LysoTracker Red DND-99 (Invitrogen) was used as a marker of lysosome or acidic

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1 information MATERIAL AND METHODS Lysosome staining LysoTracker Red DND-99 (Invitrogen) was used as a marker of lysosome or acidic compartments, according to the manufacturer s protocol. Plasmid independent autophagy induction by BPSS0180 Hela cells transiently expressing GFP-LC3 (pselect-ngfp-lc3; purchased from InvivoGen (San Diego, CA),) were grown on coverslips and transfected with constructs (pcdna 3.1D/V5-His-TOPO, Invitrogen) encoding His-tagged BPSS0180 using ŀsupplementary FuGENE HD transfection reagent (Roche). For plasmid independent autophagy induction studies in RAW264.7 cells, a DNA fragment containing the BPSS0180 gene was amplified by PCR and cloned into the mammalian expressing vector, pci-neo (Promega). RAW cells stably expressing GFP-LC3 were seeded on coverslips at 10 5 cells/well and transfected by the pci-neo-bpss0180 construct using FuGENE HD. 48 hours after transfection, cells were fixed with 4% formaldehyde solution and stained with 0.1mg/ml DAPI followed by imaging using CLSM. From the fluorescence images, the proportion of GFP-positive cells and the proportion of those exhibiting GFP puncta were determined.

2 Growth curve To understand whether the mutation of BPSS0180 might cause the slow growth of the bacteria, growth curves were determined by following the optical density (600nm) of overnight cultures. Bacteria strains were initially inoculated into LB medium containing appropriate antibiotics and grown overnight. The next day cells were diluted 1:50 dilution and grown for 3 hours in plain LB medium before further diluting the culture to achieve a starting optical density of Subsequent measurements of OD 600 were made every hour for the next 12 hours and at a final time point of 24 hours. Data are presented as OD 600 vs time. B. pseudomallei Transcriptome Profiling A detailed analysis of the B. pseudomallei microarray compendium will be presented in a separate report (Ong et al., manuscript in prep). Briefly, bacterial mrnas were profiled on a high-density B. pseudomallei tiling array representing both strands of the B. pseudomallei K96243 genome covering all 5855 annotated protein-coding genes (7.2 Mb) (Nimblegen) (50-mers, 15-base overlap). Total bacterial RNAs were isolated using Trizol, treated with TURBO DNase I (Ambion), and bacterial mrnas enriched using the MicrobExpress (Ambion) kit. Strand-specific cdnas were synthesized using SuperScript II reverse transcriptase (Invitrogen). Purified cdnas were labeled with either Cy5 or Cy3 fluorescent dyes (Cy5-ULS, Cy3-ULS, Kreatech Diagnostics), and hybridized to arrays as previously described (43). Individual Cy5 and Cy3 microarray profiles were acquired using an Axon scanner and normalized using the LOWESS algorithm. Microarray profiles were median-normalized prior to data analysis. Differentially expressed probes

3 were identified using Genespring GX11 software, using a 2.0-fold change cut-off. Log2- transformed fold change of BPSS0180 was computed by measuring the median value of fold-changes of all probes corresponding to the BPSS0180 genic region. Error bars were computed using standard deviations of transformed fold-changes of probes within genic regions. P values were computed using the Wilcoxon Sign Rank test comparing expression levels of BPSS0180 probes between test and reference conditions.

4 S. no Gene name Start Stop Protein (aa) BPSL BPSL BPSL Forward Primer Reverse Primer Product TTGTCGTCCATCGTTTCG ATGTTCCGCCCTCTCCG ATGAGCGATTGCACGAC G- S1: Primer sequences used for PCR amplification of selected genes Strand T- C- ŀtable length (bp) AGATTGTCAAGGTTGCGGTTC GGTTACGGCGCCTCATTAC- 482 GAACAGTTGCGCCGAGTC TCATCACCTCGACCAGCGTA- 471 BPSL TTGCGGAGCGTCGACAT-

5 5 GCCGAGCTGGATCAGTTGGC GAGTTCCCGATGCCTTC - BPSS GTCC- 6 GCGGGAGGCGGCGAGTT BPSS GCTGAGCCGCGCATTCG TT CGCGCGCGGCCTGGTCT GTCTGCGTGTGCGTCGCGGTTG- BPSS T ATGACCCCCAGCAGAAA GGTTTCGTCCGCGCTCCACAG- BPSS ACCG- 9 ATGAATCCATTCTTTGGC GAACGAATCAACCGAAACG BPSL G - 10 BPSS ATGCCGAAGCGTGAGGC CGGCGACAATGCGCCATC- 2985

