Akos Nyerges, Svenja Vinke, Regan Flynn, Siân V. Owen, Eleanor A. Rand, Bogdan Budnik, Eric Keen, Kamesh Narasimhan, Jorge A. Marchand, Maximilien Baas-Thomas, Min Liu, Kangming Chen, Anush Chiappino-Pepe, Fangxiang Hu, Michael Baym, and George M. Church. 7/2022. “Swapped genetic code blocks viral infections and gene transfer.” bioRxiv. Publisher's VersionAbstract
Removing cellular transfer RNAs (tRNAs), making their cognate codons unreadable, creates a genetic firewall that prevents viral replication and horizontal gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, including tRNAs, potentially rendering a genetic-code-based firewall ineffective. In this paper, we show that such horizontally transferred tRNA genes can enable viral replication in Escherichia coli cells despite the genome-wide lack of three codons and the previously essential cognate tRNAs and release factor 1. By repurposing viral tRNAs, we then develop recoded cells bearing an amino-acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino-acid-swapped genetic code renders cells completely resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. Finally, we also repurpose the third free codon to biocontain this virus-resistant host via dependence on an amino acid not found in nature.Competing Interest StatementThe authors declare competing financial interests. Harvard Medical School has filed a provisional patent application related to this work on which A.N., S.V., and G.M.C. are listed as inventors. M.L., K.C., and F.H. are employed by GenScript USA Inc., but the company had no role in designing or executing experiments. G.M.C. is a founder of the following companies in which he has related financial interests: GRO Biosciences, EnEvolv, and 64x Bio. Other potentially relevant financial interests of G.M.C. are listed at
Anurag Limdi, Siân V Owen, Cristina Herren, Richard E Lenski, and Michael Baym. 5/2022. “Parallel changes in gene essentiality over 50,000 generations of evolution.” bioRxiv.
Justin E Silpe, Joel WH Wong, Siân V Owen, Michael Baym, and Emily P. Balskus. 2/2022. “The bacterial toxin colibactin triggers prophage induction.” Nature, 603, 7900, Pp. 315–320.
Leonard Koolman, Reenesh Prakash, Yohane Diness, Chisomo Msefula, Tonney S. Nyirenda, Franziska Olgemoeller, Paul Wigley, Blanca Perez-Sepulveda, Jay C. D. Hinton, Siân V. Owen, Nicholas A. Feasey, Philip M. Ashton, and Melita A. Gordon. 2022. “Case-control investigation of invasive Salmonella disease in Malawi reveals no evidence of environmental or animal transmission of invasive strains, and supports human to human transmission.” PLOS Neglected Tropical Diseases, 16, 12, Pp. 1-17. Publisher's VersionAbstract
Background Invasive Salmonella infections cause significant morbidity and mortality in Sub-Saharan Africa. However, the routes of transmission are uncertain. We conducted a case-control study of index-case and geographically-matched control households in Blantyre, Malawi, sampling Salmonella isolates from index cases, healthy people, animals, and the household environment. Methodology Sixty index cases of human invasive Salmonella infection were recruited (March 2015-Oct 2016). Twenty-eight invasive Non-Typhoidal Salmonella (iNTS) disease and 32 typhoid patients consented to household sampling. Each index-case household was geographically matched to a control household. Extensive microbiological sampling included stool sampling from healthy household members, stool or rectal swabs from household-associated animals and boot-sock sampling of the household environment. Findings 1203 samples from 120 households, yielded 43 non-Typhoidal Salmonella (NTS) isolates from 25 households (overall sample positivity 3.6%). In the 28 iNTS patients, disease was caused by 3 STs of Salmonella Typhimurium, mainly ST313. In contrast, the isolates from households spanned 15 sequence types (STs). Two S. Typhimurium isolates from index cases closely matched isolates from their respective asymptomatic household members (2 and 3 SNP differences respectively). Despite the recovery of a diverse range of NTS, there was no overlap between the STs causing iNTS disease with any environmental or animal isolates. Conclusions The finding of NTS strains from index cases that matched household members, coupled with lack of related animal or environmental isolates, supports a hypothesis of human to human transmission of iNTS infections in the household. The breadth of NTS strains found in animals and the household environment demonstrated the robustness of NTS sampling and culture methodology, and suggests a diverse ecology of Salmonella in this setting. Healthy typhoid (S. Typhi) carrier state was not detected. The lack of S. Typhi isolates from the household environment suggests that further methodological development is needed to culture S. Typhi from the environment.
Sanne Wolput, Angela Makumi, Laura Wicke, Leonard E Bäcker, William Cenens, Yves Briers, Nicolas A Wenner, Siân V Owen, Jay CD Hinton, Rob Lavigne, and others. 2022. “Transcriptional Organization of the Salmonella Typhimurium Phage P22 pid ORFan Locus.” International Journal of Molecular Sciences, 23, 3, Pp. 1253.
