1887

Abstract

The sphingomonads encompass a diverse group of bacteria within the family , with the presence of sphingolipids on their cell surface instead of lipopolysaccharide as their main common feature. They are particularly interesting for bioremediation purposes due to their ability to degrade or metabolise a variety of recalcitrant organic pollutants. However, research and development on their full bioremediation potential has been hampered because of the limited number of tools available to investigate and modify their genome. Here, we present a markerless genome editing method for TFA, which can be further optimised for other sphingomonads. This procedure is based on a double recombination triggered by a DNA double-strand break in the chromosome. The strength of this protocol lies in forcing the second recombination rather than favouring it by pressing a counterselection marker, thus avoiding laborious restreaking or passaging screenings. Additionally, we introduce a modification with respect to the original protocol to increase the efficiency of the screening after the first recombination event. We show this procedure step by step and compare our modified method with respect to the original one by deleting , the master regulator of the general stress response in TFA. This adds to the genetic tool repertoire that can be applied to sphingomonads and stands as an efficient option for fast genome editing of this bacterial group.

Funding
This study was supported by the:
  • Biotechnology and Biological Sciences Research Council (Award BB/V007823/1)
    • Principle Award Recipient: NotApplicable
  • Junta de Andalucía (Award POSTDOC_21_00064)
    • Principle Award Recipient: InmaculadaGarcía-Romero
  • Ministerio de Ciencia, Innovación y Universidades / Agencia Estatal de Investigación y FEDER (UE) (Award MICIU/AEI/10.13039/501100011033)
    • Principle Award Recipient: FranciscaReyes-Ramírez
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/acmi/10.1099/acmi.0.000755.v3
2024-05-13
2024-05-23
Loading full text...

Full text loading...

/deliver/fulltext/acmi/6/5/acmi000755.v3.html?itemId=/content/journal/acmi/10.1099/acmi.0.000755.v3&mimeType=html&fmt=ahah

