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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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Northern blot analysis of B. subtilis 168 mtnVWXYZ region. A total of 3 μg of RNA was used. A. Northern hybridization with mtnV gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source. B. Northern hybrydization with mtnW gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source. C. Northern hybrydization with mtnZ gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source.
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Figure 3: Northern blot analysis of B. subtilis 168 mtnVWXYZ region. A total of 3 μg of RNA was used. A. Northern hybridization with mtnV gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source. B. Northern hybrydization with mtnW gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source. C. Northern hybrydization with mtnZ gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source.

Mentions: To further investigate transcription of the mtnVWXYZ genes, RNA synthesis was analyzed by Northern blotting. RNA was extracted from exponentially growing cells, in minimal medium containing either sulfate or methionine as sulfur source. As shown in Figure 3.A, a band of about 1200 nt, corresponding to the expected length of a transcript initiated at the mtnV promoter and terminating near its stop codon, was observed for the mtnV gene probe. An equal intensity of the signal was observed for mtnV transcripts prepared from cells either grown with sulfate or with methionine as sulfur source (lanes 1 and 2, Fig. 3.A).


The methionine salvage pathway in Bacillus subtilis.

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

Northern blot analysis of B. subtilis 168 mtnVWXYZ region. A total of 3 μg of RNA was used. A. Northern hybridization with mtnV gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source. B. Northern hybrydization with mtnW gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source. C. Northern hybrydization with mtnZ gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source.
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Related In: Results  -  Collection

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

Figure 3: Northern blot analysis of B. subtilis 168 mtnVWXYZ region. A total of 3 μg of RNA was used. A. Northern hybridization with mtnV gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source. B. Northern hybrydization with mtnW gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source. C. Northern hybrydization with mtnZ gene specific probe. RNA corresponding to lane 1 was obtained from a culture grown in minimal medium with sulfate as a sulfur source, and for lane 2 from a culture grown in minimal medium with methionine as a sulfur source.
Mentions: To further investigate transcription of the mtnVWXYZ genes, RNA synthesis was analyzed by Northern blotting. RNA was extracted from exponentially growing cells, in minimal medium containing either sulfate or methionine as sulfur source. As shown in Figure 3.A, a band of about 1200 nt, corresponding to the expected length of a transcript initiated at the mtnV promoter and terminating near its stop codon, was observed for the mtnV gene probe. An equal intensity of the signal was observed for mtnV transcripts prepared from cells either grown with sulfate or with methionine as sulfur source (lanes 1 and 2, Fig. 3.A).

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