FIG. 4.

Upregulation of lpsS and exo genes by SyrA requires wild type chvI. A K214T mutation in chvI was introduced by transduction into lpsS::pVO155 strains (which contain an lpsS::uidA fusion) harboring vector alone or overexpressing SyrA. (A) Expression of lpsS::uidA fusion. Strains were grown to saturation (OD₆₀₀ of 2.5) and assayed for β-glucuronidase activity as described in Materials and Methods. Error bars represent standard deviations of experiments carried out in triplicate. Activity is in Miller units. Lane 1, lpsS::pVO155/pTE3 (vector); lane 2, lpsS::pVO155/pTE3::syrA; lane 3, lpsS::pVO155 chvI(K214T)/pTE3; lane 4, lpsS::pVO155 chvI(K214T)/pTE3::syrA; lane 5, exoY::pVO155 chvI(K214T)/pTE3; lane 6, exoY::pVO155 chvI(K214T)/pTE3::syrA. (B) Measurement of expression by RT-PCR. Lane 1, cDNA prepared from chvI(K214T)/pTE3, amplified with lpsS-specific primers; lane 2, cDNA prepared from chvI(K214T)/pTE3::syrA, amplified with lpsS-specific primers; lane 3, cDNA prepared from chvI(K214T)/pTE3, amplified with exoY-specific primers; lane 4, cDNA prepared from chvI(K214T)/pTE3::syrA, amplified with exoY-specific primers; lane 5, cDNA prepared from chvI(K214T)/pTE3, amplified with rpsF-specific primers; lane 6, cDNA prepared from chvI(K214T)/pTE3::syrA, amplified with rpsF-specific primers. (C) Upregulation of succinoglycan production by SyrA requires wild-type chvI. Strains were streaked out on plates containing calcofluor to determine succinoglycan production and photographed under UV light.