It is shown that Bacteroides instead rely on a different family of RNA-binding proteins, more typical of eukaryotes than bacteria, to survive cold stress, and that these proteins promote survival under the conditions encountered during transmission.
Abstract
Bacteria use post-transcriptional regulatory mechanisms to rapidly adjust gene expression during environmental change. In the gut-associated genus Bacteroides, these mechanisms remain poorly defined as these organisms lack canonical RNA chaperones like Hfq and CsrA that coordinate post-transcriptional stress responses in many well-studied model bacteria. Most Bacteroides possess conserved RNA recognition motif-1 (RRM-1) domain-containing RNA-binding proteins (more common in eukaryotes than bacteria) that have been proposed to act as global RNA chaperones. Here, we show that these RNA binding proteins (RBPs) are central to cold stress adaptation. Simultaneous deletion of all rbp genes produces a cold-sensitive growth defect across multiple Bacteroides species, while single deletions do not, revealing conserved functional redundancy. RBP transcripts and proteins accumulate rapidly after temperature downshift, and loss of RBPs extensively reprograms the transcriptome. Cold sensitivity of Bacteroides rbp mutants is not caused by defects in ribosome assembly or rRNA maturation. Instead, we find that in Bacteroides thetaiotaomicron, RBPs act together with BT1884, the sole canonical cold shock protein possessed by this organism. The combined loss of RBPs and BT1884 produces a synthetic severe cold sensitivity phenotype, defining two functionally redundant cold stress systems belonging to unrelated protein families. Strains lacking RBPs show reduced survival under simultaneous cold and oxygen stress, the conditions Bacteroides cells are expected to encounter during host-to-host transmission. Together, these findings establish RRM-1 RBPs as non-canonical cold shock proteins that enable cold adaptation and environmental survival in Bacteroides and suggest how these organisms withstand the stresses of transmission between hosts. IMPORTANCE Bacteroides species are among the most abundant and stable members of the human gut microbiome, and they are also among the most readily transmitted between people. Reaching a new host requires surviving conditions outside the gut, including cold and oxygen exposure, yet how these bacteria withstand such stress is not well understood. Most bacteria manage stress using a well-defined set of RNA-binding proteins, but Bacteroides lack these canonical factors. We show that Bacteroides instead rely on a different family of RNA-binding proteins, more typical of eukaryotes than bacteria, to survive cold stress, and that these proteins promote survival under the conditions encountered during transmission. This work identifies a molecular system that allows an abundant and ecologically successful gut bacterium to endure the environmental challenges of moving between hosts.
Abstract Bacteria employ sophisticated post-transcriptional regulatory mechanisms to adapt to environmental changes. Carbon storage regulator A (CsrA), a highly conserved RNA-binding protein, serves as a critical post-transcriptional regulator by typically recognizing GGA-containing hairpin loops in target mRNAs and repressing translation. However, how this conserved regulator evolved diverse species-specific regulatory networks remains unclear. We developed Swarm-seq, a high-throughput platform assessing CsrA homologs across the bacterial domain for regulating flagella-dependent swarming in Bacillus subtilis. Testing over five-hundred codon-optimized csrA homologs revealed functional divergence, partitioning CsrAs into two broad classes. Class I (CsrAHp, CsrASm, RsmNPa) strongly inhibited swarming, while Class II (CsrAEc, RsmAPa) failed despite sequence conservation. This differential activity occurred despite canonical GGA motifs in flagellin (hag) transcript, suggesting evolutionary plasticity in RNA-binding specificity beyond motif recognition. Leveraging this dataset, we trained machine learning algorithms to predict CsrA functionality, experimentally validating Bdellovibrio bacteriovorus CsrA (CsrABb) and Pseudomonas putida RsmA (RsmAPp) as Class I. Our findings establish Swarm-seq as a powerful platform for characterizing CsrA homologs from genetically intractable or unculturable bacteria and demonstrate the potential for machine learning-guided discovery of functional regulatory proteins, providing insights into post-transcriptional regulatory network evolution.
