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. 1996 Apr;178(8):2375–2382. doi: 10.1128/jb.178.8.2375-2382.1996

Properties of a Bacillus subtilis polynucleotide phosphorylase deletion strain.

W Wang 1, D H Bechhofer 1
PMCID: PMC177948  PMID: 8636041

Abstract

The pnpA gene of Bacillus subtilis, which codes for polynucleotide phosphorylase (PNPase), has been cloned and employed in the construction of pnpA deletion mutants. Growth defects of both B. subtilis and Escherichia coli PNPase-deficient strains were complemented with the cloned pnpA gene. RNA decay characteristics of the B. subtilis pnpA mutant were studied, including the in vivo decay of bulk mRNA and the in vitro decay of either poly(A) or total cellular RNA. The results showed that mRNA decay in the pnpA mutant is accomplished despite the absence of the major, Pi-dependent RNA decay activity of PNPase. In vitro experiments suggested that a previously identified, Mn2+ -dependent hydrolytic activity was important for decay in the pnpA mutant. In addition to a cold-sensitive-growth phenotype, the pnpA deletion mutant was found to be sensitive to growth in the presence of tetracycline, and this was due to an increased intracellular accumulation of the drug. The pnpA deletion strain also exhibited multiseptate, filamentous growth. It is hypothesized that defective processing of specific RNAs in the pnpA mutant results in these phenotypes.

