Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates

Abstract

The growth activity of Pseudomonas putida cells colonizing the rhizosphere of barley seedlings was estimated at the single-cell level by monitoring ribosomal contents and synthesis rates. Ribosomal synthesis was monitored by using a system comprising a fusion of the ribosomal Escherichia coli rrnBP1 promoter to a gene encoding an unstable variant of the green fluorescent protein (Gfp). Gfp expression in a P. putida strain carrying this system inserted into the chromosome was strongly dependent on the growth phase and growth rate of the strain, and cells growing exponentially at rates of >0.17 h ؊1 emitted growth rate-dependent green fluorescence detectable at the single-cell level. The single-cell ribosomal contents were very heterogeneous, as determined by quantitative hybridization with fluorescently labeled rRNA probes in P. putida cells extracted from the rhizosphere of 1-day-old barley seedlings grown under sterile conditions. After this, cells extracted from the root system had ribosomal contents similar to those found in starved cells. There was a significant decrease in the ribosomal content of P. putida cells when bacteria were introduced into nonsterile bulk or rhizosphere soil, and the Gfp monitoring system was not induced in cells extracted from either of the two soil systems. The monitoring system used permitted nondestructive in situ detection of fast-growing bacterial microcolonies on the sloughing root sheath cells of 1-and 2-day-old barley seedlings grown under sterile conditions, which demonstrated that it may be possible to use the unstable Gfp marker for studies of transient gene expression in plant-microbe systems.

Evaluating rRNA as an indicator of microbial activity in environmental communities: limitations and uses

“…Two principal lines of evidence used to support rRNA as an indicator of current activity originate from earlier studies testing how rRNA scales with growth rate. First, total RNA and rRNA content correlate well with growth rate for a handful of microbes in pure culture, over a wide range of growth rates under balanced growth conditions (that is, growing in an unchanging environment) (Schaechter et al, 1958;Neidhardt and Magasanik, 1960;Rosset et al, 1966;Koch, 1970;Kemp et al, 1993;Kerkhof and Ward, 1993;Poulsen et al, 1993;Wagner, 1994;Bremer and Dennis, 1996;Ramos et al, 2000). Second, decreased rRNA content is associated with decreased growth rate for some organisms growing under specific nutrient-limiting conditions (Mandelstam and Halvorson, 1960;Davis et al, 1986;Kramer and Singleton, 1992;Tolker-Nielsen et al, 1997).…”

Biological and photochemical production of dissolved gaseous mercury in a boreal lake

“…Microbiology of the depth profile in Lake 658-The dominant actively metabolizing bacteria in the 5.5 and 12 m depth in September 2001 were identified by their 16s rRNA sequences (Ramos et al 2000). Fifty-four clones representing the community at 5.5 m depth were divided to 28 RFLP groups, and 49 clones representing the community at 12 m were divided to 27 RFLP groups.…”

Mode of action of Trichoderma asperellum SKT-1, a biocontrol agent against Gibberella fujikuroi

“…18,19) Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, also has been used for monitoring the fate and behavior of bacterial and fungal pathogens or biocontrol agents in situ. [20][21][22][23][24][25][26][27][28][29] GFP, unlike other biomarkers, 30) does not require any substrate or additional cofactors to fluoresce. Even a single cell expressing GFP can be easily detected among many cells under an epifluorescence microscope or a confocal scanning laser microscope (CSLM).…”