Genetic manipulations of nonmodel gut microbes

Abstract

Hundreds of microbiota gene expressions are significantly different between healthy and diseased humans. The “bottleneck” preventing a mechanistic dissection of how they affect host biology/disease is that many genes are encoded by nonmodel gut commensals and not genetically manipulatable. Approaches to efficiently identify their gene transfer methodologies and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. This paper will introduce a step-by-step protocol to identify gene transfer conditions and build the gene manipulation tools for nonmodel gut microbes, focusing on Gram-negative Bacteroidia and Gram-positive Clostridia organisms. This protocol enables us to identify gene transfer methods and develop gene manipulation tools without prior knowledge of their genome sequences, by targeting bacterial 16s ribosomal RNAs or expanding their compatible replication origins combined with clustered regularly interspaced short palindromic repeats machinery. Such an efficient and generalizable approach will facilitate functional studies that causally connect gut microbiota genes to host diseases.

Keywords

genetic manipulation strategies / human gut microbiota / nonmodel gut Bacteroidia / nonmodel gut Clostridia

References

[1] Yachida, Shinichi, Sayaka Mizutani, Hirotsugu Shiroma, Satoshi Shiba, Takeshi Nakajima, Taku Sakamoto, Hikaru Watanabe, et al. 2019. “Metagenomic and Metabolomic Analyses Reveal Distinct Stage-Specific Phenotypes of the Gut Microbiota in Colorectal Cancer.” Nature Medicine 25: 968-976. https://doi.org/10.1038/s41591-019-0458-7

[2] Lloyd-Price, Jason, Cesar Arze, Ashwin N. Ananthakrishnan, Melanie Schirmer, Julian Avila-Pacheco, Tiffany W. Poon, Elizabeth Andrews, et al. 2019. “Multi-Omics of the Gut Microbial Ecosystem in Inflammatory Bowel Diseases.” Nature 569: 655-662. https://doi.org/10.1038/s41586-019-1237-9

[3] Thomas, Andrew Maltez, Paolo Manghi, Francesco Asnicar, Edoardo Pasolli, Federica Armanini, Moreno Zolfo, Francesco Beghini, et al. 2019. “Metagenomic Analysis of Colorectal Cancer Datasets Identifies Cross-Cohort Microbial Diagnostic Signatures and a Link with Choline Degradation.” Nature Medicine 25: 667-678. https://doi.org/10.1038/s41591-019-0405-7

[4] Wirbel, Jakob, Paul Theodor Pyl, Ece Kartal, Konrad Zych, Alireza Kashani, Alessio Milanese, Jonas S. Fleck, et al. 2019. “Meta-Analysis of Fecal Metagenomes Reveals Global Microbial Signatures That Are Specific for Colorectal Cancer.” Nature Medicine 25: 679-689. https://doi.org/10.1038/s41591-019-0406-6

[5] Zhou, Wenyu, M. Reza Sailani, Kévin Contrepois, Yanjiao Zhou, Sara Ahadi, Shana R. Leopold, Martin J. Zhang, et al. 2019. “Longitudinal Multi-Omics of Host-Microbe Dynamics in Prediabetes.” Nature 569: 663-671. https://doi.org/10.1038/s41586-019-1236-x

[6] Qin, Junjie, Yingrui Li, Zhiming Cai, Shenghui Li, Jianfeng Zhu, Fan Zhang, Suisha Liang, et al. 2012. “A Metagenome-Wide Association Study of Gut Microbiota in Type 2 Diabetes.” Nature 490: 55-60. https://doi.org/10.1038/nature11450

[7] Cho, Ilseung, and Martin J. Blaser. 2012. “The Human Microbiome: At the Interface of Health and Disease.” Nature Reviews Genetics 13(4): 260-270. https://doi.org/10.1038/nrg3182

[8] Lloyd-Price, Jason, Galeb Abu-Ali, and Curtis Huttenhower. 2016. “The Healthy Human Microbiome.” Genome Medicine 8(1): 51. https://doi.org/10.1186/s13073-016-0307-y

[9] Taketani, Mao, Jianbo Zhang, Shuyi Zhang, Alexander J. Triassi, Yu-Ja Huang, Linda G. Griffith, and Christopher A. Voigt. 2020. “Genetic Circuit Design Automation for the Gut Resident Species Bacteroides thetaiotaomicron.” Nature Biotechnology 38(8): 962-969. https://doi.org/10.1038/s41587-020-0468-5

[10] Cañadas, Inés C., Daphne Groothuis, Maria Zygouropoulou, Raquel Rodrigues, and Nigel P. Minton. 2019. “RiboCas: A Universal CRISPR-based Editing Tool for Clostridium.” ACS Synthetic Biology 8: 1379-1390. https://doi.org/10.1021/acssynbio.9b00075

