APEX2 proximity labeling of RNA in bacteria

Summary

Rapid, spatially controlled methods are needed to investigate RNA localization in bacterial cells. APEX2 proximity labeling was shown to be adaptable to rapid RNA labeling in eukaryotic cells and, through the fusion of APEX2 to different proteins targeted to diverse subcellular locations, has been useful to identify RNA localization in these cells. Therefore, we adapted APEX2 proximity labeling of RNA to bacterial cells by generating an APEX2 fusion to the ribonuclease (RNase) E gene, which is necessary and sufficient for bacterial ribonucleoprotein (BR)-body formation. APEX2 fusion is minimally perturbative, and RNA can be rapidly labeled on the sub-minute timescale with alkyne-phenol, outpacing the rapid speed of mRNA decay in bacteria. Alkyne-phenol provides flexibility in the overall application with copper-catalyzed click chemistry for downstream processes, such as fluorescent dye azides or biotin-azides for purification. Altogether, APEX2 proximity labeling of RNA provides a useful method for studying RNA localization in bacteria.

Graphical abstract

Highlights

• •APEX2 proximity labeling can be applied to RNA in bacteria
• •APEX2 RNA labeling reactions occur on the sub-minute timescale
• •APEX2 workflow requires less material and time than the current methods
• •The alkyne-phenol APEX2 substrate provides flexibility with click chemistry

Motivation

Studies over the past several years have shown that distinct RNAs can be targeted to subcellular locations in bacterial cells. The ability to investigate localized RNAs in bacteria is currently limited to imaging-based approaches or to laborious procedures to isolate ribonucleoprotein complexes by grad-seq, HITS-CLIP, or Rloc-seq. However, a major challenge in studying mRNA localization in bacterial cells is that bacterial mRNAs typically last for only a few minutes in the cell, while experiments to investigate their localization or interaction partners can take much longer. Therefore, rapid methods of studying RNA localization are needed to address this technical challenge.

Yassine et al. adapt an APEX2-based RNA proximity labeling approach that labels RNAs in both gram-negative and gram-positive bacteria. This method involves fusing an RNA-binding protein to APEX2, which rapidly labels localized RNAs on a faster timescale than the short lifetimes of RNAs in bacteria.

Introduction

Bacterial mRNAs localize to distinct subcellular locations. At a global level, many mRNAs have been found to associate with the nucleoid, membrane, or cell poles,^1,2,3,4 and it has been hypothesized that mRNA localization can be important for proper gene expression. For example, flagellin mRNA in Campylobacter jejuni localizes to the cell poles, which may facilitate cotranslational flagellar assembly.⁵ In addition, biomolecular condensates, which are non-membrane-bound organelles often assembled through phase separation, have been found to be prevalent and important in bacterial cells, with multiple involved in RNA metabolism.^4,6 Yet the functional significance of localization of RNAs to biomolecular condensates in bacteria has just started to be explored.

GRAD-seq, HITS-CLIP, and Rloc-seq methods^1,7,8 have been successful at identifying RNAs associated with specific RNPs or localized to certain subcellular locations. However, a major difficulty is that bacterial mRNAs are very short lived, and these methods require labeling or isolation times much longer than a typical mRNA’s half-life, leading to the potential for false negatives. Therefore, rapid methods are needed to isolate the associated labile bacterial mRNAs before they are degraded.

APEX2 proximity labeling allows for spatially controlled and rapid RNA labeling,^9,10,11,12 making it a useful method to assay for localized bacterial RNAs. In this manuscript, we show that APEX2 proximity labeling can be adapted to bacterial condensates via fusion of the core bacterial ribonucleoprotein (BR)-body scaffold ribonuclease (RNase) E to APEX2. RNA can be labeled robustly by APEX2, as many bacteria natively produce heme, including Escherichia coli, Bacillus subtilis, and Caulobacter crescentus. The addition of H₂O₂- and alkyne-phenol-triggered labeling allows copper-catalyzed click chemistry of alkyne-phenol-labeled RNA with a variety of azides, such as Cy5-azide. This protocol can be used to rapidly label bacterial RNAs by APEX2 on the sub-minute timescale, which can then be rapidly purified via copper-catalyzed click chemistry conjugation of biotin-peg3-azide followed by streptavidin purification. Through the fusion of APEX2 to differently localized RNA-binding proteins (RBPs), APEX2 proximity labeling has the potential to improve our knowledge of RNA localization in bacteria.

Results

RNase E-APEX2 proximity labeling of RNA requires alkyne-phenol, H₂O₂, and APEX2

APEX2 is an engineered ascorbate peroxidase that catalyzes the creation of a free radical on the oxygen of alkyne-phenol, and this highly reactive species can react with molecules within a 10–20 nm radius^13,14 (Figure 1A). APEX2 can label RNA in eukaryotic cells, making this a useful experimental system for identifying localized RNAs.^10,12,15,16 To perform RNA proximity labeling in bacteria, we first generated a gene fusion between RNase E and APEX2. RNase E is known to phase separate into BR-bodies, biomolecular condensates that contain mRNA, and promote mRNA decay.^17,18 To determine whether APEX2 fusions are tolerated in bacteria, we examined the subcellular localization, cell fitness, and mRNA decay activity of RNase E-APEX2 fusions in the bacterium C. crescentus (Figure 1B). We observed that fusing APEX2 to RNase E led to proper expression of RNase E-APEX2 (Figure S1) and did not alter its ability to phase separate into BR-bodies (Figure 1B). In addition, since RNase E’s ability to degrade mRNAs is essential for cell growth,^18,19 we also examined the cellular fitness of the RNase E-APEX2 fusion and found that both colony-forming units (CFUs) and colony size were indistinguishable from wild type (Figure 1B). Finally, we compared the mRNA decay activity of the RNase E-APEX2 fusion and found that it has similar rates of mRNA decay as the wild type (Figure 1B), while 5S rRNA remained stable. Altogether, we could not detect any measurable differences in RNase E function when fused to APEX2. To examine whether the APEX2 fusion can be used to label RNA, we added all combinations of proximity labeling reactants (H₂O₂ and alkyne-phenol) with or without the RNase E-APEX2 fusion (Figure 1C). Each of the proximity labeling conditions were performed, and then we extracted the RNA and used copper click chemistry to conjugate a Cy5-azide to the RNA. The RNA was then deposited on a positively charged nylon membrane using a dot blot apparatus, and the Cy5 signal was measured in a fluorescent gel imager. We find that only in the presence of APEX2, H₂O₂, and alkyne-phenol do we observe RNA labeling (Figure 1C), suggesting that APEX2 proximity labeling reactions occurred. To determine whether the Cy5 signal was a result of RNA or contaminating DNA, we performed RNase A or DNase I digestions on our samples before spotting them on the dot blot (Figure 1C). Here, we see that RNase A treatment led to a complete loss of Cy5-fluorescence, while DNase I treatment led to no difference in fluorescence signal, suggesting that our Cy5 fluorescence is from RNA. We next examined the subcellular localization of APEX2 proximity labeling by incubating cells with alkyne-phenol and H₂O₂, then fixing labeled cells, conjugating Cy5 by copper click chemistry, and imaging by epifluorescence microscopy (Figure S2). Labeled RNase E-APEX2 showed distinct foci of Cy5 signal, indicating that proximity labeling likely occurs within BR-bodies. Conversely, a strain that cannot make BR-bodies (RNaseEΔCTD-APEX2) showed diffuse Cy5 signal throughout the cytoplasm (Figure S2).

