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.

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2004-05-01
2021-10-19
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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 translationin 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]
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