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emi12005-sup-0001-FigS1.tif780K

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).

emi12005-sup-0002-FigS2.tif116K

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.

emi12005-sup-0003-FigS3.tif417K

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.

emi12005-sup-0004-FigS4.tif146K

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.

emi12005-sup-0005-TableS1.pdf396K

Table S1. Bacterial strains and plasmids used in this study.

emi12005-sup-0006-TableS2.pdf287K

Table S2. Primers used in this study.

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