An Adapted Method for Cas9-mediated Editing Reveals the Species-specific Role of β-glucoside Utilization Driving Competition Between Species

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 2024 Feb 14

PMID 38353529

Authors

Eva D H Almasi

Nele Knischewski

Lisa Osbelt

Uthayakumar Muthukumarasamy

Youssef El Mouali

Elena Vialetto

Chase L Beisel

Till Strowig

Affiliations

Soon will be listed here.

Abstract

Importance: Cas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in and demonstrate after optimization its utility for gene editing in three members of the species complex (KoSC) namely , , and . We then adapted a recently developed protocol for functional complementation based on universal "bookmark" targets applicable to all tested species. In summary, species-specific adaptation of state-of-the-art genetic tools allows efficient gene deletion and complementation in type strains as well as natural isolates of KoSC members to study microbial interactions.

Citing Articles

Klebsiella oxytoca facilitates microbiome recovery via antibiotic degradation and restores colonization resistance in a diet-dependent manner.

Almasi E, Eisenhard L, Osbelt L, Lesker T, Vetter A, Knischewski N Nat Commun. 2025; 16(1):551.

PMID: 39789003 PMC: 11717976. DOI: 10.1038/s41467-024-55800-y.

Development of modular expression across phylogenetically distinct diazotrophs.

Kulakowski S, Rivier A, Kuo R, Mengel S, Eng T J Ind Microbiol Biotechnol. 2024; 51.

PMID: 39257030 PMC: 11537724. DOI: 10.1093/jimb/kuae033.

Native CRISPR-Cas-based programmable multiplex gene repression in Klebsiella variicola.

Mo Z, Lin S, Li T, Yu G, Sun Y, Zhou J Biotechnol Lett. 2024; 46(6):973-982.

PMID: 39066958 DOI: 10.1007/s10529-024-03516-w.

Klebsiella oxytoca inhibits Salmonella infection through multiple microbiota-context-dependent mechanisms.

Osbelt L, Almasi E, Wende M, Kienesberger S, Voltz A, Lesker T Nat Microbiol. 2024; 9(7):1792-1811.

PMID: 38862602 PMC: 11222139. DOI: 10.1038/s41564-024-01710-0.

References

Lai X, Davis F, Hespell R, Ingram L . Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides. Appl Environ Microbiol. 1997; 63(2):355-63. PMC: 168328. DOI: 10.1128/aem.63.2.355-363.1997. View

Rodriguez-Valverde D, Leon-Montes N, Soria-Bustos J, Martinez-Cruz J, Gonzalez-Ugalde R, Rivera-Gutierrez S . cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Tilivalline Cytotoxin. Front Microbiol. 2021; 12:743594. PMC: 8515920. DOI: 10.3389/fmicb.2021.743594. View

Waller M, Bober J, Nair N, Beisel C . Toward a genetic tool development pipeline for host-associated bacteria. Curr Opin Microbiol. 2017; 38:156-164. PMC: 5705416. DOI: 10.1016/j.mib.2017.05.006. View

Sassone-Corsi M, Chairatana P, Zheng T, Perez-Lopez A, Edwards R, George M . Siderophore-based immunization strategy to inhibit growth of enteric pathogens. Proc Natl Acad Sci U S A. 2016; 113(47):13462-13467. PMC: 5127304. DOI: 10.1073/pnas.1606290113. View

Cui L, Bikard D . Consequences of Cas9 cleavage in the chromosome of Escherichia coli. Nucleic Acids Res. 2016; 44(9):4243-51. PMC: 4872102. DOI: 10.1093/nar/gkw223. View

Sorbara M, Dubin K, Littmann E, Moody T, Fontana E, Seok R . Inhibiting antibiotic-resistant Enterobacteriaceae by microbiota-mediated intracellular acidification. J Exp Med. 2018; 216(1):84-98. PMC: 6314524. DOI: 10.1084/jem.20181639. View