Figure 1.

APEX2 proximity labeling of RNA in bacterial cells with minimal perturbation

(A) Schematic of APEX2 labeling of RNA protocol. The APEX2 protein was fused to RNase E, the major protein that scaffolds BR-bodies. Cells were incubated in medium containing alkyne-phenol, and labeling was initiated with H₂O₂. After a brief labeling reaction, RNA was extracted from the cells, and Cy5-azide was conjugated to the RNA by copper-catalyzed click chemistry.

(B) APEX2 fusion does not dramatically impact localization or function (growth rate). See also Figures S1 and S2. Left: in vivo localization of RNase E-msfGFP vs. RNase E APEX2-msfGFP shows that fusion does not impact the formation of BR-bodies. Scale bars, 1 μm. Middle: growth of RNase E-APEX2 fusion is similar to wild-type Caulobacter cells. Right: mRNA half-life measurements by RT-qPCR show that RNase E-APEX2 degrades mRNAs with a similar half-life to wild type. Data are the averages from three biological and technical replicates and error bars represent standard deviation.

(C) APEX2 labeling requires H₂O₂ and alkyne-phenol to label RNAs. RNA labeling reactions were placed on a nylon membrane to bind to the RNA in a dot blot apparatus and scanned for Cy5 fluorescence in a gel imager. As a control, the RNA samples were incubated for 2 h with DNase I and RNase A. The RNA was then precipitated before being subjected to the azide-Cy5 click chemistry reaction and was re-precipitated before being filtered on the nylon membrane in the dot blot apparatus.

Rapid labeling of cellular RNA with alkyne-phenol

One of the major challenges of studying bacterial mRNA decay is the very short lifetimes of cellular mRNAs. Most bacterial mRNAs in rapidly growing species have half-lives between 1 and 4 min,^20,21 making it hard to harvest the RNA before they are degraded. To address this technical limitation, we first optimized the alkyne-phenol concentration used for labeling cells and found that 2.5 mM alkyne-phenol robustly labels cellular RNA (Figure 2). Next, we performed a time course of H₂O₂ exposure to identify the timescale of RNA labeling, ranging from 15-s to 1-min reaction times. We found that robust labeling could be achieved in as little as 15 s, while labeling increased upon longer proximity labeling reaction times (Figure 2). Importantly, the labeling reaction occurs on the sub-minutes timescale, making this method well suited to use in exponentially growing bacterial cells.

Figure 2.

APEX2 proximity labeling of RNA works rapidly

Optimization of APEX2 labeling of RNA with alkyne-phenol. Top: alkyne-phenol titration reveals that peak labeling occurs with 2.5 mM alkyne-phenol. RNA was labeled in the scheme shown in Figure 1A, and the Cy5 intensity was measured in a gel imager. Bottom: time course of APEX2 labeling. RNA was labeled in the scheme shown in Figure 1A, and RNA labeling is apparent in as little as 15 s of H₂O₂ incubation, while peak labeling efficiency is observed at 45 s of H₂O₂ incubation. RNA blots were obtained from two biological replicates.

See also Figure S3.

RNA proximity labeling works across bacteria

To increase the applicability of this method, we sought to determine whether APEX2 can label RNA in other species of bacteria. As a key requirement of APEX2 activity is heme, we chose to fuse APEX2 to RNA degradosome scaffold proteins in E. coli (gram-negative) and B. subtilis (gram-positive) because both organisms have heme biosynthesis pathways encoded in their genomes. In E. coli, we fused APEX2 to RNase E, which scaffolds BR-bodies in this species^22,23 and localizes to membrane-anchored RNP foci.²² In B. subtilis, we fused APEX2 to RNase Y, which scaffolds BR-bodies in this species and also localizes in RNP foci on the inner membrane of the cell.^23,24 In both species, we observed RNA labeling after 45 s of H₂O₂ incubation that required the APEX2 fusion (Figure 3), suggesting that APEX2 proximity labeling of RNA can be used in diverse species of bacteria that encode heme biosynthesis pathways.

Figure 3.

APEX2 proximity labeling of RNA works across species

APEX2 requires heme for activity, so APEX2 fusions were generated in two additional species with heme biosynthesis pathways, B. subtilis (gram-positive) and E. coli (gram-negative). Both species were subjected to RNA proximity labeling in mid-exponential phase of growth in Luria Broth (LB) medium at 37°C, pre-incubated with alkyne-phenol for 30 min, and proximity labeling was triggered by a 45-s incubation with H₂O₂. RNA labeling reactions were subjected to azide-Cy5 click chemistry reactions and 5 μg RNA placed on a nylon membrane to bind to the RNA in a dot blot apparatus and scanned for Cy5 fluorescence in a gel imager. At least three biological replicates were carried out for each species.

Copper click chemistry of biotin-azide and streptavidin purification of labeled RNA

APEX2 proximity labeling of RNA is rather flexible due to the diversity of azides commercially available for copper-catalyzed click chemistry. To isolate cellular RNA for RNA sequencing (RNA-seq), we altered the azide from azide-Cy5 dye to biotin-azide to allow for streptavidin-mediated RNA purification from the cell (Figure 4A). In this approach, labeled RNA is extracted from cells and conjugated to biotin-azide using copper-catalyzed click chemistry, which allows for subsequent purification of the labeled RNA using streptavidin resin. While copper can cleave RNA, using a short incubation time for the click reaction minimizes RNA cleavage by copper (Figure S3). After click chemistry, the RNA can be purified under stringent conditions and eluted from the resin under denaturing conditions. When applied to the RNase E-APEX2 fusion presented earlier, we found that the eluted RNA profile indeed matched that of BR-body RNA isolated via density centrifugation (Figure 4B).^18,25 To compare the specificity of APEX RNA proximity labeling, we prepared eluted RNAs from BR-body + cells (JS767) and BR-Body − cells (RNaseEΔCTD-APEX2, JS803) and prepared them for RNA-seq. As a control for the initial amount of each RNA in the cell, we also performed RNA-seq on the total RNA lysates of each strain. The overall level of proximity labeling was calculated as the log2 enrichment (eluted/lysate) of each RNA measured at >50 reads/sample. We found that BR-body + samples (JS767) contained higher overall enrichment levels than BR-body − samples (JS803) (Figure S4A), whose mean enrichment was near 0, with high reproducibility between biological replicates (Figure S4B). Prior density centrifugation of BR-bodies comparing BR-body + and BR-body − strains found that BR-bodies are enriched in mRNAs, small non-coding RNAs, and antisense RNAs and depleted of tRNAs and rRNAs.¹⁸ Our proximity-labeled RNA samples showed mRNAs and small non-coding RNAs were both enriched, while antisense RNAs also appeared to be enriched; however, the p value was below a 95% confidence interval (p = 0.1). tRNAs and rRNAs were not enriched (Figure 4C). Interestingly, while 16S and 23S rRNAs were not enriched, 5S rRNA was enriched, in-line with RNase E’s known role in 5S rRNA processing^26,27 and the colocalization of BR-bodies with rRNA loci in vivo.²⁸ Importantly, while centrifugation-based isolation of BR-bodies required 200 mL of cell culture and 5 days of hands-on work to isolate the BR-body-enriched RNAs,²⁵ APEX2 proximity labeling required only 4 mL of cell culture and 3 days of hands-on work, making it higher throughput.

