1887

Abstract

The soil bacterium can utilize exogenous proline as a sole nitrogen or carbon source. The proline-inducible (formerly ) operon encodes proteins responsible for proline uptake and two-step oxidation of proline to glutamate. We now report that a gene (formerly , now designated ) located downstream of the operon is essential for cells to utilize proline as a sole nitrogen or carbon source. Disruption of the gene also abolished proline induction of transcription. expression is not subject to autoregulation and proline induction. The PrcR protein shows no significant amino acid sequence similarity to the known transcriptional activators for proline utilization genes of other bacteria, but it does show partial amino acid sequence similarity to the transcriptional regulator PucR for the purine degradation genes of . PrcR orthologues of unknown function are present in some other species. Primer-extension analysis suggests that both and are transcribed by a σ-dependent promoter. Deletion and mutation analysis revealed that an inverted repeat (5′-TTGTGG-N5-CCACAA-3′) centred at position −76 relative to the transcriptional initiation site of is essential for expression. Electrophoretic mobility shift assays showed that the purified His-tagged PrcR was capable of binding specifically to this inverted repeat. Altogether, these results suggest that PrcR is a PucR-type transcriptional activator that mediates expression of the operon in response to proline availability.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.054197-0
2011-12-01
2019-12-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/12/3370.html?itemId=/content/journal/micro/10.1099/mic.0.054197-0&mimeType=html&fmt=ahah

