Swarming by Pseudomonas syringae B728a requires gacS (lemA) and gacA but not the acyl-homoserine lactone biosynthetic gene ahlI

Abstract

Pseudomonas syringae pv. syringae B728a, a causal agent of bacterial brown spot on snap beans, swarms with a characteristic dendritic pattern on semisolid (0.4%) agar plates. Filamentation of swarming cells of B728a was not observed. Mutations in either the gacS (formerly lemA) or gacA gene of B728a eliminate the ability of this P. syringae isolate to swarm without obvious effects on bacterial motility. Three field isolates showed a similar dependence on gacS for swarming. Since gacS and gacA mutants are known to be deficient in N-acyl-L-homoserine lactone (acyl-HSL) production, a mutant was constructed by disruption of the ahlI gene of B728a. This mutant did not make any acyl-HSL detectable by the Agrobacterium traG::lacZ reporter system, yet was unaffected in its ability to swarm. Other phenotypes of gacS and gacA mutations were similarly unaffected in the ahlI mutant.

FIG. 1

(A) Swarming ability of B728a and mutant derivatives. Cells were inoculated with an applicator stick to the center of an SWM plate (5 g of peptone, 3 g of yeast extract, and 4 g of granulated agar per liter; all medium components are from Difco) supplemented with 5 μg of tetracycline per ml to provide plasmid selection. Results were obtained after approximately 48 h of incubation at 28°C. NPS3136 (25) and BGACX (23) are gacS and gacA insertion mutants, respectively. The ability to swarm is restored to the appropriate mutant by plasmid-borne copies of gacS (i.e., pEMH97) (11) and gacA (i.e., pSyrgac23) (20), but not by the plasmid vector pLAFR3. The surface colony sizes of NPS3136(pLAFR3) and BGACX(pLAFR3) are about the same, with the larger apparent diameter of BGACX(pLAFR3) resulting from growth inside the agar matrix (see text). SWM medium in all swarming figures in this report was also supplemented with 1% potato infusion broth; this was done for consistency with other work in our laboratory and has no effect on swarming. (B) Swarming ability of field isolates and gacS mutant derivatives (19). Strains were inoculated on SWM medium (described above [B]) without tetracycline. Pictured are results after approximately 48 h of incubation at 28°C. Most of the visible growth of the gacS mutants represents subsurface spreading within the agar matrix, with the lighter spot at the center being the surface colony.

FIG. 2

Swarming ability of strain BHSL. The mutant was inoculated on an SWM plate as described above (Fig. 1A). The results shown were obtained after approximately 48 h at 28°C. (B) Foliar symptoms produced by the infiltration of the strains indicated into primary leaves of snap bean cultivar “Bush Blue Lake 274.” Bacterial cultures were adjusted to 10⁶ CFU/ml in sterile water and infiltrated as previously described (25). The leaf was digitally imaged 4 days postinoculation.