Table 1.
Bacterial strains and plasmids.
Fig 1.
Screening of Pseudomonas syringae pv. phaseolicola 1448A transposon disruption mutants.
Tn mutant colonies (48) were inoculated onto; A. standard agar plates and changes in colony morphology recorded after 72hr, B. soft agar plates and reduction in swarming ability recorded after 72hr. Selected mutants are highlighted with red arrows. WT, wild type.
Fig 2.
Examples of phenotypes of selected individual Pseudomonas syringae pv. phaseolicola transposon disruption mutants.
Colony size selected mutants were individually streaked onto agar plates and colonies observed after 48 h incubation at the same magnification. Individual swarming mutants were inoculated into the centre of a 9 cm soft agar plate and observed after 144 h incubation. 14-, Pph 1448A; 13-, Pph 1302A; WT, wild type.
Table 2.
Number of selected Pseudomonas syringae pv. phaseolicola transposon disruption mutants from 960 screened for each phenotype.
Fig 3.
Pseudomonas syringae pv. phaseolicola transposon disruption mutants selected in a biofilm attachment assay.
Transposon mutants and Pph 1302A wild type strain (WT) were cultured static in 10 ml broths. After seven days crystal violet was used to measure (OD570) the attachment of the cells to the culture vessel surface. Error bars represent standard error of the mean of three biological experimental replicates. *above bars indicate a significant difference compared to WT at p<0.05 assessed by a Student’s t-test.
Fig 4.
Insertion points of transposon hits on a map of the circular chromosome of Pseudomonas syringae pv. phasiolicola 1448A.
Outer two circles show positions of protein-coding genes on the plus and minus strands. Red dashes, Pph 1448A Tn hits (56); blue dashes, Pph 1302A Tn hits (41). The genome map was generated by using the DNAplotter [28].
Table 3.
Pseudomoas syringae pv. phaseolicola transposon insertions mutants identified in the flagellar and associated chemotaxis genes.
Fig 5.
In vitro growth of Pseudomonas syringae pv. phaseolicola transposon mutants.
Transposon mutants and wild type (WT) strains were inoculated in LB broth with shaking for 24 hr and OD600 recorded. Phenotypic screens: Sw, swarming reduction; S, small colony; L, large colony; Bf, biofilm formation. 14-, Pph 1448A; 13-, Pph 1302A. Error bars represent standard error of the mean of three biological experimental replicates. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.
Table 4.
Characteristics of selected Pseudomonas syringae pv. phaseolicola transposon disruption mutants.
Fig 6.
Complementation of selected Pseudomonas syringae pv. phaseolicola transposon disruption mutants.
Genes identified through transposon (Tn) insertion were cloned into a broad host range vector and transformed into their respective mutant strain (TnC). An empty vector was also transformed into the strains to use as a control (TnE). A number of tests were carried out with these strains: A. in vitro growth in LB broth after 16hrs; B. in planta growth in bean cultivar Canadian Wonder after 2 days; C. colony size after 2 days incubation, shown at the same magnification; D. swarming ability in soft agar after 5 days incubation, shown at the same magnification. 14-, Pph 1448A; 13- Pph 1302A; CHP, conserved hypothetical protein. *above bars indicate significant differences compared to WT at p<0.05 assessed with Students t-test.