@article { doi:10.1126/science.add1417,
	title = {Changing fitness effects of mutations through long-term bacterial evolution},
	journal = {Science},
	volume = {383},
	number = {6681},
	year = {2024},
	pages = {eadd1417},
	abstract = {The distribution of fitness effects of new mutations shapes evolution, but it is challenging to observe how it changes as organisms adapt. Using Escherichia coli lineages spanning 50,000 generations of evolution, we quantify the fitness effects of insertion mutations in every gene. Macroscopically, the fraction of deleterious mutations changed little over time whereas the beneficial tail declined sharply, approaching an exponential distribution. Microscopically, changes in individual gene essentiality and deleterious effects often occurred in parallel; altered essentiality is only partly explained by structural variation. The identity and effect sizes of beneficial mutations changed rapidly over time, but many targets of selection remained predictable because of the importance of loss-of-function mutations. Taken together, these results reveal the dynamic{\textemdash}but statistically predictable{\textemdash}nature of mutational fitness effects. The benefits and costs of mutations that undergo natural selection can change depending on genetic interactions with subsequent mutations. In an enduring experiment, 12 lineages of Escherichia coli have been maintained for more than 75,000 generations, with each generation sampled and preserved. Couce et al. made transposon insertion libraries in ancestral and evolved strains taken at the 50,000 generation point and measured fitness in competition experiments using these samples. The numbers of beneficial mutations rapidly tailed off during long-term passage, with parallel changes in fitness cost and gene essentiality occurring across the lineages. The authors found nonessential genes that became essential and essential genes that became nonessential in all lineages. Predictability stemmed from the importance of loss-of-function mutations that scale with the length of the target genes. {\textemdash}Caroline Ash Predictable and parallel changes occur in the fitness effects of mutations in Escherichia coli over 50,000 generations.},
	doi = {10.1126/science.add1417},
	url = {https://www.science.org/doi/abs/10.1126/science.add1417},
	author = {Alejandro Couce and Limdi, Anurag and Melanie Magnan and Owen, Si{\^a}n V. and Cristina M. Herren and Richard E. Lenski and Olivier Tenaillon and Baym, Michael}
}
@article {quinones-olvera_diverse_2024,
	title = {Diverse and abundant phages exploit conjugative plasmids.},
	journal = {Nature communications},
	volume = {15},
	number = {1},
	year = {2024},
	note = {Place: England},
	pages = {3197},
	abstract = {Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily~promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.},
	keywords = {*Bacteriophages/genetics, Biological Evolution, biotechnology, Cell Membrane, Wastewater},
	issn = {2041-1723},
	doi = {10.1038/s41467-024-47416-z},
	author = {Quinones-Olvera, Natalia and Owen, Si{\^a}n V. and McCully, Lucy M. and Marin, Maximillian G. and Rand, Eleanor A. and Fan, Alice C. and Dosumu, Oluremi J. Martins and Paul, Kay and Casta{\~n}o, Cleotilde E. Sanchez and Petherbridge, Rachel and Paull, Jillian S. and Baym, Michael}
}
@article {dulberger_mycobacterial_2023,
	title = {Mycobacterial nucleoid-associated protein Lsr2 is required for productive mycobacteriophage infection},
	journal = {Nature Microbiology},
	year = {2023},
	note = {Publisher: Nature Publishing Group},
	pages = {1{\textendash}16},
	abstract = {Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.},
	keywords = {Bacteriophages, Phage biology},
	issn = {2058-5276},
	doi = {10.1038/s41564-023-01333-x},
	url = {https://www.nature.com/articles/s41564-023-01333-x},
	author = {Dulberger, Charles L. and Guerrero-Bustamante, Carlos A. and Owen, Si{\^a}n V. and Wilson, Sean and Wuo, Michael G. and Garlena, Rebecca A. and Serpa, Lexi A. and Russell, Daniel A. and Zhu, Junhao and Braunecker, Ben J. and Squyres, Georgia R. and Baym, Michael and Kiessling, Laura L. and Garner, Ethan C. and Rubin, Eric J. and Hatfull, Graham F.}
}
@article {nyerges_swapped_2023,
	title = {A swapped genetic code prevents viral infections and gene transfer},
	journal = {Nature},
	volume = {615},
	number = {7953},
	year = {2023},
