An RNA-binding protein acts as a major post-transcriptional modulator in Bacillus anthracis

Abstract

HitRS is a two-component system that responds to cell envelope damage in the human pathogen Bacillus anthracis. Here we identify an RNA-binding protein, KrrA, that regulates HitRS function by modulating the stability of the hitRS mRNA. In addition to hitRS, KrrA binds to over 70 RNAs and, directly or indirectly, affects the expression of over 150 genes involved in multiple processes, including genetic competence, sporulation, RNA turnover, DNA repair, transport, and cellular metabolism. KrrA does not exhibit detectable nuclease activity in vitro, and thus the mechanism by which it modulates mRNA stability remains unclear.

Fig. 1. An unbiased genetic selection strategy identifies KrrA as a regulatory factor in HitRS signaling.

A Schematic of the genetic selection strategy: a strain containing ermC driven by a HitR promoter (P_hitermC) was plated on medium containing toxic levels of erythromycin and colonies that arose represented bacteria that acquired mutations that constitutively activate the P_hit promoter. B All 21 erythromycin-resistant suppressors isolated from genetic selections exhibit frameshift mutations at five different positions within krrA. C, D Growth kinetics of B. anthracis WT, WT P_hitermC, ΔkrrA P_hitermC, ΔkrrA ΔhitRS P_hitermC, and the complemented strain ΔkrrA P_hitermC pOS1.P_lgtkrrA-3xFLAG in vehicle (C) or 20 µg ml^-1 erythromycin (D) were monitored for 24 h. The experiments were conducted at least three times. Data shown are averages of three biological replicates (mean ± SD).

Fig. 2. The HitRS signaling system is finely tuned by KrrA through modulating mRNA stability.

A To examine the effects of krrA deletion on the stability of hitPRS transcripts, northern blot analysis was carried out in WT and ΔkrrA in the presence or absence of ‘205 using a pool of four sequence-specific probes against hitPRS. The housekeeping gene 16 S rRNA serves as a sample loading control. The experiments were performed at least three times and representative images are shown. Source data are provided as a Source Data file. The mRNA stability of hitR (B, C) and gyrA (D, E) was examined in two sets of strains: B. anthracis WT P_hitermC and B26 suppressor mutant (B, D), and WT and ΔkrrA (C, E). Relative abundance of hitR (B, C), or gyrA (D, E) was quantified using qPCR. The housekeeping gene gyrA serves as a negative control. The mRNA half-life was determined using a single exponential decay model. Data shown are three biological replicates (mean ± SD). Significant differences are determined by two-tailed t tests.

Fig. 3. KrrA directly binds to many RNA targets in vivo, including hitPRS.

A To evaluate the RNA-binding ability of KrrA, a CLIP assay (UV crosslinkingg immunoprecipitation) was carried out in WT and ΔkrrA pOS1.P_lgtkrrA-3xFLAG strains in the presence or absence of ‘205 treatment with or without UV crosslinkingg. The immunoprecipitated KrrA-3xFLAG protein was detected by Western blot using an anti-FLAG antibody (top panel). This serves as a control to evaluate the immunoprecipitation efficiency. The immunoprecipitated and radioactively labeled RNA-KrrA complexes were detected by phosphorimaging (bottom panel). The experiments were conducted at least three times and representative images are shown. Source data are provided as a Source Data file. B Overview of KrrA-RNA-binding profiles revealed by formaldehyde cross-linking RNA immunoprecipitation coupled with Illumina sequencing (fRIP-seq) under vehicle or ‘205-treated conditions. C Genes identified in the RIP-seq analysis with significantly increased enrichment grouped into predicted functional categories. D Volcano plot showing KrrA-binding RNA transcripts enriched following ‘205 treatment compared to vehicle. Transcripts that were most significantly enriched upon ‘205 treatment are highlighted in blue. The log₂(fold change) cutoff is ≥2 and the P value cutoff is <0.05, both of which are indicated by gray lines. E A zoom-in example of KrrA binding to the hitPRS transcripts identified by fRIP-seq. Two biological replicates were included for each condition. S/N denotes the signal-to-noise ratio for peak calling. F KrrA specifically binds to hitR transcript under vehicle conditions and transcripts of the entire hitPRS operon upon ‘205 treatment. ND not detectable. G KrrA binding to the transcripts of hitPRS was confirmed by fRIP-qPCR under vehicle or ‘205-treated conditions. The gene gyrA was used as a control. The data are expressed as the mean ± SD (n = 3). Significant differences are determined by two-tailed t tests. Source data are provided as a Source Data file.

