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. 2019 Jan 30;10:85. doi: 10.3389/fmicb.2019.00085

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Copyright © 2019 Jin, Zhang, Zhu, Peng, Weng, Zhao, Liu, Bai, Cheng, Jin and Wu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The decrease of cAMP is not caused by changed expression of CyaA, CyaB or CpdA. (A) Relative mRNA levels of cyaA, cyaB, and cpdA. Total RNA of indicated strains was isolated and the mRNA levels of these genes were determined by real time PCR with rpsL serving as an internal control. ns, not significant, ^∗p < 0.05 by Student’s t-test. (B) Indicated strains containing a cyaA-Flag (cyaB-Flag or cpdA-Flag) driven by its native promoter were grown at 37°C with or without 5 mM EGTA until OD₆₀₀ of 1.0. Samples from equal number of bacteria were separated by SDS-PAGE and probed with an anti-Flag antibody or an anti-RNA polymerase beta subunit antibody. (C,D) Indicated strains containing a cyaA-Flag or cyaB-Flag driven by a tac promoter or the empty vector pMMB67EH were grown to an OD₆₀₀ of 1.0 in LB with 1 mM IPTG with or without 5 mM EGTA. Proteins from equivalent bacterial cells of indicated strains were separated by SDS-PAGE and probed with an anti-ExoS antibody, an anti-Flag antibody or an anti-RNA polymerase beta subunit antibody. S, supernatant; P, pellet.