	note = {Number: 7953 Publisher: Nature Publishing Group},
	pages = {720{\textendash}727},
	abstract = {Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer1{\textendash}6. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus7{\textendash}9, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop 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 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. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature10. Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.},
	keywords = {biotechnology, Microbial genetics, Molecular Biology, synthetic biology},
	issn = {1476-4687},
	doi = {10.1038/s41586-023-05824-z},
	url = {https://www.nature.com/articles/s41586-023-05824-z},
	author = {Nyerges, Akos and Vinke, Svenja and Flynn, Regan and Owen, Si{\^a}n V. and Rand, Eleanor A. and Budnik, Bogdan and Keen, Eric and Narasimhan, Kamesh and Marchand, Jorge A. and Baas-Thomas, Maximilien and Liu, Min and Chen, Kangming and Chiappino-Pepe, Anush and Hu, Fangxiang and Baym, Michael and George M. Church}
}
@article {Nyerges2022.07.08.499367,
	title = {Swapped genetic code blocks viral infections and gene transfer},
	journal = {bioRxiv},
	year = {2022},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {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 http://arep.med.harvard.edu/gmc/tech.html.},
	doi = {10.1101/2022.07.08.499367},
	url = {https://www.biorxiv.org/content/early/2022/07/10/2022.07.08.499367},
	author = {Nyerges, Akos and Vinke, Svenja and Flynn, Regan and Owen, Si{\^a}n V. and Rand, Eleanor A. and Budnik, Bogdan and Keen, Eric and Narasimhan, Kamesh and Marchand, Jorge A. and Baas-Thomas, Maximilien and Liu, Min and Chen, Kangming and Chiappino-Pepe, Anush and Hu, Fangxiang and Baym, Michael and George M. Church}
}
@article {limdi2022parallel,
	title = {Parallel changes in gene essentiality over 50,000 generations of evolution},
	journal = {bioRxiv},
	year = {2022},
	publisher = {Cold Spring Harbor Laboratory},
	author = {Limdi, Anurag and Owen, Si{\^a}n V and Herren, Cristina and Lenski, Richard E and Baym, Michael}
}
@article {silpe2022bacterial,
	title = {The bacterial toxin colibactin triggers prophage induction},
	journal = {Nature},
	volume = {603},
	number = {7900},
	year = {2022},
	pages = {315{\textendash}320},
	publisher = {Nature Publishing Group},
	author = {Silpe, Justin E and Wong, Joel WH and Owen, Si{\^a}n V and Baym, Michael and Emily P. Balskus}
}
@article {10.1371/journal.pntd.0010982,
	title = {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},
	journal = {PLOS Neglected Tropical Diseases},
	volume = {16},
	number = {12},
	year = {2022},
	month = {12},
	pages = {1-17},
	publisher = {Public Library of Science},
	abstract = {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.},
	doi = {10.1371/journal.pntd.0010982},
	url = {https://doi.org/10.1371/journal.pntd.0010982},
	author = {Koolman, Leonard and Prakash, Reenesh and Diness, Yohane and Msefula, Chisomo and Nyirenda, Tonney S. and Olgemoeller, Franziska and Wigley, Paul and Perez-Sepulveda, Blanca and Hinton, Jay C. D. and Owen, Si{\^a}n V. and Feasey, Nicholas A. and Ashton, Philip M. and Gordon, Melita A.}
}
@article {wolput2022transcriptional,
	title = {Transcriptional Organization of the Salmonella Typhimurium Phage P22 pid ORFan Locus},
	journal = {International Journal of Molecular Sciences},
	volume = {23},
	number = {3},
	year = {2022},
	pages = {1253},
	publisher = {Multidisciplinary Digital Publishing Institute},
	author = {Wolput, Sanne and Makumi, Angela and Wicke, Laura and B{\"a}cker, Leonard E and Cenens, William and Briers, Yves and Wenner, Nicolas A and Owen, Si{\^a}n V and Hinton, Jay CD and Lavigne, Rob and others}
}
@article {owen2021detection,
	title = {Detection of bacteriophages: sequence-based systems},
	journal = {Bacteriophages: Biology, Technology, Therapy},
	year = {2021},
	pages = {621{\textendash}644},
	publisher = {Springer International Publishing},
	author = {Owen, Si{\^a}n V and Perez-Sepulveda, Blanca M and Adriaenssens, Evelien M}
}
@article {OWEN2021,
	title = {Prophages encode phage-defense systems with cognate self-immunity},
	journal = {Cell Host \& Microbe},
	year = {2021},
	abstract = {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.},
	keywords = {aba, abortive infection, Bacteria, BstA, lysogeny, phage defense, phage resistance, prophage, self-immunity, Superinfection},
	issn = {1931-3128},