Figure 4.

APEX2-proximity-labeled RNA can be isolated by streptavidin purification

(A) Schematic of the conjugation of biotin-azide to clicked alkyne-phenol and the resulting streptavidin purification.

(B) Streptavidin purification of biotinylated RNAs. Agilent TapeStation RNA profiles of the lysates and elution fractions of biotinylated-proximity-labeled RNAs. The bottom two bands are tRNAs, which are known to be highly depleted in BR-bodies from differential-centrifugation-based isolation of BR-bodies.^18,25

(C) RNA-seq enrichment analysis of proximity-labeled RNA from BR-body + (RNaseE-APEX2, JS767) and BR-body− (RNaseEΔCTD-APEX2, JS803) cells. Log₂ ratio of elution/lysate from each sample is plotted for each RNA quantified. Data points were obtained from two biological replicates for each strain. Colored horizontal bars indicate the median values for each distribution. t test with uneven variance was used to compare the distributions of each RNA type, and RNA types with p < 0.05 are shown.

See also Figure S4.

Discussion

RNP complexes are important regulators of RNA biology, including transcription, RNA processing, transport, translation, and decay.^4,29,30,31 These complexes have been increasingly found to localize into biomolecular condensate structures that facilitate the spatial organization of the stages in the mRNA life cycle via phase separation.^{4,17,18,32,33,34,35} As the realization that RNA localization is important in bacteria has grown, methods to identify the population of localized RNAs have been developed, yet these methods have been limited due to the long timescale of the procedures compared to the short mRNA lifetimes in bacteria. APEX2 can rapidly label RNA and can be easily genetically fused to genes whose proteins have known patterns of subcellular localization. APEX2 proximity labeling of RNA requires small amounts of cells, can be completed in a few hours by a single scientist, and yields sufficient RNA for downstream analysis. The reactivity radius of APEX2 is estimated to be 10–20 nm,³⁶ suggesting it gives high spatial precision. Genetic fusion of APEX2 is easy to generate, APEX2 RNA labeling works across heme-producing bacteria (C. crescentus, B. subtilis, and E. coli), and APEX2 is similar in size to a fluorescent protein and has no observable functional impacts when fused to RNase E, so we anticipate that this is unlikely to disrupt target protein function. The flexibility of click chemistry allows the conjugation of a wide array of clickable fluorophores or clickable affinity substrates as biotin or azide resin. Altogether, APEX2 proximity labeling of RNA will help to accelerate the discovery of localized RNAs in bacteria.

Limitations of the study

This work establishing RNA proximity labeling in bacteria is limited to experiments on a handful of RBPs. Therefore, it is not yet clear how robustly this method can be applied to different RBPs. While APEX2 is the approximate size of GFP, which has been utilized extensively, it is not yet clear whether fusion with APEX2 may lead to alterations in protein function/RNA-binding specificity, so users should take caution and validate their interactions with alternative methods. In addition, our examination of RNA labeling specificity has been limited to C. crescentus BR-bodies, where APEX2 proximity labeling identified similar types of RNAs enriched compared to BR-bodies isolated by density centrifugation.¹⁸ However, since this methodology has not been extensively tested among the vast diversity of bacterial RBPs, it is therefore important to include controls such as comparison of RBP-APEX2-labeled RNAs to free APEX2. In addition, while alkyne-phenol was cell permeable in the three species tested, it is unclear if alkyne-phenol can permeate across the vast diversity of bacterial envelopes.

Resource availability

Lead contact

Requests for additional information, resources, and reagents should be directed to and fulfilled by Jared M. Schrader (jaschrad@iu.edu).

Materials availability

The APEX2 gene can be obtained from Addgene (Cat# 129640). All other strains generated in this study are available from the lead contact without restriction.

Data and code availability

• •The dataset generated in this study was deposited in Figshare (https://figshare.com/articles/preprint/APEX2_proximity_labeling_of_RNA_in_bacteria/29525762?file=57828331). Raw sequencing reads and processed RNA-seq data were deposited to the NCBI GEO database with accession number GSE297938 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE297938).
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

Acknowledgments

We thank Tamara Hendrickson for equipment, reagents, and hosting the Schrader lab after it was destroyed in a fire. We thank Dr. Anat Nussbaum-Shochat for technical help with the construction of ES413 E. coli strain. NIH grants R35GM124733 to J.M.S. and R35GM131783 to D.B.K; WSU Career Chair Award to J.M.S.; IU startup funds to J.M.S.; and NIH T32GM142519-03 to H.Y. Research in the OAC lab was supported by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities (grant no.: 1274/19). O.A.-C. is an incumbent of the Dr. Jacob Grunbaum Chair in Medical Sciences.

