Pseudomonad Cyanogenesis Figure 5. Suppression of B. subtilis biofilm formation on A. thaliana Col-0 roots by indirect exposure of the pseudomonad strains and cyanide (A) and the effect of indirect exposure of the pseudomonad strains and cyanide on single cell growth of B. subtilis (B). The images show complete suppression of B. subtilis biofilm formation when exposed to the indirect exposure of the strains PAO1, PA14, CHAO and HCN when compared to control plants not exposed to either bacterial culture or HCN and DhcnB mutants PAO6344 and CHAO77. The data also shows extensive colonization and biofilm formation by B. subtilis with indirect bacterial exposure from the hcnB mutants PAO6344 and CHAO77. The images were representative of the roots from six independent plants imaged. doi:10.1371/journal.pone.0002073.g005 nad strains resulted in acute primary root growth inhibition. However, a reduced growth inhibition was observed with the cyanogenic mutants PAO6344 and CHAO77, giving an indica- tion for the involvement of cyanide in the growth inhibitory effect. Our observation was partially supported by the fact that cyanide is reported as a strong virulence factor of P. aeruginosa [1]. Similarly, the compartment plate assays demonstrated that indirect exposure of the strains exhibited a similar growth inhibitory phenotype as observed previously with the direct plate assays. In addition, exogenously supplied cyanide in the form of KCN and HCN caused similar root inhibitory effects. The cyanide in pseudomo- nads is produced by the oxidative decarboxylation of glycine by a membrane bound three-subunit enzyme encoded by hcnABC [20]. The reduced growth inhibitory effect observed with the indirect exposure of the two cyanide synthase mutant strains, one each from P. aeruginosa and P. fluorescens, indicated the direct role of cyanogenesis. However, the primary root growth inhibitory effect observed with the direct plate assay may be due to the interference of other virulence factors in the pseudomonads, in addition to cyanide. This observation was well supported with the quantitative data on cyanide production recorded in different pseudomonad cultures in the present study. The observed indirect effects in PLoS ONE | www.plosone.org 7 April 2008 | Volume 3 | Issue 4 | e2073