Jared T. Winkelman, Ethan Yarberry, Georgia Fanouraki et al.· Nucleic Acids Research· 0 citations
Bacterial gene expression depends on the coordinated regulation of RNA synthesis, processing, and decay. The conserved RNA degradosome scaffold, Ribonuclease E (RNase E), assembles into phase-separated bacterial ribonucleoprotein bodies (BR-bodies) through its C-terminal intrinsically disordered region (IDR). This IDR also scaffolds recruitment of degradosome client proteins that carry out post-transcriptional gene regulation. Whether BR-bodies regulate virulence programs to support infection, however, is largely undefined. We show that RNase E of the intracellular pathogen Brucella ovis forms RNA-dependent condensates in vivo and phase-separates with RNA in vitro, with the IDR necessary and sufficient for BR-body assembly. Deleting the IDR (rne(ΔIDR)) did not impair growth but sensitized Brucella to host-relevant oxidative and cell-envelope stressors. Transcriptome-wide profiling that simultaneously resolved mRNA decay and processing revealed that BR-bodies primarily accelerate mRNA turnover while stabilizing a distinct subset of transcripts. Notably, BR-bodies control the processing and levels of virB type IV secretion system (T4SS) mRNA and the turnover of its key activators, linking condensate-based RNA regulation to a core virulence pathway. Consistent with virB dysregulation, the rne(ΔIDR) mutant was severely attenuated in mammalian macrophages. To test whether phase separation is sufficient for BR-body function, we replaced the B. ovis IDR with the highly divergent Caulobacter crescentus IDR. This chimera assembled BR-bodies but rescued fitness incompletely, fully restoring oxidative-stress resistance but not cell-envelope stress resistance or intracellular fitness. BR-bodies therefore link RNA metabolism to Brucella stress resistance and infection, and their full function requires both phase separation and additional native IDR-specific activities such as degradosome interactions. Importance Biomolecular condensates are non-membrane bound organelles that organize biological processes across all domains of life, but their role in bacterial infection is not well characterized. We show that Brucella, an intracellular bacterial pathogen and causative agent of the disease brucellosis, relies on biomolecular condensates called BR-bodies to control RNA stability and regulate gene expression. Without BR-bodies, Brucella is sensitized to host-relevant chemical stresses, cannot properly regulate essential infection machinery, and has severely diminished fitness within mammalian host cells. These results indicate that control of RNA levels by biomolecular condensates is required for Brucella survival within animal hosts, providing evidence that phase separation is a fundamental mechanism underlying Brucella adaptation to the hostile environment encountered during infection.
Kaveendya S. Mallikaarachchi, Rosemary Northcote, Thomas Kim et al.· bioRxiv· 1 citation
It is proposed that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts have evolved structural features that fine-tune protein levels that may be prevalent in bacteria which exhibit uncoupled transcription and translation.
Zachory M. Park, Christina R. Savage, Amanda R. Decker-Farrell et al.· Cell Reports· 0 citations
N6-methyladenosine (m6A) is a widespread RNA modification that regulates RNA metabolism in eukaryotes, but its distribution and function in bacteria remain poorly defined. Here, we apply GLORI sequencing to generate single-base resolution transcriptome-wide m6A maps in seven bacterial species. We identify 2,845 m6A sites during exponential growth and find extensive condition-dependent methylation dynamics in three strains. In Pseudomonas syringae, m6A remodeling is associated with virulence-related pathways. Comparative analyses reveal 455 conserved m6A site pairs enriched in genes required for growth, energy metabolism, and transmembrane transport. Integrating methylation, transcript abundance, and RNA stability analyses shows that m6A is associated with reduced mRNA abundance and increased RNA stability. We further identify the rRNA methyltransferases RlmF and RlmJ as bacterial mRNA m6A writers. Together, these findings provide a quantitative atlas of bacterial m6A and establish a foundation for understanding its regulatory and evolutionary roles.
Youyue Li, Letong Xu, Na Liu et al.· Cell Reports· 0 citations
This is the first comprehensive study to detail changes in chromatin, histone acetylation, and transcription during nitrogen starvation, highlighting the importance of Esa1 and H4Ac in this process.
Uzair Khan, Krystal Cvetkovski, Matthew Werick et al.· bioRxiv· 0 citations
Toxoplasma gondii, a highly successful apicomplexan parasite, primarily relies on post-transcriptional mechanisms to regulate mRNA stability and translation during rapid life-cycle stage transitions and to adapt to diverse host environments. While RNA-binding proteins (RBPs) are crucial for these regulatory processes, their specific roles in mRNA translation, storage, and degradation during life-stage transitions and under physiological stress in Toxoplasma remain poorly understood. Here, we identified the PUF family of RBPs and characterized two conserved members, TgPuf1 and TgPuf2. We examined their expression, localization, RNA-binding activity, essentiality during asexual stages in cell culture and mouse host, responses to stress conditions, and roles in transcript regulation. Gene-knockout studies in cell-culture showed that TgPuf1 modestly supports parasite fitness under both normal and stress conditions, while TgPuf2 appears largely dispensable. Mice infected with Puf1-deleted tachyzoites showed delayed mortality compared with wild-type, whereas neither Puf1 nor Puf2 deletion affected bradyzoite development. Both TgPuf proteins bind to a conserved RNA sequence known as PUF Recognition Elements (PREs), associate with ribonucleoprotein complexes, and interact with the deadenylase enzyme TgPop2. Using synthetic RNA reporter systems, we demonstrated that, upon interaction with TgPop2, TgPuf proteins stimulate the removal of the poly(A) tail from RNA targets, thereby promoting RNA degradation. The inability to generate the double knockout is adequately addressed using TgPuf1-mAID in the delta TgPuf2 background, indicating that individual Puf proteins are dispensable; however, the lack of both results in severe growth defects. Overall, these findings suggest that PUF proteins regulate transcript levels in Toxoplasma through a deadenylation-dependent mechanism.
Chittiraju Khandavalli, Rajkumar Gurupwar, Abhijit S. Deshmukh· Journal of Biological Chemis...· 0 citations