6 G ATGCAACGCTGTCTCGC GCTATAGCCGTAGCAGCGTCTGA BPSS CCAC ATGTCGCGTCAGATTCG TAGTCCCGTACCGCTCGCCTTC- BPSS ATTTTG- 13 ATGCATCGCCAGGCACGC- 606 BPSS ATGTCATACATCATCGCG GGCC ATGAAAATCGCCATCGTC GGCCTGCTGATGTTCGATAA BPSL CCGTTAGACGGTTGCCAGA BPSL ATGGCAGGCTCGCGAAT A- 16 BPSL CGACGCGAGCCGGCTTCAG - 951

7 2 ATGTCGTCCCCCCGTTT GTCC- 17 CGTCTTCGAGCGTGTCTG BPSL ATGCAGGCATTCCGTTG C - 18 TCGTGCGTGTCATTTCCAC BPSL ATGACGCAAAGCAGCCA CT- 19 GCGGGTTCATTCATGGTAGT BPSL ATGACCCATCCTCTGTTC AC TCGAACCTTTGCCGTGA CGTTAAAATTTTTGAATGGACGTG BPSL G CAGTTCAAGGTCGCGTGAT BPSL ATGCCTTTCGTCACGATC -

8 22 TCGATCATGCCGATCCA GCATCGACGCTCGCAAC BPSS C- 23 ACA CCG CTA GCC CTG CTG ATGAGCAAGAGCGAGAT BPSS CGAC- 24 CCATGAAGTGATGGGGGATG CAGGCAGGGAGGAAGCA BPSS T- 25 TCCCGCCCACTACTTGAC BPSS GGCTTCAAAATGCAGAG AACT- 26 TTCGAACGCATAGTCGATACT ATGACGGACCCATTTTC C- BPSS G- 27 BPSL ATGATCCGTCTTGCGAC ATTC- GGGTTGAAACGGATGCAC- 2630

9 CCGGCTTGACCTTCATGG BPSS GTGCTGGTGGGCAGTCT G GTGCGGGGCGATCTCAT GTATCTCGTCAAGCGCATTC BPSS G ATGAAGCAAACGAAGCA GAACTTGTGCCGAATTCCGACGA- BPSS TCTCGC-

10 Table S2. Conditions inducing BPSS0180 Regulation Conditions inducing BPSS0180 Up regulation Test conditions Reference conditions Fold Duration of Temp of Reference Duration of Temp of change Specific condition condition Minimal medium (Chemically Defined Medium (CDM), Mid-log phase) 8hrs 37 C LB media 8hrs 37 C Minimal medium(chemically Defined Medium (CDM), Stationary phase) 17hrs 37 C LB media 22hrs 37 C ph Acidic condition (ph4.0 (HCl), 1XDPBS media) 16hrs 37 C 1XDPBS media 16hrs 37 C Nutrient Deprivation (Deionized water) 1hr 37 C LB media 24hrs 37 C Oxidative stress (100mM Hydrogen peroxide, LB medium 10min 37 C LB media 20min 37 C

11 Conditions inducing BPSS0180 Down regulation Test conditions Reference conditions Fold Duration of Temp of Reference Duration of Temp of change Specific condition condition Taurine as sulfur source (250uM Taurine, Modified M63 media, Mid Log phase) 24hrs 37 C LB media 8hrs 37 C Taurine as sulfur source (250uM Taurine, Modified M63 media, Stationary phase) 48hrs 37 C LB media 30hrs 37 C

12 S1: BPSS0180 induced punctuate-like structures colocalizing with lysosomes. A, Representative confocal images of Hela cells transfected with empty vector (green) or GFP-tagged BPSS0180 (green), ŀfig. or treated with rapamycin. Cells were fixed, permeabilized, and stained for nucleus (blue) and lysotracker red, a marker of lysosomes or acidic compartments (red). Arrows indicate punctate-like structures colocalized with lysosomes. Colocalization of GFP puncta with lysosomes was defined by the presence of green puncta induced by GFP-tagged BPSS0180 (green), which were fully overlaid by intense lysotracker red staining. Scale bars represent 10 µm. B, Quantitative analysis of cells with GFP puncta colocalized with lysosomes. Asterisk (*) indicates P<0.05 relative to cells transfected with empty vector.

13 Fig. S2: BPSS0180 induces autophagy in phagocytic cells (RAW264.7). A, Representative confocal images of RAW264.7 macrophage cells transfected with empty vector (green) or BPSS0180 (green), treated with rapamycin. Cells were fixed, permeabilized, and stained with DAPI for the nucleus (blue) and LC3 (red). Arrows indicate punctate-like structures colocalized with LC3. Colocalization of puncta with LC3 was defined by the presence of green puncta induced by GFP-tagged BPSS0180 (green), which were fully overlaid by intense Alexa flour-594conjugated anti-lc3 red staining. Scale bars represent 10 µm.