-
Science (New York, N.Y.) Mar 2020Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic...
Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
Topics: CRISPR-Cas Systems; Humans; Open Reading Frames; Operon; Peptides; Protein Biosynthesis; RNA, Messenger; Ribosomes; Transcriptome
PubMed: 32139545
DOI: 10.1126/science.aay0262 -
Nature Jun 2020The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids...
The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile, promote hepatocellular carcinoma and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. ). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.
Topics: Animals; Bile Acids and Salts; Clostridium; Deoxycholic Acid; Gastrointestinal Microbiome; Hydroxylation; Lithocholic Acid; Male; Metabolic Engineering; Metabolic Networks and Pathways; Mice; Operon; Symbiosis
PubMed: 32555455
DOI: 10.1038/s41586-020-2396-4 -
Journal of Bacteriology Jan 2021The division and cell wall () cluster is a highly conserved region of the bacterial genome consisting of genes that encode several cell division and cell wall synthesis... (Review)
Review
The division and cell wall () cluster is a highly conserved region of the bacterial genome consisting of genes that encode several cell division and cell wall synthesis factors, including the central division protein FtsZ. The region immediately downstream of encodes the genes and is conserved across diverse lineages of Gram-positive bacteria and In some organisms, this region remains part of the cluster, but in others, it appears as an independent operon. A well-studied protein coded from this region is the positive FtsZ regulator SepF (YlmF), which anchors FtsZ to the membrane. Recent developments have shed light on the importance of SepF in a range of species. Additionally, new studies are highlighting the importance of the other conserved genes in this neighborhood. In this minireview, we aim to bring together the current research linking the region to cell division and highlight further questions surrounding these conserved genes.
Topics: Bacterial Proteins; Cell Division; Cytoskeletal Proteins; Gene Expression Regulation, Bacterial; Operon
PubMed: 32900832
DOI: 10.1128/JB.00460-20 -
MSystems Dec 2022Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their host's CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often colocalize...
Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their host's CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often colocalize to form operons. Here, we present AcaFinder as the first Aca genome mining tool. AcaFinder can (i) predict Acas and their associated operons using guilt-by-association (GBA); (ii) identify homologs of known Acas using an HMM (Hidden Markov model) database; (iii) take input genomes for potential prophages, CRISPR-Cas systems, and self-targeting spacers (STSs); and (iv) provide a standalone program (https://github.com/boweny920/AcaFinder) and a web server (http://aca.unl.edu/Aca). AcaFinder was applied to mining over 16,000 prokaryotic and 142,000 gut phage genomes. After a multistep filtering, 36 high-confident new Aca families were identified, which is three times that of the 12 known Aca families. Seven new Aca families were from major human gut bacteria (, , and ) and their phages, while most known Aca families were from and . A complex association network between Acrs and Acas was revealed by analyzing their operonic colocalizations. It appears very common in evolution that the same genes can recombine with different genes and to form diverse operon combinations. At least four bioinformatics programs have been published for genome mining of Acrs since 2020. In contrast, no bioinformatics tools are available for automated Aca discovery. As the self-transcriptional repressor of operons, Aca can be viewed as anti-anti-CRISPRs, with great potential in the improvement of CRISPR-Cas technology. Although all the 12 known Aca proteins contain a conserved helix-turn-helix (HTH) domain, not all HTH-containing proteins are Acas. However, HTH-containing proteins with adjacent Acr homologs encoded in the same genetic operon are likely Aca proteins. AcaFinder implements this guilt-by-association idea and the idea of using HMMs of known Acas for homologs into one software package. Applying AcaFinder in screening prokaryotic and gut phage genomes reveals a complex operonic colocalization network between different families of Acrs and Acas.
Topics: Humans; CRISPR-Cas Systems; Bacteria; Bacteriophages; Operon; Prophages
PubMed: 36413017
DOI: 10.1128/msystems.00817-22 -
Microbiology Spectrum Aug 2022Overproduction of the exopolysaccharide alginate contributes to the pathogenicity and antibiotic tolerance of Pseudomonas aeruginosa in chronic infections. The second...
