CHARACTERIZING A NEW MOLECULAR MECHANISM OF TRANSMISSIBLE PERSISTENCE IN SALMONELLA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007215
• Project No.: NYC-189438
• Project Start Date: Oct 1, 2015
• Project End Date: Sep 30, 2018

Project Director
Peters, JO, E.

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Microbiology

Non Technical Summary
Food poisoning is a major food safety issue in New York State. Salmonellosis is the most frequent type of food poisoning. For the first time, we have found that a molecular system that allows bacteria to form a semi-dormant state or "persistent state" can be spread on mobile "jumping genes" called transposons. This system was found in many types of Salmonella associated with serious food poisoning outbreaks including, Salmonella Heidelberg, Montevideo, Tennessee and Senftenberg. One of the reasons that this is a serious concern is that protocols set up to sanitize and control for growth of bacteria are established with strains of bacteria that may not have this persistence system. It is important to know if the genes that allow persistence are allowing pathogens to subvert sanitation procedures. However, its spread on mobile elements is consistent with an important role for this genetic element and the genes it encodes in food poisoning. The persistence genes are encoded on a newly appreciated member of a group of transposons that is very efficiently transmitted between bacteria called a heteromeric transposase element. In our work we will characterize the persistence genes to determine how they change the ability of the bacterial strain to withstand sanitary treatments, treatments with biocides, and treatments with antibiotics. We will also determine if this putative "persistence" system affects how the element itself spreads between bacteria. Ultimately, these findings may lead to new sanitation and monitoring procedures to adequately protect the New York State food production system.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4099	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

food safety

salmonella

resistance

sanitation methods

Goals / Objectives
Goal 1 - Determine if the genes allow for a greater percentage of persistent cells that are resistance to standard treatments used to sanitize for bacteria. The himA himB system was the first confirmed molecular system allowing the formation of persister cells. This system in Salmonella and other bacteria is homologous, but also shows some interesting differences from the original system that necessitates further investigation. Goal 2 - Determine if the persistence genes also alter the ability of the genetic element to move between strains of Salmonella and potentially other strains of pathogenic bacteria. Interestingly, the persistence genes are in a region of the genetic element that is typically involved in controlling movement of the element. Genetic elements are sensitive to the metabolic state of the cell. It is essential to know if activation of the persister system activates spread of the element.

Project Methods
Both goals require establishing the expression of the genes. We will clone the genes with the native promoter in the highly tractable E. coli system using vectors that are functional in the laboratory strain of Salmonella Typhimurium. These are non-pathogenic strains that are easy to manipulate in the laboratory. We will also delete the genes instrain Salmonella Montevideo background. Specifically to address Goal 1 we will determine the percentage of persister cells in the population using standard published assays. This will include determining the percentage survival with dilutions of bleach, hydrogen peroxide, and the effect of desiccation. The percentage of survivors will also be tested with individual antibiotics. In all cases the results will be compared to controls without this system. Typically, two genes are needed for persister systems, but if we can knock out activity in Salmonella Montevideo background, but not establish it in the heterologous system we will investigate if other genes on the transposon are also required. To address Goal 2 we will utilize standard transposition assay that will be set up using the Tn6230 transmissible genetic element of interest. Briefly, the element will be situated on a plasmid that does not replicate in the test strain, but can be introduced efficiently using conjugation. Successful transposition events will result in strains resistant to antibiotic. Once the system is established the role of the persister system will be tested. We will look for changes in frequency and targeting. Targeting can be accessed via arbitrary PCR.

Progress 10/01/15 to 09/30/18

Outputs
Target Audience:We had significant results over the three years of funding. Specifically in the third year of funding we had a high profile publication accepted to Nature Microbiology and an invited analysis was submitted in December 2018 to the high-profile journal Nature Reviews Microbiology . I also presented our results at numerous national and international meetings. In each of these cases the target audience was scientists and decision makers who are interested in the role of mobile genetic elements in the spread of antibiotic resistance and the general tolerance of bacteria to sanitation procedures. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and two undergraduate students have been working on this project. They have been trained in basic molecular microbiology and bioinformatics techniques. How have the results been disseminated to communities of interest?In addition to the publications, and presentations, information has been made freely available for download to investigators anywhere in the world for the first two publications. The research and associated datasets from the recently submitted third publication will also be freely available once it is published. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We carried out a comprehensive bioinformatics analysis of elements carrying the persistence genes and found them widespread across mobile genetic elements in diverse bacteria. Both the distribution of mobile genetic elements and these genes were much greater than previously known, and well beyond the original identification in Salmonella. Unexpectedly, analysis of these elements revealed an exciting convergence between these genes, genes involved in movement of the genetic element, and a set of genes used to protect bacterial from other mobile genetic elements (called CRISPR/Cas systems). This information was of great interest resulting in a broadly downloaded publication in the Proceedings of the National Academy of Science (as well as carried on social media and the subject of a podcast through the American Society of Microbiology). Expression of the genes had no phenotype in isolation and we continue to investigate the role in mobilization of the genetic element. We believe that the CRISPR/Cas system is being utilized as a mechanism of targeting the mobile element to other genetic elements that would allow is spread across diverse bacteria. We continue to piece together the role of these systems in the mobilization of genetic information, but this work has already significantly increased our understanding of how genes are spread between bacteria and could also provide an important new tool for genome modification in bacteria, plants and animals. Based on the widespread nature of the elements, we worked with other investigators to established a system inspired by the transposon and CRISPR relationship that was accepted in 2018 and published in Nature Microbiology in January 2019. An expanded analysis of these elements was submitted for publication in December 2018.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: JM Peters, B-M Koo, R Patino, GE Heussler, CC Hearne, J Qu, YF Inclan, JS Hawkins, CHS Lu, MR Silvis, MM Harden, H Osadnik, JE Peters, JN Engel, RJ Dutton, AD Grossman, CA Gross, and OS Rosenberg (2019) Enabling Genetic Analysis of Diverse Bacteria with Mobile-CRISPRi. Nature Microbiology. 1-7
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: G Faure, SA Shmakov, WX Yan, DR Cheng, DA Scott, JE Peters, KS Makarova, and EV Koonin (2018) Analysis: CRISPR in mobile genetic elements: counter-defense and beyond. Nature Reviews Microbiology Invited article - under review 12/18/2018
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Sept 2018 - Netherlands - "Tn7 elements: Highly controlled and targeted transposition" Bi-annual Workshop on DNA transactions - Invited Speaker
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Nov 2018 - Cold Spring Harbor Laboratory - Recruitment of CRISPR-Cas systems by Tn7-like transposons - Transposons - Invited Speaker