	doi = {https://doi.org/10.1016/j.chom.2021.09.002},
	url = {https://www.sciencedirect.com/science/article/pii/S1931312821004182},
	author = {Owen*, Si{\^a}n V. and Wenner*, Nicolas and Dulberger, Charles L. and Rodwell, Ella V. and Bowers-Barnard, Arthur and Quinones-Olvera, Natalia and Rigden, Daniel J. and Rubin, Eric J. and Garner, Ethan C. and Baym, Michael and Hinton, Jay C. D.}
}
@article {10.1371/journal.ppat.1008763,
	title = {Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa},
	journal = {PLOS Pathogens},
	volume = {16},
	number = {8},
	year = {2020},
	month = {08},
	pages = {1-30},
	publisher = {Public Library of Science},
	abstract = {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.},
	doi = {10.1371/journal.ppat.1008763},
	url = {https://doi.org/10.1371/journal.ppat.1008763},
	author = {Honeycutt, Jared D. and Wenner, Nicolas and Yan Li and Brewer, Susan M. and Massis, Liliana M. and Brubaker, Sky W. and Chairatana, Phoom and Owen, Si{\^a}n V. and Canals, Roc{\'\i}o and Hinton, Jay C. D. and Monack, Denise M.}
}
@article {Qian1135,
	title = {Barcoded microbial system for high-resolution object provenance},
	journal = {Science},
	volume = {368},
	number = {6495},
	year = {2020},
	pages = {1135{\textendash}1140},
	publisher = {American Association for the Advancement of Science},
	abstract = {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.},
	issn = {0036-8075},
	doi = {10.1126/science.aba5584},
	url = {https://science.sciencemag.org/content/368/6495/1135},
	author = {Qian, Jason and Lu, Zhi-xiang and Mancuso, Christopher P. and Jhuang, Han-Ying and del Carmen Barajas-Ornelas, Roc{\'\i}o and Sarah A. Boswell and Fernando H. Ram{\'\i}rez-Guadiana and Jones, Victoria and Sonti, Akhila and Sedlack, Kole and Artzi, Lior and Jung, Giyoung and Arammash, Mohammad and Pettit, Mary E. and Melfi, Michael and Lorena Lyon and Owen, Si{\^a}n V. and Baym, Michael and Khalil, Ahmad S. and Pamela A. Silver and Rudner, David Z. and Michael Springer}
}
@article {Preciado-Llanes202007472,
	title = {Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease},
	journal = {Proceedings of the National Academy of Sciences},
	year = {2020},
	publisher = {National Academy of Sciences},
	abstract = {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.},
	issn = {0027-8424},
	doi = {10.1073/pnas.2007472117},
	url = {https://www.pnas.org/content/early/2020/08/07/2007472117},
	author = {Preciado-Llanes, Lorena and Aulicino, Anna and Canals, Roc{\'\i}o and Moynihan, Patrick J. and Zhu, Xiaojun and Jambo, Ndaru and Nyirenda, Tonney S. and Kadwala, Innocent and Sousa Ger{\'o}s, Ana and Owen, Si{\^a}n V. and Jambo, Kondwani C. and Kumwenda, Benjamin and Veerapen, Natacha and Besra, Gurdyal S. and Gordon, Melita A. and Hinton, Jay C. D. and Napolitani, Giorgio and Salio, Mariolina and Alison Simmons}
}
@article {647187,
	title = {A window into lysogeny: revealing temperate phage biology with transcriptomics},
	journal = {Microbial Genomics},
	year = {2020},
	publisher = {Microbiology Society,},
	abstract = {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 {\textquoteleft}accessory{\textquoteright} or {\textquoteleft}moron{\textquoteright} 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 Salmonella 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.},
	keywords = {transcriptomics},
	doi = {https://doi.org/10.1099/mgen.0.000330},
	url = {https://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000330},
	author = {Owen*, Si{\^a}n V. and Canals, Roc{\'\i}o and Wenner, Nicolas and Hammarl{\"o}f, Disa L. and Kr{\"o}ger, Carsten and Hinton, Jay C. D.}
}
@article {Canals689075,
	title = {The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid},
	journal = {PLOS Pathogens},
	year = {2019},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African\&nbsp;Salmonella enterica\&nbsp;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\&nbsp;in vitro\&nbsp;and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two\&nbsp;in vitro\&nbsp;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\&nbsp;S. Typhimurium ST19 and\&nbsp;S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one\&nbsp;in vitro\&nbsp;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\&nbsp;in vitro. None of these genes were unique to\&nbsp;S. Typhimurium D23580, consistent with a high conservation of gene function between\&nbsp;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\&nbsp;had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr\&nbsp;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.},
