Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

LoaP is a broadly conserved antiterminator protein that regulates antibiotic gene clusters in Bacillus amyloliquefaciens

Nature Microbiology volume 2, Article number: 17003 (2017) Cite this article

Subjects

Abstract

A valuable resource available in the search for new natural products is the diverse microbial life that spans the planet. A large subset of these microorganisms synthesize complex specialized metabolites exhibiting biomedically important activities. A limiting step to the characterization of these compounds is an elucidation of the genetic regulatory mechanisms that oversee their production. Although proteins that control transcription initiation of specialized metabolite gene clusters have been identified, those affecting transcription elongation have not been broadly investigated. In this study, we analysed the phylogenetic distribution of the large, widespread NusG family of transcription elongation proteins and found that it includes a cohesive outgroup of paralogues (herein coined LoaP), which are often positioned adjacent or within gene clusters for specialized metabolites. We established Bacillus amyloliquefaciens LoaP as a paradigm for this protein subgroup and showed that it regulated the transcriptional readthrough of termination sites located within two different antibiotic biosynthesis operons. Both of these antibiotics have been implicated in plant-protective activities, demonstrating that LoaP controls an important regulon of specialized metabolite genes for this microorganism. These data therefore reveal transcription elongation as a point of regulatory control for specialized metabolite pathways and introduce a subgroup of NusG proteins for this purpose.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: LoaP is required for expression of the B. amyloliquefaciens difficidin gene cluster.
Figure 2: LoaP promotes readthrough of intrinsic terminator sites.
Figure 3: LoaP mediates transcription antitermination in reporter constructs.
Figure 4: LoaP expression affects transcription of both difficidin and macrolactin operons.
Figure 5: LoaP-dependent production of difficidin and macrolactin.
Figure 6: Large-scale phylogenetic analysis of NusG family proteins reveals several subclasses of specialized paralogues.

Similar content being viewed by others

References

  1. Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Sidebottom, A. M. & Carlson, E. E. A reinvigorated era of bacterial secondary metabolite discovery. Curr. Opin. Chem. Biol. 24, 104–111 (2015).

    Article  CAS  PubMed  Google Scholar 

  3. Walsh, C. Antibiotics: Actions, Origins, Resistance (ASM, 2013).

    Google Scholar 

  4. Waters, L. S. & Storz, G. Regulatory RNAs in bacteria. Cell 136, 615–628 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Weisberg, R. A. & Gottesman, M. E. Processive antitermination. J. Bacteriol. 181, 359–367 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Roberts, J. W., Shankar, S. & Filter, J. J. RNA polymerase elongation factors. Annu. Rev. Microbiol. 62, 211–233 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Santangelo, T. J. & Artsimovitch, I. Termination and antitermination: RNA polymerase runs a stop sign. Nat. Rev. Microbiol. 9, 319–329 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ray-Soni, A., Bellecourt, M. J. & Landick, R. Mechanisms of bacterial transcription termination: all good things must end. Annu. Rev. Biochem. 85, 319–347 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Gusarov, I. & Nudler, E. The mechanism of intrinsic transcription termination. Mol. Cell 3, 495–504 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Greenblatt, J., McLimont, M. & Hanly, S. Termination of transcription by nusA gene protein of Escherichia coli. Nature 292, 215–220 (1981).