Author contributions

H.Y. performed RNA labeling experiments. J.M.S. and H.Y. designed the study and wrote the paper. H.Y. generated C. crescentus APEX2 fusions strains. D.B.K. generated B. subtilis APEX2 fusions strains. E.S., O.G., and O.A.-C. generated E. coli APEX2 fusion strains.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
DYKDDDDK Tag (D6W5B) Rabbit mAb	Cell Signaling Technology	Cat# 14793S
Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP	Invitrogen	Cat# 31460
Bacterial and virus strains
Caulobacter crescentus NA1000	Lucy Shapiro’s Lab T00841	N/A
Js767 (C. crescentus RNE-Apex2FlgC-2 kanR)	This study	N/A
Js768 (C. crescentus RNE-Apex2Flg-EGFPC-2 kanR)	This study	N/A
Js87 (C. crescentus RNE-msfGFPC-4 gentR)	Al-Husini et al.17	N/A
LS4379 (C. crescentus hfq-m2)	Lucy Shapiro’s Lab	N/A
Js803 (RNEΔCTD-Apex2FlgC-2 kanR)	This study	N/A
B. subtilis DK1042 comIQ12L	Konkol et al.37 T0001037	N/A
DB2579 (B. subtilis amyE::rny-APEX2 specRampR)	This study	N/A
Escherichia coli MG1655	Orna Amster-Choder’s Lab T00007	N/A
Kti162 (E. coli rne-mCherry)	Strahl et al.22	N/A
ES413 (E. coli rne-mcherry-Apex2 kanR)	This study	N/A
Chemicals, peptides, and recombinant proteins
Alkyne-phenol	MedChemExpress	Cat# HY-131442
HYDROGEN PEROXIDE SOLN., 50%, ACS RGT.	Sigma Aldrich	Cat# 7722-84-1
Sodium ascorbate	Sigma Aldrich	Cat# 134-03-2
TRIzol	Thermo Fisher Scientific	Cat# 15596018
Chloroform	Thermo Fisher ScientificThermo Fisher Scientific	Cat# AC423550010
2-PROPANOL, ANHYDROUS	Sigma Aldrich	Cat# 67-63-0
Glycogen, RNA grade	Thermo Fisher Scientific	Cat# RO551
ETHANOL-D6 (D, 99%), ANHYD.	Sigma Aldrich	Cat# 1516-08-1
Sodium Acetate (3 M), pH 5.5, RNase-free	Thermo Fisher Scientific	Cat# AM9740
Biotin-peg3-Azide	Vector Laboratories	CAS Number: CCT-AZ104
Cy5 Azide	Clickchemistrytools	Cat# AZ118-1
BrightStar™-Plus Positively Charged Nylon Membrane, 30 cm × 45 cm	Thermo Fisher Scientific	Cat# AM10102
UltraPure™ SSC, 20X	Thermo Fisher Scientific	Cat# 15557044
SODIUM HYDROXIDE, PELLETS	Sigma Aldrich	Cat# 1310-73-2
EDTA (0.5 M), pH 8.0, RNase-free	Thermo Fisher Scientific	Cat# AM9261
Sodium Dodecyl Sulfate (98.5%)	Sigma Aldrich	Cat# L3771-500G
Bromophenol Blue	Sigma Aldrich	Cat# B0126-25G
Glycerol	Sigma Aldrich	Cat# 56-81-5
THPTA	Vectorslab	SKU: CCT-1010-100
Copper(II) sulfate pentahydrate	Sigma Aldrich	Cat# 7758-99-8
Bovine Serum Albumin	Sigma Aldrich	Cat# 9048-46-8
Thermo Scientific™ Pierce™ ECL Western Blotting Substrate	Fisher Scientific	Cat# PI32109
Heparin sodium salt from porcine intestinal mucosa	Sigma Aldrich	Cat# 9041-08-1
Tris (1 M), pH 7.0, RNase-free	Thermo Fisher Scientific	Cat# AM9851
Tris (Tris Base)	GoldBio	Cat# T-400-500
Hydrochloric acid	Carolina	Cat# 867792
SODIUM CHLORIDE, CRYSTAL	Sigma Aldrich	Cat# 7647-14-5
Tween 20	Sigma Aldrich	Cat# 9005-64-5
PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa	Thermo Fisher Scientific	Cat# 26620
Urea (ACS)	Fisher Chemical	Cat# U15-3
PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-free	Thermo Fisher Scientific	Cat# AM9624
Agar	Fisher Chemical	Cat# DF0001-17-0
Bactopeptone	Thermo Fisher Scientific	Cat# 211677
Yeast extract	Sigma Aldrich	Cat# 92144-500G-F
UltraPure™ Ethidium Bromide, 10 mg/mL	Thermo Fisher Scientific	Cat# 15585011
Thermo Scientific™ TriTrack DNA Loading Dye (6X)	Thermo Fisher Scientific	Cat#.:FERR1161
Thermo Scientific™ GeneRuler 1 kb Plus DNA Ladder, ready-to-use	Thermo Fisher Scientific	Cat# FERSM1334
Magnesium sulfate	Sigma Aldrich	Cat# M7506-1KG
Calcium chloride (97%)	Sigma Aldrich	Cat# 746495-500G
Kanamycin	Thermo Fisher Scientific	Cat# 11815032
Potassium phosphate dibasic anhydrous	Fisher Chemical	Cat# P288-500
Ammonium Chloride (99.5%)	Sigma Aldrich	Cat# A9434-1KG
Sodium phosphate dibasic	Sigma Aldrich	Cat# S7907-1KG
Glucose (99.5%)	Sigma Aldrich	Cat# G8270-1KG
Luria Broth Base	Thermo Fisher Scientific	Cat# 12795084
Iron (II) sulfate heptahydrate	Sigma Aldrich	Cat# F8633-250G
FD Kpn1	Thermo Fisher Scientific	Cat# FD0524
FD EcoR1	Thermo Fisher Scientific	Cat# FD0274
FD EcoRV	Thermo Fisher Scientific	Cat# FD0303
FD Dpn1	Thermo Fisher Scientific	Cat# ER1701
Hifi DNA Assembly Master Mix	NEB	Cat# E2621L
T4 DNA Ligase	Thermo Fisher Scientific	Cat# EL0011
Nalidixic Acid	Fisher scientific	Cat# 50488853
Gentamycin Sulfate	Sigma Aldrich	Cat# 345814-1GM
Agarose	Alkali	Cat# A7705
RNAprotect Bacteria Reagent	Qiagen	Cat# 76506
Rifampicin	Sigma Aldrich	Cat# 557303-1GM
Pierce™ ECL Western Blotting Substrate	Thermo Fisher Scientific	Cat# 32106
Glycine (99%)	Sigma Aldrich	Cat# G8898
Methanol (NF), Fisher Chemical™	Fisher Scientific	Cat# 67-56-1
PVDF Transfer Membranes, 0.2 μm	Thermo Fisher Scientific	Cat# 88520
DTT	Sigma Aldrich	Cat# 3483-12-3
Phenol Solution	Sigma Aldrich	Cat# 108-95-2
Glass Beads, acid-washed 106μm (∼140 U.S. sieve)	Sigma Aldrich	Cat# G4649
Phenol:Chloroform 5:1	sigmaladrich	Cat# P1944
Lithium Chloride Solution	Sigma Aldrich	Cat# 7447-41-8
Formaldehyde, 4% in PBS	Thermo Fisher Scientific	Cat# J60401.AK
Iodoacetamide	sigmaladrich	Cat# I1149-25G
Critical commercial assays
GeneJET Gel Extraction Kit	Thermo Fisher Scientific	Cat# K0692
GeneJET Plasmid Miniprep Kit	Thermo Fisher Scientific	Cat# K0503
GeneJET PCR Purification Kit	Thermo Fisher Scientific	Cat# K0702
Deposited data
Dataset generated	This paper	Figshare https://doi.org/10.1101/2024.09.18.612050 https://figshare.com/articles/preprint/APEX2_proximity_labeling_of_RNA_in_bacteria/29525762?file=57828331
Raw sequencing reads and processed RNA-seq data	This paper	NCBI GEO database; GSE297938https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE297938
Oligonucleotides
Oligonucleotides used	This study	see Table S1
Recombinant DNA
pFlgC-2	Thanbichler et al.38	N/A
pRNE-YFPC-1	Al-Husini et al.17	N/A
Apex2FlgC-2	This paper	N/A
RNE-Apex2FlgC-2	This paper	N/A
RNE-Apex2Flg-EGFPC-2	This paper	N/A
RNEΔCTD-Apex2FlgC-2	This paper	N/A
pcDNA5/FRT/TO APEX2-GFP	Addgene	Cat# 129640
pDP647 (amyE::Phypsank-rny-apex2 spec amp)	This paper	N/A
pDR111 (amyE::Physpank spec amp)	Gift from David Rudner	N/A
DNA sequence of rne-Apex2-KmR	Twist Bioscience	see Table S2
Software and algorithms
ImageJ	ImageJ	https://imagej.net/software/fiji/downloads
iBright analysis software	Thermo Fisher Scientific	https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/ibright-western-imager.html

Experimental model and study participant details

Cell growth conditions

Caulobacter crescentus NA1000, Js767, Js768, Js803, Js87, and Ls4379 were grown in Peptone Yeast Extract (PYE) media at 28°C. When applicable, the following antibiotics were added to solid media: Nalidixic acid (Nal; 20 μg/mL), Kanamycin (Kan; 25 μg/mL), and Gentamicin (Gent; 5 μg/mL). When applicable, the following antibiotics were added to liquid media: Kan (5 μg/mL), and Gent (0.5 μg/mL).

