1887

Abstract

The genome of strain 168 encodes ten rRNA () operons. We previously reported that strains with only a single operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single operon ( or ). The suppressor mutants for strain RIK656 with a single operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the operon. Suppressor mutants were also isolated from the six other strains with single operons ( or ). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that operon copy number has a major role in cellular processes such as cell growth and sporulation.

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2016-01-01
2021-07-25
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References

  1. Anderson R. P., Roth J. R. 1977; Tandem genetic duplications in phage and bacteria. Annu Rev Microbiol 31:473–505 [View Article][PubMed]
    [Google Scholar]
  2. Anderson R. P., Roth J. R. 1978; Tandem genetic duplications in Salmonella typhimurium: amplification of the histidine operon. J Mol Biol 126:53–71 [View Article][PubMed]
    [Google Scholar]
  3. Anderson R. P., Roth J. R. 1979; Gene duplication in bacteria: alteration of gene dosage by sister-chromosome exchanges. Cold Spring Harb Symp Quant Biol 43:1083–1087 [View Article][PubMed]
    [Google Scholar]
  4. Ashikaga S., Nanamiya H., Ohashi Y., Kawamura F. 2000; Natural genetic competence in Bacillus subtilis natto OK2. J Bacteriol 182:2411–2415 [View Article][PubMed]
    [Google Scholar]
  5. Ashizawa Y., Yokochi T., Ogata Y., Shobuike Y., Kato J., Ikeda H. 1999; Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype. J Mol Biol 289:447–458 [View Article][PubMed]
    [Google Scholar]
  6. Ben-Yehuda S., Rudner D. Z., Losick R. 2003; RacA, a bacterial protein that anchors chromosomes to the cell poles. Science 299:532–536 [View Article][PubMed]
    [Google Scholar]
  7. Bierne H., Ehrlich S. D., Michel B. 1997; Deletions at stalled replication forks occur by two different pathways. EMBO J 16:3332–3340 [View Article][PubMed]
    [Google Scholar]
  8. Chen K., Wallis J. W., McLellan M. D., Larson D. E., Kalicki J. M., Pohl C. S., McGrath S. D., Wendl M. C., Zhang Q., other authors. 2009; BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Nat Methods 6:677–681 [View Article][PubMed]
    [Google Scholar]
  9. Chibazakura T., Yamashita S., Yoshikawa H., Kawamura F., Takahashi H., Saito H. 1988; The multicopy spo0F gene inhibits an enhancement of the spo0A transcription at the early stage of sporulation in Bacillus subtilis . J Gen Appl Microbiol 34:451–455 [View Article]
    [Google Scholar]
  10. Condon C., French S., Squires C., Squires C. L. 1993; Depletion of functional ribosomal RNA operons in Escherichia coli causes increased expression of the remaining intact copies. EMBO J 12:4305–4315[PubMed]
    [Google Scholar]
  11. Condon C., Liveris D., Squires C., Schwartz I., Squires C. L. 1995; rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation. J Bacteriol 177:4152–4156[PubMed]
    [Google Scholar]
  12. Ellwood M., Nomura M. 1980; Deletion of a ribosomal ribonucleic acid operon in Escherichia coli . J Bacteriol 143:1077–1080[PubMed]
    [Google Scholar]
  13. Errington J. 1993; Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev 57:1–33[PubMed]
    [Google Scholar]
  14. Errington J., Daniel R. A., Scheffers D. J. 2003; Cytokinesis in bacteria. Microbiol Mol Biol Rev 67:52–65 [View Article][PubMed]
    [Google Scholar]
  15. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., other authors. 1995; The minimal gene complement of Mycoplasma genitalium . Science 270:397–403 [View Article][PubMed]
    [Google Scholar]
