RNase E and HupB dynamics foster mycobacterial cell homeostasis and fitness

. 2022 Apr 12;25(5):104233. doi: 10.1016/j.isci.2022.104233

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Mouse monoclonal anti-6xHist tag (H3)	Santa Cruz Biotechnology	Cat# sc-8036; RRID: AB_627727
Mouse monoclonal anti-Ag85 (HYT 27)	Santa Cruz Biotechnology	Cat# sc-57611; RRID: AB_628733
ECL HRP-conjugated anti-mouse m-IgGκ	Santa Cruz Biotechnology	Cat# sc-516102; RRID: AB_2687626
Rabbit monoclonal anti-GFP (3E6)	Thermo Fisher	Cat# A-11120; RRID: AB_221568
Anti·His HRP Conjugate (RGSHHHH)	QIAGEN	Cat# 652503; RRID: AB_2734520
ECL HRP-conjugated anti-rabbit	Amersham	Cat# NA934; RRID: AB_772206
GFP-Trap_A for immunoprecipitation	Chromotek	Cat# gta-20; RRID: AB_2631357

Bacterial strains

Escherichia coli DH5α	Invitrogen	Cat# 12297016
Mycobacterium smegmatis mc²155	Lab collection	ATCC 700084
ATCC®700084 - pAG202; inducible strain overexpressing wild type Rne_6X-His-tag	This paper	AGS2
ATCC®700084 – pDV201; attB::rne-linker-mKate2	This paper	DVS2
ATCC®700084 – pAG212; attB::rne-linker-mKate2	This paper	AGS13
AGS13 – pAG219, attB::rne-linker-mKate2_attT::deaD-linker-mCitrine	This paper	AGS20
AGS13 – pAG215, attB::rne-linker-mKate2_attT::rraA-linker-mCitrine	This paper	AGS16
AGS13 – pAG218, attB::rne-linker-mKate2_ attT_obg-linker-mCitrine	This paper	AGS19
AGS13 – pAG220, attB::rne-linker-mKate2_ attT::relA-linker-mCitrine	This paper	AGS21
AGS13 – pAG221 attB::rne-linker-mKATE2_attT::ppk-linker-mCitrine	This paper	AGS22
AGS13 – pAG217 attB::rne-linker-mKate2_attT::uvrA-linker-mCitrine	This paper	AGS18
AGS13 – pAG213 attB::rne-linker-mKate2_ attT::lsr2-linker-mCitrine	This paper	AGS23
AGS13 – pAG214 attB::rne-linker-mKate2_attT::infA-linker-mCitrine	This paper	AGS15
AGS13 – pAG216 attB::rne-linker-mKate2_attT::hupB-linker-mCitrine	This paper	AGS17
ATCC®700084 – pGM255; rne (MSMEG_4626) CRISPRi/dCas9	This paper	mc²155/pGM255
ATCC®700084 – pGM257; scrambled CTsgRNA/dCas9	This paper	mc²155/pGM257
AGS2 – pAG216; attT::hupB-linker-mCitrine	This paper	AGS33
mc²155/pGM255 - pAG216; attT::hupB-linker-mCitrine	This paper	AGS32
Mycobacterium tuberculosis Erdman	Lab collection	ATCC 35801
ATCC®35801 – gfp::rpsJ (gfp chromosomal integration by two-step homologous recombination using pGM213)	This paper	GMT14
ATCC®35801 – gfp::rpsJ_attB::rne-linker-mCherry	This paper	GMT14/pGM301
ATCC®35801 – pGM301 – pGM305; attB::rne-linker-mCherry_attT::hupB-linker-sfgfp	This paper	GMT25
ATCC®35801 – pGM305 – pGM306; attT::hupB-linker-sfgfp;	This paper	GMT26
ATCC®35801 – pGM305 – pGM309; CRISPRi/dCas9_attT::hupB-linker- sfgfp	This paper	GMT27

Chemicals, peptides, and recombinant proteins

LB Broth Base	Invitrogen	Cat# 12780052
Agar powder	Invitrogen	Cat# 30391049
Middlebrook 7H9 Broth	BD Difco	Cat# DF0713-17-9
Middlebrook 7H10 Agar	BD Difco	Cat# DF0627-17-4
Middlebrook OADC Growth Supplement	BD Difco	Cat# BD212351
Glycerol	Sigma-Aldrich	Cat# G5516
Tween 80	Sigma-Aldrich	Cat# P5188
Bovine Serum Albumin (BSA) Fraction V	Sigma-Aldrich	Cat# A9647
Sodium chloride	Sigma-Aldrich	Cat# S9888
Dextrose	Sigma-Aldrich	Cat# G8270
Sucrose	Sigma-Aldrich	Cat# 84100
Hartmans-de Bont minimal medium for mycobacteria	(Smeulders et al., 1999)	N/A
Sodium propionate	Sigma-Aldrich	Cat# P1880
Kanamycin solution	Sigma-Aldrich	Cat# K0254
Hygromycin B solution	Sigma-Aldrich	Cat# H0654
Isoniazid	Sigma-Aldrich	Cat# I3377
Rifampicin	EUROPEAN PHARMACOPOEIA	Cat# R0700000
Mitomycin C	Sigma-Aldrich	Cat#M4287
FtsZ inhibitor: Methyl [(4-nitro-2,1,3-benzothiadiazol-5-yl)thio]acetate	Lab collection (Chiarelli et al., 2020)	C109
Chloramphenicol	Sigma-Aldrich	Cat# C0378
M5 inhibitor: ethyl N-[(1-{[5-chloro-3-(trifluoromethyl)-2-pyridyl]amino}-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)carbonyl]carbamate	Maybridge	KM08782

