Beyond antibiotic resistance: the whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria

. Author manuscript; available in PMC: 2025 Apr 18.

Published in final edited form as: Cell Chem Biol. 2024 Jan 23;31(4):669–682.e7. doi: 10.1016/j.chembiol.2023.12.020

Key Resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-FLAG M2 antibody	Sigma Aldrich	Cat# F1804
Bacterial and virus strains
E. coli NEB 5-alpha	New England Biolabs	C2987H
M. tuberculosis H37Rv	Christopher Sassetti (UMass Worcester)	NA
M. smegmatis mc² 155	Sarah Fortune (Harvard)	NA
$Δ$ whiB7 M. smegmatis mc² 155	Carl Nathan (Weill Cornell)	Schrader et al.²⁶
Chemicals, peptides, and recombinant proteins
Amikacin	Sigma Aldrich	Cat# A0365900
Anhydrotetracycline	Fisher Scientific	Cat# AC233135000
Azithromycin dihydrate	Sigma Aldrich	Cat# PZ0007
Bedaquiline	AdooQ Biosciences	Cat# A12327–5
Bortezomib	Sigma Aldrich	Cat# 5043140001
Capreomycin Sulfate	Sigma Aldrich	Cat# PHR1716
Chloramphenicol		Cat# C-105
Clarithromycin	Sigma Aldrich	Cat# C9742
Clindamycin hydrochloride	GoldBio	Cat# C-175–10
Ecumicin	Sanghyun Cho (Univ. Illinois at Chicago College of Pharmacy)	Gao et al²⁹
Ethambutol dihydrochloride	Sigma Aldrich	Cat# E4630
Fusidic acid sodium salt	Sigma Aldrich	Cat# F0881
Isoniazid	Sigma Aldrich	Cat# I3377
Kanamycin	GoldBio	Cat# K-120–50
Levofloxacin	Sigma Aldrich	Cat# 28266
Lincomycin hydrochloride	Sigma Aldrich	Cat# 62143
Linezolid	Sigma Aldrich	Cat# SML1290
PBTZ169	Cayman Chemical	Cat# 22202
PA-824 (Pretomanid)	Sigma Aldrich	Cat# SML1290
Rifampicin	TCI	Cat# R0079
Spectinomycin dihydrochloride	GoldBio	Cat# S-140–5
Streptomycin sulfate	GoldBio	Cat# S-150–100
Tigecycline hydrate	Sigma Aldrich	Cat# PZ0021
Vancomycin hydrochloride	Sigma Aldrich	Cat# V2002
Zeocin	Alfa Aesar	Cat# J67140–8EQ
6-FABA	Sigma Aldrich	Cat# 443026
Bacto^™ casamino acids	Thermo Fisher	Cat# 223050
L-alanine	Sigma Aldrich	Cat# 1250–100GM
L-aspartic acid	Sigma Aldrich	Cat# A7219–100G
L-glutamic acid	Sigma Aldrich	Cat# G1501–100G
L-lysine	Sigma Aldrich	Cat# L5626–100G
L-methionine	Sigma Aldrich	Cat# M9625–100G
L-threonine	Sigma Aldrich	Cat# T8625–100G
L-tryptophan	Sigma Aldrich	Cat# T0254–100G
Critical commercial assays
NEB HiFi 2X Master Mix (Gibson assembly)	New England Biolabs	Cat# E2621L
NEB Q5 2X Master Mix (molecular cloning)	New England Biolabs	Cat# M0492L
NEBUltra II Q5 Master Mix (NGS PCR)	New England Biolabs	Cat# M0544L
NextSeq High Output Kit v2.5 (75 Cycles)	Illumina	Cat# 20024906
NovaSeq 6000 S2 kit	Illumina	Cat# 20028316
SuperScript IV First-Strand Synthesis System	Thermo Fisher	Cat# 18–091-050
SYBR^™ Green PCR master mix	Thermo Fisher	Cat# 4364344
T4 DNA ligase	NEB	Cat# M0202T