Overproduction of the exopolysaccharide alginate contributes to the pathogenicity and antibiotic tolerance of Pseudomonas aeruginosa in chronic infections. The second messenger, c-di-GMP, is a positive regulator of the production of various biofilm matrix components and is known to regulate alginate synthesis at the posttranslational level in P. aeruginosa. We provide evidence that c-di-GMP also regulates transcription of the alginate operon in P. aeruginosa. Previous work has shown that transcription of the alginate operon is regulated by nine different proteins, AmrZ, AlgP, IHFα, IHFβ, CysB, Vfr, AlgR, AlgB, and AlgQ, and we investigated if some of these proteins function as a c-di-GMP effector. We found that deletion of , , , and had only a marginal effect on the transcription of the alginate operon. Deletion of and led to decreased transcription of the alginate operon, and the dependence of the c-di-GMP level was less pronounced, indicating that Vfr and CysB could be partially required for c-di-GMP-mediated regulation of alginate operon transcription. Our experiments indicated that the AmrZ, AlgR, and AlgB proteins are absolutely required for transcription of the alginate operon. However, differential radial capillary action of ligand assay (DRaCALA) and site-directed mutagenesis indicated that c-di-GMP does not bind to any of the AmrZ, AlgR, and AlgB proteins. The proliferation of alginate-overproducing P. aeruginosa variants in the lungs of cystic fibrosis patients often leads to chronic infection. The alginate functions as a biofilm matrix that protects the bacteria against host immune defenses and antibiotic treatment. Knowledge about the regulation of alginate synthesis is important in order to identify drug targets for the development of medicine against chronic P. aeruginosa infections. We provide evidence that c-di-GMP positively regulates transcription of the alginate operon in P. aeruginosa. Moreover, we revisited the role of the known alginate regulators, AmrZ, AlgP, IHFα, IHFβ, CysB, Vfr, AlgR, AlgB, and AlgQ, and found that their effect on transcription of the alginate operon is highly varied. Deletion of , , , or only had a marginal effect on transcription of the alginate operon, whereas deletion of or led to decreased transcription and deletion of , , or abrogated transcription.
Topics: Alginates; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Operon; Pseudomonas aeruginosa
PubMed: 35862969
DOI: 10.1128/spectrum.00675-22 -
Methods (San Diego, Calif.) Apr 2020An operon is a set of neighboring genes in a genome that is transcribed as a single polycistronic message. Genes that are part of the same operon often have related... (Review)
Review
An operon is a set of neighboring genes in a genome that is transcribed as a single polycistronic message. Genes that are part of the same operon often have related functional roles or participate in the same metabolic pathways. The majority of all bacterial genes are co-transcribed with one or more other genes as part of a multi-gene operon. Thus, accurate identification of operons is important in understanding co-regulation of genes and their functional relationships. Here, we present a computational system that uses RNA-seq data to determine operons throughout a genome. The system takes the name of a genome and one or more files of RNA-seq data as input. Our method combines primary genomic sequence information with expression data from the RNA-seq files in a unified probabilistic model in order to identify operons. We assess our method's ability to accurately identify operons in a range of species through comparison to external databases of operons, both experimentally confirmed and computationally predicted, and through focused experiments that confirm new operons identified by our method. Our system is freely available at https://cs.wellesley.edu/~btjaden/Rockhopper/.