Fig. 4. KrrA binds to hitR transcript in vitro.

A, B To examine whether KrrA binds to hitR transcript in vitro, an electrophoretic mobility gel shift assay was carried out using in vitro-transcribed and radioactively labeled hitR transcript (A) and control RNA (B) in the presence of increasing concentration of KrrA protein as indicated. The experiments were conducted at least three times and representative images are shown.

Fig. 5. KrrA-mediated RNA regulation negatively affects the abundance of numerous transcripts.

Volcano plot showing transcriptomic comparison between B. anthracis ΔkrrA and WT following vehicle (A) or 50 µM ‘205 treatment (B). Genes with significantly different expressions are highlighted in cyan (A) or orange (B). The fold change cutoff is ≥3 and the P value cutoff is <0.05, both of which are indicated by gray lines. C Overview of KrrA targets revealed by RNA-seq under vehicle or ‘205-treated conditions. D Genes identified in the RNA-seq analysis with significantly differential expression grouped into predicted functional categories. Inset: Efficiency of sporulation and germination was further examined in B. anthracis WT and ΔkrrA. Data shown are three independent experiments with three replicates each time (mean ± SEM; n = 9). Significant differences are determined by two-tailed t tests. E The key stages in the cycle of sporulation and germination showing all genes with significantly elevated expression identified from RNA-seq under vehicle- (cyan), ‘205-treated (orange), or both conditions (purple). Other genes that have not been characterized for any specific stage are listed in the box. Additional genes with minor changes in expression but identified from RIP-seq are shown in gray. The genes enriched by KrrA immunoprecipitation (RIP-seq) are denoted by asterisk.

Fig. 6. The HitRS activator ‘205 diverts cellular metabolism from aerobic respiration to fermentation.

A Volcano plot showing transcriptomic comparison between ‘205 (50 µM) and vehicle treatments of B. anthracis WT. Genes significantly upregulated or downregulated following ‘205 treatment are highlighted in orange or blue, respectively. The fold change cutoff is ≥3 and the P value cutoff is <0.05, both of which are indicated by gray lines. B Genes identified in the RNA-seq analysis with significantly differential expression grouped into predicted functional categories. C A subset of notable metabolic pathways that showed significantly different expression following ‘205 treatment: glycolysis, gluconeogenesis, and TCA cycle. Products of genes with increased expression are labeled in orange while the ones with decreased expression are labeled in blue. The fold change of each specific gene is indicted: +, upregulation; −, downregulation. D Ethanol concentration was quantified in the spent media of WT cell cultures in the absence or presence of 50 µM ‘205. ‘205 drives glucose metabolism towards pyruvate fermentation, resulting in higher level of ethanol compared to vehicle control. Data shown are three independent experiments with three replicates each time (mean ± SEM; n = 9). Significant differences are determined by two-tailed t tests.

Fig. 7. Combined fRIP-seq and RNA-seq datasets uncover the broad effects in gene expression and coregulatory gene networks modulated by KrrA function.

A The transcript abundance of 73 direct targets was significantly affected by KrrA regulation and these direct RNA targets are grouped into various functional categories. B Combined fRIP-seq and RNA-seq data showing global changes modulated by KrrA function. Each bar represents a gene with significantly differential abundance. Each level of color gradation represents a fivefold change. Genes with ≥15-fold change are represented with bars that hit the maximum limit. The bars in different colors show genes with differential abundance under vehicle (green), ‘205-treated (red), or both conditions (blue). The triangles indicate the KrrA-regulated transcripts that are involved in TCS signaling (black), competence (red), sporulation (green), and germination (blue). C Overview of KrrA targets revealed by comparing the datasets of fRIP-seq and RNA-seq.