Soil Colonization and the discovery of adnA
In the environment the ability of organisms such as P. fluorescens to survive and persist in the soil gives them a competitive advantage over other microorganisms. Compeau et al. 1988. developed a soil growth assay using sterile gamma irradiated soil. P. fluorescens Pf0-1 was isolated during that study and has been used extensively to examine the genes involved in soil colonization (Deflaun et al., 1990; Deflaun et al., 1994; Marshall et al., 2001). Two transposon insertion mutants, Pf0-5 and Pf0-10, were identified to be deficient in adhesion to quartz sand, soil, and seeds (Deflaun et al., 1990; Deflaun et al., 1994). Casaz et al. 2001. identified the site of insertion of the transposon was in the same gene, named adnA for deficient in adhesion, which encodes the transcriptional regulator AdnA. adnA mutants are also deficient in colonization, spread, and persistence in soil (Marshall et al., 2001).
AdnA a homolog of FleQ :
AdnA is a homolog of FleQ (83% identity, (Casaz et al., 2001.)), the master regulator of flagellar biogenesis in Pseudomonas aeruginosa (Arora et al., 1997; Dasgupta et al., 2003). Transcriptional regulation of flagella biogenesis in P. aeruginosa is separated into a four-tiered regulatory circuit (Dasgupta et al., 2003). This regulation cascade involves several transcription regulators and sigma factors: FleQ (Arora et al., 1997., Dasgupta et al., 2003), FleSR (Ritchings, et. al. 1995., Dasgupta, et al. 2003.), FliA (σ28) (Dasgupta et al., 2002), σ54 (Starnbach & Lory, 1992), and σ70 (Dasgupta et al., 2002; Totten et al., 1990), which are all required for the regulation of flagellar associated genes.
It is not fully understood if FleQ is under the control of environmental stimuli or that the transcription of fleQ is enhanced by any regulatory proteins. However, it is known that σ70 is required for transcription of fleQ (Dasgupta et al., 2002). Also, FleR, FliA, and FleQ do not have an effect on the transcription of the fleQ gene (Dasgupta et al., 2003). Several proteins have been shown to negatively regulate fleQ either by binding to the DNA upstream of the gene or interacting directly with FleQ. AlgT has been seen to negatively effect the synthesis of flagella in P. aeruginosa, but only in a mucA- background (Garrett et al., 1999; Tart et al., 2005; Tart et al., 2006). This effect on flagellar synthesis by AlgT is mediated through the transcriptional regulator AmrZ (AlgZ) (Tart et al., 2005; Tart et al., 2006), which has been shown to bind to the fleQ promoter in vitro (Tart et al., 2006). Another protein, FleN has been shown to regulate flagellar number in P. aeruginosa (Dasgupta et al., 2000). FleN was shown to interact directly with FleQ using a yeast two hybrid system, however this interaction has no effect on DNA binding ability of FleQ (Dasgupta & Ramphal, 2001). Vfr a homolog of CRP in Escherichia coli was also seen to bind the fleQ promoter (Dasgupta et al., 2002). Other factors such as activators may be involved in the regulation of fleQ, but none have been found.
Above is a motility assay of Pf0-1 (left) and Pf0-2x (adnA-, right) performed on 0.3% Pseudomonas minimal medium (PMM) grown at 30 degrees Celsius for ~20 hours. The adnA mutant (right) is non-motile, which is due to the lack of flagella (shown below).
Novel genes involved in motility:
In recent years previously unknown genes involved in motility have been found. The fliT/fleP gene is a gene in P. fluorescens/P. aeruginosa located in the same region, but each has a different function in these species (Capdevila et al., 2004.). fleP is regulated by FleQ in P. aeruginosa (Dasgupta et al., 2003.). The fliT homolog in P. fluorescens Pf0-1 has been identified, using a microarray, to be under the control of AdnA. FliT has been shown to be involved in motility in P. fluorescens F113. Further investigations are being done looking into other novel genes that may be involved in motility, which could help deduce their functions. The genes being examined for thier roles in motility and then soil colonization are controlled by AdnA and most of these are not present in P. aeruginosa.
The role of AdnA in biofilm formation has also been examined. adnA mutants are deficient in biofilm formation on surfaces such as borosilicate glass (Casaz et al., 2001, Robleto et al., 2003), polypropoylene, and polystyrene (Robleto et al., 2003). The defect in biofilm formation is linked to the lack of flagella, which are required for enhanced attachment to these surfaces (Casaz et al., 2001, Robleto et al., 2003).
Pictures will be added.
Environmentally induced genes:
Two P. fluorescens strains are being used to evaluate genes induced in their natural environment either in the plant rhizosphere (SBW25, Rainey, 1999), on the plant surface (SBW25, Giddens et al., 2007), or in the soil (Pf0-1, Silby & Levy. 2004). Utilization of these three methods has identified over 200 genes that are induced in the natural environment of P. fluorescens. Giddens et al. were able to develop a regulation hierarchy model for several of these genes. This work is ongoing by many labs and may help to improve the beneficial qualities of many P. fluorescens strains.