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cmi12221-sup-0001-figs1.tif1418KFig. S1. Coomassie stained gel used for nanoLC-MS/MS analysis. Detergent extraction of cell envelopes of M. marinum wild type (wt) and secA2 mutant in duplo (sec_I and sec_II). Fractions detergent pellet and detergent supernatant in a 1:1 ratio are loaded on SDS-PAGE and stained by Coomassie.
cmi12221-sup-0002-figs2.tif1015K

Fig. S2. Expression levels of HA-tagged constructs.

A. Immunoblots of total lysates from M. marinum wild type (wt), secA2 mutant (sec) and complemented secA2 mutant (c) expressing IipA-HA behind hsp60 promoter in liquid culture.

B. Immunoblots of total lysates obtained directly from plate-grown strains expressing HA-tagged constructs with dppA, iipA and MMAR_3060 behind their own promoter. Note that the expression of Mm3060 in the plate-grown bacteria seems higher than in culture (Fig. 2). Blots were probed with an antiserum against the HA epitope. Anti-GroEL2 is used as loading control.

cmi12221-sup-0003-figs3.tif156KFig. S3. Quantification: three putative substrates are dependent on SecA2 for stability and/or processing. Relative staining levels of different fractions containing HA-labelled proteins, quantified by densitometric analysis after immunoblotting with an antiserum directed against the HA epitope, expressed as a fraction of wild type total lysate levels. The following fractions of M. marinum E11 wild type (wt), secA2 mutant (SecA2) and complemented mutant (comp) were loaded on a polyacrylamide gel: total lysate (T), cytosol (cyt), cell envelopes (CE), detergent-treated CE pellet (DP) and detergent-treated CE supernatant (DS). Strains contained HA-labelled (A) DppA, (B) MMAR_3060 or (C) IipA respectively. Quantification of the DppA and MMAR_3060 signal was performed on the observed band, whereas for IipA the whole lane was quantified. Results represent the mean of three independent experiments with standard deviation.
cmi12221-sup-0004-figs4.tif58KFig. S4. Quantification: SecA2-dependent cell wall localization of PknG. Relative staining levels of PknG (upper band), quantified by densitometric analysis after immunoblotting with an antiserum directed PknG, expressed as a fraction of wild type total lysate levels. The following fractions of M. marinum E11 wild type (wt), secA2 mutant (secA2) and complemented mutant (comp) were loaded on a polyacrylamide gel: total lysate (T), cytosol (cyt) and cell envelopes (CE). Results represent the mean of three independent experiments with standard deviation.
cmi12221-sup-0005-figs5.tif957KFig. S5. Effects of SecA2 on antibiotic susceptibility. Meropenem E-test result of a 1:1 mix of M. marinum wild type (wt) containing the pSMT3-eGFP vector and the secA2 mutant (secA2) containing the pSMT3- mCherry vector monitored by fluorescent microscopy. At increasing meropenem concentration (top), growth of the secA2 mutant (right panel) is more restricted than that of wild type bacteria (middle panel). This result is representative for 2 independent experiments.
cmi12221-sup-0006-figs6.tif275K

Fig. S6. A. Fractionation of M. bovis BCG. Total lysate (T), cytosol (cyt) and cell envelopes (CE) were loaded on a polyacrylamide gel in 1:1:1 ratio, transferred to a nitrocellulose membrane and immunostained using antisera against EccC5, PknG and GroEL2.

B. Intracellular replication of secA2 mutant. J774 macrophages were infected at an moi of 0.1 with M. marinum wild type (wt + pMV), M. marinum WT overexpressing PknGBCG (wt + pMV::PknG), the secA2 mutant (secA2 + pMV) and the secA2 mutant overexpressing PknGBCG (secA2 + pMV::PknG). The number of cfu was determined by lysing infected J774 cells and plating lysates at the indicated time-points. Graph represents mean ± SEM from three independent experiments.

C. Quantification of infection of embryos with wild type M. marinum (wt + pMV) and wild type M. marinum overexpressing PknGBCG (wt + PknG) at 5 dpi. Each data point represents the number of cfu per embryo, as determined by plating and the bars indicate means after log transformation. Mean ± standard deviation of inocula were 134 ± 19 and 127 ± 28 cfu respectively. The graphs represent data of 4 to 5 independent experiments.

cmi12221-sup-0007-tables1.xls883KTable S1. Proteins identified by nanoLC-MS/MS analysis. Supplied separately as Microsoft Excel file.
cmi12221-sup-0008-tables2.doc36KTable S2. Primers used in this study.

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