9% clay, with a pH level of 8.3; for a more detailed description of soil properties see [24]) in a 35 ml Pyrex test tube. Prior to inoculation Nevada soil was sifted with 1 mm2 screen. Inoculation resulted in a wetting event. Soil water

content throughout the experiment selleck chemicals llc varied from fully saturated conditions (0 kPa) to permanent wilting point (-1500 kPa). Tubes were capped. Growth and persistence in soil depends on functional DapB (Figure 1). Strains that grow in soil carry promoters in the genomic fragment which activate dapB transcription, thus rescuing the no-growth phenotype. To carry out two rounds of seven- day soil exposure, a soil sample of 1 g from inoculated soil was recovered, suspended in 9 mL dH2O, and 1mL of suspension was used to inoculate a further 5 g of soil. Bacteria were allowed to grow in this soil for an additional 7 days. Figure 1 Growth and persistence in Nevada arid soil of P. fluorescens Pf0-1 carrying mutations in arid soil-induced genes relative to wild-type Pf0-1 and Pf0-1Δ dapB . A. When inoculated at relatively high density, the sif2 (Pfl01_2143) mutant fails to maintain the population density reached by wild-type Pf0-1 while the sif10 (Pfl01_5595) mutant shows no aberrant phenotype. B. When inoculated at relatively lower density, the sif10 (Pfl01_5595) mutant fails to establish the same population level as wild-type click here Pf0-1, whereas the sif2 (Pfl01_2143) mutant is

indistinguishable from wild-type. In both panels, error bars represent 4 replications. Error bars represent standard errors. Anova for these experiments indicates significant values at P ≤0.01. For the experiments in 1A, difference values between

any two means that were greater than 0.11 (day1), 0.05 (day3) and 0.08 (day7) denoted statistical significance. For the experiments in 1B, difference values between any two means that were greater than 0.07 (day1), 0.07 (day3) and 0.11 (day7) denoted statistical significance. After the second 7-day period, a suspension was made from 1 g of soil (as described above), diluted, selleck compound and plated onto Pseudomonas minimal medium supplemented with diaminopimelic acid (DAP) and X-gal, and ampicillin and tetracycline to select IVET strains. Control plates indicated that these conditions were effective at inhibiting growth of indigenous bacteria. White colonies presumed to contain soil-activated promoters fused to dapB were chosen for further study. We surmised that blue colonies carry fusions active in both soil and laboratory; these were not studied further. Sequence and promoter analysis DNA sequences from the 30 soil induced fragments (sif) were blasted against the Pf0-1 annotated genome. Based on their match to the annotated genome, sifs were grouped into metabolism, transport, regulation and poorly characterized genes categories (Table 3). In addition to BLAST analysis, promoter scans of the regions upstream of sifs were conducted using PromScan (http://​molbiol-tools.