These authors contributed equally to this work.
A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis
Article first published online: 24 OCT 2012
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd
Special Issue: Environmental Ecology of Pathogens and Resistances
Volume 15, Issue 2, pages 557–569, February 2013
How to Cite
Zhang, W., Wang, Y., Song, Y., Wang, T., Xu, S., Peng, Z., Lin, X., Zhang, L. and Shen, X. (2013), A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis. Environmental Microbiology, 15: 557–569. doi: 10.1111/1462-2920.12005
- Issue published online: 28 JAN 2013
- Article first published online: 24 OCT 2012
- Accepted manuscript online: 28 SEP 2012 06:05AM EST
- Manuscript Accepted: 23 SEP 2012
- Manuscript Received: 5 MAY 2012
- National Natural Science Foundation of China. Grant Numbers: 31170100, 31170121, 31270078
- Specialized Research Fund for the Doctoral Program of Higher Education of China. Grant Number: 20110204120023
Fig. S1. Putative OmpR binding sites O1, O2 and O3 (marked in red) in the T6SS4 promoter region. The ATG start codon of the first ORF of T6SS4 operon was also marked in red. Letters underlined denote the sequences used to replace the three binding regions (M1, M2 and M3).
Fig. S2. A. Standard curves made for changes in the excitation/emission wavelengths for GFP fluorescence at different pHs. pH dependence of fluorescence of GFPmut3* in wild-type strain YpIII, ΔclpV4, Δhcp4 and ΔompR were measured. The fluorescence intensity was measured in a microplate fluorometer as described in Experimental procedures. pHi and pHo were equilibrated by incubation with carbonyl cyanide m-chlorophenyl-hydrazone (1 mM).
B. Standard curve made for changes in the ratio of 480 nm (peak fluorescene) and 435 nm excitation wavelengths for BCECF at different pHs. pH dependence of fluorescence of BCECF in wild-type strain YpIII containing plasmid pBS was measured. pHi and pHo were equilibrated by incubation with K+ and nigericin as described in Experimental procedures.
Fig. S3. The ATPase activity of ClpV4 is responsible for Hcp4 secretion. Complementation of the ΔclpV4 secretion defect by plasmid-encoded ClpV4 and ClpV4M using Hcp4-specific antibodies. The metabolic protein phosphoglucose isomerase (Pgi) was probed as a loading control. The Pgi protein was not detected in the culture supernatant, indicating that the secretion of Hcp4 was not a consequence of cell lysis. Sup: culture supernatant; Pellet: total cell pellet.
Fig. S4. A. Western blot analysis of VgrG4His production and secretion in the wild type, hcp4 and clpV4 mutants containing pBS-VgrG4His after growing to early stationary phase at 26°C using an anti-His monoclonal antibody.
B. Overexpression of vgrG4GFP prevents secretion of Hcp4. Western blot detection of Hcp4 expression and secretion in the strains YpIII wild type, YpIII(pBS-vgrG4GFP) and YpIII(pBS-vgrG4His). The metabolic protein phosphoglucose isomerase (Pgi) was probed as a loading control. The Pgi protein was not detected in the culture supernatant, indicating that the secretion of Hcp4 was not a consequence of cell lysis. Sup: culture supernatant; Pellet: total cell pellet.
Table S1. Bacterial strains and plasmids used in this study.
Table S2. Primers used in this study.
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