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The methionine salvage pathway in Bacillus subtilis.

Sekowska A, Danchin A - BMC Microbiol. (2002)

Bottom Line: Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling.In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway.A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: HKU-Pasteur Research Centre, Dexter HC Man Building, 8, Sassoon Road, Pokfulam, Hong Kong, China. sekowska@hkucc.hku.hk

ABSTRACT

Background: Polyamine synthesis produces methylthioadenosine, which has to be disposed of. The cell recycles it into methionine through methylthioribose (MTR). Very little was known about MTR recycling for methionine salvage in Bacillus subtilis.

Results: Using in silico genome analysis and transposon mutagenesis in B. subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins. The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression. Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling.

Conclusions: A complete methionine salvage pathway exists in B. subtilis. This pathway is chemically similar to that in K. pneumoniae, but recruited different proteins to this purpose. In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway. A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs. In addition to methionine salvage, this pathway protects B. subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane).

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Location of transposon (Tn10) insertions in the mtn region. One insertion was localized 73 bp upstream of the translational start point of the mtnK gene [6], four were located into mtnW and six into the mtnY gene. The insertion situated 353 bp downstream of the mtnW translation start point (strain BSHP7064) and one situated 556 bp downstream of the mtnY translation start point (strain BSHP7065) are shown in the figure.
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Figure 1: Location of transposon (Tn10) insertions in the mtn region. One insertion was localized 73 bp upstream of the translational start point of the mtnK gene [6], four were located into mtnW and six into the mtnY gene. The insertion situated 353 bp downstream of the mtnW translation start point (strain BSHP7064) and one situated 556 bp downstream of the mtnY translation start point (strain BSHP7065) are shown in the figure.

Mentions: The MTR analog trifluormethylthioribose (3F-MTR) is toxic if the methyl sulfur moiety of the molecule is recycled [9]. This molecule was therefore an excellent candidate to explore the steps needed for MTR recycling to methionine. Mutants were obtained by transformation of a wild type strain with a random transposon library, selecting for growth in the presence of 3F-MTR in the presence of sulfate as sulfur source. The mutants were subsequently tested for growth on plates lacking sulfur source but supplemented with MTR: only those that could not grow were retained for further study. In order to ascertain that the resistant phenotype was not coming from secondary mutations but was directly related to the transposon insert, the chromosome DNA was extracted from each putative mutant and back transformed into a wild type strain selecting for the transposon antibiotic marker. The 3F-MTR and MTR phenotypes were subsequently tested and only those mutants that passed the test were retained. The insertion positions of the transposons were then sequenced. As shown in Figure 1 we recovered mutants in several genes located in the close vicinity of each other. One mutant was located at the mtnK locus (previously named ykrT[6]), four were located into ykrW and six into the ykrY gene. One clone with transposon insertion into the ykrW gene (strain BSHP7064, insertion situated 353 bp downstream of ykrW translation start point) and one into the ykrY gene (strain BSHP7065, insertion situated 556 bp downstream of ykrY translation start point) were retained for further studies.


The methionine salvage pathway in Bacillus subtilis.

Sekowska A, Danchin A - BMC Microbiol. (2002)

Location of transposon (Tn10) insertions in the mtn region. One insertion was localized 73 bp upstream of the translational start point of the mtnK gene [6], four were located into mtnW and six into the mtnY gene. The insertion situated 353 bp downstream of the mtnW translation start point (strain BSHP7064) and one situated 556 bp downstream of the mtnY translation start point (strain BSHP7065) are shown in the figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113757&req=5

Figure 1: Location of transposon (Tn10) insertions in the mtn region. One insertion was localized 73 bp upstream of the translational start point of the mtnK gene [6], four were located into mtnW and six into the mtnY gene. The insertion situated 353 bp downstream of the mtnW translation start point (strain BSHP7064) and one situated 556 bp downstream of the mtnY translation start point (strain BSHP7065) are shown in the figure.
Mentions: The MTR analog trifluormethylthioribose (3F-MTR) is toxic if the methyl sulfur moiety of the molecule is recycled [9]. This molecule was therefore an excellent candidate to explore the steps needed for MTR recycling to methionine. Mutants were obtained by transformation of a wild type strain with a random transposon library, selecting for growth in the presence of 3F-MTR in the presence of sulfate as sulfur source. The mutants were subsequently tested for growth on plates lacking sulfur source but supplemented with MTR: only those that could not grow were retained for further study. In order to ascertain that the resistant phenotype was not coming from secondary mutations but was directly related to the transposon insert, the chromosome DNA was extracted from each putative mutant and back transformed into a wild type strain selecting for the transposon antibiotic marker. The 3F-MTR and MTR phenotypes were subsequently tested and only those mutants that passed the test were retained. The insertion positions of the transposons were then sequenced. As shown in Figure 1 we recovered mutants in several genes located in the close vicinity of each other. One mutant was located at the mtnK locus (previously named ykrT[6]), four were located into ykrW and six into the ykrY gene. One clone with transposon insertion into the ykrW gene (strain BSHP7064, insertion situated 353 bp downstream of ykrW translation start point) and one into the ykrY gene (strain BSHP7065, insertion situated 556 bp downstream of ykrY translation start point) were retained for further studies.

Bottom Line: Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling.In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway.A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: HKU-Pasteur Research Centre, Dexter HC Man Building, 8, Sassoon Road, Pokfulam, Hong Kong, China. sekowska@hkucc.hku.hk

ABSTRACT

Background: Polyamine synthesis produces methylthioadenosine, which has to be disposed of. The cell recycles it into methionine through methylthioribose (MTR). Very little was known about MTR recycling for methionine salvage in Bacillus subtilis.

Results: Using in silico genome analysis and transposon mutagenesis in B. subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins. The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression. Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling.

Conclusions: A complete methionine salvage pathway exists in B. subtilis. This pathway is chemically similar to that in K. pneumoniae, but recruited different proteins to this purpose. In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway. A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs. In addition to methionine salvage, this pathway protects B. subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane).

Show MeSH
Related in: MedlinePlus