Critical commercial assays

Q5® Site-Directed Mutagenesis Kit	NEB	E0554S
HisPur^TM Cobalt Superflow Agarose	Thermo Fisher	252229

Deposited data

Whole-cell mass spectrometry-based proteomics of M. smegmatis rne overexpressing (AGS2), silenced (mc²155/pGM255) and control strains	This paper (Table S1), PRIDE repository (Perez-Riverol et al., 2019)	ProteomeXchange ID: PXD025674
Whole-cell mass spectrometry-based proteomics of RNase E-His pull-down from M. smegmatis rne overexpressing (AGS2) and control strain	This paper (Table S2) PRIDE repository (Perez-Riverol et al., 2019)	ProteomeXchange ID: PXD025674

Experimental models: Cell lines

RAW 264.7 murine macrophages	Lab collection	ATCC TIB-71

Oligonucleotides

see Table S3

Recombinant DNA

pCR2.1-TOPO TA cloning plasmid, Amp^R, Km^R	Invitrogen	pCR2.1-TOPO
pMYC mycobacterial expression vector, for C-term fusion of 6X-His-tag, Hyg^R	Addgene (Beckham et al., 2020)	# 42192
pJG1100 – Suicide vector expressing Km^R, Hyg^R resistance cassettes and sacB marker	Lab collection	pJG1100
pTTP1A-based integrative vector, containing Tweety phage integration site (attT), Km^R	Addgene (Pham et al., 2007)	# 91721
mCitrine-N1, containing mEYFP, Km^R	Addgene (Griesbeck et al., 2001)	# 54594
pLJR962, tetracycline-inducible dCas9 attB-integrative vector for M. smegmatis gene silencing, Km^R	Addgene (Rock et al., 2017)	# 115162
pLJR965, tetracycline-inducible dCas9 attB-integrative vector for M. tuberculosis gene silencing, Km^R	Addgene (Rock et al., 2017)	# 115163
pMYC rne (MSMEG_4626), acetamide-inducible Rne-6xHist tag	This paper	pAG202
pMV361-based integrative vector, containing attB phage attachment site, Km^R	Lab collection (Peña et al., 1996)	pND200
pND200 expressing mCherry_wt from UV15 strong promoter, Hyg^R	Lab collection	pGM218
pND200 expressing P_native-rne-linker-mKate2, Km^R (MSMEG_4626)	This paper	pDV201
pGM218 expressing P_native-rne-linker-mKate2, Hyg^R (MSMEG_4626)	This paper	pAG212
pTTP1A expressing P_native-deaD-linker-mCitrine, Km^R	This paper	pAG219
pTTP1A expressing P_native-rraA-linker-mCitrine, Km^R	This paper	pAG215
pTTP1A expressing P_native-obg-linker-mCitrine, Km^R	This paper	pAG218
pTTP1A expressing P_native-relA-linker-mCitrine, Km^R	This paper	pAG220
pTTP1A expressing P_native-ppk-linker-mCitrine, Km^R	This paper	pAG221
pTTP1A expressing P_native-uvrA-linker-mCitrine, Km^R	This paper	pAG217
pTTP1A expressing P_native-lsr2-linker-mCitrine, Km^R	This paper	pAG213
pTTP1A expressing P_native-infA-linker-mCitrine, Km^R	This paper	pAG214
pTTP1A expressing P_native-hupB-linker-mCitrine, Km^R	This paper	pAG216
pGM218 expressing P_native-rne-linker-mCherry, Hyg^R (rv2444c)	This paper	pGM301
pJG1100-based vector for chromosomal knock-in of gfp-linker fused in frame to rpsJ (rv0700) start codon	This paper	pGM213
pTTP1A expressing P_native-hupB-linker-sfgfp, Km^R	This paper	pGM305
pLJR962 modified with Hyg^R cassette	This paper	pGM256
pGM256 carrying sgRNA for rne (MSMEG_4626) silencing, Hyg^R	This paper	pGM255
pGM256 carrying a scrambled CTsgRNA, Hyg^R	This paper	pGM257
pLJR965 modified with Hyg^R cassette	This paper	pGM309
pGM309 carrying sgRNA for rne (rv2444c) silencing, Hyg^R	This paper	pGM306

Software and algorithms

Mycobrowser	(Kapopoulou et al., 2011)	https://mycobrowser.epfl.ch/
Cytoscape Version 3.8.2	Cytoscape	https://cytoscape.org/
PANTHER Classification System	(Mi et al., 2013)	http://pantherdb.org
ImageJ 1.51s	(Schneider et al., 2012)	https://imagej.net/Fiji
Prism Versions 8.4	GraphPad	https://www.graphpad.com/scientific-software/prism/
RStudio Version 1.1.423	RStudio, Inc	https://rstudio.com/pri
Python Version 3.8.3	Python	https://www.python.org/

Other

Custom R script for single-cell image data post-processing	(Manina et al., 2019)	TL_PostProc_Script_AG-IT.R
Custom Python script for single-cell image data post-processing	This paper	post_process_fluorescence.py
Custom ImageJ macros for analysis of infected macrophages	This paper	InfectedCells_Analysis_GM.ijm