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Selected References

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  1. ACS G., REICH E., VALANJU S. RNA METABOLISM OF B. SUBTILIS. EFFECTS OF ACTINOMYCIN. Biochim Biophys Acta. 1963 Sep 17;76:68–79. [PubMed] [Google Scholar]
  2. Bai U., Mandic-Mulec I., Smith I. SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction. Genes Dev. 1993 Jan;7(1):139–148. doi: 10.1101/gad.7.1.139. [DOI] [PubMed] [Google Scholar]
  3. Bechhofer D. H., Zen K. H. Mechanism of erythromycin-induced ermC mRNA stability in Bacillus subtilis. J Bacteriol. 1989 Nov;171(11):5803–5811. doi: 10.1128/jb.171.11.5803-5811.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Breidt F., Dubnau D. Identification of cis-acting sequences required for translational autoregulation of the ermC methylase. J Bacteriol. 1990 Jul;172(7):3661–3668. doi: 10.1128/jb.172.7.3661-3668.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Causton H., Py B., McLaren R. S., Higgins C. F. mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures. Mol Microbiol. 1994 Nov;14(4):731–741. doi: 10.1111/j.1365-2958.1994.tb01310.x. [DOI] [PubMed] [Google Scholar]
  6. Cheng J., Guffanti A. A., Krulwich T. A. The chromosomal tetracycline resistance locus of Bacillus subtilis encodes a Na+/H+ antiporter that is physiologically important at elevated pH. J Biol Chem. 1994 Nov 4;269(44):27365–27371. [PubMed] [Google Scholar]
  7. Cohen S. N. Surprises at the 3' end of prokaryotic RNA. Cell. 1995 Mar 24;80(6):829–832. doi: 10.1016/0092-8674(95)90284-8. [DOI] [PubMed] [Google Scholar]
  8. Deutscher M. P. Promiscuous exoribonucleases of Escherichia coli. J Bacteriol. 1993 Aug;175(15):4577–4583. doi: 10.1128/jb.175.15.4577-4583.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deutscher M. P., Reuven N. B. Enzymatic basis for hydrolytic versus phosphorolytic mRNA degradation in Escherichia coli and Bacillus subtilis. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3277–3280. doi: 10.1073/pnas.88.8.3277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donovan W. P., Kushner S. R. Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1986 Jan;83(1):120–124. doi: 10.1073/pnas.83.1.120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dubnau D., Davidoff-Abelson R. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol. 1971 Mar 14;56(2):209–221. doi: 10.1016/0022-2836(71)90460-8. [DOI] [PubMed] [Google Scholar]
  12. Duffy J. J., Chaney S. G., Boyer P. D. Incorporation of water oxygens into intracellular nucleotides and RNA. I. Predominantly non-hydrolytic RNA turnover in Bacillus subtilis. J Mol Biol. 1972 Mar 14;64(3):565–579. doi: 10.1016/0022-2836(72)90083-6. [DOI] [PubMed] [Google Scholar]
  13. Grant S. G., Jessee J., Bloom F. R., Hanahan D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4645–4649. doi: 10.1073/pnas.87.12.4645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gryczan T. J., Israeli-Reches M., Dubnau D. Induction of macrolide-lincosamide-streptogramin B resistance requires ribosomes able to bind inducer. Mol Gen Genet. 1984;194(3):357–361. doi: 10.1007/BF00425544. [DOI] [PubMed] [Google Scholar]
  15. Hajnsdorf E., Braun F., Haugel-Nielsen J., Régnier P. Polyadenylylation destabilizes the rpsO mRNA of Escherichia coli. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3973–3977. doi: 10.1073/pnas.92.9.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hue K. K., Bechhofer D. H. Effect of ermC leader region mutations on induced mRNA stability. J Bacteriol. 1991 Jun;173(12):3732–3740. doi: 10.1128/jb.173.12.3732-3740.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hue K. K., Cohen S. D., Bechhofer D. H. A polypurine sequence that acts as a 5' mRNA stabilizer in Bacillus subtilis. J Bacteriol. 1995 Jun;177(12):3465–3471. doi: 10.1128/jb.177.12.3465-3471.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Itaya M., Kondo K., Tanaka T. A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome. Nucleic Acids Res. 1989 Jun 12;17(11):4410–4410. doi: 10.1093/nar/17.11.4410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ives C. L., Bott K. F. Cloned Bacillus subtilis chromosomal DNA mediates tetracycline resistance when present in multiple copies. J Bacteriol. 1989 Apr;171(4):1801–1810. doi: 10.1128/jb.171.4.1801-1810.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Luttinger A., Hahn J., Dubnau D. Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis. Mol Microbiol. 1996 Jan;19(2):343–356. doi: 10.1046/j.1365-2958.1996.380907.x. [DOI] [PubMed] [Google Scholar]
  22. McLaren R. S., Newbury S. F., Dance G. S., Causton H. C., Higgins C. F. mRNA degradation by processive 3'-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo. J Mol Biol. 1991 Sep 5;221(1):81–95. [PubMed] [Google Scholar]
  23. McMurry L. M., Levy S. B. Tn5 insertion in the polynucleotide phosphorylase (pnp) gene in Escherichia coli increases susceptibility to antibiotics. J Bacteriol. 1987 Mar;169(3):1321–1324. doi: 10.1128/jb.169.3.1321-1324.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mitra S., Hue K., Bechhofer D. H. In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase. Mol Microbiol. 1996 Jan;19(2):329–342. doi: 10.1046/j.1365-2958.1996.378906.x. [DOI] [PubMed] [Google Scholar]
  25. Márquez L. M., Helmann J. D., Ferrari E., Parker H. M., Ordal G. W., Chamberlin M. J. Studies of sigma D-dependent functions in Bacillus subtilis. J Bacteriol. 1990 Jun;172(6):3435–3443. doi: 10.1128/jb.172.6.3435-3443.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. O'Hara E. B., Chekanova J. A., Ingle C. A., Kushner Z. R., Peters E., Kushner S. R. Polyadenylylation helps regulate mRNA decay in Escherichia coli. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1807–1811. doi: 10.1073/pnas.92.6.1807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shishido K., Noguchi N., Kim C., Ando T. Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis. Plasmid. 1983 Nov;10(3):224–234. doi: 10.1016/0147-619x(83)90036-7. [DOI] [PubMed] [Google Scholar]
  28. Ulmanen I., Lundström K., Lehtovaara P., Sarvas M., Ruohonen M., Palva I. Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. J Bacteriol. 1985 Apr;162(1):176–182. doi: 10.1128/jb.162.1.176-182.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weinrauch Y., Msadek T., Kunst F., Dubnau D. Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis. J Bacteriol. 1991 Sep;173(18):5685–5693. doi: 10.1128/jb.173.18.5685-5693.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Xu F., Cohen S. N. RNA degradation in Escherichia coli regulated by 3' adenylation and 5' phosphorylation. Nature. 1995 Mar 9;374(6518):180–183. doi: 10.1038/374180a0. [DOI] [PubMed] [Google Scholar]

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