B. subtilis DK1042³⁷ and DB2579 were grown in Luria Broth (LB) media at 37°C. When applicable, the following antibiotics were added to solid and liquid media: Spectinomycin (Spec; 100 μg/mL).

E. coli MG1655, Kti162, and ES413 were grown in Luria Broth (LB) media at 37°C. When applicable, the following antibiotics were added to solid and liquid media: Kan (30 μg/mL).

Plasmid sequences were verified via Sanger sequencing (genewiz).

Protein expressions were verified by western blotting or fluorescent imaging.

Method details

Plasmid construction

pAPEX2-FlgC-2 Kan^R

The pFlgC-2 vector³⁸ was PCR amplified using primers HY1F & HY1R (Table S1). APEX2 was PCR amplified using primers HY2F & HY2R (Table S1) from the addgene template #129640. The amplicons were run on a 1% agarose gel and gel extracted using a GeneJET Gel Extraction Kit. The purified vector was Dpn1 treated then column purified using the GeneJET PCR Purification Kit. The Apex2FlgC-2 Kan^R plasmid was assembled via Gibson assembly (NEB) and transformed into chemically competent DHbeta10 E. coli and plated on LB + Kan (50 μg/mL) plates. The resulting KanR colonies were minipreped using the GeneJET Plasmid Miniprep Kit and screened via restriction digestion (EcoR1) and the insert sequence was verified by Sanger sequencing (Genewiz).

pRNE-Apex2-FlgC-2 Kan^R

The last 534 RNase E (RNE) base pairs were obtained by digesting pRNE-YFPC-1³⁸ with Nde1 and Kpn1. APEX2FlgC-2 was digested with Nde1 and Kpn1. The digestion reactions were run on a 1% agarose gel and the RNE fragment and Apex2-FlgC-2 vector were gel extracted using a GeneJET Gel Extraction Kit. The RNE fragment and Apex2FlgC-2 vector were ligated using T4 ligase, transformed into chemically competent DHbeta10 E. coli, and selected on LB + Kan plates. The resulting KanR colonies were minipreped using the GeneJET Plasmid Miniprep Kit and screened via restriction digestion (EcoRV) and the insert sequence was verified by Sanger sequencing (Genewiz).

pRNE-Apex2FlgC-EGFPC-2 Kan^R

The RNE-Apex2FlgC-2 vector was PCR amplified using primers HY3F & HY3R (Table S1). The EGFP insert was PCR amplified using primers HY4F & HY4R (Table S1) from the addgene template #129640. The amplicons were run on a 1% agarose gel and gel extracted using a GeneJET Gel Extraction Kit. The purified vector was Dpn1 treated then column purified using the GeneJET PCR Purification Kit. The RNE-Apex2FlgC-EGFPC-2 plasmid was assembled via Gibson assembly (NEB) and transformed into chemically competent DHbeta10 E. coli and selected on LB + Kan plates. The resulting KanR colonies were minipreped using the GeneJET Plasmid Miniprep Kit and screened via restriction digestion (EcoRV) and the insert sequence was verified by Sanger sequencing (Genewiz).

pRNEΔCTD-Apex2FlgC-2 Kan^R

The pApex2-FlgC-2 vector³⁸ was PCR amplified using primers HY5F & HY5R (Table S1). RNE-NTD was PCR amplified using primers HY6F & HY6R (Table S1) from NA1000 cells. The amplicons were run on a 1% agarose gel and gel extracted using a GeneJET Gel Extraction Kit. The purified vector was Dpn1 treated then column purified using the GeneJET PCR Purification Kit. The pRNE(NTD)-Apex2FlgC-2 Kan^R plasmid was assembled via Gibson assembly (NEB) and transformed into chemically competent DHbeta10 E. coli and plated on LB + Kan (50 μg/mL) plates. The resulting KanR colonies were minipreped using the GeneJET Plasmid Miniprep Kit and screened via restriction digestion (Kpn1) and the insert sequence was verified by Sanger sequencing (Genewiz).

pDP647 amyE::Phypsank-rny-apex2 spec^R amp^R

To generate the B. subtilis inducible Rny-APEX2 construct pDP647, the rny gene was amplified from wild-type DK1042 DNA with primers 8688/8689 (Table S1) and apex2 was amplified from plasmid pAPEXC-2 with primers 8692/8693 (Table S1). Next, the rny amplicon was digested with SalI and NheI, the apex2 amplicon was digested with NheI and SphI and the two fragments were simultaneously ligated into the SalI and SphI sites of pDR111 that carries a polylinker downstream of the IPTG-inducible P_hyspank promoter, the gene encoding the LacI repressor, and a spectinomycin resistance cassette between the arms of the amyE gene (generous gift of David Rudner, Harvard Medical School).

Strain construction

Js767: NA1000 rne:rne-apex2-flg Kan^R

The RNE-Apex2FlgC2 plasmid was recombined into the rne locus in NA1000 via mating and the selection was carried out on PYE + Nal (20 μg/mL) + Kan (25 μg/mL) plates. The resulting KanR colonies were first grown in PYE + Kan (5 μg/mL) cultures and then screened by PCR.

Js803: NA1000 rne:rneΔCTD-apex2-flg Kan^R

The RNEΔCTD-Apex2FlgC2 plasmid was recombined into the rne locus in NA1000 via mating and the selection was carried out on PYE + Nal (20 μg/mL) + Kan (25 μg/mL) plates. The resulting KanR colonies were first grown in PYE + Kan (5 μg/mL) cultures and then screened by PCR.

Js768: NA1000 rne:rne-apex2-flg-egfp Kan^R

The RNE-Apex2FlgC-EGFPC-2 plasmid was recombined into the rne locus in NA1000 via mating and the selection was carried out on PYE + Kan plates. The resulting KanR colonies were first grown in PYE + Kan cultures and then screened by PCR.

DB2579 amyE:rny-APEX2 spec^R amp^R

The pDP647 plasmid was transformed into DK1042³⁷ cells, and selected on plates containing LB and 100 μg/mL Spectinomycin. The resulting colonies were then screened for the integration by PCR.

ES413: rne:rne-mcherry-flg-Apex2 kan^R

The in vitro synthesized sequence rne-Apex2-KmR (Table S2) was PCR amplified using F-rne-mch and R-rne primers (Table S1) and recombined into the rne locus in Kti162²² by lambda red system,³⁹ the selection was carried out on LB + Kan (30 μg/mL) plates. The rne-mcherry-flag-Apex2 kan^R was then moved to MG1655 by P1 transduction.⁴⁰ The resulting Kan^R colonies were grown in LB + Kan, screened by PCR and verified by Sanger sequencing (The Center for Genomic Technologies – Huji).

In vivo RNA proximity labeling

APEX2, alkyne-phenol, and H₂O₂ are needed for RNA proximity labeling.