[11] Guo, Chun-Jun, Breanna M. Allen, Kamir J. Hiam, Dylan Dodd, Will Van Treuren, Steven Higginbottom, Kazuki Nagashima, et al. 2019. “Depletion of Microbiome-Derived Molecules in the Host Using Clostridium Genetics.” Science 366: eaav1282. https://doi.org/10.1126/science.aav1282

[12] Wu, Meng, Nathan P. McNulty, Dmitry A. Rodionov, Matvei S. Khoroshkin, Nicholas W. Griffin, Jiye Cheng, Phil Latreille, et al. 2015. “Genetic Determinants of In Vivo Fitness and Diet Responsiveness in Multiple Human Gut Bacteroides.” Science 350(6256): aac5992. https://doi.org/10.1126/science.aac5992

[13] Mimee, Mark, Alex C. Tucker, Christopher A. Voigt, and Timothy K. Lu. 2015. “Programming a Human Commensal Bacterium, Bacteroides thetaiotaomicron, to Sense and Respond to Stimuli In the Murine Gut Microbiota.” Cell Systems 1: 62-71. https://doi.org/10.1016/j.cels.2015.06.001

[14] Goodman, Andrew L., Nathan P. McNulty, Yue Zhao, Douglas Leip, Robi D. Mitra, Catherine A. Lozupone, Rob Knight, and Jeffrey I. Gordon. 2009. “Identifying Genetic Determinants Needed to Establish a Human Gut Symbiont in Its Habitat.” Cell Host & Microbe 6: 279-289. https://doi.org/10.1016/j.chom.2009.08.003

[15] Heap, John T., Oliver J. Pennington, Stephen T. Cartman, Glen P. Carter, and Nigel P. Minton. 2007. “The ClosTron: A Universal Gene Knock-Out System for the Genus Clostridium.” Journal of Microbiological Methods 70: 452-464. https://doi.org/10.1016/j.mimet.2007.05.021

[16] Salyers, Abigail A., Nadja Shoemaker, Andrew Cooper, John D'Elia, and Joseph A. Shipman. 1999. “8 Genetic Methods for Bacteroides Species.” Methods in Microbiology 29: 229-249. https://doi.org/10.1016/S0580-9517(08)70119-3

[17] Whitaker, Weston R., Elizabeth Stanley Shepherd, and Justin L. Sonnenburg. 2017. “Tunable Expression Tools Enable Single-Cell Strain Distinction in the Gut Microbiome.” Cell 169: 538-546.e12. https://doi.org/10.1016/j.cell.2017.03.041

[18] Lim, Bentley, Michael Zimmermann, Natasha A. Barry, and Andrew L. Goodman. 2017. “Engineered Regulatory Systems Modulate Gene Expression of Human Commensals in the Gut.” Cell 169: 547-558.e15. https://doi.org/10.1016/j.cell.2017.03.045

[19] García-Bayona, Leonor, and Laurie E. Comstock. 2019. “Streamlined Genetic Manipulation of Diverse Bacteroides and Parabacteroides Isolates from the Human Gut Microbiota.” mBio 10(4): e01762-19. https://doi.org/10.1128/mbio.01762-19

[20] Bencivenga-Barry, Natasha A., Bentley Lim, Carmen M. Herrera, M. Stephen Trent, and Andrew L. Goodman. 2020. “Genetic Manipulation of Wild Human Gut Bacteroides.” Journal of Bacteriology 202(3): e00544-19. https://doi.org/10.1128/jb.00544-19

[21] Martens, Eric C., Herbert C. Chiang, and Jeffrey I. Gordon. 2008. “Mucosal Glycan Foraging Enhances Fitness and Transmission of a Saccharolytic Human Gut Bacterial Symbiont.” Cell Host & Microbe 4: 447-457. https://doi.org/10.1016/j.chom.2008.09.007

[22] Garnier, Thierry, and Stewart T. Cole. 1988. “Identification and Molecular Genetic Analysis of Replication Functions of the Bacteriocinogenic Plasmid pIP404 From Clostridium perfringens.” Plasmid 19: 151-160. https://doi.org/10.1016/0147-619X(88)90053-4

[23] Trieu-Cuot, Patrick, Cécile Carlier, Claire Poyart-Salmeron, and Patrice Courvalin. 1991. “Shuttle Vectors Containing a Multiple Cloning Site and a La &Amp; a Escherichia coli to Gram-Positive Bacteria Gene for Conjugal Transfer of DNA from Escherichia coli to Gram-Positive Bacteria.” Gene 102: 99-104. https://doi.org/10.1016/0378-1119(91)90546-N