BsmBI-HF restriction enzyme	NEB	Cat# R0739L
Deposited data
Raw sequencing data from the whiB7 induction screen (Fig. 2) and the $Δ$ whiB7 vulnerability screen (Fig. 4).	This study	SRA: PRJNA970266 and PRJNA970343
Oligonucleotides
sgRNAs and RT-qPCR primers (Table S2)	This study	NA
Recombinant DNA
plRL91 (plAW145) Empty complementation vector used in M. smegmatis for figure(s) 1B,E	This study	https://benchling.com/s/seq-l2e2KoT2t1rryGetcqEk?m=slm-buY07uBNcN8k2Ftj0UHx
plRL125 backbone used for cloning of M. tuberculosis mScarlet reporter construct (not used directly in experiments)	This study	https://benchling.com/s/seq-mslTEj8tckVmDo7agJvm?m=slm-3looaqQ5LvmWaqUuDvJB
plRL284 (plNP441) zeoR driven by EM7 promoter used in M. smegmatis for figure(s) 1B	This study	https://benchling.com/s/seq-keovdkto7gS8Pvlffwc1?m=slm-QxR93n3NeuO6SocRtVZh
plRL285 (plNP442) zeoR driven by M. smegmatis whiB7 + 500 bp region used in M. smegmatis for figure(s) 1A–C, 2A–C	This study	https://benchling.com/s/seq-BuEHqVYrpKdaPOgbM1LG?m=slm-YGryGwZumq25G32nLEqJ
plRL286 (plNP445) mScarlet driven by p300 promoter used in M. smegmatis for figure(s) 1E	This study	https://benchling.com/s/seq-mdAXDB8tz9PX5jircmcG?m=slm-z645sCvy9h2xhj3CayMJ
plRL287 (plNP446) mScarlet driven by M. smegmatis whiB7 + 500 bp region used in M. smegmatis for figure(s) 1A,D-F, S1A, S2C,D,F, 3A,B	This study	https://benchling.com/s/seq-xVp5VXajLMpA6ZUtM9CU?m=slm-gXTXKm1xNzchZnpYKKIW
plRL277 (plNP466) mScarlet driven by M. tuberculosis whiB7 + 500 bp region used in M. tuberculosis for figure(s) 1F, S1B, S2E, 3A,C	This study	https://benchling.com/s/seq-kaaIOaUpb4hbZUCmR4aC?m=slm-AGfDgX4AjN4Z3uamM1ru
plRL288 (plNP623) whiB7 driven by WT regulatory region used in M. smegmatis for figure(s) 3E,F, S4A–C	This study	https://benchling.com/s/seq-lgjYeAJP4iqefoWNX96K?m=slm-rvpRpiZHaybrmqgOoJ8C
plRL289 (plNP624) whiB7 driven by regulatory region with His-less uORF used in M. smegmatis for figure(s) 3E,F, S4A–C	This study	https://benchling.com/s/seq-KrKP84U6kGmwRF88Q354?m=slm-6Kt3A5yWd5SuOVxPCriT
plRL290 (plNP625) whiB7 driven by regulatory region with Trp-less uORF used in M. smegmatis for figure(s) 3E,F, S4A–C	This study	https://benchling.com/s/seq-JBPYxdqzMde5aW3c3WKq?m=slm-5yTQ43IYoqMZwsHo1IDr
plRL291 (plNP672) M. smegmatis whiB7 driven by Mtb regulatory region (+500 bp) used in M. smegmatis for figure(s) S4C,D	This study	https://benchling.com/s/seq-Td5eHorcOa20WK251v8H?m=slm-t2pWnjdRICdO4W8YA2Js
plRL292 (plNP687) M. smegmatis whiB7 driven by Mtb regulatory region (+500 bp) with Ala13Pro mutation in uORF used in M. smegmatis for figure(s) S4C,D	This study	https://benchling.com/s/seq-yoQpkJEjxgkuXqiQuUxK?m=slm-77arP3sIM71P9o9afYsg
