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SUPPLEMENTAL INFORMATION SUPPLEMENTAL EXPERIMENTAL PROCEDURES

MSMEG_4247 Gene Deletion — The 5' and 3' regions (~1 kb) of MSMEG_4247 gene were PCR-amplified from genomic DNA (primers 005/006 and 007/008, Table S1). The amplified fragments were ligated with a kanamycin-resistance cassette, excised from pHBJ395 (1) using HindIII and EcoRI, and inserted into vector pHBJ428 (carrying the streptomycin resistance gene and the sacB gene for selection) (1) at the SpeI and NotI sites. The resultant construct, designated pYAB029, was electroporated into M. smegmatis strain mc2155, and mutants were selected as before (2), by resistance to kanamycin and sucrose (loss of sacB), and sensitivity to streptomycin.

Southern Blot Analysis — Aliquots of 15 µg of EcoNI-digested DNA were electrophoresed in a 0.8% agarose gel, blotted onto Hybond N+ nylon membrane (Amersham Biosciences), and hybridized as previously described (2). The probe was PCR-amplified from genomic DNA (primers CH036/CH037).

Rv2181 Gene Deletion and Complementation — The 5ʹ′ and 3ʹ′ regions (~1 kb) of the Rv2181 gene were PCR-amplified from genomic DNA (CH001/CH005 and CH006/CH007, Table S1). The 5ʹ′ fragment was initially cloned into the XbaI/HindIII sites of pBluescript II KS+, and then digested with NotI and HindIII. PCR fragments were then ligated into a thermosensitive vector pPR23-1 (3) at the NotI and SpeI sites together with a kanamycin-resistance cassette excised by HindIII and EcoRI from pHBJ395 (1). The resultant construct (pYAB266) was electroporated into M. tuberculosis strain H37Rv. The Rv2181 deletion mutant was selected by resistance to kanamycin, sucrose (loss of sacB gene) and high temperature (non-autonomous replication at 39°C). For integrative Rv2181 expression vectors driven by the endogenous promoter, the region spanning Rv2179c-Rv2181 was first amplified using the primers 243/244, and cloned into the EcoRV site of pYAB184 by blunt-end ligation, resulting in pYAB203. The HindIII/NspI fragment was then removed, resulting in pYAB228 that carries Rv2181 with its 242 bp upstream sequence. For an integrative Phsp60-driven expression vector, Rv2181 was amplified using primers 055/056 (Table S1), cloned into the MscI/EcoRI sites of pHBJ334, excised by XbaI/NheI, and sub-cloned into pYAB184 at EcoRV site by blunt-end ligation, resulting in pYAB230.

SUPPLEMENTAL REFERENCES 1. Jeevarajah, D., Patterson, J. H., Taig, E., Sargeant, T., McConville, M. J., and