  16. Gaur N. K., Dubnau E., Smith I. 1986; Characterization of a cloned Bacillus subtilis gene that inhibits sporulation in multiple copies. J Bacteriol 168:860–869[PubMed]
    [Google Scholar]
  17. Gaur N. K., Cabane K., Smith I. 1988; Structure and expression of the Bacillus subtilis sin operon. J Bacteriol 170:1046–1053[PubMed]
    [Google Scholar]
  18. Grossman A. D. 1995; Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis . Annu Rev Genet 29:477–508 [View Article][PubMed]
    [Google Scholar]
  19. Hanada K., Ukita T., Kohno Y., Saito K., Kato J., Ikeda H. 1997; RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli . Proc Natl Acad Sci U S A 94:3860–3865 [View Article][PubMed]
    [Google Scholar]
  20. Heath J. D., Weinstock G. M. 1991; Tandem duplications of the lac region of the Escherichia coli chromosome. Biochimie 73:343–352 [View Article][PubMed]
    [Google Scholar]
  21. Hoch J. A. 1993; Regulation of the phosphorelay and the initiation of sporulation in Bacillus subtilis . Annu Rev Microbiol 47:441–465 [View Article][PubMed]
    [Google Scholar]
  22. Ikeda H., Aoki K., Naito A. 1982; Illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules. Proc Natl Acad Sci U S A 79:3724–3728 [View Article][PubMed]
    [Google Scholar]
  23. Kawamura F., Shimotsu H., Saito H., Hirochika H., Kobayashi Y. 1981; Cloning of spo0 genes with bacteriophage and plasmid vectors in Bacillus subtilis . In Sporulation and Germination pp 109–113 Edited by Levinson H. S., Sonenshein A. L., Tipper D. J. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  24. Klappenbach J. A., Dunbar J. M., Schmidt T. M. 2000; rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol 66:1328–1333 [CrossRef]
    [Google Scholar]
  25. Kunst F., Ogasawara N., Moszer I., Albertini A. M., Alloni G., Azevedo V., Bertero M. G., Bessieres P., Bolotin A., other authors. 1997; The complete genome sequence of the gram-positive bacterium Bacillus subtilis . Nature 390:249–256 [CrossRef]
    [Google Scholar]
  26. Lee Z.M.-P., Bussema C. III, Schmidt T. M. 2009; rrnDB: documenting the number of rRNA and tRNA genes in bacteria and archaea. Nucleic Acids Res 37:(Database)D489–D493 [View Article][PubMed]
    [Google Scholar]
  27. Leighton T. J., Doi R. H. 1971; The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis . J Biol Chem 246:3189–3195[PubMed]
    [Google Scholar]
  28. Li H., Durbin R. 2009; Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25:1754–1760 [View Article][PubMed]
    [Google Scholar]
  29. Losick R., Stragier P. 1992; Crisscross regulation of cell-type-specific gene expression during development in B. subtilis . Nature 355:601–604 [View Article][PubMed]
    [Google Scholar]
  30. Loughney K., Lund E., Dahlberg J. E. 1983; Deletion of an rRNA gene set in Bacillus subtilis . J Bacteriol 154:529–532[PubMed]
    [Google Scholar]
  31. Nanamiya H., Sato M., Masuda K., Sato M., Wada T., Suzuki S., Natori Y., Katano M., Akanuma G., Kawamura F. 2010; Bacillus subtilis mutants harbouring a single copy of the rRNA operon exhibit severe defects in growth and sporulation. Microbiology 156:2944–2952 [View Article][PubMed]
    [Google Scholar]
  32. Natori Y., Tagami K., Murakami K., Yoshida S., Tanigawa O., Moh Y., Masuda K., Wada T., Suzuki S., other authors. 2009; Transcription activity of individual rrn operons in Bacillus subtilis mutants deficient in (p)ppGpp synthetase genes, relA, yjbM, and ywaC . J Bacteriol 191:4555–4561 [View Article][PubMed]
    [Google Scholar]
  33. Nomura M. 1999; Engineering of bacterial ribosomes: replacement of all seven Escherichia coli rRNA operons by a single plasmid-encoded operon. Proc Natl Acad Sci U S A 96:1820–1822 [View Article][PubMed]
