影响淋球菌的生理学和毒力决定因素的表达。通过青霉素敏感的P9-2菌株和耐青霉素的KW1菌株在植入豚鼠胁腹的聚丙烯腔室中存活的能力来评估其毒力。最初在Manchee等人（《FEMS微生物学通讯》7，115 - 118，1980）定义的培养基中进行的连续培养实验表明，生长实际上受胱氨酸限制，而非预期的葡萄糖限制。令人惊讶的是，半胱氨酸未被完全代谢，铵盐仍有剩余。葡萄糖的摩尔生长产量（YGlc）为65 g干重/mol，45%代谢的葡萄糖碳转化为生物量。淋球菌在超过100代的生长过程中虽保留P+O-表型，但以各种剂量接种时在皮下腔室中均不能存活。当胱氨酸浓度增加而葡萄糖浓度分别降低时，生长变为葡萄糖限制型，KW1菌株的YGlc增加到108 g/mol，75%代谢的葡萄糖碳转化为生物量。然而，生长17代后，只有2%的淋球菌保留P+O-表型，P-O-细菌占主导。尽管如此，这些细菌在腔室模型中具有毒力，P9-2菌株也是如此，其在葡萄糖限制的连续培养过程中也仅保留2%的P+O-表型。相比之下，在长期的胱氨酸或氧气限制生长过程中P+O-表型得以保留，但只有后者具有毒力。膜提取物的SDS-PAGE证实，从葡萄糖限制培养物中选出的不透明菌落（O+）含有一种热可修饰蛋白（蛋白II），而透明菌落类型则缺乏此类蛋白。从皮下腔室中回收的有毒力淋球菌的初始表型为P+O-，但几天后不透明变体占主导。在氧气限制的连续培养过程中显然诱导出一种40 kDa的外膜蛋白，而在胱氨酸限制生长过程中不存在一种44 kDa的蛋白。