Billman-Jacobe, H. (2004) J Bacteriol 186, 6792-6799

2. Morita, Y. S., Sena, C. B., Waller, R. F., Kurokawa, K., Sernee, M. F., Nakatani, F., Haites,

R. E., Billman-Jacobe, H., McConville, M. J., Maeda, Y., and Kinoshita, T. (2006) J Biol

Chem 281, 25143-25155

3. Pelicic, V., Jackson, M., Reyrat, J. M., Jacobs, W. R., Jr., Gicquel, B., and Guilhot, C. (1997)

Proc Natl Acad Sci U S A 94, 10955-10960

4. Notredame, C., Higgins, D. G., and Heringa, J. (2000) J Mol Biol 302, 205-217

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SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1. Generation of the MSMEG_4247 deletion mutant (∆MSMEG_4247). (A) Strategies for disrupting the MSMEG_4247 gene. Black arrow represents the MSMEG_4247 gene. Gray arrows represent the flanking genes. E, EcoNI sites. Kan, kanamycin-resistance cassette. Gray bar represents the region of DNA recognized by the Southern blot probe used in panel B. (B) Southern blot using EcoNI-digested genomic DNA from wild-type (lane 1) and ∆MSMEG_4247 mutant (lane 2). Supplemental Figure S2. Expression levels of MSMEG_4247 do not affect the profiles of PIMs and phospholipids. (A) PIM or (B) phospholipid profile of ∆MSMEG_4247 mutants complemented by various expression vectors was analyzed by HPTLC. PIMs and phospholipids were visualized by orcinol staining and molybdenum blue staining, respectively. Lane 1, wild-type; lane 2, ∆MSMEG_4247; lanes 3 and 4, two clones of ∆MSMEG_4247+Phsp60MSMEG_4247 (pYAB250); lanes 5 and 6, two clones of ∆MSMEG_4247+P4247MSMEG_4247 (pYAB247) and lanes 7 and 8, two clones of ∆MSMEG_4247 transfected with empty integrative vector (pYAB184). CL, cardiolipin; PE, phosphatidylethanolamine. Note that the levels of AcPIM2 were slightly reduced in ΔMSMEG_4247+Phsp60MSMEG_4247 strains (panel A, lanes 3 and 4), possibly suggesting that over-expression of MSMEG_4247 enhances LM/LAM biosynthesis and slightly depletes the AcPIM2 pool. Supplemental Figure S3. Over-expression of D45A-mutagenized MSMEG_4247 in ∆MSMEG_4247 mutants does not affect the profiles of LM and LAM. (A) T-Coffee alignment (4) of a region of MSMEG_4247 homologs from various mycobacteria. MT, M. tuberculosis; MB, M. bovis; MAP, M. avium paratuberculosis; ML, M. leprae and MS, M. smegmatis. Aspartic acid at position 45 (D45) of M. smegmatis protein is indicated by an arrow. D58 of M. smegmatis PimE (PimE-MS) aligns with D45 of MSMEG_4247. (B) LM/LAM profiles of ∆MSMEG_4247 mutants transfected with Phsp60-driven expression vectors for D45A-mutagenized MSMEG_4247 were analyzed by SDS-PAGE and visualized by carbohydrate staining. Lane 1, wild-type; lane 2, ∆MSMEG_4247; lanes 3 and 4, ∆MSMEG_4247 transfected with pYAB251, an empty episomal vector; lanes 5 and 6, ∆MSMEG_4247 transfected with pYAB250, an episomal vector to express MSMEG_4247 driven by Phsp60; lanes 7 and 8, ∆MSMEG_4247 transfected with pYAB255, an episomal vector to express MSMEG_4247 D45A mutant driven by Phsp60. (C) MSMEG_4247 expression examined by Western blotting using anti-MSMEG_4247 antibody. Lanes are arranged in the same order as panel B. Loading was adjusted to 5-µg protein per lane for lanes 1- 4 or 0.25 µg per lane for lanes 5-8. Note that MSMEG_4247 D45A is expressed as efficient as wild-type MSMEG_4247. Supplemental Figure S4. Intensity profiles of lanes 2 (gray line), 4 (black line), and 6 (white line) in panel B of Figure 4. The black and white arrowheads indicate the positions of wild-type LAM and LM, respectively. Supplemental Figure S5. Generation of M. tuberculosis Rv2181 deletion mutants. (A) Schematic of the Rv2181 genomic region showing the strategy of targeted gene deletion and the binding sites of PCR primers used in panel B. (B) The Rv2181 gene deletion was confirmed by PCR. Lane M, molecular weight markers; lane 1, wild-type; lane 2, ∆Rv2181 mutant. Sizes of fragments expected from the indicated primer combinations are shown in panel A.

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SUPPLEMENTAL TABLE S1

PCR primers used in this study. Restriction enzyme sites are underlined.

Names Sequences MSMEG_4247 deletion (5ʹ′) (005/006)

GAGCACTAGTCGGACCAGTACACGTCGGACAGATG (SpeI) GTCCAAGCTTCCGGATCGGTTCGAGATAGATCACC (HindIII)

MSMEG_4247 deletion (3ʹ′) (007/008)

CGAGGAATTCACGCTGAACACCAACCAGAACATCG (EcoRI) GGAGACGGCGGCCGCACGGCAGGCTCTACAAGGGCAAGAC (NotI)

MSMEG_4247 Southern blot (CH036/CH037)

TGGTAACTGCCGGTAAAGCGC CGCATGAACCAGTTCGCCTCG

MSMEG_4247 cloning (053/054)

CTAAGCGGCAGTCGCCCCGCG GCGAATTCAGCCGGCCTCGCCGCGTG (EcoRI)

MSMEG_4247 cloning (312/313)

CATCGATCGATGCGGATGTAGCGCACCAC (ClaI) GCTCTTCTAGATCCTGAACGGGCCGGTCA (XbaI)

MSMEG_4247 D45A (320/321)

GCCCTACCGCATCGCCATCGACGTGTACC GGTACACGTCGATGGCGATGCGGTAGGGC

MSMEG_4241 cloning (329/330)

ACTTGGGATCCATGACACCGACGGAAACCCA (BamHI) TCGGTTCTAGACTATTGACGGCTCGCCGT (XbaI)

Rv2181 deletion (5ʹ′) (CH001/CH005)

CCCAAGCTTTCATCAGCACCACATTGATCTGAC (HindIII) GCTCTAGATACCGACGTGCGTCCGTCTT (XbaI)

Rv2181 deletion (3ʹ′) (CH006/CH007)

CGGAATTCCGGCGCTGCCGCCTTGAACACAGACCAGAA (EcoRI) GGACTAGTATCGACCGCACCAATGCCGACT (SpeI)

∆Rv2181 PCR (CH018, CH020, CH022)

CCACCCAGTTGGCGCCTTG GCCATGTAAGCCCACTGCAAGC GGTGGCGGATAGCTTCTACCTTCC

Rv2181 cloning (055/056)

CTGCATGGCGGGCGCCCGAGGTGGGCAG CGGAATTCTAGTCAGCTGGCCGTCGGCG (EcoRI)

Rv2181 cloning (243/244)

AATATTTGACTGTCCGTCTCCTCAGCG (SspI) CACGTGAATCCCCGAGACAGCCGC (PmlI)

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