    Article  CAS  PubMed  Google Scholar 

  11. Mondal, S., Yakhnin, A. V., Sebastian, A., Albert, I. & Babitzke, P. NusA-dependent transcription termination prevents misregulation of global gene expression. Nat. Microbiol. 1, 15007 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Breaker, R. R. Prospects for riboswitch discovery and analysis. Mol. Cell 43, 867–879 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Proshkin, S., Mironov, A. & Nudler, E. Riboswitches in regulation of Rho-dependent transcription termination. Biochim. Biophys. Acta 1839, 974–977 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Chowdhury, S. P., Hartmann, A., Gao, X. & Borriss, R . Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42—a review. Front. Microbiol. 6, 780 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Chen, X.-H. et al. Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42. J. Bacteriol. 188, 4024–4036 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Fan, B . et al. dRNA-Seq reveals genomewide TSSs and noncoding RNAs of plant beneficial rhizobacterium Bacillus amyloliquefaciens FZB42. PLoS ONE 10, e0142002 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Irnov, I., Sharma, C. M., Vogel, J. & Winkler, W. C. Identification of regulatory RNAs in Bacillus subtilis. Nucleic Acids Res. 38, 6637–6651 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nakamura, K. et al. Sequence-specific error profile of Illumina sequencers. Nucleic Acids Res. 39, e90 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Irnov, I. & Winkler, W. C. A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales. Mol. Microbiol. 76, 559–575 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Chen, X. H. et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J. Biotechnol. 140, 27–37 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Finn, R. D., Clements, J. & Eddy, S. R. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 39, W29–W37 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tomar, S. K. & Artsimovitch, I. NusG-Spt5 proteins—universal tools for transcription modification and communication. Chem. Rev. 113, 8604–8619 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chatzidaki-Livanis, M., Weinacht, K. G. & Comstock, L. E. Trans locus inhibitors limit concomitant polysaccharide synthesis in the human gut symbiont Bacteroides fragilis. Proc. Natl Acad. Sci. USA 107, 11976–11980 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. King, R. A., Banik-Maiti, S., Jin, D. J. & Weisberg, R. A. Transcripts that increase the processivity and elongation rate of RNA polymerase. Cell 87, 893–903 (1996).

    Article  CAS  PubMed  Google Scholar 

  25. Artsimovitch, I. & Landick, R. The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand. Cell 109, 193–203 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. NandyMazumdar, M. & Artsimovitch, I. Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins—shifting shapes and paradigms. BioEssays 37, 324–334 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Paitan, Y., Orr, E., Ron, E. Z. & Rosenberg, E. A NusG-like transcription anti-terminator is involved in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus. FEMS Microbiol. Lett. 170, 221–227 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Behnken, S., Lincke, T., Kloss, F., Ishida, K. & Hertweck, C. Antiterminator-mediated unveiling of cryptic polythioamides in an anaerobic bacterium. Angew. Chem. Int. Ed. 51, 2425–2428 (2012).

    Article  CAS  Google Scholar 

  29. Yakhnin, A. V. & Babitzke, P. NusG/Spt5: are there common functions of this ubiquitous transcription elongation factor? Curr. Opin. Microbiol. 18, 68–71 (2014).

    Article  CAS  PubMed  Google Scholar 

  30. Yakhnin, A. V., Murakami, K. S. & Babitzke, P . NusG is a sequence-specific RNA polymerase pause factor that binds to the non-template DNA within the paused transcription bubble. J. Biol. Chem. 291, 5299–5308 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Crickard, J. B., Fu, J. & Reese, J. C. Biochemical analysis of yeast suppressor of Ty 4/5 (Spt4/5) reveals the importance of nucleic acid interactions in the prevention of RNA polymerase II arrest. J. Biol. Chem. 291, 9853–9870 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Thapar, R., Denmon, A. P. & Nikonowicz, E. P. Recognition modes of RNA tetraloops and tetraloop-like motifs by RNA-binding proteins. Wiley Interdiscip. Rev. RNA 5, 49–67 (2014).

