High-throughput analysis of  Yersinia pseudotuberculosis  gene essentiality in optimised in vitro conditions, and implications for the speciation of  Yersinia pestis

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Abstract

Background

Yersinia pseudotuberculosis is a zoonotic pathogen, causing mild gastrointestinal infection in humans. From this comparatively benign pathogenic species emerged the highly virulent plague bacillus, Yersinia pestis, which has experienced significant genetic divergence in a relatively short time span. Much of our knowledge of Yersinia spp. evolution stems from genomic comparison and gene expression studies. Here we apply transposon-directed insertion site sequencing (TraDIS) to describe the essential gene set of Y. pseudotuberculosis IP32953 in optimised in vitro growth conditions, and contrast these with the published essential genes of Y. pestis.

Results

The essential genes of an organism are the core genetic elements required for basic survival processes in a given growth condition, and are therefore attractive targets for antimicrobials. One such gene we identified is yptb3665, which encodes a peptide deformylase, and here we report for the first time, the sensitivity of Y. pseudotuberculosis to actinonin, a deformylase inhibitor. Comparison of the essential genes of Y. pseudotuberculosis with those of Y. pestis revealed the genes whose importance are shared by both species, as well as genes that were differentially required for growth. In particular, we find that the two species uniquely rely upon different iron acquisition and respiratory metabolic pathways under similar in vitro conditions.

Conclusions

The discovery of uniquely essential genes between the closely related Yersinia spp. represent some of the fundamental, species-defining points of divergence that arose during the evolution of Y. pestis from its ancestor. Furthermore, the shared essential genes represent ideal candidates for the development of novel antimicrobials against both species.

Electronic supplementary material

The online version of this article (10.1186/s12866-018-1189-5) contains supplementary material, which is available to authorized users.

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Most cited references 75

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Genome sequence of Yersinia pestis, the causative agent of plague.

J. Parkhill, B W Wren, N. R. Thomson … (2001)

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.

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Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.

Gemma Langridge, Minh-Duy Phan, Daniel J. Turner … (2009)

Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.

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Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution.

I Kobayashi (2001)

Restriction-modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.

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Author and article information

Contributors

Samuel J. Willcocks:

ORCID: http://orcid.org/0000-0002-0756-4859

sam.willcocks@lshtm.ac.uk

Richard A. Stabler: Richard.stabler@lshtm.ac.uk

Helen S. Atkins: hsatkins@mail.dstl.gov.uk

Petra F. Oyston: pcoyston@mail.dstl.gov.uk

Brendan W. Wren: +44 (0) 2079272288 , brendan.wren@lshtm.ac.uk

Journal

Journal ID (nlm-ta): BMC Microbiol

Journal ID (iso-abbrev): BMC Microbiol

Title: BMC Microbiology

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2180

Publication date (Electronic): 31 May 2018

Publication date PMC-release: 31 May 2018

Publication date Collection: 2018

Volume: 18

Electronic Location Identifier: 46

Affiliations

[1 ]ISNI 0000 0004 0425 469X, GRID grid.8991.9, Department of Pathogen Molecular Biology, , London School of Hygiene and Tropical Medicine, ; Keppel Street, London, WC1E 7HT UK

[2 ]Microbiology, CBR Division, DSTL Porton Down, Salisbury, SP4 0JQ UK

Author information

Samuel J. Willcocks http://orcid.org/0000-0002-0756-4859

Article

Publisher ID: 1189

DOI: 10.1186/s12866-018-1189-5

PMC ID: 5984423

PubMed ID: 29855259

SO-VID: ed3cd8bc-9881-4a4b-8bb5-56482037c241

License:

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 5 September 2017

Date accepted : 18 May 2018

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100010418, Defence Science and Technology Laboratory;

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ScienceOpen disciplines: Microbiology & Virology

Keywords: yersinia,tradis,tnseq,essential genes

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ScienceOpen disciplines: Microbiology & Virology

Keywords: yersinia, tradis, tnseq, essential genes

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High-throughput analysis of Yersinia pseudotuberculosis gene essentiality in optimised in vitro conditions, and implications for the speciation of Yersinia pestis

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Role of Microbes in Soil Fertility and Human Health

Most cited references 75

Genome sequence of Yersinia pestis, the causative agent of plague.

Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.

Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution.

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