1887

Abstract

Several genome engineering methods have been developed for . However, they suffer from limitations such as extensive cloning, multiple steps, successful expression of heterologous genes via plasmid etc. Here, we report a rapid method for performing genomic deletions/disruptions in spp. using heterologous linear DNA. The method is cost effective and less labour intensive. The applicability of the method was demonstrated by successful disruption of and orphan . None of the disrupted genes were found to be essential for the viability of the cell. Disruption of orphan and resulted in elongated cells and short rods, respectively. This is the first report demonstrating disruption of and orphan genes by electroporation of heterologous linear DNA in spp.

Funding
This study was supported by the:
  • Science and Engineering Research Board
    • Principle Award Recipient: SrivastavaPreeti
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001028
2021-02-02
2021-10-19
Loading full text...

Full text loading...

References

  1. de Carvalho CCCR, da Fonseca MMR. The remarkable Rhodococcus erythropolis . Appl Microbiol Biotechnol 2005; 67:715–726 [View Article][PubMed]
    [Google Scholar]
  2. Ichiyama S, Shimokata K, Tsukamura M. Carotenoid pigments of genus Rhodococcus . Microbiol Immunol 1989; 33:503–508 [View Article][PubMed]
    [Google Scholar]
  3. Finnerty WR. The biology and genetics of the genus Rhodococcus . Annu Rev Microbiol 1992; 46:193–218 [View Article][PubMed]
    [Google Scholar]
  4. Bicca FC, Fleck LC, Ayub MAZ. Production of biosurfactant by hydrocarbon degrading Rhodococcus ruber and Rhodococcus erythropolis . Rev Microbiol 1999; 30:231–236 [View Article]
    [Google Scholar]
  5. Shavandi M, Mohebali G, Haddadi A, Shakarami H, Nuhi A. Emulsification potential of a newly isolated biosurfactant-producing bacterium, Rhodococcus sp. strain TA6. Colloids Surf B Biointerfaces 2011; 82:477–482 [View Article][PubMed]
    [Google Scholar]
  6. Linder R. Rhodococcus equi and Arcanobacterium haemolyticum: two "coryneform" bacteria increasingly recognized as agents of human infection. Emerg Infect Dis 1997; 3:145153 [View Article][PubMed]
    [Google Scholar]
  7. Weinstock DM, Brown AE. Rhodococcus equi: an emerging pathogen. Clin Infect Dis 2002; 34:1379–1385 [View Article][PubMed]
    [Google Scholar]
  8. Rückert C, Birmes FS, Müller C, Niewerth H, Winkler A et al. Complete genome sequence of Rhodococcus erythropolis BG43 (DSM 46869), a degrader of Pseudomonas aeruginosa quorum sensing signal molecules. J Biotechnol 2015; 211:99–100 [View Article][PubMed]
    [Google Scholar]
  9. McLeod MP, Warren RL, Hsiao WWL, Araki N, Myhre M et al. The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proc Natl Acad Sci U S A 2006; 103:15582–15587 [View Article][PubMed]
    [Google Scholar]
  10. Larkin MJ, Kulakov LA, Allen CC. Genomes and plasmids in Rhodococcus . Biology of Rhodococcus Springer; 2010 pp 73–90
    [Google Scholar]
  11. Singhi D, Goyal A, Gupta G, Yadav A, Srivastava P. Rhodoccoccus erythropolis is different from other members of Actinobacteriaria: monoploidy, overlapping replication cycle, and unique segregation pattern. J Bacteriol 2019; 201: 15 12 2019 [View Article][PubMed]
    [Google Scholar]
  12. Sharan SK, Thomason LC, Kuznetsov SG, Court DL. Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 2009; 4:206–223 [View Article][PubMed]
    [Google Scholar]
  13. Datta S, Costantino N, Court DL. A set of recombineering plasmids for gram-negative bacteria. Gene 2006; 379:109–115 [View Article][PubMed]
    [Google Scholar]
  14. Yang XW, Model P, Heintz N. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 1997; 15:859–865 [View Article][PubMed]
    [Google Scholar]
  15. Wang Y, Zhang Z-T, Seo S-O, Lynn P, Lu T et al. Bacterial genome editing with CRISPR-Cas9: deletion, integration, single nucleotide modification, and desirable "Clean" mutant selection in Clostridium beijerinckii as an example. ACS Synth Biol 2016; 5:721–732 [View Article][PubMed]
    [Google Scholar]
  16. Aubert DF, Hamad MA, Valvano MA. A Markerless Deletion Method for Genetic Manipulation of Burkholderia cenocepacia and Other Multidrug-Resistant Gram-Negative Bacteria Host-Bacteria Interactions: Springer; 2014 pp 311–327
    [Google Scholar]
  17. van der Geize R, Hessels GI, van Gerwen R, van der Meijden P, Dijkhuizen L. Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Δ1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker. FEMS Microbiol Lett 2001; 205:197–202 [View Article][PubMed]
    [Google Scholar]