Uzairue L, Rabaan A, Adewumi F, Okolie O, Folorunso J, Bakhrebah M . Global Prevalence of Colistin Resistance in from Bloodstream Infection: A Systematic Review and Meta-Analysis. Pathogens. 2022; 11(10). PMC: 9607870. DOI: 10.3390/pathogens11101092. View

McConville T, Giddins M, Uhlemann A . An efficient and versatile CRISPR-Cas9 system for genetic manipulation of multi-drug resistant . STAR Protoc. 2021; 2(1):100373. PMC: 7941085. DOI: 10.1016/j.xpro.2021.100373. View

Sassone-Corsi M, Nuccio S, Liu H, Hernandez D, Vu C, Takahashi A . Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature. 2016; 540(7632):280-283. PMC: 5145735. DOI: 10.1038/nature20557. View

10.

Rousset F, Cabezas-Caballero J, Piastra-Facon F, Fernandez-Rodriguez J, Clermont O, Denamur E . The impact of genetic diversity on gene essentiality within the Escherichia coli species. Nat Microbiol. 2021; 6(3):301-312. DOI: 10.1038/s41564-020-00839-y. View

11.

Asri N, Ahmad S, Mohamud R, Mohd Hanafi N, Mohd Zaidi N, Irekeola A . Global Prevalence of Nosocomial Multidrug-Resistant : A Systematic Review and Meta-Analysis. Antibiotics (Basel). 2021; 10(12). PMC: 8698758. DOI: 10.3390/antibiotics10121508. View

12.

Jiang W, Bikard D, Cox D, Zhang F, Marraffini L . RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31(3):233-9. PMC: 3748948. DOI: 10.1038/nbt.2508. View

13.

Pyne M, Moo-Young M, Chung D, Chou C . Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli. Appl Environ Microbiol. 2015; 81(15):5103-14. PMC: 4495200. DOI: 10.1128/AEM.01248-15. View

14.

Luo M, Leenay R, Beisel C . Current and future prospects for CRISPR-based tools in bacteria. Biotechnol Bioeng. 2015; 113(5):930-43. PMC: 4816669. DOI: 10.1002/bit.25851. View

15.

Oliveira R, Ng K, Correia M, Cabral V, Shi H, Sonnenburg J . Klebsiella michiganensis transmission enhances resistance to Enterobacteriaceae gut invasion by nutrition competition. Nat Microbiol. 2020; 5(4):630-641. DOI: 10.1038/s41564-019-0658-4. View

16.

Park S, Ko K . Improvement of transformation efficiency in hypermucoviscous Klebsiella pneumoniae using citric acid. J Microbiol Methods. 2023; 205:106673. DOI: 10.1016/j.mimet.2023.106673. View

17.

Zhao D, Feng X, Zhu X, Wu T, Zhang X, Bi C . CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. Sci Rep. 2017; 7(1):16624. PMC: 5709385. DOI: 10.1038/s41598-017-16998-8. View

18.

Unterhauser K, Poltl L, Schneditz G, Kienesberger S, Glabonjat R, Kitsera M . enterotoxins tilimycin and tilivalline have distinct host DNA-damaging and microtubule-stabilizing activities. Proc Natl Acad Sci U S A. 2019; 116(9):3774-3783. PMC: 6397511. DOI: 10.1073/pnas.1819154116. View

19.

Mimee M, Tucker A, Voigt C, Lu T . Programming a Human Commensal Bacterium, , to Sense and Respond to Stimuli in the Murine Gut Microbiota. Cell Syst. 2016; 1(1):62-71. PMC: 4762051. DOI: 10.1016/j.cels.2015.06.001. View

20.

Kienesberger S, Cosic A, Kitsera M, Raffl S, Hiesinger M, Leitner E . Enterotoxin tilimycin from gut-resident Klebsiella promotes mutational evolution and antibiotic resistance in mice. Nat Microbiol. 2022; 7(11):1834-1848. PMC: 9613472. DOI: 10.1038/s41564-022-01260-3. View