	doi = {10.1101/689075},
	url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007948},
	author = {Canals, Roc{\'\i}o and Chaudhuri, Roy R. and Steiner, Rebecca E. and Owen, Si{\^a}n V. and Quinones-Olvera, Natalia and Gordon, Melita A. and Baym, Michael and Ibba, Michael and Hinton, Jay C. D.}
}
@article {10.1371/journal.pbio.3000059,
	title = {Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580},
	journal = {PLOS Biology},
	volume = {17},
	number = {1},
	year = {2019},
	month = {01},
	pages = {1-32},
	publisher = {Public Library of Science},
	abstract = {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.},
	doi = {10.1371/journal.pbio.3000059},
	url = {https://doi.org/10.1371/journal.pbio.3000059},
	author = {Canals, Roc{\'\i}o and Hammarl{\"o}f, Disa L. and Kr{\"o}ger, Carsten and Owen, Si{\^a}n V. and Fong, Wai Yee and Lacharme-Lora, Lizeth and Zhu, Xiaojun and Wenner, Nicolas and Carden, Sarah E. and Honeycutt, Jared and Monack, Denise M. and Kingsley, Robert A. and Brownridge, Philip and Chaudhuri, Roy R. and Rowe, Will P. M. and Predeus, Alexander V. and Hokamp, Karsten and Gordon, Melita A. and Hinton, Jay C. D.}
}
@article {lacharme2019use,
	title = {The use of chicken and insect infection models to assess the virulence of African Salmonella Typhimurium ST313},
	journal = {PLOS Neglected Tropical Diseases},
	volume = {13},
	number = {7},
	year = {2019},
	pages = {e0007540},
	publisher = {Public Library of Science},
	abstract = {Over recent decades, Salmonella infection research has predominantly relied on murine infection models. However, in many cases the infection phenotypes of Salmonella pathovars in mice do not recapitulate human disease. For example, Salmonella Typhimurium ST313 is associated with enhanced invasive infection of immunocompromised people in Africa, but infection of mice and other animal models with ST313 have not consistently reproduced this invasive phenotype. The introduction of alternative infection models could help to improve the quality and reproducibility of pathogenesis research by facilitating larger-scale experiments. To investigate the virulence of S. Typhimurium ST313 in comparison with ST19, a combination of avian and insect disease models were used. We performed experimental infections in five lines of inbred and one line of outbred chickens, as well as in the alternative chick embryo and Galleria mellonella wax moth larvae models. This extensive set of experiments identified broadly similar patterns of disease caused by the African and global pathovariants of Salmonella Typhimurium in the chicken, the chicken embryo and insect models. A comprehensive analysis of all the chicken infection experiments revealed that the African ST313 isolate D23580 had a subtle phenotype of reduced levels of organ colonisation in inbred chickens, relative to ST19 strain 4/74. ST313 isolate D23580 also caused reduced mortality in chicken embryos and insect larvae, when compared with ST19 4/74. We conclude that these three infection models do not reproduce the characteristics of the systemic disease caused by S. Typhimurium ST313 in humans.},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/31348776},
	author = {Lacharme-Lora*, Lizeth and Owen*, Si{\^a}n V and Richard Blundell and Canals, Roc{\'\i}o and Wenner, Nicolas and Perez-Sepulveda, Blanca and Fong, Wai Yee and Gilroy, Rachel and Wigley, Paul and Hinton, Jay CD}
}
@article {hammarlof2018role,
	title = {Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella},
	journal = {Proceedings of the National Academy of Sciences},
	year = {2018},
	pages = {201714718},
	publisher = {National Academy of Sciences},
	abstract = {Salmonella enterica\&nbsp;serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of\&nbsp;Salmonella\&nbsp;genomes has revealed that ST313 is closely related to the ST19 group of\&nbsp;S\&nbsp;Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only \~{}1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African\&nbsp;Salmonella\&nbsp;from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of\&nbsp;Salmonella\&nbsp;We identified a SNP in the promoter of the\&nbsp;pgtE\&nbsp;gene that caused high expression of the PgtE virulence factor in African\&nbsp;S.\&nbsp;Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African\&nbsp;S\&nbsp;Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.},