  18. DeLorenzo DM, Rottinghaus AG, Henson WR, Moon TS. Molecular toolkit for gene expression control and genome modification in Rhodococcus opacus PD630. ACS Synth Biol 2018; 7:727–738 [View Article][PubMed]
    [Google Scholar]
  19. Liang Y, Jiao S, Wang M, Yu H, Shen Z. A CRISPR/Cas9-based genome editing system for Rhodococcus ruber th. Metab Eng 2020; 57:13–22 [View Article][PubMed]
    [Google Scholar]
  20. Roberts MAJ, Wadhams GH, Hadfield KA, Tickner S, Armitage JP. ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes. Proc Natl Acad Sci U S A 2012; 109:6698–6703 [View Article][PubMed]
    [Google Scholar]
  21. Ikeda M, Sato T, Wachi M, Jung HK, Ishino F et al. Structural similarity among Escherichia coli FtsW and RodA proteins and Bacillus subtilis SpoVE protein, which function in cell division, cell elongation, and spore formation, respectively. J Bacteriol 1989; 171:6375–6378 [View Article][PubMed]
    [Google Scholar]
  22. Sieger B, Schubert K, Donovan C, Bramkamp M. The lipid II flippase RodA determines morphology and growth in Corynebacterium glutamicum . Mol Microbiol 2013; 90:966–982 [View Article][PubMed]
    [Google Scholar]
  23. Emami K, Guyet A, Kawai Y, Devi J, Wu LJ et al. RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway. Nature Microbiology 2017; 2:1–9 [View Article]
    [Google Scholar]
  24. Fekete RA, Chattoraj DK. A cis-acting sequence involved in chromosome segregation in Escherichia coli . Mol Microbiol 2005; 55:175–183 [View Article][PubMed]
    [Google Scholar]
  25. Srivastava P, Fekete RA, Chattoraj DK. Segregation of the replication terminus of the two Vibrio cholerae chromosomes. J Bacteriol 2006; 188:1060–1070 [View Article][PubMed]
    [Google Scholar]
  26. Murarka P, Bagga T, Singh P, Rangra S, Srivastava P. Isolation and identification of a TetR family protein that regulates the biodesulfurization operon. AMB Express 2019; 9:71 [View Article][PubMed]
    [Google Scholar]
  27. Singhi D, Jain A, Srivastava P. Localization of low copy number plasmid pRC4 in replicating rod and non-replicating cocci cells of Rhodococcus erythropolis PR4. PLoS One 2016; 11:e0166491 [View Article][PubMed]
    [Google Scholar]
  28. Yan X, Yu H-J, Hong Q, Li S-P. Cre/Lox system and PCR-based genome engineering in Bacillus subtilis . Appl Environ Microbiol 2008; 74:5556–5562 [View Article][PubMed]
    [Google Scholar]
  29. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 2000; 97:6640–6645 [View Article][PubMed]
    [Google Scholar]
  30. Ringgaard S, Schirner K, Davis BM, Waldor MK. A family of ParA-like ATPases promotes cell pole maturation by facilitating polar localization of chemotaxis proteins. Genes Dev 2011; 25:1544–1555 [View Article][PubMed]
    [Google Scholar]
  31. Lutkenhaus J. The ParA/MinD family puts things in their place. Trends Microbiol 2012; 20:411–418 [View Article][PubMed]
    [Google Scholar]
  32. Thompson SR, Wadhams GH, Armitage JP. The positioning of cytoplasmic protein clusters in bacteria. Proc Natl Acad Sci U S A 2006; 103:8209–8214 [View Article]
    [Google Scholar]
  33. Donovan C, Schwaiger A, Krämer R, Bramkamp M. Subcellular localization and characterization of the ParAB system from Corynebacterium glutamicum . J Bacteriol 2010; 192:3441–3451 [View Article][PubMed]
    [Google Scholar]
  34. Begg KJ, Spratt BG, Donachie WD. Interaction between membrane proteins PBP3 and rodA is required for normal cell shape and division in Escherichia coli . J Bacteriol 1986; 167:1004–1008 [View Article][PubMed]
    [Google Scholar]
  35. Henriques AO, Glaser P, Piggot PJ, Moran CP. Control of cell shape and elongation by the rodA gene in Bacillus subtilis . Mol Microbiol 1998; 28:235–247 [View Article][PubMed]
    [Google Scholar]
  36. Thibessard A, Fernandez A, Gintz B, Leblond-Bourget N, Decaris B. Effects of rodA and pbp2b disruption on cell morphology and oxidative stress response of Streptococcus thermophilus CNRZ368. J Bacteriol 2002; 184:2821–2826 [View Article][PubMed]
    [Google Scholar]
  37. Bendezú FO, de Boer PAJ, lethality C. Conditional lethality, division defects, membrane involution, and endocytosis in MRE and mrd shape mutants of Escherichia coli . J Bacteriol 2008; 190:1792–1811 [View Article][PubMed]
    [Google Scholar]
  38. Vinella D, Joseleau-Petit D, Thévenet D, Bouloc P, D'Ari R. Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression. J Bacteriol 1993; 175:6704–6710 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001028
Loading
/content/journal/micro/10.1099/mic.0.001028
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited this month Most Cited RSS feed

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