Gram-negative bacteria

Gram-negative bacteria were grown overnight in liquid media at their optimal growth temperature. Once the cultures reached an optical density (OD) of ∼0.3–0.6, 2.5 mM of alkyne-phenol was added and incubated for 30 min in a shaker incubator at 200 rpm. A 100 mM H₂O₂ solution (994 μL H₂O + 6 μL H₂O₂, 50%) was then freshly prepared. 1 mM of H₂O₂ was then added for 45 s as the cultures were still shaking. The labeling experiment was carried out again with specific omissions as controls: no alkyne-phenol added, no H₂O₂ added, no alkyne-phenol and H₂O₂ added, different concentrations of alkyne-phenol added (0.5 mM and 1 mM), and different H₂O₂ exposures (15 s, 30 s, and 60 s). 4 mL of cell culture were then quickly transferred into two 2 mL Eppendorf tubes and centrifuged at 14,000 rpm for 10–15 s. The supernatant was quickly discarded, and the cell pellets were resuspended in 1 mL of 65°C pre-warmed TRIzol. Because proximity labeling requires the folded APEX2 protein, its denaturation in TRIzol can be used to quench the proximity labeling reactions. RNA extraction followed by ethanol precipitation was then carried out. 55 μL of a 10 mM Tris-HCl pH 7.0 (no EDTA) solution was added to dissolve the air-dried RNA pellets. The samples were vortexed briefly to aid resuspension. 5 μL were taken from the samples and placed in new tubes for Agilent TapeStation analyses and cell lysate RNA sequencing.

Gram-positive bacteria

Gram-positive bacteria were grown overnight in liquid media at their optimal growth temperature. Once the cultures reached an optical density (OD) of ∼0.3–0.6, the cultures were re-inoculated into fresh media at an OD of ∼0.05 supplemented with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce the expression of RNY-APEX2. Once the cultures reached an OD of ∼0.4–0.5, 2.5 mM of alkyne-phenol was added and incubated for 30 min in a shaker incubator at 200 rpm. A 100 mM H₂O₂ solution was then freshly prepared.1 mM of H₂O₂ was then added for 45 s as the cultures were still shaking. An equal amount of cold methanol (pre-stored at −80°C) was then added to the culture solution. The mixture was then quickly vortexed for 5 s and centrifuged at 14,000 rpm for 10–15 s. The cell pellets were resuspended in LETS buffer (1M Tris-HCl, 1M LiCl, 0.5M EDTA, and 10% SDS; for a 4 mL of gram-positive cultures, the cell pellets were dissolved in 550 μL of LETS buffer). Both the LETS buffer and bead:phenol solutions were pre-warmed at 75°C before being used. The solution was then transferred to a tube containing glass beads + phenol (for a 4 mL of gram-positive culture, 320 μL of glass beads +380 μL of phenol were used). The mixture was vortexed for 3 min. Chloroform was then added to the mixture and the solution was vortexed for 30 s (for a 4 mL of gram-positive culture, 400 μL of chloroform was added to the cells + beads solution). The samples were then centrifuged at 3200 x g for 10 min. The top aqueous layer was then transferred to a fresh tube containing a phenol:chloroform solution (for a 4 mL of gram-positive culture, 550 μL of phenol:chloroform was used). The phenol:chloroform solution was pre-warmed at 75°C before being used. The mixture was then vortexed for 3 min, and the sample was centrifuged at 3200 x g for 10 min. The top aqueous layer was then transferred to a fresh tube and RNA precipitation was carried out. 55 μL of a 10 mM Tris-HCl pH 7.0 (no EDTA) solution was added to dissolve the air-dried RNA pellets. The samples were vortexed briefly to aid resuspension.

Copper-catalyzed click chemistry reactions

Copper-catalyzed click chemistry reactions were then conducted on the labeled RNA. The reactions have a 300 μL total volume and contained: 50 μL of 100 ng/uL RNA (5 μg) + 175 μL H₂O + 12 μL of 125 mM sodium ascorbate +3 μL of 10 mM Cy5-Azide +60 μL of a mix of 2.5 mM Cu(II)SO₄/12.5 mM THPTA (tris-hydroxypropyltriazolylmethylamine). The reagents were added in the order they are listed. The click reaction was vortexed for 5 s and incubated for 10 min at room temperature away from light exposure. Afterward, RNA precipitation was conducted and the RNA pellets were resuspended in 50 μL of 10 mM Tris-HCl pH 7.0 and 0.1 mM EDTA. The samples were then subjected to dot blot filtration on a nylon membrane following the manual’s instructions of BIO RAD’s Bio-Dot and Bio-Dot SF Microfiltration Apparatus. A nylon membrane was pre-wetted in a 6X SSC solution for at least 10 min. The pre-wetted nylon membrane was then assembled into the dot blot apparatus. Vacuuming was applied to the apparatus and tighter sealing was implemented. The wells were then washed 3 times with 1 mL of an ice-cold mixture of 10 mM NaOH/1 mM EDTA. Afterward, the 5 μg RNA samples were directly applied to the wells (no RNA dissolution was performed). The wells were washed 3 times with 1 mL of an ice-cold mixture of 10 mM NaOH/1 mM EDTA. The blotted membrane was washed 3 times for 5 min with a 30 mL solution containing 2X SSC and 0.1% SDS. Fluorescent blot imaging was then visualized using an iBright imaging system at a fluorescent light exposure of 50 ms under the Cy5 channel.

RNase A & DNase I treatments

5 μg RNA (50 μL of 100 ng/μL of RNA) collected from a proximity labeling reaction (4 mL of Js767 cells grown in M2G media with 2.5 mM alkyne phenol, 45 s H₂O₂) were subjected to either an RNase A (5 μL of 10 mg/mL RNase A) or DNase I (5 μL of 1 U/μL DNase I) treatment. The treated and mock samples were incubated for 2 h at 37°C. Following the enzyme or mock treatments, the RNA was precipitated and resuspended in 50 μL of 10 mM Tris-HCl pH 7.0 and then subjected to the Copper catalyzed click-chemistry reaction with Cy5-azide. Following the 10-min click reaction incubation, the RNA was then precipitated and subjected to dot blot filtration as previously described.

Streptavidin capture of biotin labeled RNA

To enrich the transcriptome of BR-bodies, RNA labeled with alkyne-phenol was subjected to the following click chemistry reaction: 50 μL of RNA (total RNA volume) + 175 μL H2O + 12 μL of 125 mM sodium ascorbate +3 μL of 10 mM Biotin-peg3-azide +60 μL of a mix of 2.5 mM Cu(II)SO₄/12.5 mM THPTA. The reagents were added in order as they are listed. The click reaction was vortexed briefly and incubated for 10 min at room temperature away from light exposure. RNA precipitation was then conducted and the air-dried RNA pellets were resuspended in 100 μL B&W buffer (5 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5 mM EDTA, 0.1% v/v tween 20). The RNA samples were then immediately used for biotinylated RNA enrichment.