    [Google Scholar]
  34. Pfannes K. R., Vogl K., Overmann J. 2007; Heterotrophic symbionts of phototrophic consortia: members of a novel diverse cluster of Betaproteobacteria characterized by a tandem rrn operon structure. Environ Microbiol 9:2782–2794 [View Article][PubMed]
    [Google Scholar]
  35. Rainey F. A., Ward-Rainey N. L., Janssen P. H., Hippe H., Stackebrandt E. 1996; Clostridium paradoxum DSM 7308T contains multiple 16S rRNA genes with heterogeneous intervening sequences. Microbiology 142:2087–2095 [View Article][PubMed]
    [Google Scholar]
  36. Reams A. B., Neidle E. L. 2004; Gene amplification involves site-specific short homology-independent illegitimate recombination in Acinetobacter sp. strain ADP1. J Mol Biol 338:643–656 [View Article][PubMed]
    [Google Scholar]
  37. Robinson J. T., Thorvaldsdóttir H., Winckler W., Guttman M., Lander E. S., Getz G., Mesirov J. P. 2011; Integrative genomics viewer. Nat Biotechnol 29:24–26 [View Article][PubMed]
    [Google Scholar]
  38. Romero D., Palacios R. 1997; Gene amplification and genomic plasticity in prokaryotes. Annu Rev Genet 31:91–111 [View Article][PubMed]
    [Google Scholar]
  39. Sambrook J., Russell D. 2001 Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor; NY: Cold Spring Harbor Laboratory
    [Google Scholar]
  40. Schaeffer P., Millet J., Aubert J. P. 1965; Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A 54:704–711 [View Article][PubMed]
    [Google Scholar]
  41. Shimizu H., Yamaguchi H., Ikeda H. 1995; Molecular analysis of lambda bio transducing phage produced by oxolinic acid-induced illegitimate recombination in vivo . Genetics 140:889–896[PubMed]
    [Google Scholar]
  42. Shimizu H., Yamaguchi H., Ashizawa Y., Kohno Y., Asami M., Kato J., Ikeda H. 1997; Short-homology-independent illegitimate recombination in Escherichia coli: distinct mechanism from short-homology-dependent illegitimate recombination. J Mol Biol 266:297–305 [View Article][PubMed]
    [Google Scholar]
  43. Shiraishi K., Imai Y., Yoshizaki S., Tadaki T., Ogata Y., Ikeda H. 2006; The role of UvrD in RecET-mediated illegitimate recombination in Escherichia coli . Genes Genet Syst 81:291–297 [View Article][PubMed]
    [Google Scholar]
  44. Stevenson B. S., Schmidt T. M. 2004; Life history implications of rRNA gene copy number in Escherichia coli . Appl Environ Microbiol 70:6670–6677 [View Article][PubMed]
    [Google Scholar]
  45. Tagami K., Nanamiya H., Kazo Y., Maehashi M., Suzuki S., Namba E., Hoshiya M., Hanai R., Tozawa Y., other authors. 2012; Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp0 mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome. MicrobiologyOpen 1:114–134 [CrossRef]
    [Google Scholar]
  46. Ukita T., Ikeda H. 1996; Role of the recJ gene product in UV-induced illegitimate recombination at the hotspot. J Bacteriol 178:2362–2367[PubMed]
    [Google Scholar]
  47. Wannarat W., Motoyama S., Masuda K., Kawamura F., Inaoka T. 2014; Tetracycline tolerance mediated by gene amplification in Bacillus subtilis . Microbiology 160:2474–2480 [View Article][PubMed]
    [Google Scholar]
  48. Yamaguchi H., Yamashita T., Shimizu H., Ikeda H. 1995; A hotspot of spontaneous and UV-induced illegitimate recombination during formation of λ bio transducing phage. Mol Gen Genet 248:637–643 [View Article][PubMed]
    [Google Scholar]
  49. Yano K., Wada T., Suzuki S., Tagami K., Matsumoto T., Shiwa Y., Ishige T., Kawaguchi Y., Masuda K., other authors. 2013; Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis . Microbiology 159:2225–2236 [View Article][PubMed]
    [Google Scholar]
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