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:We had significant results during our second year of funding with a high-profile PNAS publication (http://www.pnas.org/content/114/35/E7358.full). This collaborative work allowed us to define the distribution of our transmissible persistence candidate genes across almost 50,000 prokaryotic genomes. According to the Altmetircs report it was in the top 5% of all research outputs scored by Altmetric with the abstract downloaded 8,542 times, the full text downloaded 1,725 times, and the pdf version of the document downloaded 1,113 times. Based on this work I was invited to speak at the Molecular Genetic of Bacteria and Phages Conference in Madison, Wisconsin in Summer 2017 and also the Mobile Genetic Element conference in Woods Hole, MA in Fall 2017. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student and two undergraduate have been working on this project. They have been trained in basic molecular microbiology techniques. How have the results been disseminated to communities of interest?This information has been made freely available for download to investigators anywhere in the world. Additionally, the article was made freely available. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work towards the completion of the goals described in the grant. Our work in year two has established the most broadly relevant genes. The context of these genes suggests cloning strategies that will be the most productive for stable and controlled expression. This work will also benefit from a series of strains we engineered to allow controlled expression. Multiple important expression systems utilize catabolic pathways that are only induced when a particular carbon sources is available. One issue with these systems is that transport of the sugar can be inefficient thereby preventing a tunable response across the population. We were able to clone gratuitous transport systems into our strains and show that we could get a linear response over orders of magnitude of induction and strong repression. In this last year, we will combine the candidate genes we identified with our computational analysis with the expression system to complete our analysis.

Impacts
What was accomplished under these goals? Working from our collaboration established in the first year of funding we were able to move the research to publication in a high-profile article published in PNAS. I am the lead author and co-corresponding author on the paper. This work has allowed us to put transmissible persistence in the context of all sequenced prokaryotic genomes (almost 50,000 complete and partial genomes). This analysis focused initially on neighbor analysis where the genomes were queried for the transposon genes that are distinct to these elements. Genes in the neighborhood of the transposon genes, including the persistence genes of interest, could then be subjected to phylogenetic reconstruction to suggest functional linkages between the transposon genes and the persistence genes. This pool of data will form much of the work in the remaining year, but as a side benefit from this work we also made an important and unexpected discovery. We were able to show phylogenetically the transposon genes were also linked to important gene modification system in prokaryotes, CRISPR/Cas systems. These systems are defense systems that protect prokaryotic organisms from mobile elements, such as mobile plasmids and bacterial viruses. Our work strongly support a model where these systems are being used by the transposon to direct insertion into other mobile elements that will allow them to travel between bacteria. This is an important component for how persistence spreads. However, it also suggested an important application where easy-to-produce synthetic guide RNAs could potentially be used to direct a DNA segment of interest to essentially any location in the cell. We processed a pre-patent with the technology transfer office to protect this potentially important technology for Cornell University.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: JE Peters, KS Makarova, S Shmakov, and EV Koonin (2017) Recruitment of CRISPR-Cas systems by Tn7-like transposons. Proc. Natl. Acad. Sci. USA 114:E7358-E7366

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student and two undergraduate have been working on this project. They have been trained in basic molecular microbiology techniques. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue with our cloning work. Some of the genes have been difficult to clone and express. We will utilize the expression system that we developed during the first year of funding. We will also utilize new advanced mechanisms of genome assembly to produce the needed constructs. Regulation in this system is very important and it is possible that expressing the components of the system individually does not allow the appropriate coordination between the various aspects of the system. Continued bioinformatics comparisons will also help us understand which components are most important in these systems. We will also include previously studied persistence systems in our cloning as a further control for our work.

Impacts
What was accomplished under these goals? During the first year of work on this project we have been individually cloning the genes in the persistence system. One of the exciting things about this system is that while the himA component is well studied (and highly homologous to these other systems), the himB component seems to be unique to the systems found common in food pathogens like Salmonella. Therefore, a number of candidate himB systems are under investigation. As part of this work we are developing a novel expression system that utilizes bacteriophage control systems. This system will ensure that the system is only activated when we need it to be. This system should be useful for other groups working in bacteria as well. Excitingly, a bioinformatics analysis that we are working on in collaboration with the Koonin Group at the National Institutes of Health suggests that this system is much more common than we appreciate in a variety of food pathogens.

Publications