High-capacity magnetic streptavidin beads (Vector Labs) were washed three times with 1 mL B&W buffer, twice with 1 mL of a mix of 0.1 M NaOH/0.05 M NaCl, and once with 1 mL 0.1 M NaCl at room temperature. The beads were then blocked using a blocking buffer (1 mg/mL BSA +1 mg/mL heparin salt, dissolved in B&W buffer) at room temperature for 2 h. The beads were then washed three times with 1 mL of B&W buffer under gentle vortexing. Afterward, the 100 μL RNA solution was added to the beads along with an extra 100 μL of B&W buffer. The RNA and beads were allowed to mix at room temperature for one hour. The RNA-loaded beads were washed three times with 1 mL of B&W buffer, twice with 1 mL of a PBS solution containing 4 M urea +0.1% SDS, and twice with 1 mL of just PBS at room temperature. To elute the enriched RNA, 900 μL of 65°C pre-warmed TRIzol was added to the beads and the samples were incubated at 65°C for 10 min. Afterward, 200 μL of chloroform was added and the samples were inverted 6–7 times and then were incubated for 5 min at room temperature. The samples were centrifuged at 14,000 rpm for 10 min at 4°C. The upper aqueous layer was transferred to a new Eppendorf tube and the enriched RNA was precipitated as previously described. The air-dried RNA pellets were resuspended in 10 μL of 10 mM Tris-HCl pH 7.0 and 0.1 mM EDTA. 4 μL were then used for Agilent TapeStation analysis. The remaining volume could be utilized for the follow-up RNA-seq experiments.

RNA quality assessment

Js767 (RNE-Apex2FlgC-2) was grown in PYE at 28°C overnight and re-inoculated into 5 mL M2G the next day. Once the cultures reached an OD of 1.2, 2.5 mM of alkyne-phenol was added and incubated for 30 min in a shaker incubator at 28°C at 200 rpm. 1 mM of H₂O₂ was added to the culture for 45 s. Total RNA was then collected and precipitated from 4 mL of culture as previously described using TRIzol. Afterward, 50 μL of 100 ng/μL of alkylated RNA were subjected to the following copper catalyzed click-chemistry reaction: 50 μL of 100 ng/μL RNA (5 μg) + 175 μL H₂O + 12 μL of 125 mM sodium ascorbate +3 μL of 10 mM Cy5-Azide +15 μL of 100 mM aminoguanidine hydrochloric acid +60 μL of a mix of 2.5 mM Cu(II)SO₄/12.5 mM THPTA. The click reaction was incubated for 10 min at room temperature away from light exposure. The RNA from the click reaction was then precipitated and the air-dried RNA pellet was resuspended in 50 μL of 10 mM Tris-HCl pH 7.0 and 0.1 mM EDTA. 250 ng of RNA from the Js767 RNA lysate and the click reaction RNA were sent for Agilent TapeStation analysis.

RNA precipitation

RNA precipitation was carried out by adding, in order, 2 μL of 1 mg/mL glycogen, 1/10 sample volume of 3 M sodium acetate pH 5.5, and 1 sample volume of isopropanol. The sample mixtures were vigorously vortexed and stored at −80°C overnight. The samples were spun for 1 h at 14,000 rpm. The supernatant was then decanted and 1 mL of ice-cold 80% ethanol was added. The samples were spun for 15 min at 14,000 rpm. The ethanol was pipetted out and the samples were then spun for 1 min at 14,000 rpm. Any remaining ethanol was pipetted out and the pellets were allowed to air-dry until they became translucent. The RNA was then resuspended in the buffer of choice.

Western blots

The following buffers were initially prepared.

• (1)10X transfer buffer: 144 grams of glycine and 30.2 grams of Tris-Base were dissolved in 900 mL of ddH2O. The buffer volume was adjusted to 1 L of ddH2O and then filtered utilizing a Nalgene Rapid-flow sterile disposable filter unit.
• (2)10X TBS: 24 grams of Tris-Base and 88 grams of NaCl were dissolved in 900 mL of ddH2O. The pH of the buffer was adjusted to 7.6 utilizing hydrochloric acid. The buffer volume was adjusted to 1 L of ddH2O and then sterilized utilizing a Nalgene Rapid-flow sterile disposable filter unit.
• (3)1X TBST: 10X TBS was diluted 10-fold with ddH2O to make 1X TBS. 0.1% Tween 20 was then added to the 1X TBS solution to make 1X TBST.

NA1000, LS4379 (hfq-m2), and Js767(RNE-Apex2FlgC-2) were inoculated in PYE and incubated overnight at 28°C until they reached log phase (OD ∼0.3–0.6). 1 mL was taken from each culture, placed in 1.5 mL Eppendorf tubes, and centrifuged for 2 min at 6,000 rpm. The liquid cultures were discarded, and the cells were resuspended in 4X SDS loading dye. 125 μL of 4X SDS loading dye was added to cultures with an OD∼0.5. The samples were boiled at 95°C for 5 min then vortexed vigorously. The vortexed samples were quickly spun down and placed on ice. 5 μL of a pre-stained PageRuler marker and 20 μL of lysate from each sample were loaded onto an SDS-PAGE gel. 1X transferring buffer was freshly prepared (20 mL of 10X transfer buffer +20 mL of methanol in 160 mL ddH2O) while the samples were running on the gel. A PVDF transferring membrane was wetted in methanol and 6 blotting papers were wetted using 1X transferring buffer for at least 10 min. Once the lysate samples on the SDS-PAGE were properly resolved, the gel was placed on top of 3 pre-wetted blotting papers, and 2 mL of 1X transferring buffer was poured on the gel. The PVDF membrane was briefly plunged into the 1X transferring buffer and then placed on top of the gel. The remaining 3 blotting papers were placed on top of the PVDF membrane. The transferring of the lysates from the SDS-PAGE gel to the PVDF membrane occurred by utilizing the BIO-RAD Trans-Blot Turbo Transfer system (1 amps, 2.5 volts, 15 min). Once the transfer was completed, the PVDF membrane was placed in a new container containing 5% BSA (blocking solution). The container was nutated for 1 h at room temperature. Afterward, the 5% BSA blocking solution was discarded and the PVDF membrane was washed 5 times with ddH2O for 5 min per wash. The primary antibody (DYKDDDDK anti-flag tag; binds to the same epitope as Sigma-Aldrich Anti-FLAG M2 antibody) was then diluted (1:10000) into a fresh 5% BSA solution. Once the last ddH2O wash was completed, the primary antibody solution was poured on the PVDF membrane, and the container was nutated at 4°C overnight. The following day, the primary antibody solution was discarded and the PVDF membrane was washed 5 times with TBST for 5 min per wash. The secondary antibody (goat anti-rabbit secondary antibody, HRP conjugated) was then diluted (1:10000) into a fresh 5% BSA solution. Once the last TBST wash was completed, the secondary antibody solution was poured on the PVDF membrane, and the container was nutated at room temperature for 1 h avoiding light exposure. The secondary antibody solution was then discarded and the PVDF membrane was washed 5 times with TBST for 5 min per wash. Following the final washing procedure, a pierce ECL western blotting substrate solution was prepared (1 mL of reagent 1 + 1 mL of reagent 2 were mixed and vigorously vortexed). The PVDF membrane was placed on Seram wrap and the 2 mL substrate solution was poured on top of the membrane. The substrate was incubated on the PVDF membrane at room temperature for 5 min avoiding light exposure. The membrane was then placed into an iBright imaging system and the signal was detected under the Chemi-blot filter.