Topics: Gene Regulatory Networks; Genome, Bacterial; Genomics; Models, Genetic; Operon; RNA-Seq; Transcription, Genetic
PubMed: 30953757
DOI: 10.1016/j.ymeth.2019.03.026 -
Frontiers in Cellular and Infection... 2019Streptococci, including the dental pathogen , undergo cell-to-cell signaling that is mediated by small peptides to control critical physiological functions such as... (Review)
Review
Streptococci, including the dental pathogen , undergo cell-to-cell signaling that is mediated by small peptides to control critical physiological functions such as adaptation to the environment, control of subpopulation behaviors and regulation of virulence factors. One such model pathway is the regulation of genetic competence, controlled by the ComRS signaling system and the peptide XIP. However, recent research in the characterization of this pathway has uncovered novel operons and peptides that are intertwined into its regulation. These discoveries, such as cell lysis playing a critical role in XIP release and importance of bacterial self-sensing during the signaling process, have caused us to reevaluate previous paradigms and shift our views on the true purpose of these signaling systems. The finding of new peptides such as the ComRS inhibitor XrpA and the peptides of the RcrRPQ operon also suggests there may be more peptides hidden in the genomes of streptococci that could play critical roles in the physiology of these organisms. In this review, we summarize the recent findings in regarding the integration of other circuits into the ComRS signaling pathway, the true mode of XIP export, and how the RcrRPQ operon controls competence activation. We also look at how new technologies can be used to re-annotate the genome to find new open reading frames that encode peptide signals. Together, this summary of research will allow us to reconsider how we perceive these systems to behave and lead us to expand our vocabulary of peptide signals within the genus .
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genome, Bacterial; Operon; Peptides; Quorum Sensing; Signal Transduction; Streptococcus mutans
PubMed: 31245303
DOI: 10.3389/fcimb.2019.00194 -
Infection and Immunity Sep 2021Riboflavin is an essential micronutrient, but its transport and utilization have remained largely understudied among pathogenic spirochetes. Here, we show that Borrelia...
Riboflavin is an essential micronutrient, but its transport and utilization have remained largely understudied among pathogenic spirochetes. Here, we show that Borrelia burgdorferi, the zoonotic spirochete that causes Lyme disease, is able to import riboflavin via products of its -like operon as well as synthesize flavin mononucleotide and flavin adenine dinucleotide despite lacking canonical genes for their synthesis. Additionally, a mutant deficient in the -like operon is resistant to the antimicrobial effect of roseoflavin, a natural riboflavin analog, and is attenuated in a murine model of Lyme borreliosis. Our combined results indicate not only that are riboflavin and the maintenance of flavin pools essential for B. burgdorferi growth but also that flavin utilization and its downstream products (e.g., flavoproteins) may play a more prominent role in B. burgdorferi pathogenesis than previously appreciated.
Topics: Animals; Bacterial Proteins; Borrelia burgdorferi; Female; Lyme Disease; Mammals; Mice; Mice, Inbred C3H; Operon; Riboflavin
PubMed: 34310888
DOI: 10.1128/IAI.00307-21 -
International Journal of Molecular... Dec 2021Brucellosis is a highly prevalent zoonotic disease caused by . spp. are gram-negative facultative intracellular parasitic bacteria. Its intracellular survival and... (Review)
Review
Brucellosis is a highly prevalent zoonotic disease caused by . spp. are gram-negative facultative intracellular parasitic bacteria. Its intracellular survival and replication depend on a functional virB system, an operon encoded by VirB1-VirB12. Type IV secretion system (T4SS) encoded by the virB operon is an important virulence factor of . It can subvert cellular pathway and induce host immune response by secreting effectors, which promotes replication in host cells and induce persistent infection. Therefore, this paper summarizes the function and significance of the VirB system, focusing on the structure of the VirB system where VirB T4SS mediates biogenesis of the endoplasmic reticulum (ER)-derived replicative containing vacuole (rBCV), the effectors of T4SS and the cellular pathways it subverts, which will help better understand the pathogenic mechanism of and provide new ideas for clinical vaccine research and development.
Topics: Animals; Brucella; Brucellosis; Host-Pathogen Interactions; Humans; Operon; Type IV Secretion Systems; Virulence Factors
PubMed: 34948430
DOI: 10.3390/ijms222413637 -
Cell Reports Dec 2020Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR...
Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2', 3'-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5' tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair.
Topics: Humans; Operon; RNA; RNA, Transfer
PubMed: 33357439
DOI: 10.1016/j.celrep.2020.108527