Case study: Introduction of phenazine genes into Pseudomonas spp.
Phenazines are colourful, redox-active antibiotics produced by members of some fluorescent Pseudomonas spp. and a few other bacterial genera (Mavrodi et al., 2006). Phenazines are produced in the rhizosphere (Mavrodi et al., 2012), where they are involved in the suppression of plant pathogens (Chin-A-Woeng et al., 2003; Mavrodi et al., 2006; Thomashow et al., 1990), can act as electron shuttles (Hernandez et al., 2004; Rabaey et al., 2005) and contribute to the ecology (Maddula et al., 2008; Mazzola et al., 1992), physiology and morphology (Dietrich et al., 2008; Price-Whelan, 2006) of the strains that produce them. Expression of the core seven-gene phenazine (phz) biosynthesis operon (phzABCDEFG) is controlled in pseudomonads by homoserine lactone (HSL)-mediated quorum sensing (Mavrodi et al., 2006). Phenazines and quorum sensing are required for the establishment and development of biofilms on surfaces, seeds and roots (Maddula et al., 2008; Mavrodi et al., 2006). In the rhizosphere, expression of phz genes can be induced by homoserine lactones produced by heterologous isolates (Pierson et al., 1998; Pierson and Pierson, 2007) or quenched by HSL-degrading rhizosphere inhabitants (Morello et al., 2004).
A disarmed TnJ vector (pUT: Ptac-phzABCDEFG), originally constructed by L.S. Thomashow and colleagues, has been used extensively to stably introduce a single copy of the phenazine-1-carboxylic acid biosynthesis genes (isolated from Pseudomonas fluorescens 2-79) under the control of a Ptac promotor into Pseudomonas spp. from sources worldwide to improve biocontrol activity. Strains transformed with the phz locus also serve as model organisms to determine the impact of transgenes on the ecological fitness and the impact of recombinant strains and on the indigenous rhizosphere microbial community (Ryan et al., 2009). For example, the phz operon was introduced into Pseudomonas brassicacearum (formerly Pseudomonas fluorescens) Q8r1-96
(Loper et al., 2012), a strain that naturally produces the antibiotic DAPG and suppresses Take-all disease of wheat. Several recombinants of Q8r1-96 were selected (Z30-97, Z32-97, Z33-97 and Z34-97) and all produced greater amounts of PCA than strain 2-79, the source of the phz operon, because the genes were under the control of a constitutive promotor. Surprisingly however, addition of the phz genes also caused elevated production of DAPG in all of the transgenic strains as compared to the wild type Q8r1-96. Although the transgenic strains were no more suppressive of Take-all and Pythium root rot than Q8r1-96, they showed remarkable suppression of Rhizoctonia root rot at a dose of only 100 CFU seed-1, which was 100 to 1 000 times less than the dose required for similar disease control by the wild type Q8r1-96 (Huang et al., 2004).
In a similar study, Pseudomonas fluorescens SBW25 was transformed with the mini-TnJ vector carrying the phz genes and the transgenic strains gained enhanced ability to suppress Pythium ultimum damping-off disease of pea when compared to the wild-type strains SBW25 and 2-79 (source of the phz operon) (Timms-Wilson et al., 2000).
Some of the best studies of the population dynamics and non-target effects of transgenic BCAs in the field have been conducted with Pseudomonas putida strain WCS358r engineered to produce either PCA or DAPG by using the mini-TnJ vector system described above (Glandorf et al. 2001; Leeflang et al. 2002; Viebahn et al. 2003). PCA was shown to be produced in the rhizosphere by the transgenic strain, and both cultivation-dependent and independent methods indicated that the wild-type and transgenic strains had transient effects on the composition of the rhizosphere fungal and bacterial microflora of wheat. The effects of the transgenic strains sometimes were longer lasting than those of WCS358r, and differed from year to year and study to study. These results were similar to those of others conducted under controlled or field conditions and were not surprising given that strain WCS358r and other BCAs often establish high population sizes soon after inoculation, and then the densities decline over time and distance from the inoculum source. In addition, introduced BCAs do not become uniformly dispersed throughout the rhizosphere or among roots of the same or different plants. Collectively, these and other studies of the non-target effects of wild-type and recombinant BCAs indicate that even though the introduced bacteria have definite impacts on non-target microbial communities, the effects vary from study to study and are transient (Ryan et al., 2009).