Siân V Owen, Blanca M Perez-Sepulveda, and Evelien M Adriaenssens. 2021. “Detection of bacteriophages: sequence-based systems.” Bacteriophages: Biology, Technology, Therapy, Pp. 621–644.
Siân V. Owen*, Nicolas Wenner*, Charles L. Dulberger, Ella V. Rodwell, Arthur Bowers-Barnard, Natalia Quinones-Olvera, Daniel J. Rigden, Eric J. Rubin, Ethan C. Garner, Michael Baym, and Jay C. D. Hinton. 2021. “Prophages encode phage-defense systems with cognate self-immunity.” Cell Host & Microbe. Publisher's VersionAbstract
Summary Temperate phages are pervasive in bacterial genomes, existing as vertically inherited islands termed prophages. Prophages are vulnerable to predation of their host bacterium by exogenous phages. Here, we identify BstA, a family of prophage-encoded phage-defense proteins in diverse Gram-negative bacteria. BstA localizes to sites of exogenous phage DNA replication and mediates abortive infection, suppressing the competing phage epidemic. During lytic replication, the BstA-encoding prophage is not itself inhibited by BstA due to self-immunity conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella, and Escherichia prophages is generally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, preventing exploitation by variant BstA-encoding phages. The BstA protein allows prophages to defend host cells against exogenous phage attack without sacrificing the ability to replicate lytically.
Jason Qian, Zhi-xiang Lu, Christopher P Mancuso, Han-Ying Jhuang, Roc{\'ıo del Carmen Barajas-Ornelas, Sarah A Boswell, Fernando H Ram{\'ırez-Guadiana, Victoria Jones, Akhila Sonti, Kole Sedlack, and others. 2020. “Barcoded microbial system for high-resolution object provenance.” Science, 368, 6495, Pp. 1135–1140.
Lorena Preciado-Llanes, Anna Aulicino, Roc{\'ıo Canals, Patrick J Moynihan, Xiaojun Zhu, Ndaru Jambo, Tonney S Nyirenda, Innocent Kadwala, Ana Sousa Gerós, Siân V Owen, and others. 2020. “Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease.” Proceedings of the National Academy of Sciences, 117, 34, Pp. 20717–20728.
Jared D Honeycutt, Nicolas Wenner, Yan Li, Susan M Brewer, Liliana M Massis, Sky W Brubaker, Phoom Chairatana, Sian V Owen, Rocio Canals, Jay CD Hinton, and others. 2020. “Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa.” PLoS pathogens, 16, 8, Pp. e1008763.
Jared D. Honeycutt, Nicolas Wenner, Yan Li, Susan M. Brewer, Liliana M. Massis, Sky W. Brubaker, Phoom Chairatana, Siân V. Owen, Rocío Canals, Jay C. D. Hinton, and Denise M. Monack. 2020. “Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa.” PLOS Pathogens, 16, 8, Pp. 1-30. Publisher's VersionAbstract
Author summary Salmonella Typhimurium will generally cause acute gut infections in humans. However, S. Typhimurium strains causing severe, systemic infections have emerged in sub-Saharan Africa and are phylogenetically distinct from other S. Typhimurium strains. Our comparative genomic analysis revealed S. Typhimurium sequence-type 313 (ST313) from Africa have notable sequence variations within the macA and macB genes. These genes are already known to play a role in Salmonella pathogenesis and are otherwise conserved in Salmonella and many other Gram-negative bacteria. We show that regulation of macAB transcription depends, in part, on the key Salmonella virulence system PhoP/Q and that expression of MacAB improves Salmonella resistance to an antimicrobial peptide. African macAB variants interfere with this antimicrobial peptide resistance function and can alter Salmonella replication within macrophages. Using competitive infection experiments in mice, we see that these macAB variants influence fitness in the mammalian gut and systemic sites, with African S. Typhimurium reliant upon its macAB genotype for systemic infection of susceptible hosts. These results suggest that the evolution of African S. Typhimurium has been shaped by human populations with impaired ability to control intracellular Salmonella infections.
Sian V Owen, Rocio Canals, Nicolas Wenner, Disa L Hammarlöf, Carsten Kröger, and Jay CD Hinton. 2020. “A window into lysogeny: revealing temperate phage biology with transcriptomics.” Microbial Genomics.
Jason Qian, Zhi-xiang Lu, Christopher P. Mancuso, Han-Ying Jhuang, Rocío del Carmen Barajas-Ornelas, Sarah A. Boswell, Fernando H. Ramírez-Guadiana, Victoria Jones, Akhila Sonti, Kole Sedlack, Lior Artzi, Giyoung Jung, Mohammad Arammash, Mary E. Pettit, Michael Melfi, Lorena Lyon, Siân V. Owen, Michael Baym, Ahmad S. Khalil, Pamela A. Silver, David Z. Rudner, and Michael Springer. 2020. “Barcoded microbial system for high-resolution object provenance.” Science, 368, 6495, Pp. 1135–1140. Publisher's VersionAbstract
Under adverse environmental conditions, some microorganisms form spores that provide robust protection for genetic material. Qian et al. developed a system in which DNA barcodes are encapsulated inside nongerminating microbial spores and can be dispersed on objects or in the environment (see the Perspective by Nivala). These barcoded spores provide a durable, specific marker that can be read out quickly with simple equipment. When applied to soil, the spores can be transferred to and from objects around them, enabling tracking at meter-scale resolution. On plant leaves, the spores are not readily transferred, and the authors demonstrate a potential use for tracking agricultural products.Science, this issue p. 1135; see also p. 1058Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, a Cas13a RNA-guided nucleic acid detection assay, facilitating its implementation in a wide range of applications.