Dilution plates

NA1000 & Js767 (RNE-Apex2FlgC-2) were grown in PYE overnight at 28°C. The overnight cultures were diluted into fresh media and incubated at 28°C until the cells reached an OD of ∼0.3–0.6. Cells were then diluted to an OD = 0.05 in PYE and 4 10-fold serial dilutions were made. 5 μL were spotted from each dilution onto PYE plates. The plates were incubated at 28°C for two days, and images were taken using an iBright imaging system.

Fluorescent cell imaging

Js87 (RNE-msfGFP) & Js768 (RNE-Apex2FlgC-EGFPC-2) were grown in PYE + gent (0.5 μg/mL) and PYE + Kan, respectively, overnight at 28°C. Cells were then fixed on M2G + 1.5% agarose pads placed on microscope slides (3051, Thermo Fisher Scientific). Imaging was performed on an epifluorescence microscope with a 100× objective. Specifically, Nikon elements software was used to control a Nikon Eclipse NI-E equipped with a CoolSNAP MYO-CCD camera and a 100× Oil CFI Plan Fluor (Nikon) objective to capture the images.

In vivo imaging of proximity labeling

Js767 (RNE-Apex2FlgC-2) and Js803 (RNE-NTD-APEX2FlgC-2) were grown in PYE overnight at 28°C. The overnight cultures were diluted into fresh media and incubated at 28°C until the cells reached an OD of ∼0.3–0.6. 2.5 mM of alkyne-phenol was added to 1 mL of cells and incubated for 30 min in a shaker incubator at 200 rpm. A 100 mM H₂O₂ solution was then freshly prepared. 1 mM of H₂O₂ was then added for 45 s as the cultures were still shaking. Cells were then quickly transferred to a 2 mL Eppendorf tube and centrifuged at 14,000 rpm for 10–15 s. Cell pellets were washed once with 500 μL PBS, then fixed with 300 μL of 4% formaldehyde for 30 min at 28°C. Cells were then pelleted and washed once with 500 μL PBS. Cell pellets were resuspended in 300 μL of ice-cold 70% ethanol and incubated for 30 min on ice. The cells were then pelleted and washed once with 500 μL PBS and resuspended in 300 μL 0.1 mM iodoacetamide and incubated for 30 min at 28°C. After pelleting the cells and washing them with 500 μL PBS, cells were resuspended in a freshly prepared Azide-Cy5 cocktail (25 μM Azide-Cy5, 2.5 mM sodium ascorbate in PBS, 250 μM THPTA, 50 μM CuSO₄, and PBS was added until the total volume reached was 300 μL) and incubated for 30 min at room temperature away from light exposure. The Azide-Cy5 cocktail was incubated for 10 min on ice prior to application. The cells were then pelleted and washed twice with 500 μL PBS, then resuspended in 20–200 μL PBS depending on the size of the cell pellet. Cells were then fixed on M2G + 1.5% agarose pads placed on microscope slides (3051, Thermo Fisher Scientific). Imaging was performed on an epifluorescence microscope with a 100× oil immersion objective. Specifically, Nikon elements software was used to control a Nikon Eclipse NI-E equipped with a CoolSNAP MYO-CCD camera and a 100× Oil CFI Plan Fluor (Nikon) objective to capture the images.

Quantification and statistical analysis

mRNA half-lives measurement

NA1000 and Js767 (RNE-Apex2FlgC-2) were grown in liquid PYE at 28°C overnight. Cells were then serially re-inoculated into liquid M2G and incubated until overnight log phase (OD ∼0.3–0.6) was reached. Log phase cultures were then re-inoculated to an OD = 0.05 in 25 mL liquid M2G. Before adding rifampicin, at time point 0, 1 mL of cells were added to 2 mL of RNAprotect Bacterial reagent (Qiagen) and vortexed for 5 s 200 μg/mL of Rifampicin was administered to the cultures and 1 mL of cells were extracted and added to 2 mL of RNAprotect Bacterial reagent at each of the following time points (followed by 5 s vortexing): 1, 2, 4, and 8 min. Cells were incubated at room temperature in the RNAprotect Bacterial reagent for 5 min before being spun at 5000 rpm for 5 min. The bacterial pellets were resuspended in 1 mL of 65°C pre-heated TRizol (Ambion) and incubated at 65°C for 10 min 200 μL of chloroform was then added and the samples were incubated at room temperature for 5 min. Subsequently, the samples were centrifuged at 14,000 rpm for 10 min at 4°C. The aqueous layer was removed and placed in a new 1.5 mL Eppendorf tube. The RNA was then precipitated and the pellets were resuspended in 50 μL elution buffer (10 mM Tris-HCl, pH = 7.0, 0.1 mM EDTA). PCR tubes were filled with a master mix that contained 0.4 μM of ctrA forward primer and 0.4 μM of ctrA reverse primer (Table S1), 1X Luna Universal One-Step Reaction Mix, 1X Luna WarmStart RT Enzyme Mix, and water. 100 ng/μL of RNA template was additionally aliquoted into each of the PCR tubes. The samples were mixed well and quickly spun down. A QuantStudio Real-Time PCR apparatus was utilized to conduct and examine the qRT-PCR experiments. The same qRT-PCR experiment was done using instead the 5S forward and reverse primers. To determine the mRNA-decay rates, we fitted a linear curve to the ln (fraction RNA remaining) at each time point. Using a standard curve, the Ct was converted into the quantity of RNA. Each time point’s 5S rRNA amount was divided by the amount of 5S rRNA at time point 0. The number obtained at each time point was then used to normalize the amount of ctrA at each time point. The natural log of % RNA remaining found in each sample was divided by the natural log of RNA at time point 0. The slopes of the linear curve fit were then converted into mRNA half-life using the following equation: mRNA half-life = −ln(2)/slope. RNA quantified at each time point was obtained from the average of three biological replicates, with three technical replicates each.

RNA-seq

Duplicate biological samples were prepared for RNA-seq of the Full-length RNase E-APEX2 fusion (JS767) and the RNase E-NTD-APEX2 (JS803). Lysates from each sample, as well as the eluted RNA from affinity purification were prepared for RNA-seq. RNA-seq was performed by IU-Bloomington CGB genomics service facility using the Illumina Tru-seq stranded HT kit. Raw sequencing reads were deposited to the NCBI GEO database with accession number GSE297938. Data analysis was performed similar to Al-Husini et al. Mol Cell 2020.¹⁸ RNA reads were stripped of adapters, and tRNA and rRNA reads were aligned to unique file with duplicate copies deleted using bowtie,⁴¹ and reads not aligning to tRNA and rRNA were aligned to the C. crescentus transcriptional units⁴² using bowtie. Genes with fewer than 50 reads mapping to them were omitted from analysis, and the RPKM values were calculated for each sample. The log2 ratio between the RPKM values in the elution and lysate samples were then calculated on all genes with ≥50 reads in both elution and lysate samples. Samples with log2(elution/lysate) measurements in all four biological samples were used for comparison for BR-body proximity labeling enrichment. To calculate which RNA types were enriched in BR-bodies, the distribution of enrichment measurements log2(elution RPKM/lysate RPKM) were subjected to a T-test with unequal variance between the BR-body + (JS767) and BR-body – (JS803) strains and the resulting p-values were reported.

Published: October 20, 2025