References

  1. Takeuchi M, Hamana K, Hiraishi A. Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 2001; 51:1405–1417 [View Article] [PubMed]
    [Google Scholar]
  2. Kosako Y, Yabuuchi E, Naka T, Fujiwara N, Kobayashi K. Proposal of Sphingomonadaceae fam. nov., consisting of Sphingomonas Yabuuchi et al. 1990, Erythrobacter Shiba and Shimidu 1982, Erythromicrobium Yurkov et al. 1994, Porphyrobacter Fuerst et al. 1993, Zymomonas Kluyver and van Niel 1936, and Sandaracinobacter Yurkov et al. 1997, with the type genus Sphingomonas Yabuuchi et al. 1990. Microbiol Immunol 2000; 44:563–575 [View Article] [PubMed]
    [Google Scholar]
  3. Glaeser SP, Kämpfer P. The family Sphingomonadaceae. In Rosenberg E. ed The Prokaryotes Berlin, Heidelberg: Springer; 2014 pp 641–707 [View Article]
    [Google Scholar]
  4. Balkwill DL, Fredrickson JK, Romine MF. Sphingomonas and related genera. In Dworkin M. ed The Prokaryotes New York: Springer; 2006 pp 605–629 [View Article]
    [Google Scholar]
  5. Battu L, Reddy MM, Goud BS, Ulaganathan K, Ulaganathan K. Assembly of genomic reads of elite indica rice cultivar onto 2101 reference bacterial genomes for identification of co-sequenced endophytic bacteria. Data Brief 2017; 12:305–312 [View Article] [PubMed]
    [Google Scholar]
  6. Battu L, Reddy MM, Goud BS, Ulaganathan K, Kandasamy U. Genome inside genome: NGS based identification and assembly of endophytic Sphingopyxis granuli and Pseudomonas aeruginosa genomes from rice genomic reads. Genomics 2017; 109:141–146 [View Article] [PubMed]
    [Google Scholar]
  7. Gatheru Waigi M, Sun K, Gao Y. Sphingomonads in microbe-assistedphytoremediation: tackling soil pollution. Trends Biotechnol 2017; 35:883–899 [View Article] [PubMed]
    [Google Scholar]
  8. García-Romero I, Pérez-Pulido AJ, González-Flores YE, Reyes-Ramírez F, Santero E et al. Genomic analysis of the nitrate-respiring Sphingopyxis granuli (formerly Sphingomonas macrogoltabida) strain TFA. BMC Genomics 2016; 17:93 [View Article] [PubMed]
    [Google Scholar]
  9. Floriano B, Santero E, Reyes-Ramírez F. Biodegradation of tetralin: genomics, gene function and regulation. Genes 2019; 10:339 [View Article] [PubMed]
    [Google Scholar]
  10. Romine MF, Stillwell LC, Wong KK, Thurston SJ, Sisk EC et al. Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199. J Bacteriol 1999; 181:1585–1602 [View Article] [PubMed]
    [Google Scholar]
  11. Wittich RM, Wilkes H, Sinnwell V, Francke W, Fortnagel P. Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1. Appl Environ Microbiol 1992; 58:1005–1010 [View Article] [PubMed]
    [Google Scholar]
  12. Copley SD, Rokicki J, Turner P, Daligault H, Nolan M et al. The whole genome sequence of Sphingobium chlorophenolicum L-1: insights into the evolution of the pentachlorophenol degradation pathway. Genome Biol Evol 2012; 4:184–198 [View Article] [PubMed]
    [Google Scholar]
  13. Aulestia M, Flores A, Mangas EL, Pérez-Pulido AJ, Santero E et al. Isolation and genomic characterization of the ibuprofen-degrading bacterium Sphingomonas strain MPO218. Environ Microbiol 2021; 23:267–280 [View Article] [PubMed]
    [Google Scholar]
  14. Ohtsubo Y, Nagata Y, Numata M, Tsuchikane K, Hosoyama A et al. Complete genome sequence of Sphingopyxis macrogoltabida type strain NBRC 15033, originally isolated as a polyethylene glycol degrader. Genome Announc 2015; 3:e01401-15 [View Article] [PubMed]
    [Google Scholar]
  15. Yamatsu A, Matsumi R, Atomi H, Imanaka T. Isolation and characterization of a novel poly(vinyl alcohol)-degrading bacterium, Sphingopyxis sp. PVA3. Appl Microbiol Biotechnol 2006; 72:804–811 [View Article] [PubMed]
    [Google Scholar]
  16. Kawai F. Sphingomonads involved in the biodegradation of xenobiotic polymers. J Ind Microbiol Biotechnol 1999; 23:400–407 [View Article] [PubMed]
    [Google Scholar]
  17. Masai E, Shinohara S, Hara H, Nishikawa S, Katayama Y et al. Genetic and biochemical characterization of a 2-pyrone-4, 6-dicarboxylic acid hydrolase involved in the protocatechuate 4, 5-cleavage pathway of Sphingomonas paucimobilis SYK-6. J Bacteriol 1999; 181:55–62 [View Article] [PubMed]
    [Google Scholar]
  18. García-Romero I, Förstner KU, Santero E, Floriano B. SuhB, a small non-coding RNA involved in catabolite repression of tetralin degradation genes in Sphingopyxis granuli strain TFA. Environ Microbiol 2018; 20:3671–3683 [View Article] [PubMed]
