Use of Green Fluorescent Protein To Tag and Investigate Gene Expression in Marine Bacteria

Abstract

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 thenpt-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 thegfp 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 toN′-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.

Effect of integration of a GFP reporter gene on fitness of Ralstonia eutropha during growth with 2,4‐dichlorophenoxyacetic acid

“…Green fluorescent protein (GFP)‐expressing strains are now frequently used for tracking specific microbial strains in ecosystems. Bacteria marked with gfp have been employed to study the distribution and dynamics of bacterial populations in soils (Errampalli et al ., 1998; Tresse et al ., 1998), aqueous systems (Stretton et al ., 1998), rhizospheres (Gage et al ., 1996; Bloemberg et al ., 1997; Tombolini et al ., 1997), activated‐sludge (Eberl et al ., 1997; Olofsson et al ., 1998) and biofilms (Möller et al ., 1998; Skillman et al ., 1998). Furthermore, GFPs can be used to visualize gene expression (Cubitt et al ., 1995; Andersen et al ., 1998; Jaspers et al., 2001).…”

Biological activity and colonization pattern of the bioluminescence-labeled plant growth-promoting bacterium Kluyvera ascorbata SUD165/26

“…Thus, K. ascorbata SUD165/26 was labeled with green £uorescent protein (GFP) from the jelly¢sh Aquoria victoria, and with luciferase (Lux) from the bacterium Vibrio harveyi. GFP and Lux are both very sensitive, easily detected and can be quanti¢ed with a spectro£uorimeter or a luminometer [13,14], thereby providing a simple and accurate means of monitoring and localizing selected bacteria in a complex environment.…”

Colonization of siliques and seeds of rapid cycling Brassica oleracea plants by Xanthomonas campestris pv. campestris after spray-inoculation of flower clusters

“…10% of the cells had lost the plasmid (data not shown). Other studies using bacteria tagged with a plasmidbased gfp construct also demonstrated that during colonization of substrates, part of the population lost their ability to express GFP (Gage et al 1996;Stretton et al 1998). To guarantee stable expression in long term in planta studies, the gene encoding GFP should be introduced into the bacterial chromosome, which is more stable than those plasmidborne (Errampalli et al 1999).…”