1887

Abstract

The 60 nucleotides encoding the signal peptide of the membrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression of from either a T7- or a P-driven system in . Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem–loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of the transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26780-0
2004-05-01
2019-10-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/5/mic1501427.html?itemId=/content/journal/micro/10.1099/mic.0.26780-0&mimeType=html&fmt=ahah

References

  1. Bolivar, F. & Backman, K. ( 1979; ). Plasmids of Escherichia coli as cloning vectors. Methods Enzymol 68, 245–267.
    [Google Scholar]
  2. Chang, A. C. Y. & Cohen, S. N. ( 1978; ). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134, 1141–1156.
    [Google Scholar]
  3. Deana, A., Ehrlich, R. & Reiss, C. ( 1998; ). Silent mutations in the Escherichia coli ompA leader peptide region strongly affect transcription and translation in vivo. Nucleic Acids Res 26, 4778–4782.[CrossRef]
    [Google Scholar]
  4. Ehlert, K., Höltje, J.-V. & Templin, M. F. ( 1995; ). Cloning and expression of a murein hydrolase lipoprotein from Escherichia coli. Mol Microbiol 16, 761–768.[CrossRef]
    [Google Scholar]
  5. Höltje, J.-V. ( 1998; ). Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 62, 181–203.
    [Google Scholar]
  6. Iost, I. & Dreyfus, M. ( 1995; ). The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation. EMBO J 14, 3252–3261.
    [Google Scholar]
  7. Iost, I., Guillerez, J. & Dreyfus, M. ( 1992; ). Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo. J Bacteriol 174, 619–622.
    [Google Scholar]
  8. Jacobs, C., Frère, J.-M. & Normark, S. ( 1997; ). Cytosolic intermediates for cell wall biosynthesis and degradation control inducible β-lactam resistance in gram-negative bacteria. Cell 88, 823–832.[CrossRef]
    [Google Scholar]
  9. Jennings, G. T., Savino, S., Marchetti, E. & 8 other authors ( 2002; ). GNA33 from Neisseria meningitidis serogroup B encodes a membrane-bound lytic transglycosylase (MltA). Eur J Biochem 269, 3722–3731.[CrossRef]
    [Google Scholar]
  10. Katz, L. & Burge, C. B. ( 2003; ). Widespread selection for local RNA secondary structure in coding regions of bacterial genes. Genome Res 13, 2042–2051.[CrossRef]
    [Google Scholar]
  11. Kraft, A. R., Prabhu, J., Ursinus, A. & Höltje, J.-V. ( 1999; ). Interference with murein turnover has no effect on growth but reduces beta-lactamase induction in Escherichia coli. J Bacteriol 181, 7192–7198.
    [Google Scholar]
  12. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–684.[CrossRef]
    [Google Scholar]
  13. Lommatzsch, J., Templin, M. F., Kraft, A. R., Vollmer, W. & Höltje, J.-V. ( 1997; ). Outer membrane localization of murein hydrolases: MltA, a third lipoprotein lytic transglycosylase in Escherichia coli. J Bacteriol 179, 5465–5470.
    [Google Scholar]
  14. Lyakhov, D. L., He, B., Zhang, X., Studier, F. W., Dunn, J. J. & McAllister, W. T. ( 1998; ). Pausing and termination by bacteriophage T7 RNA polymerase. J Mol Biol 280, 201–213.[CrossRef]
    [Google Scholar]
  15. Nanninga, N. ( 1998; ). Morphogenesis of Escherichia coli. Microbiol Mol Biol Rev 62, 110–129.
    [Google Scholar]
  16. Pizza, M., Scarlato, V., Masignani, V. & 33 other authors ( 2000; ). Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287, 1816–1820.[CrossRef]
    [Google Scholar]
  17. Romeis, T. & Höltje, J.-V. ( 1994; ). Specific interaction of penicillin-binding proteins 3 and 7/8 with the soluble lytic transglycosylase in Escherichia coli. J Biol Chem 269, 21603–21607.
    [Google Scholar]
  18. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  19. Studier, F. W. ( 1991; ). Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219, 37–44.[CrossRef]
    [Google Scholar]
  20. Studier, F. W. & Moffat, B. A. ( 1986; ). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113–130.[CrossRef]
    [Google Scholar]
  21. Thaller, M. C., Berlutti, F., Schippa, S., Lombardi, G. & Rossolini, G. M. ( 1994; ). Characterization and sequence of PhoC, the principal phosphate-irrepressible acid phosphatase of Morganella morganii. Microbiology 140, 1341–1350.[CrossRef]
    [Google Scholar]
  22. Thaller, M. C., Berlutti, F., Schippa, S., Selan, L. & Rossolini, G. M. ( 1998; ). Bacterial acid phosphatase gene fusions useful as targets for cloning-dependent insertional inactivation. Biotechnol Prog 14, 241–247.[CrossRef]
    [Google Scholar]
  23. Vollmer, W., von Rechenberg, M. & Höltje, J.-V. ( 1999; ). Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli. J Biol Chem 274, 6726–6734.[CrossRef]
    [Google Scholar]
  24. von Rechenberg, M., Ursinus, A. & Höltje, J.-V. ( 1996; ). Affinity chromatography as a means to study multienzyme complexes involved in murein synthesis. Microb Drug Resist 2, 155–157.[CrossRef]
    [Google Scholar]
  25. Wood, W. B. ( 1966; ). Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol 16, 118–133.[CrossRef]
    [Google Scholar]
  26. Zhang, X. & Studier, F. W. ( 1997; ). Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J Mol Biol 269, 10–27.[CrossRef]
    [Google Scholar]
  27. Zuker, M. ( 1989; ). On finding all suboptimal foldings of an RNA molecule. Science 244, 48–52.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26780-0
Loading
/content/journal/micro/10.1099/mic.0.26780-0
Loading

Data & Media loading...

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