	url = {https://www.pnas.org/content/115/11/E2614.long},
	author = {Hammarl{\"o}f*, Disa L and Kr{\"o}ger*, Carsten and Owen*, Si{\^a}n V and Canals, Roc{\'\i}o and Lacharme-Lora, Lizeth and Wenner, Nicolas and Schager, Anna E and Wells, Timothy J and Henderson, Ian R and Wigley, Paul and others}
}
@mastersthesis {634163,
	title = {Exploring the prophage biology of Salmonella enterica serovar Typhimurium ST313},
	year = {2017},
	type = {PhD thesis},
	abstract = {In the past 30 years, Salmonella bloodstream infections have become a significant health problem in sub-Saharan Africa and are responsible for the deaths of ~390,000 people each year. The disease is largely caused by a recently described sequence type of Salmonella Typhimurium: ST313. Comparative genomic analysis showed that the ST313 lineage is closely-related to the ancestral gastroenteritis-associated Typhimurium sequence type ST19, but carries a distinct prophage repertoire. I hypothesised that prophages contribute to the biology of this clinically-relevant ST. In this thesis I show that the African ST313 representative strain D23580 contains 5 full length prophages. Prophage BTP1 and BTP5 are undescribed, novel prophages specific to African ST313 strains, whilst Gifsy-2, ST64B and Gifsy-1 are well-characterised prophages found in other strains of S. Typhimurium. Of the five prophages, only BTP1 and BTP5 showed evidence for functional phage production, and mutations responsible for the inactivation of Gifsy-2, ST64B and Gifsy-1 were identified. The BTP1 prophage spontaneously induced at a prolific rate, estimated to result in the phage-mediated lysis of approximately 0.2\% of the lysogenic cell population. A GFP reporter system was developed to visualise the spontaneous induction of the BTP1 prophage at the single-cell level. Though the BTP5 phage could not be studied using traditional plaque assay methodology, there was evidence that the BTP5 prophage was capable of forming viable BTP5 phage that could lysogenise na{\"\i}ve hosts. I analysed the genomes of recently discovered ST313 isolates from the UK and show that ST313 in the UK represents a distinct population of antibiotic susceptible strains associated with gastrointestinal infection. Additionally, analysis of the UK-ST313 genomes indicated that the BTP1 and BTP5 prophages were acquired independently by the two African ST313 lineages, showing convergent evolution to acquire and conserve the BTP1 and BTP5 prophages. Finally I present evidence that the prophages, in particular BTP5, effect the global gene expression of ST313, and the ST313-td gene of BTP1 mediates lysogenic conversion by functioning as a superinfection immunity factor against infection by Salmonella phage P22. The implications of these findings for understanding the pathogen in terms of ecological niche, host range and invasiveness in humans is discussed.},
	url = {https://livrepository.liverpool.ac.uk/3011773/},
	author = {Owen*, Si{\^a}n V.}
}
@article {owen2017characterization,
	title = {Characterization of the prophage repertoire of African Salmonella Typhimurium ST313 reveals high levels of spontaneous induction of novel phage BTP1},
	journal = {Frontiers in microbiology},
	volume = {8},
	year = {2017},
	pages = {235},
	publisher = {Frontiers},
	abstract = {In the past 30 years,\&nbsp;Salmonella\&nbsp;bloodstream infections have become a significant health problem in sub-Saharan Africa and are responsible for the deaths of an estimated 390,000 people each year. The disease is predominantly caused by a recently described sequence type of\&nbsp;Salmonella\&nbsp;Typhimurium: ST313, which has a distinctive set of prophage sequences. We have thoroughly characterized the ST313-associated prophages both genetically and experimentally. ST313 representative strain D23580 contains five full-length prophages: BTP1, Gifsy-2D23580, ST64BD23580, Gifsy-1D23580, and BTP5. We show that common\&nbsp;S.\&nbsp;Typhimurium prophages Gifsy-2, Gifsy-1, and ST64B are inactivated in ST313 by mutations. Prophage BTP1 was found to be a functional novel phage, and the first isolate of the proposed new species "Salmonella virus BTP1", belonging to the\&nbsp;P22virus\&nbsp;genus. Surprisingly, \~{}109\&nbsp;BTP1 virus particles\&nbsp;per\&nbsp;ml were detected in the supernatant of non-induced, stationary-phase cultures of strain D23580, representing the highest spontaneously induced phage titer so far reported for a bacterial prophage. High spontaneous induction is shown to be an intrinsic property of prophage BTP1, and indicates the phage-mediated lysis of around 0.2\% of the lysogenic population. The fact that BTP1 is highly conserved in ST313 poses interesting questions about the potential fitness costs and benefits of novel prophages in epidemic\&nbsp;S.\&nbsp;Typhimurium ST313.},
