scholarly journals Validation that human microbiome phages use alternative genetic coding with TAG stop read as Q

2022 ◽  
Author(s):  
Samantha Peters ◽  
Adair L Borges ◽  
Richard J Giannone ◽  
Michael Morowitz ◽  
Jill Banfield ◽  
...  

Metagenomic findings suggesting that bacteriophages (phages) can use genetic codes different from those of their host bacteria reveal a new dimension of phage-host interaction dynamics. Whereas reassignment of stop codons to code for amino acids has been predicted, there has been no proteomic validation of alternative coding in phages. In fact, one code where the stop codon TAG is reassigned to glutamine (code 15) has never been experimentally validated in any biological system. Here, we characterized stop codon reassignment in two crAss-like phages found in the human gut microbiome using LC-MS/MS-based metaproteomics. The proteome data from several phage structural proteins clearly demonstrates reassignment of the TAG stop codon to glutamine, establishing for the first time the expression of genetic code 15.

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 546 ◽  
Author(s):  
David Schwark ◽  
Margaret Schmitt ◽  
John Fisk

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.


2020 ◽  
Vol 7 ◽  
Author(s):  
Xiao Chen ◽  
Chundi Wang ◽  
Bo Pan ◽  
Borong Lu ◽  
Chao Li ◽  
...  

Peritrichs are one of the largest groups of ciliates with over 1,000 species described so far. However, their genomic features are largely unknown. By single-cell genomic sequencing, we acquired the genomic data of three sessilid peritrichs (Cothurnia ceramicola, Vaginicola sp., and Zoothamnium sp. 2). Using genomic data from another 53 ciliates including 14 peritrichs, we reconstructed their evolutionary relationships and confirmed genome skimming as an efficient approach for expanding sampling. In addition, we profiled the stop codon usage and programmed ribosomal frameshifting (PRF) events in peritrichs for the first time. Our analysis reveals no evidence of stop codon reassignment for peritrichs, but they have prevalent +1 or -1 PRF events. These genomic features are distinguishable from other ciliates, and our observations suggest a unique evolutionary strategy for peritrichs.


2013 ◽  
Vol 91 (3) ◽  
pp. 155-164 ◽  
Author(s):  
Lijun Xu ◽  
Yanrong Hao ◽  
Cui Li ◽  
Quan Shen ◽  
Baofeng Chai ◽  
...  

One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.


RNA ◽  
2009 ◽  
Vol 15 (5) ◽  
pp. 889-897 ◽  
Author(s):  
H. Vallabhaneni ◽  
H. Fan-Minogue ◽  
D. M. Bedwell ◽  
P. J. Farabaugh

2011 ◽  
Vol 11 (1) ◽  
Author(s):  
Louise J Johnson ◽  
James A Cotton ◽  
Conrad P Lichtenstein ◽  
Greg S Elgar ◽  
Richard A Nichols ◽  
...  

Science ◽  
2021 ◽  
Vol 372 (6546) ◽  
pp. 1057-1062
Author(s):  
Wesley E. Robertson ◽  
Louise F. H. Funke ◽  
Daniel de la Torre ◽  
Julius Fredens ◽  
Thomas S. Elliott ◽  
...  

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)—making their cognate codons unreadable—might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.


2020 ◽  
Author(s):  
Andrey N Shkoporov ◽  
Ekaterina V Khokhlova ◽  
Niamh Stephens ◽  
Cara Hueston ◽  
Samuel Seymour ◽  
...  

The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of classical lysogeny, these viruses demonstrate unexplained long-term persistence in the human gut microbiome, dominating the virome in some individuals. Here we demonstrate that rapid phase variation of alternate capsular polysaccharides plays an important role in dynamic equilibrium between phage sensitivity and resistance in B. intestinalis cultures, allowing phage and bacteria to multiply in parallel. The data also suggests the role of concomitant phage persistence mechanisms associated with delayed lysis of infected cells, such as carrier state infection. From an ecological and evolutionary standpoint this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other 'benign' forms of phage infection when the host is stably present at high abundance.


2021 ◽  
Author(s):  
Leyuan Li ◽  
Zhibin Ning ◽  
Xu Zhang ◽  
James Butcher ◽  
Caitlin Simopoulos ◽  
...  

Functional redundancy is a key property of ecosystems and represents the fact that phylogenetically unrelated taxa can play similar functional roles within an ecosystem. The redundancy of potential functions of human microbiome has been recently quantified using metagenomics data. Yet, the redundancy of functions which are actually expressed within the human microbiome remains largely unexplored. Here, we quantify the protein-level functional redundancy in the human gut microbiome using metaproteomics and network approaches. In particular, our ultra-deep metaproteomics approach revealed high protein-level functional redundancy and high nestedness in proteomic content networks - bipartite graphs that connect taxa with their expressed functions. We further examined multiple metaproteomics datasets and showed that various environmental factors, including individuality, biogeography, xenobiotics, and disease, significantly altered the protein-level functional redundancy. Finally, by projecting the bipartite proteomic content networks into unipartite weighted genus networks, functional hub genera across individual microbiomes were discovered, suggesting that there may be a universal principle of functional organization in microbiome assembly.


mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Maria I. Rojas ◽  
Giselle S. Cavalcanti ◽  
Katelyn McNair ◽  
Sean Benler ◽  
Amanda T. Alker ◽  
...  

ABSTRACT Many commensal bacteria antagonize each other or their host by producing syringe-like secretion systems called contractile injection systems (CIS). Members of the Bacteroidales family have been shown to produce only one type of CIS—a contact-dependent type 6 secretion system that mediates bacterium-bacterium interactions. Here, we show that a second distinct cluster of genes from Bacteroidales bacteria from the human microbiome may encode yet-uncharacterized injection systems that we term Bacteroidales injection systems (BIS). We found that BIS genes are present in the gut microbiomes of 99% of individuals from the United States and Europe and that BIS genes are more prevalent in the gut microbiomes of healthy individuals than in those individuals suffering from inflammatory bowel disease. Gene clusters similar to that of the BIS mediate interactions between bacteria and diverse eukaryotes, like amoeba, insects, and tubeworms. Our findings highlight the ubiquity of the BIS gene cluster in the human gut and emphasize the relevance of the gut microbiome to the human host. These results warrant investigations into the structure and function of the BIS and how they might mediate interactions between Bacteroidales bacteria and the human host or microbiome. IMPORTANCE To engage with host cells, diverse pathogenic bacteria produce syringe-like structures called contractile injection systems (CIS). CIS are evolutionarily related to the contractile tails of bacteriophages and are specialized to puncture membranes, often delivering effectors to target cells. Although CIS are key for pathogens to cause disease, paradoxically, similar injection systems have been identified within healthy human microbiome bacteria. Here, we show that gene clusters encoding a predicted CIS, which we term Bacteroidales injection systems (BIS), are present in the microbiomes of nearly all adult humans tested from Western countries. BIS genes are enriched within human gut microbiomes and are expressed both in vitro and in vivo. Further, a greater abundance of BIS genes is present within healthy gut microbiomes than in those humans with with inflammatory bowel disease (IBD). Our discovery provides a potentially distinct means by which our microbiome interacts with the human host or its microbiome.


Sign in / Sign up

Export Citation Format

Share Document