**Background:** Regulation of messenger RNA stability is critical for programmed gene expression in bacteria, enabling adaptation to changing environments. While mRNA degradation in bacteria was traditionally thought to initiate via endonucleolytic cleavage followed by 3′–5′ degradation, the discovery of 5′–3′ exonucleolytic activity of RNase J in Bacillus subtilis raised questions about how cotranslational mRNA degradation shapes the bacterial degradome. In eukaryotes, the 5′–3′ exonuclease Xrn1 follows the last translating ribosome, producing an in vivo toeprint of its position. Whether similar mechanisms exist across diverse bacterial species with divergent mRNA degradation machinery remained unknown.
**Methods:** The authors developed an optimized 5′ monophosphorylated (5′P) mRNA decay intermediate sequencing approach (5PSeq) and applied it to 96 bacterial species spanning 58 genera, including model organisms (B. subtilis, E. coli), and complex microbiomes (vaginal swabs, human faecal samples, faecal cultures, compost). They analysed 5′P degradome patterns in open reading frames (ORFs), measuring 3-nucleotide periodicity via Fast Fourier Transform (FFT), ribosome protection at start/stop codons, and codon-specific stalling. Perturbation experiments included antibiotic treatments (chloramphenicol [CAM] at 100 μg/ml, mupirocin [MUP] at 65 μg/ml, doxycycline [DOX] at 5 μg/ml, erythromycin [ERY] at 5 μg/ml), heat shock (60–65°C for 10–15 min), salt stress (1 M NaCl for 10 min), low nutrient (0.5× LB), and stationary phase (up to 8 days). RNase knockout strains (rnjA, rnjB, rny) in B. subtilis were used to dissect nuclease contributions. In vitro RNA fragmentation served as negative control. Polyribosome fractionation confirmed association of 5′P intermediates with translating ribosomes.
**Key Results:** In B. subtilis, 5′P reads showed a clear 3-nucleotide periodicity with preference for the second nucleotide of each codon (F1), and accumulated 11 nt upstream of translation start sites and 14 nt upstream of stop sites—3 nt smaller than the eukaryotic protection size, consistent with bacterial ribosome size differences. In vitro fragmentation eliminated >99.5% of this periodicity (FFT signal dropping to 0.11). RNase JA deletion (rnjA) abolished the pattern, confirming RNase J drives cotranslational 5′–3′ degradation. Species with RNase J (Bacillota, Cyanobacteria, some Pseudomonadota) showed similar 3-nt periodicity, though Synechocystis sp. PCC 6803 showed a displaced protection pattern (−12 nt from start, −15 nt from stop). Species lacking 5′–3′ exonucleases (E. coli, Bacteroidota) showed endonucleolytic cleavage sites concentrated around start/stop codons, with subtle or absent periodicity. Sequence motif analysis revealed conserved cleavage preferences: RNase J-containing species showed homogeneous motifs; RNase E/G species preferred −1U/+1A; RNase Y species preferred +1A. CAM treatment increased ribosome protection patterns in RNase J-containing species, with 5′P accumulation in 5′ regions of ORFs. MUP treatment caused Ile-specific ribosome stalls (14 nt upstream of Ile codons) in L. plantarum, L. reuteri, and B. subtilis. CAM induced context-specific stalls at Ala and Ser (8 nt upstream) in species with RNase J. Heat shock and stationary phase caused Tyr, His, and Arg codon stalls in L. plantarum, and Gln and Arg stalls in B. subtilis. Principal component analysis of ribosome protection phenotypes separated control, stressed, and drug-treated samples. In complex faecal microbiomes, 5PSeq detected species-specific responses: MUP caused Ile stalls in Enterococcus faecalis, Clostridium tyrobutiricum, and Anaeroglobus geminatus; CAM caused Ala stalls; DOX caused accumulation at −11 nt from start codons (initiation stalling); ERY caused stalling at (R/K)×(R/K) motifs and Pro codons. Effects were observed as early as 5 min post-treatment. The method detected MUP-induced Ile pauses even at 1:10,000 dilution (0.01% abundance) in mixed Lactobacillus species. The degradome atlas across 96 species revealed that 3-nt periodicity is widespread but varies by taxonomy and RNase complement.
**Clinical Implications:** This work establishes metadegradome sequencing as a powerful tool for studying posttranscriptional regulation in bacteria without requiring culturing or subcellular fractionation. The method can detect rapid (within 5 min) species-specific translational responses to antibiotics in complex microbiomes, potentially enabling monitoring of drug effects in situ. The ability to infer ribosome dynamics and amino acid limitations from stored RNA samples makes it applicable to clinical biobanks. The atlas of 96 species provides a reference for future studies of RNA decay mechanisms, and the approach could be used to characterize posttranscriptional regulation in unculturable species, which comprise more than half of human bacterial communities. The interactive website (http://metadegradome.pelechanolab.com) facilitates further exploration of these data.