[24] Tardif, C., H. Maamar, M. Balfin, and J. P. Belaich. 2001. “Electrotransformation Studies in Clostridium cellulolyticum.” Journal of Industrial Microbiology and Biotechnology 27: 271-274. https://doi.org/10.1038/sj.jim.7000081

[25] McAllister, Kathleen N., Laurent Bouillaut, Jennifer N. Kahn, William T. Self, and Joseph A. Sorg. 2017. “Using CRISPR-Cas9-mediated Genome Editing to Generate C. difficile Mutants Defective in Selenoproteins Synthesis.” Scientific Reports 7: 14672. https://doi.org/10.1038/s41598-017-15236-5

[26] Zetsche, Bernd, Jonathan S. Gootenberg, Omar O. Abudayyeh, Ian M. Slaymaker, Kira S. Makarova, Patrick Essletzbichler, Sara E. Volz, et al. 2015. “Cpf1 Is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System.” Cell 163: 759-771. https://doi.org/10.1016/j.cell.2015.09.038

[27] Kim, Seong Keun, Haseong Kim, Woo-Chan Ahn, Kwang-Hyun Park, Eui-Jeon Woo, Dae-Hee Lee, and Seung-Goo Lee. 2017. “Efficient Transcriptional Gene Repression by Type V-A CRISPR-Cpf1 from Eubacterium eligens.” ACS Synthetic Biology 6: 1273-1282. https://doi.org/10.1021/acssynbio.6b00368

[28] Tang, Xu, Levi G. Lowder, Tao Zhang, Aimee A. Malzahn, Xuelian Zheng, Daniel F. Voytas, Zhaohui Zhong, et al. 2017. “Correction: A CRISPR-Cpf1 System for Efficient Genome Editing and Transcriptional Repression in Plants.” Nature Plants 3(3): 17103. https://doi.org/10.1038/nplants.2017.103

[29] Hong, Wei, Jie Zhang, Guzhen Cui, Luxin Wang, and Yi Wang. 2018. “Multiplexed CRISPR-Cpf1-mediated Genome Editing in Clostridium difficile Toward the Understanding of Pathogenesis of C. difficile Infection.” ACS Synthetic Biology 7: 1588-1600. https://doi.org/10.1021/acssynbio.8b00087

[30] Hur, Junho K., Kyoungmi Kim, Kyung Wook Been, Gayoung Baek, Sunghyeok Ye, Junseok W. Hur, Seuk-Min Ryu, Youn Su Lee, and Jin-Soo Kim. 2016. “Targeted Mutagenesis in Mice by Electroporation of Cpf1 Ribonucleoproteins.” Nature Biotechnology 34(8): 807-808. https://doi.org/10.1038/nbt.3596

[31] Zhang, Xiaochun, Jingman Wang, Qiuxiang Cheng, Xuan Zheng, Guoping Zhao, and Jin Wang. 2017. “Multiplex Gene Regulation by CRISPR-ddCpf1.” Cell Discov 3: 17018. https://doi.org/10.1038/celldisc.2017.18

[32] Breunig, Christopher T., Tamara Durovic, Andrea M. Neuner, Valentin Baumann, Maximilian F. Wiesbeck, Anna Köferle, Magdalena Götz, Jovica Ninkovic, and Stefan H. Stricker. 2018. “One Step Generation of Customizable gRNA Vectors for Multiplex CRISPR Approaches Through String Assembly gRNA Cloning (STAgR).” PLoS ONE 13: e0196015. https://doi.org/10.1371/journal.pone.0196015

[33] Kim, Won-Nyeong, Hye-Jeong Kim, Young-Soo Chung, and Hyun-Uk Kim. 2021. “Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean.” Plants 10: 2542. https://doi.org/10.3390/plants10112542

[34] Jin, Wen-Bing, Ting-Ting Li, Da Huo, Sophia Qu, Xin V. Li, Mohammad Arifuzzaman, Svetlana F. Lima, et al. 2022. “Genetic Manipulation of Gut Microbes Enables Single-Gene Interrogation in a Complex Microbiome.” Cell 185: 547-562.e22. https://doi.org/10.1016/j.cell.2021.12.035

[35] Yang, Pengjie, Jinzhong Tian, Lu Zhang, Hui Zhang, Gaohua Yang, Yimeng Ren, Jingyuan Fang, Yang Gu, and Weihong Jiang. 2024. “A Toolbox for Genetic Manipulation in Intestinal Clostridium Symbiosum.” Synthetic and Systems Biotechnology 9: 43-54. https://doi.org/10.1016/j.synbio.2023.12.005

RIGHTS & PERMISSIONS

2024 The Authors. iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.