Stretton S, Techkarnjanaruk S, McLennan A M, Goodman A E
School of Biological Sciences, Flinders University of South Australia, Adelaide, Australia.
Appl Environ Microbiol. 1998 Jul;64(7):2554-9. doi: 10.1128/AEM.64.7.2554-2559.1998.
Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87-94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or the npt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when the gfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed to N'-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.
之前构建的用于土壤细菌的两种广宿主范围载体（A.G. Matthysse、S. Stretton、C. Dandie、N.C. McClure和A.E. Goodman，《FEMS微生物学快报》145:87 - 94，1996年）通过落射荧光显微镜进行评估，以用于标记三种海洋细菌物种。在弧菌属菌株S141细胞中，从lac或npt - 2启动子均可观察到gfp以均匀的强度水平表达，而在嗜冷杆菌属菌株SW5H细胞中，仅从npt - 2启动子才能观察到gfp在不同强度水平的表达。当从任一启动子表达gfp基因时，在第三种细菌——假交替单胞菌属菌株S91中未检测到绿色荧光蛋白（GFP）荧光。构建了一个新的mini - Tn10 - kan - gfp转座子，以进一步研究海洋细菌荧光标记的可能性。mini - Tn10 - kan - gfp的插入在所有三种海洋物种中均以高频产生随机稳定突变体。利用该转座子，分离出了强表达和弱表达的S91启动子。与在基本培养基中生长时相比，当S91（mini - Tn10 - kan - gfp）细胞在丰富培养基中生长时，通过落射荧光显微镜观察到的GFP明显减少。mini - Tn10 - kan - gfp用于创建一个S91几丁质酶阴性、GFP阳性的突变体。chi - gfp融合蛋白的表达在暴露于N - 乙酰葡糖胺或附着于几丁质颗粒的细胞中被诱导。通过激光扫描共聚焦显微镜观察发现，由几丁质酶阴性、GFP + S91细胞的微菌落组成的生物膜位于距天然几丁质基质几微米处。用GFP标记细菌菌株能够在原位实时观察活的单细胞并监测其基因表达。