    [Google Scholar]
  19. Zhu L, Wu X, Li O, Qian C, Gao H. Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461. PLoS One 2012; 7:e35099 [View Article] [PubMed]
    [Google Scholar]
  20. Li A, Hu T, Luo H, Alam N-U, Xin J et al. A carotenoid- and poly-β-hydroxybutyrate-free mutant strain of Sphingomonas elodea ATCC 31461 for the commercial production of gellan. mSphere 2019; 4:e00668-19 [View Article] [PubMed]
    [Google Scholar]
  21. Kontur WS, Bingman CA, Olmsted CN, Wassarman DR, Ulbrich A et al. Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the β-aryl ether bond of lignin. J Biol Chem 2018; 293:4955–4968 [View Article] [PubMed]
    [Google Scholar]
  22. Cai C, Xu Z, Li J, Zhou H, Jin M. Developing Rhodococcus opacus and Sphingobium sp. coculture systems for valorization of lignin-derived dimers. Biotechnol Bioeng 2022; 119:3162–3177 [View Article] [PubMed]
    [Google Scholar]
  23. Basta T, Keck A, Klein J, Stolz A. Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains. J Bacteriol 2004; 186:3862–3872 [View Article] [PubMed]
    [Google Scholar]
  24. Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Markerless gene deletion system for sphingomonads. Appl Environ Microbiol 2012; 78:3774–3777 [View Article] [PubMed]
    [Google Scholar]
  25. Song D, Chen X, Yao H, Kong G, Xu M et al. The variations of native plasmids greatly affect the cell surface hydrophobicity of sphingomonads. mSystems 2023; 8:e0086223 [View Article] [PubMed]
    [Google Scholar]
  26. Lu H, Huang Y. Transcriptome analysis of Novosphingobium pentaromativorans US6-1 reveals the Rsh regulon and potential molecular mechanisms of N-acyl-l-homoserine lactone accumulation. Int J Mol Sci 2018; 19:2631 [View Article] [PubMed]
    [Google Scholar]
  27. Cecil JH, Garcia DC, Giannone RJ, Michener JK. Rapid, parallel identification of catabolism pathways of lignin-derived aromatic compounds in Novosphingobium aromaticivorans. Appl Environ Microbiol 2018; 84:22 [View Article] [PubMed]
    [Google Scholar]
  28. Gottschlich L, Geiser P, Bortfeld-Miller M, Field CM, Vorholt JA. Complex general stress response regulation in Sphingomonas melonis Fr1 revealed by transcriptional analyses. Sci Rep 2019; 9:9404 [View Article] [PubMed]
    [Google Scholar]
  29. Fujita M, Sakumoto T, Tanatani K, Yu H, Mori K et al. Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds. Sci Rep 2020; 10:12177 [View Article] [PubMed]
    [Google Scholar]
  30. Yu Z, Hu Z, Xu Q, Zhang M, Yuan N et al. The LuxI/LuxR-type quorum sensing system regulates degradation of polycyclic aromatic hydrocarbons via two mechanisms. Int J Mol Sci 2020; 21:5548 [View Article] [PubMed]
    [Google Scholar]
  31. Higuchi Y, Sato D, Kamimura N, Masai E. Roles of two glutathione S-transferases in the final step of the β-aryl ether cleavage pathway in Sphingobium sp. strain SYK-6. Sci Rep 2020; 10:20614 [View Article] [PubMed]
    [Google Scholar]
  32. Liang J, Xu J, Zhao W, Wang J, Chen K et al. Benzo[a]pyrene might be transported by a TonB-dependent transporter in Novosphingobium pentaromativorans US6-1. J Hazard Mater 2021; 404:124037 [View Article]
    [Google Scholar]
  33. Presley GN, Werner AZ, Katahira R, Garcia DC, Haugen SJ et al. Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound. Metab Eng 2021; 65:1–10 [View Article] [PubMed]
    [Google Scholar]
  34. Huang J, Chen D, Kong X, Wu S, Chen K et al. Coinducible catabolism of 1-Naphthol via synergistic regulation of the initial hydroxylase genes in Sphingobium sp. strain B2. Appl Environ Microbiol 2021; 87:11 [View Article] [PubMed]
    [Google Scholar]
  35. Gao C, Zeng Y-H, Li C-Y, Li L, Cai Z-H et al. Bisphenol A biodegradation by Sphingonomas sp. YK5 is regulated by acyl-homoserine lactone signaling molecules. Sci Total Environ 2022; 802:149898 [View Article] [PubMed]
    [Google Scholar]
  36. Fujita M, Yano S, Shibata K, Kondo M, Hishiyama S et al. Functional roles of multiple Ton complex genes in a Sphingobium degrader of lignin-derived aromatic compounds. Sci Rep 2021; 11:22444 [View Article] [PubMed]
    [Google Scholar]
