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. 2012 Aug;78(16):5753–5762. doi: 10.1128/AEM.01040-12

Fig 3.

Fig 3

Detection of proline released from B. subtilis cells by bioassays. (A) Five-microliter aliquots of JH642 (opuE+), BLOB9 [Δ(opuE::tet)1], and BLOB26 [Δ(opuE::tet)1 × pBLOB15.2 (opuE+ sapB+)] were spotted onto a lawn of an E. coli lacIZYA+ proline auxotrophic derivative (proC46::Tn5) of strain MG1655 seeded in top agar. The MMA plates and top agar contained 0.8 M NaCl to osmotically stress B. subtilis and thereby trigger osmoadaptive proline synthesis; IPTG and X-Gal were included in the plates to induce the lac operon and to visualize β-galactosidase activity of this indicator strain. Cross-feeding of the E. coli Pro− auxotroph by proline released from the B. subtilis strain BLOB9 [Δ(opuE::tet)1] is visible as a blue halo around the spotted B. subtilis cells. (B) The E. coli proline auxotrophic strain RC711 (proA23) was plated onto a lawn of MMA plates containing 0.8 M NaCl, and B. subtilis colonies were replica plated onto this cell lawn. Cross-feeding of strain RC711 by proline released by the opuE mutant strain BLOB9 is evident from growth of E. coli cells around individual B. subtilis colonies. (C) Blue halo bioassay with strains JH642 (opuE+), BLOB9 [Δ(opuE::tet)1] and the mechanosensitive (msc) channel quadruple mutants SMB80 (opuE+) and TMB105 [Δ(opuE::tet)1], and the E. coli proC46::Tn5 proline auxotrophic indicator strain.