	url = {https://dx.doi.org/10.3389/fmicb.2017.00235},
	author = {Owen*, Si{\^a}n V and Wenner, Nicolas and Canals, Roc{\'\i}o and Makumi, Angela and Hammarl{\"o}f, Disa L and Gordon, Melita A and Aertsen, Abram and Feasey, Nicholas A and Hinton, Jay CD}
}
@article {faulkner2017direct,
	title = {Direct characterization of the native structure and mechanics of cyanobacterial carboxysomes},
	journal = {Nanoscale},
	volume = {9},
	number = {30},
	year = {2017},
	pages = {10662{\textendash}10673},
	publisher = {Royal Society of Chemistry},
	abstract = {Carboxysomes are proteinaceous organelles that play essential roles in enhancing carbon fixation in cyanobacteria and some proteobacteria. These self-assembling organelles encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase using a protein shell structurally resembling an icosahedral viral capsid. The protein shell serves as a physical barrier to protect enzymes from the cytosol and a selectively permeable membrane to mediate transport of enzyme substrates and products. The structural and mechanical nature of native carboxysomes remain unclear. Here, we isolate functional β-carboxysomes from the cyanobacterium Synechococcus elongatus PCC7942 and perform the first characterization of the macromolecular architecture and inherent physical mechanics of single β-carboxysomes using electron microscopy, atomic force microscopy (AFM) and proteomics. Our results illustrate that the intact β-carboxysome comprises three structural domains, a single-layered icosahedral shell, an inner layer and paracrystalline arrays of interior Rubisco. We also observe the protein organization of the shell and partial β-carboxysomes that likely serve as the β-carboxysome assembly intermediates. Furthermore, the topography and intrinsic mechanics of functional β-carboxysomes are determined in native conditions using AFM and AFM-based nanoindentation, revealing the flexible organization and soft mechanical properties of β-carboxysomes compared to rigid viruses. Our study provides new insights into the natural characteristics of β-carboxysome organization and nanomechanics, which can be extended to diverse bacterial microcompartments and are important considerations for the design and engineering of functional carboxysomes in other organisms to supercharge photosynthesis. It offers an approach for inspecting the structural and mechanical features of synthetic metabolic organelles and protein scaffolds in bioengineering.},
	url = {https://pubs.rsc.org/en/content/articlelanding/2017/nr/c7nr02524f$\#$!divAbstract},
	author = {Faulkner, Matthew and Rodriguez-Ramos, Jorge and Dykes, Gregory F and Owen, Si{\^a}n V and Casella, Selene and Simpson, Deborah M and Beynon, Robert J and Liu, Lu-Ning}
}
@article {ashton2017public,
	title = {Public health surveillance in the UK revolutionises our understanding of the invasive Salmonella Typhimurium epidemic in Africa},
	journal = {Genome medicine},
	volume = {9},
	number = {1},
	year = {2017},
	pages = {92},
	publisher = {BioMed Central},
	abstract = {BackgroundThe ST313 sequence type of\&nbsp;Salmonella\&nbsp;Typhimurium causes invasive non-typhoidal salmonellosis and was thought to be confined to sub-Saharan Africa. Two distinct phylogenetic lineages of African ST313 have been identified.MethodsWe analysed the whole genome sequences of\&nbsp;S. Typhimurium isolates from UK patients that were generated following the introduction of routine whole-genome sequencing (WGS) of\&nbsp;Salmonella enterica\&nbsp;by Public Health England in 2014.ResultsWe found that 2.7\% (84/3147) of\&nbsp;S. Typhimurium from patients in England and Wales were ST313 and were associated with gastrointestinal infection. Phylogenetic analysis revealed novel diversity of ST313 that distinguished UK-linked gastrointestinal isolates from African-associated extra-intestinal isolates. The majority of genome degradation