  37. Linz AM, Ma Y, Perez JM, Myers KS, Kontur WS et al. Aromatic dimer dehydrogenases from Novosphingobium aromaticivorans reduce monoaromatic diketones. Appl Environ Microbiol 2021; 87:e0174221 [View Article] [PubMed]
    [Google Scholar]
  38. Higuchi Y, Kamimura N, Takenami H, Kikuiri Y, Yasuta C et al. The catabolic system of acetovanillone and acetosyringone in Sphingobium sp. strain SYK-6 useful for upgrading aromatic compounds obtained through chemical lignin depolymerization. Appl Environ Microbiol 2022; 88:e0072422 [View Article] [PubMed]
    [Google Scholar]
  39. Wang Z, Zeng Y, Cheng K, Cai Z, Zhou J. The quorum sensing system of Novosphingobium sp. ERN07 regulates aggregate formation that promotes cyanobacterial growth. Sci Total Environ 2022; 851:158354 [View Article]
    [Google Scholar]
  40. Jiang W, Zhang M, Gao S, Zhu Q, Qiu J et al. Comparative genomic analysis of carbofuran-degrading sphingomonads reveals the carbofuran catabolism mechanism in Sphingobium sp. strain CFD-1. Appl Environ Microbiol 2022; 88:e0102422 [View Article] [PubMed]
    [Google Scholar]
  41. Martínez-García E, de Lorenzo V. Engineering multiple genomic deletions in gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol 2011; 13:2702–2716 [View Article] [PubMed]
    [Google Scholar]
  42. Wirth NT, Kozaeva E, Nikel PI. Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection. Microb Biotechnol 2020; 13:233–249 [View Article] [PubMed]
    [Google Scholar]
  43. Leal-Morales A, Pulido-Sánchez M, López-Sánchez A, Govantes F. Transcriptional organization and regulation of the Pseudomonas putida flagellar system. Environ Microbiol 2022; 24:137–157 [View Article] [PubMed]
    [Google Scholar]
  44. de Dios R, Santero E, Reyes-Ramírez F. The functional differences between paralogous regulators define the control of the general stress response in Sphingopyxis granuli TFA. Environ Microbiol 2022; 24:1918–1931 [View Article] [PubMed]
    [Google Scholar]
  45. Kohlstedt M, Weimer A, Weiland F, Stolzenberger J, Selzer M et al. Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties. Metab Eng 2022; 72:337–352 [View Article] [PubMed]
    [Google Scholar]
  46. Owen SV, Wenner N, Dulberger CL, Rodwell EV, Bowers-Barnard A et al. Prophages encode phage-defense systems with cognate self-immunity. Cell Host Microbe 2021; 29:1620–1633 [View Article] [PubMed]
    [Google Scholar]
  47. Nies SC, Alter TB, Nölting S, Thiery S, Phan ANT et al. High titer methyl ketone production with tailored Pseudomonas taiwanensis VLB120. Metab Eng 2020; 62:84–94 [View Article] [PubMed]
    [Google Scholar]
  48. Hobmeier K, Goëss MC, Sehr C, Schwaminger S, Berensmeier S et al. Anaplerotic pathways in Halomonas elongata: the role of the sodium gradient. Front Microbiol 2020; 11:561800 [View Article] [PubMed]
    [Google Scholar]
  49. de Dios R, Gadar K, McCarthy RR. A high-efficiency scar-free genome-editing toolkit for Acinetobacter baumannii. J Antimicrob Chemother 2022; 77:3390–3398 [View Article] [PubMed]
    [Google Scholar]
  50. de Dios R, Rivas-Marin E, Santero E, Reyes-Ramírez F. Two paralogous EcfG σ factors hierarchically orchestrate the activation of the general stress response in Sphingopyxis granuli TFA. Sci Rep 2020; 10:5177 [View Article] [PubMed]
    [Google Scholar]
  51. González-Flores YE, de Dios R, Reyes-Ramírez F, Santero E. The response of Sphingopyxis granuli strain TFA to the hostile anoxic condition. Sci Rep 2019; 9:6297 [View Article] [PubMed]
    [Google Scholar]
  52. Kast P. pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones. Gene 1994; 138:109–114 [View Article] [PubMed]
    [Google Scholar]
  53. Trebosc V, Gartenmann S, Royet K, Manfredi P, Tötzl M et al. A novel genome-editing platform for drug-resistant Acinetobacter baumannii reveals an AdeR-unrelated tigecycline resistance mechanism. Antimicrob Agents Chemother 2016; 60:7263–7271 [View Article] [PubMed]
    [Google Scholar]
  54. Martínez-García E, Fraile S, Algar E, Aparicio T, Velázquez E et al. SEVA 4.0: an update of the standard european vector architecture database for advanced analysis and programming of bacterial phenotypes. Nucleic Acids Res 2023; 51:D1558–D1567 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.0.000755.v3
Loading
/content/journal/acmi/10.1099/acmi.0.000755.v3
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error