USE OF GREEN FLUORESCENT PROTEIN TO TAG AND INVESTIGATE GENE-EXPRESSION IN MARINE-BACTERIA

Two broad-host-range vectors previously constructed for use in soil ba cteria (A. G. Matthysse, S, Stretton, C, Dandle, N, C, McClure, and A. E, Goodman, FEMS Microbiol, Lett, 145:87-94, 1996) were assessed by e pifluorescence microscopy for use in tagging three marine bacterial sp ecies. Expression of gfp could be visualized in Vibrio sp, strain S141 cells at uniform levels of intensity from either the Inc or the npt-2 promoter, whereas expression of gfp could be visualized in Psychrobac ter 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-g fp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-ka n-gfp generated random stable mutants at high frequencies with all thr ee 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 wer e grown in rich medium compared to that when cells were grown in minim al medium, Mini-Tn10-kan-gfp was used to create an S91 chitinase-negat ive, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed to N'-acetylglucosamine or attached to chitin partic les. By laser scanning confocal microscopy, biofilms consisting of mic rocolonies of chi-negative, GFP(+) S91 cells were found to be localize d several microns from a natural chitin substratum, Tagging bacterial strains with GFP enables visualization of, as well as monitoring of ge ne expression in, living single cells in situ and in real time.