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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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Identification of the mtn region promoters by primer extension. A. Identification of the transcription start site of the mtnU operon. The size of the extended product is compared to a DNA-sequencing ladder of the mtnU promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. B. Identification of the transcription start site of the mtnV gene. The size of the extended product is compared to a DNA-sequencing ladder of the mtnV promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. C. Identification of the transcription start site of the mtnKS gene. The size of the extended product is compared to a DNA-sequencing ladder of the mtnKS promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. D. Identification of the transcription start site of the mtnWXYZ operon. The size of the extended product is compared to a DNA-sequencing ladder of the mtnWXYZ promoter region. Primer extension and sequencing reaction were performed with the same primer. Two +1 sites are marked by arrows. E. Sequences of the corresponding promoter regions. Promoter sites are in capital letters and underlined (-35 and -10 boxes), and the transcription start sites are indicated by broken arrows (+1).
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Figure 2: Identification of the mtn region promoters by primer extension. A. Identification of the transcription start site of the mtnU operon. The size of the extended product is compared to a DNA-sequencing ladder of the mtnU promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. B. Identification of the transcription start site of the mtnV gene. The size of the extended product is compared to a DNA-sequencing ladder of the mtnV promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. C. Identification of the transcription start site of the mtnKS gene. The size of the extended product is compared to a DNA-sequencing ladder of the mtnKS promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. D. Identification of the transcription start site of the mtnWXYZ operon. The size of the extended product is compared to a DNA-sequencing ladder of the mtnWXYZ promoter region. Primer extension and sequencing reaction were performed with the same primer. Two +1 sites are marked by arrows. E. Sequences of the corresponding promoter regions. Promoter sites are in capital letters and underlined (-35 and -10 boxes), and the transcription start sites are indicated by broken arrows (+1).

Mentions: Several genes in the region have been shown by Henkin and co-workers to be expressed from promoters regulated by the S-box attenuation system [7]. This is the case of mtnKS and mtnWXYZ transcription units. Some of the genes, however, are not regulated in this way. Expression of the mtnU and mtnV genes is not subject to that regulation since no S-box is present in their leader transcript. As shown in Figure 2A the promoter of mtnU is located 35 nt from the translation start point. Its start was found to lie 5 nt downstream from a putative -10 box identified in the sequence (TTAAAT). Upstream from this box separated by 18 nt is a -35 box (ATGATA) with sequence similar to the consensus sequence TTGACA that is typical of B. subtilis sigmaA-dependent promoters [11].


The methionine salvage pathway in Bacillus subtilis.

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

Identification of the mtn region promoters by primer extension. A. Identification of the transcription start site of the mtnU operon. The size of the extended product is compared to a DNA-sequencing ladder of the mtnU promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. B. Identification of the transcription start site of the mtnV gene. The size of the extended product is compared to a DNA-sequencing ladder of the mtnV promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. C. Identification of the transcription start site of the mtnKS gene. The size of the extended product is compared to a DNA-sequencing ladder of the mtnKS promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. D. Identification of the transcription start site of the mtnWXYZ operon. The size of the extended product is compared to a DNA-sequencing ladder of the mtnWXYZ promoter region. Primer extension and sequencing reaction were performed with the same primer. Two +1 sites are marked by arrows. E. Sequences of the corresponding promoter regions. Promoter sites are in capital letters and underlined (-35 and -10 boxes), and the transcription start sites are indicated by broken arrows (+1).
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Related In: Results  -  Collection

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

Figure 2: Identification of the mtn region promoters by primer extension. A. Identification of the transcription start site of the mtnU operon. The size of the extended product is compared to a DNA-sequencing ladder of the mtnU promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. B. Identification of the transcription start site of the mtnV gene. The size of the extended product is compared to a DNA-sequencing ladder of the mtnV promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. C. Identification of the transcription start site of the mtnKS gene. The size of the extended product is compared to a DNA-sequencing ladder of the mtnKS promoter region. Primer extension and sequencing reaction were performed with the same primer. The +1 site is marked by an arrow. D. Identification of the transcription start site of the mtnWXYZ operon. The size of the extended product is compared to a DNA-sequencing ladder of the mtnWXYZ promoter region. Primer extension and sequencing reaction were performed with the same primer. Two +1 sites are marked by arrows. E. Sequences of the corresponding promoter regions. Promoter sites are in capital letters and underlined (-35 and -10 boxes), and the transcription start sites are indicated by broken arrows (+1).
Mentions: Several genes in the region have been shown by Henkin and co-workers to be expressed from promoters regulated by the S-box attenuation system [7]. This is the case of mtnKS and mtnWXYZ transcription units. Some of the genes, however, are not regulated in this way. Expression of the mtnU and mtnV genes is not subject to that regulation since no S-box is present in their leader transcript. As shown in Figure 2A the promoter of mtnU is located 35 nt from the translation start point. Its start was found to lie 5 nt downstream from a putative -10 box identified in the sequence (TTAAAT). Upstream from this box separated by 18 nt is a -35 box (ATGATA) with sequence similar to the consensus sequence TTGACA that is typical of B. subtilis sigmaA-dependent promoters [11].

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