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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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Toxicity of MTR for BSHP7014 strain. Strain BSHP7014 (mtnW::lacZ amyE::pxyl mtnXYZ) was grown on ED1 minimal medium plates in the presence of sulfate as sulfur source (panel A) or in the absence of any added sulfur source (agar as sole sulfur source, panel B). Xylose was added to the medium in order to trigger the expression of mtnXYZ from the pxyl promoter. 10 μl of methionine (met) or MTR was adsorbed on paper discs and plates were incubated over-night at 37°C. Methionine was used as a control for growth and/or toxicity of the sulfur source.
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Figure 7: Toxicity of MTR for BSHP7014 strain. Strain BSHP7014 (mtnW::lacZ amyE::pxyl mtnXYZ) was grown on ED1 minimal medium plates in the presence of sulfate as sulfur source (panel A) or in the absence of any added sulfur source (agar as sole sulfur source, panel B). Xylose was added to the medium in order to trigger the expression of mtnXYZ from the pxyl promoter. 10 μl of methionine (met) or MTR was adsorbed on paper discs and plates were incubated over-night at 37°C. Methionine was used as a control for growth and/or toxicity of the sulfur source.

Mentions: Two genes in the pathway are dispensable, mtnV and mtnX. The first one encodes a transaminase of which there are nine putative paralogs in the genome of B. subtilis (YwfG, AlaT, AspB, PatA, YhdR, YdfD, PatB, YisV, and HisC). In the same way, MtnX (YkrX) is a member of the phosphatase family pfam00702 ([13], Fig. 6), and therefore of a ubiquitous class of hydrolases (several phosphatase genes in particular are present in the genome of B. subtilis). This is likely to account for the lack of phenotype under our growth conditions. Inactivation of mtnZ provides only a very weak, residual growth on MTR. Inactivation of mtnK, mtnS, mtnY and mtnW result in resistance to 3F-MTR and lack of growth on MTR. Inactivation of mtnW with a polar effect on the distal genes (by insertion of a disrupting plasmid) has a phenotype similar to that of mtnY (i.e. lack of growth on MTR, and lack of influence of MTR on sulfate supplemented plates). In contrast, we discovered that MTR is toxic when the distal genes are present (when used as sole sulfur source or even in the presence of sulfate, see Fig. 7). Because of the weak phenotype of a mtnZ mutant and the absence of phenotype of a mtnX mutant, we can be confident that MtnY acts before MtnW (this is a regular feature in operons, where it is often observed that the more distal genes code for proteins acting in the more proximate steps of the pathway).


The methionine salvage pathway in Bacillus subtilis.

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

Toxicity of MTR for BSHP7014 strain. Strain BSHP7014 (mtnW::lacZ amyE::pxyl mtnXYZ) was grown on ED1 minimal medium plates in the presence of sulfate as sulfur source (panel A) or in the absence of any added sulfur source (agar as sole sulfur source, panel B). Xylose was added to the medium in order to trigger the expression of mtnXYZ from the pxyl promoter. 10 μl of methionine (met) or MTR was adsorbed on paper discs and plates were incubated over-night at 37°C. Methionine was used as a control for growth and/or toxicity of the sulfur source.
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Related In: Results  -  Collection

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

Figure 7: Toxicity of MTR for BSHP7014 strain. Strain BSHP7014 (mtnW::lacZ amyE::pxyl mtnXYZ) was grown on ED1 minimal medium plates in the presence of sulfate as sulfur source (panel A) or in the absence of any added sulfur source (agar as sole sulfur source, panel B). Xylose was added to the medium in order to trigger the expression of mtnXYZ from the pxyl promoter. 10 μl of methionine (met) or MTR was adsorbed on paper discs and plates were incubated over-night at 37°C. Methionine was used as a control for growth and/or toxicity of the sulfur source.
Mentions: Two genes in the pathway are dispensable, mtnV and mtnX. The first one encodes a transaminase of which there are nine putative paralogs in the genome of B. subtilis (YwfG, AlaT, AspB, PatA, YhdR, YdfD, PatB, YisV, and HisC). In the same way, MtnX (YkrX) is a member of the phosphatase family pfam00702 ([13], Fig. 6), and therefore of a ubiquitous class of hydrolases (several phosphatase genes in particular are present in the genome of B. subtilis). This is likely to account for the lack of phenotype under our growth conditions. Inactivation of mtnZ provides only a very weak, residual growth on MTR. Inactivation of mtnK, mtnS, mtnY and mtnW result in resistance to 3F-MTR and lack of growth on MTR. Inactivation of mtnW with a polar effect on the distal genes (by insertion of a disrupting plasmid) has a phenotype similar to that of mtnY (i.e. lack of growth on MTR, and lack of influence of MTR on sulfate supplemented plates). In contrast, we discovered that MTR is toxic when the distal genes are present (when used as sole sulfur source or even in the presence of sulfate, see Fig. 7). Because of the weak phenotype of a mtnZ mutant and the absence of phenotype of a mtnX mutant, we can be confident that MtnY acts before MtnW (this is a regular feature in operons, where it is often observed that the more distal genes code for proteins acting in the more proximate steps of the pathway).

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