References

  1. Atkinson M. R. , Wray L. V. Jr , Fisher S. H. . ( 1990; ). Regulation of histidine and proline degradation enzymes by amino acid availability in Bacillus subtilis . . J Bacteriol 172:, 4758–4765.[PubMed]
    [Google Scholar]
  2. Beier L. , Nygaard P. , Jarmer H. , Saxild H. H. . ( 2002; ). Transcription analysis of the Bacillus subtilis PucR regulon and identification of a cis-acting sequence required for PucR-regulated expression of genes involved in purine catabolism. . J Bacteriol 184:, 3232–3241. [CrossRef] [PubMed]
    [Google Scholar]
  3. Belitsky B. R. . ( 2011; ). Indirect repression by Bacillus subtilis CodY via displacement of the activator of the proline utilization operon. . J Mol Biol (in press). [CrossRef]
    [Google Scholar]
  4. Belitsky B. R. , Brill J. , Bremer E. , Sonenshein A. L. . ( 2001; ). Multiple genes for the last step of proline biosynthesis in Bacillus subtilis . . J Bacteriol 183:, 4389–4392. [CrossRef] [PubMed]
    [Google Scholar]
  5. Bremer E. . ( 2002; ). Adaptation to changing osmolarity. . In Bacillus subtilis and Its Closest Relatives: from Genes to Cells, pp. 385–391. Edited by Sonenshein A. L. , Hoch J. A. , Losick R. M. . . Washington, DC:: American Society for Microbiology;.[CrossRef]
    [Google Scholar]
  6. Chang S. , Cohen S. N. . ( 1979; ). High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. . Mol Gen Genet 168:, 111–115. [CrossRef] [PubMed]
    [Google Scholar]
  7. Cho K. , Winans S. C. . ( 1996; ). The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp-like protein and is not autoregulated. . Mol Microbiol 22:, 1025–1033. [CrossRef] [PubMed]
    [Google Scholar]
  8. Commichau F. M. , Stülke J. . ( 2008; ). Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression. . Mol Microbiol 67:, 692–702. [CrossRef] [PubMed]
    [Google Scholar]
  9. Contente S. , Dubnau D. . ( 1979; ). Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation. . Mol Gen Genet 167:, 251–258. [CrossRef] [PubMed]
    [Google Scholar]
  10. Dodd I. B. , Egan J. B. . ( 1990; ). Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. . Nucleic Acids Res 18:, 5019–5026. [CrossRef] [PubMed]
    [Google Scholar]
  11. Fedhila S. , Msadek T. , Nel P. , Lereclus D. . ( 2002; ). Distinct clpP genes control specific adaptive responses in Bacillus thuringiensis . . J Bacteriol 184:, 5554–5562. [CrossRef] [PubMed]
    [Google Scholar]
  12. Fried M. , Crothers D. M. . ( 1981; ). Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. . Nucleic Acids Res 9:, 6505–6525. [CrossRef] [PubMed]
    [Google Scholar]
  13. Gu D. , Zhou Y. , Kallhoff V. , Baban B. , Tanner J. J. , Becker D. F. . ( 2004; ). Identification and characterization of the DNA-binding domain of the multifunctional PutA flavoenzyme. . J Biol Chem 279:, 31171–31176. [CrossRef] [PubMed]
    [Google Scholar]
  14. Higuchi R. , Krummel B. , Saiki R. K. . ( 1988; ). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. . Nucleic Acids Res 16:, 7351–7367. [CrossRef] [PubMed]
    [Google Scholar]
  15. Inoue T. , Cech T. R. . ( 1985; ). Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase. . Proc Natl Acad Sci U S A 82:, 648–652. [CrossRef] [PubMed]
    [Google Scholar]
  16. Keuntje B. , Masepohl B. , Klipp W. . ( 1995; ). Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein. . J Bacteriol 177:, 6432–6439.[PubMed]
    [Google Scholar]
  17. Kilstrup M. , Jessing S. G. , Wichmand-Jørgensen S. B. , Madsen M. , Nilsson D. . ( 1998; ). Activation control of pur gene expression in Lactococcus lactis: proposal for a consensus activator binding sequence based on deletion analysis and site-directed mutagenesis of purC and purD promoter regions. . J Bacteriol 180:, 3900–3906.[PubMed]
    [Google Scholar]
  18. Lee J. H. , Choi S. H. . ( 2006; ). Coactivation of Vibrio vulnificus putAP operon by cAMP receptor protein and PutR through cooperative binding to overlapping sites. . Mol Microbiol 60:, 513–524. [CrossRef] [PubMed]
    [Google Scholar]
  19. Lee Y. H. , Nadaraia S. , Gu D. , Becker D. F. , Tanner J. J. . ( 2003; ). Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein. . Nat Struct Biol 10:, 109–114. [CrossRef] [PubMed]
    [Google Scholar]
  20. Menzel R. , Roth J. . ( 1981; ). Purification of the putA gene product. A bifunctional membrane-bound protein from Salmonella typhimurium responsible for the two-step oxidation of proline to glutamate. . J Biol Chem 256:, 9755–9761.[PubMed]
    [Google Scholar]
  21. Muro-Pastor A. M. , Ostrovsky P. , Maloy S. . ( 1997; ). Regulation of gene expression by repressor localization: biochemical evidence that membrane and DNA binding by the PutA protein are mutually exclusive. . J Bacteriol 179:, 2788–2791.[PubMed]
    [Google Scholar]
  22. Nakada Y. , Nishijyo T. , Itoh Y. . ( 2002; ). Divergent structure and regulatory mechanism of proline catabolic systems: characterization of the putAP proline catabolic operon of Pseudomonas aeruginosa PAO1 and its regulation by PruR, an AraC/XylS family protein. . J Bacteriol 184:, 5633–5640. [CrossRef] [PubMed]
    [Google Scholar]
  23. O’Kane C. , Stephens M. A. , McConnell D. . ( 1986; ). Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis . . J Bacteriol 168:, 973–981.[PubMed]
    [Google Scholar]
  24. Ostrovsky de Spicer P. , Maloy S. . ( 1993; ). PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator. . Proc Natl Acad Sci U S A 90:, 4295–4298. [CrossRef] [PubMed]
    [Google Scholar]
  25. Sambrook J. , Russell D. . ( 2001; ). Molecular Cloning: a Laboratory Manual, , 3rd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  26. Schultz A. C. , Nygaard P. , Saxild H. H. . ( 2001; ). Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. . J Bacteriol 183:, 3293–3302. [CrossRef] [PubMed]
    [Google Scholar]
  27. Soto M. J. , Jiménez-Zurdo J. I. , van Dillewijn P. , Toro N. . ( 2000; ). Sinorhizobium meliloti putA gene regulation: a new model within the family Rhizobiaceae . . J Bacteriol 182:, 1935–1941. [CrossRef] [PubMed]
    [Google Scholar]
  28. Tanner J. J. . ( 2008; ). Structural biology of proline catabolism. . Amino Acids 35:, 719–730. [CrossRef] [PubMed]
    [Google Scholar]
  29. Yuan G. , Wong S. L. . ( 1995; ). Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE). . J Bacteriol 177:, 5427–5433.[PubMed]
    [Google Scholar]
  30. Zuber P. , Losick R. . ( 1983; ). Use of a lacZ fusion to study the role of the spoO genes of Bacillus subtilis in developmental regulation. . Cell 35:, 275–283. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.054197-0
Loading
/content/journal/micro/10.1099/mic.0.054197-0
Loading

Data & Media loading...

Supplements

Supplementary Table S1 

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