plRL293 (plNP688) M. smegmatis whiB7 driven by Mtb regulatory region (+500 bp) with His22fs delG mutation in uORF used in M. smegmatis for figure(s) S4C,D	This study	https://benchling.com/s/seq-gJKRIhRdHgzMIiD6GPeL?m=slm-lofzbUxFg7018X3ZCi4U
plRL294 (plNP689) M. smegmatis whiB7 driven by Mtb regulatory region (+500 bp) with His22ProPro mutation in uORF used in M. smegmatis for figure(s) S4C,D	This study	https://benchling.com/s/seq-B8dUoil9DphongRVKHeX?m=slm-vSbXazg2ubnQcyiZ1vSC
plRL295 (plNP690) M. smegmatis whiB7 driven by Mtb regulatory region (+500 bp) with Val1Gly start lost uORF mutation in uORF used in M. smegmatis for figure(s) S4C	This study	https://benchling.com/s/seq-lgrqU4siiz9Vu3gnapEi?m=slm-L9pSJKkjCOFmWvnoGPoJ
plRL296 (plNP691) M. smegmatis whiB7 driven by Mtb regulatory region (+500 bp) with His22fs (insCTAT) mutation in uORF used in M. smegmatis for figure(s) S4C,D	This study	https://benchling.com/s/seq-zOy0ar8rwDLYOYhx33fo?m=slm-Wy14qhxXIg2IGvTmDtw3
plRL62 Tweety integrase suicide vector used in M. smegmatis for figure(s) 1A–F, S1A, 2A, S2C–F, 3A,B,E,F, S4A–C	This study	https://benchling.com/s/seq-LtN3e9WbixCmPd0VOCAa
plRL40 Giles integrase suicide vector used in M. tuberculosis for figure(s) 1F, S1B, S2E, 3A,C	This study	https://benchling.com/s/seq-2PVXvHq0CfNPVacwz1Ku
plRL19 (plJR1949) L5 integrase suicide vector used in M. smegmatais and M. tuberculosis for figure(s) 1C,E, S2A–F, 3A,F, S4B, 4C,D, S5A–C, 5A–D	Bosch et al. ³⁰	Addgene Cat# 163634
plRL58 CRISPRi plasmid M. tuberculosis KD experiments (co-transformed with plRL19)	Bosch et al. ³⁰	Addgene Cat# 166886
plRL61 CRISPRi plasmid M. smegmatis KD experiments (co-transformed with plRL19)	Bosch et al. ³⁰	Addgene Cat# 163633
Software and algorithms
ChemiDoc imaging system	BioRad	Cat# 12003153
Prism 9	GraphPad	https://www.graphpad.com/features
Python	Python software foundation	https://www.python.org/downloads/
Rstan (version 2.19.3)	Stan Development Team, 2020	https://mc-stan.org/users/interfaces/rstan.html
SciPy (version 1.2.2)	Virtanen et al. ⁵¹	https://scipy.org/install/
Stan (version 2.19.3)	Stan Development Team, 2021	https://mc-stan.org/users/interfaces/
statsmodels (version 0.10.1)	Seabold and Perktold⁵²	https://www.statsmodels.org/stable/install.html
Subread aligner (version 1.6.0)	Liao et al. ⁵³	https://sourceforge.net/projects/subread/files/subread-2.0.6//
Spark^® Plate reader	Tecan	https://lifesciences.tecan.com/
QuantStudio^™ 5 qPCR system	Thermo Fisher	Cat# A28140
Vulnerability analysis pipeline (original code)	This paper and Bosch et al. ³⁰	Github: https://github.com/rock-lab/whiB7KO_screen_2023/ and https://doi.org/10.5281/zenodo.10384792