    Article  CAS  PubMed  Google Scholar 

  33. Huang, H.-C., Nagaswamy, U. & Fox, G. E. The application of cluster analysis in the intercomparison of loop structures in RNA. RNA 11, 412–423 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Fiore, J. L. & Nesbitt, D. J. An RNA folding motif: GNRA tetraloop–receptor interactions. Q. Rev. Biophys. 46, 223–264 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. Cilley, C. D. & Williamson, J. R. Structural mimicry in the phage ϕ21 N peptide–boxB RNA complex. RNA 9, 663–676 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Behnken, S. & Hertweck, C. Cryptic polyketide synthase genes in non-pathogenic Clostridium spp. PLoS ONE 7, e29609 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  PubMed  Google Scholar 

  38. Jarmer, H., Berka, R., Knudsen, S. & Saxild, H. H. Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions. FEMS Microbiol. Lett. 206, 197–200 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Bhavsar, A. P., Zhao, X. & Brown, E. D. Development and characterization of a xylose-dependent system for expression of cloned genes in Bacillus subtilis: conditional complementation of a teichoic acid mutant. Appl. Environ. Microbiol. 67, 403–410 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ramakers, C., Ruijter, J. M., Deprez, R. H. L. & Moorman, A. F. M. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62–66 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Matz, M. V., Wright, R. M. & Scott, J. G. No control genes required: Bayesian analysis of qRT–PCR data. PLoS ONE 8, e71448 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Aronesty, E. Comparison of sequencing utility programs. Open Bioinform. J. 7, 1–8 (2013).

    Article  Google Scholar 

  43. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinforma. Oxf. Engl. 25, 1754–1760 (2009).

    Article  CAS  Google Scholar 

  44. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Armougom, F. et al. Expresso: automatic incorporation of structural information in multiple sequence alignments using 3D-Coffee. Nucleic Acids Res. 34, W604–W608 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–2690 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Price, M. N., Dehal, P. S. & Arkin, A. P. Fasttree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinform. Oxf. Engl. 26, 2460–2461 (2010).

    Article  CAS  Google Scholar 

  51. Weber, T. et al. antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 43, W237–W243 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Landy, M. & Warren, G. H. Bacillomycin; an antibiotic from Bacillus subtilis active against pathogenic fungi. Proc. Soc. Exp. Biol. Med. 67, 539–541 (1948).

    Article  CAS  PubMed  Google Scholar 

  53. Chen, X. H. et al. Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens are efficient in controlling fire blight disease. J. Biotechnol. 140, 38–44 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Research on this project was supported by the National Science Foundation (MCB1051440 to W.C.W. and NSF-CAREER MCB1253215 to P.S.).

Author information

Authors and Affiliations

  1. Department of Cell Biology and Molecular Genetics, The University of Maryland, 3112 Biosciences Research Building, College Park, 20742, Maryland, USA

    Jonathan R. Goodson, Steven Klupt & Wade C. Winkler

  2. Department of Biochemistry and Biophysics, Texas A&M University, TAMU 2128 – Rm 435, College Station, 77843, Texas, USA

    Chengxi Zhang & Paul Straight

Authors
  1. Jonathan R. Goodson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Steven Klupt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chengxi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paul Straight
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wade C. Winkler
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The experimental plan was devised by J.R.G., P.S. and W.C.W. All authors assisted in the collection and interpretation of data. J.R.G., P.S. and W.C.W. wrote the manuscript.

Corresponding authors

Correspondence to Paul Straight or Wade C. Winkler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–7. (PDF 5249 kb)

Supplementary Tables 1–5

Table 1: List of significantly differentially expressed genes in loaP deletion strain compared to wild type. Table 2: List of significantly differentially expressed genes in loaP complementation strain compared to wild type. Table 3: Quantification of HPLC analysis of polyketide antibiotics bacillaene, macrolactin and difficidin. Table 4: Information about all B. amyloliquefaciens and plasmids used in this study. Table 5: Complete list of oligonucleotide sequences used for each amplicon in qRT-PCR experiments. (XLSX 59 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Goodson, J., Klupt, S., Zhang, C. et al. LoaP is a broadly conserved antiterminator protein that regulates antibiotic gene clusters in Bacillus amyloliquefaciens. Nat Microbiol 2, 17003 (2017). https://doi.org/10.1038/nmicrobiol.2017.3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2017.3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing