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Bacterial Expression System> | Cell Reports 27, 2468–2479, May 21, 2019 2479 Rabbit polyclonal anti-FLAG Rabbit polyclonal anti-HtrA Mouse polyclonal anti-RpoB Bacterial Strains Escherichia coli TOP10 Msm mc2 155 Sigma Aldrich Helene Botella and Sabine Ehrt N/A ThermoFisher Scientific Cat#MA1-25425 Cat#89494-724 Cat#F7425 ThermoFisher Scientific (formerly Invitrogen) Laboratory of Barry Bloom Cat#404010 N/A N/A STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-Strep-tag II (NWSHPQFEK) GenScript Full list of strains in Table S2 This paper Chemicals, Peptides, and Recombinant Proteins MagStrep type3 XT Beads, 5% suspension IBA Life Sciences Cat#2-4090-002 Anti-FLAG M2 Magnetic Beads FLAG peptide Concanavalin A from Canavalia ensiformis (Jack bean) peroxidase conjugate Sigma Aldrich Sigma Aldrich Sigma Aldrich Cat#M8823 Cat#F3290 Cat#L6397 Pierce Avidin Agarose 10X Buffer BXT Deposited Data ThermoFisher Scientific Cat#20219 IBA Life Sciences Cat#2-1042-025 Full, uncropped Western blots This paper https://doi.... Membranes were blotted with rabbit a-FLAG (Sigma Aldrich) at 1:1000 in TBST + 5% milk, rabbit a-Strep (Genscript) at 1:1000 in TBST + 3% BSA, mouse a-RpoB (ThermoFisher Scientific) at 1:1000 in TBST + 5% milk, and rabbit a-HtrA (a generous gift from Helene Botella and Sabine Ehrt) at 1:1000 in TBST + 5% milk. | Get A Quote |
Mycobacterium tuberculosis, the causative agent of tuberculosis, withstands diverse environmental stresses in the host. The periplasmic protease HtrA is required only to survive extreme conditions in most bacteria but is predicted to be essential for normal growth in mycobacteria. We confirm that HtrA is indeed essential in Mycobacterium smegmatis and interacts with another essential protein of unknown function, LppZ. However, the loss of any of three unlinked genes, including those encoding Ami3, a peptidoglycan muramidase, and Pmt, a mannosyltransferase, suppresses the essentiality of both HtrA and LppZ, indicating the functional relevance of these genes' protein products. Our data indica... More