Lorena Preciado-Llanes, Anna Aulicino, Rocío Canals, Patrick J. Moynihan, Xiaojun Zhu, Ndaru Jambo, Tonney S. Nyirenda, Innocent Kadwala, Ana Sousa Gerós, Siân V. Owen, Kondwani C. Jambo, Benjamin Kumwenda, Natacha Veerapen, Gurdyal S. Besra, Melita A. Gordon, Jay C. D. Hinton, Giorgio Napolitani, Mariolina Salio, and Alison Simmons. 2020. “Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease.” Proceedings of the National Academy of Sciences. Publisher's VersionAbstract
Nontyphoidal Salmonella serotypes are a common cause of self-limiting diarrhoeal illnesses in healthy adults. However, recently, a highly invasive multidrug resistant Salmonella Typhimurium sequence type 313 has emerged as a major cause of morbidity and mortality in sub-Saharan Africa, particularly in children and immunosuppressed individuals. In this paper we describe escape from MAIT cell recognition as an additional mechanism of immune evasion of S. Typhimurium ST313. As MAIT cells represent an early defense mechanism against pathogens at mucosal surfaces, and their frequency and function are altered in immunosuppressed individuals in sub-Saharan Africa, harnessing their function may offer an important therapeutic strategy to improve mucosal immunity.Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB. This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.All data have been made available in the manuscript.
Siân V. Owen*, Rocío Canals, Nicolas Wenner, Disa L. Hammarlöf, Carsten Kröger, and Jay C. D. Hinton. 2020. “A window into lysogeny: revealing temperate phage biology with transcriptomics.” Microbial Genomics. Publisher's VersionAbstract
Prophages are integrated phage elements that are a pervasive feature of bacterial genomes. The fitness of bacteria is enhanced by prophages that confer beneficial functions such as virulence, stress tolerance or phage resistance, and these functions are encoded by ‘accessory’ or ‘moron’ loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, it is challenging to identify novel prophage accessory loci from DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and represent functionally important accessory loci. Using this approach, we identify a novel antisense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.
Rocío Canals, Roy R. Chaudhuri, Rebecca E. Steiner, Siân V. Owen, Natalia Quinones-Olvera, Melita A. Gordon, Michael Baym, Michael Ibba, and Jay C. D. Hinton. 9/27/2019. “The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid.” PLOS Pathogens. Publisher's VersionAbstract

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of Gram-negative and Gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.

Roc{\'ıo Canals, Disa L Hammarlöf, Carsten Kröger, Siân V Owen, Wai Yee Fong, Lizeth Lacharme-Lora, Xiaojun Zhu, Nicolas Wenner, Sarah E Carden, Jared Honeycutt, and others. 2019. “Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580.” PLoS biology, 17, 1, Pp. e3000059.
Roc{\'ıo Canals, Roy R Chaudhuri, Rebecca E Steiner, Siân V Owen, Natalia Quinones-Olvera, Melita A Gordon, Michael Baym, Michael Ibba, and Jay CD Hinton. 2019. “The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid.” PLoS pathogens, 15, 9, Pp. e1007948.
Lizeth Lacharme-Lora, Siân V Owen, Richard Blundell, Roc{\'ıo Canals, Nicolas Wenner, Blanca Perez-Sepulveda, Wai Yee Fong, Rachel Gilroy, Paul Wigley, and Jay CD Hinton. 2019. “The use of chicken and insect infection models to assess the virulence of African Salmonella Typhimurium ST313.” PLoS neglected tropical diseases, 13, 7, Pp. e0007540.
Rocío Canals, Disa L. Hammarlöf, Carsten Kröger, Siân V. Owen, Wai Yee Fong, Lizeth Lacharme-Lora, Xiaojun Zhu, Nicolas Wenner, Sarah E. Carden, Jared Honeycutt, Denise M. Monack, Robert A. Kingsley, Philip Brownridge, Roy R. Chaudhuri, Will P. M. Rowe, Alexander V. Predeus, Karsten Hokamp, Melita A. Gordon, and Jay C. D. Hinton. 2019. “Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580.” PLOS Biology, 17, 1, Pp. 1-32. Publisher's VersionAbstract
Comparative transcriptomic analysis of two contrasting Salmonella enterica Typhimurium isolates across 16 in vitro conditions and within macrophages reveals the mechanism of African Salmonella metabolic defect and a novel bacterial plasmid maintenance system.