of African ST313 lineage 2 was conserved in the UK-ST313, but the African lineages carried a characteristic prophage and antibiotic resistance gene repertoire. These findings suggest that a strong selection pressure exists for certain horizontally acquired genetic elements in the African setting. One UK-isolated lineage 2 strain that probably originated in Kenya carried a chromosomally located\&nbsp;bla\&nbsp;CTX-M-15, demonstrating the continual evolution of this sequence type in Africa in response to widespread antibiotic usage.ConclusionsThe discovery of ST313 isolates responsible for gastroenteritis in the UK reveals new diversity in this important sequence type. This study highlights the power of routine WGS by public health agencies to make epidemiologically significant deductions that would be missed by conventional microbiological methods. We speculate that the niche specialisation of sub-Saharan African ST313 lineages is driven in part by the acquisition of accessory genome elements.},
	url = {https://genomemedicine.biomedcentral.com/articles/10.1186/s13073-017-0480-7},
	author = {Ashton*, Philip M and Owen*, Si{\^a}n V and Kaindama, Lukeki and Rowe, Will PM and Lane, Chris R and Larkin, Lesley and Nair, Satheesh and Jenkins, Claire and de Pinna, Elizabeth M and Feasey, Nicholas A and others}
}
@article {connor2016s,
	title = {What{\textquoteright}s in a name? Species-wide whole-genome sequencing resolves invasive and noninvasive lineages of Salmonella enterica serotype Paratyphi B},
	journal = {MBio},
	volume = {7},
	number = {4},
	year = {2016},
	pages = {e00527{\textendash}16},
	publisher = {American Society for Microbiology},
	abstract = {For 100\&nbsp;years, it has been obvious that\&nbsp;Salmonella enterica\&nbsp;strains sharing the serotype with the formula 1,4,[5],12:b:1,2{\textemdash}now known as Paratyphi B{\textemdash}can cause diseases ranging from serious systemic infections to self-limiting gastroenteritis. Despite considerable predicted diversity between strains carrying the common Paratyphi B serotype, there remain few methods that subdivide the group into groups that are congruent with their disease phenotypes. Paratyphi B therefore represents one of the canonical examples in\&nbsp;Salmonella\&nbsp;where serotyping combined with classical microbiological tests fails to provide clinically informative information. Here, we use genomics to provide the first high-resolution view of this serotype, placing it into a wider genomic context of the\&nbsp;Salmonella enterica\&nbsp;species. These analyses reveal why it has been impossible to subdivide this serotype based upon phenotypic and limited molecular approaches. By examining the genomic data in detail, we are able to identify common features that correlate with strains of clinical importance. The results presented here provide new diagnostic targets, as well as posing important new questions about the basis for the invasive disease phenotype observed in a subset of strains.},
	url = {https://mbio.asm.org/content/7/4/e00527-16},
	author = {Connor, Thomas R and Owen, Si{\^a}n V and Langridge, Gemma and Connell, Steve and Nair, Satheesh and Reuter, Sandra and Dallman, Timothy J and Corander, Jukka and Tabing, Kristine C and Le Hello, Simon and others}
}
@article {srikumar2015rna,
	title = {RNA-seq brings new insights to the intra-macrophage transcriptome of Salmonella Typhimurium},
	journal = {PLoS pathogens},
	volume = {11},
	number = {11},
	year = {2015},
	pages = {e1005262},
	publisher = {Public Library of Science},
	abstract = {Salmonella enterica\&nbsp;serovar Typhimurium is arguably the world{\textquoteright}s best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of\&nbsp;Salmonella\&nbsp;during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from\&nbsp;Salmonella\&nbsp;vaccine strains. A majority (88\%) of the 280\&nbsp;S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31\&nbsp;S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during\&nbsp;in vitro\&nbsp;growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage\&nbsp;S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.},
	url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1005262},
	author = {Srikumar, Shabarinath and Kr{\"o}ger, Carsten and H{\'e}brard, Magali and Colgan, Aoife and Owen, Si{\^a}n V and Sivasankaran, Sathesh K and Cameron, Andrew DS and Hokamp, Karsten and Hinton, Jay CD}
}
