Protein secretion zones during overexpression of amylase within the Gram-positive cell wall
Abstract Background Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE with...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-10-01
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| Series: | BMC Biology |
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| Online Access: | https://doi.org/10.1186/s12915-023-01684-1 |
| _version_ | 1797556479103860736 |
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| author | Manuel Strach Felicitas Koch Svenja Fiedler Klaus Liebeton Peter L. Graumann |
| author_facet | Manuel Strach Felicitas Koch Svenja Fiedler Klaus Liebeton Peter L. Graumann |
| author_sort | Manuel Strach |
| collection | DOAJ |
| description | Abstract Background Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis. Results We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term “secretion zones”. Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system. Conclusions Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus. |
| first_indexed | 2024-03-10T17:02:27Z |
| format | Article |
| id | doaj.art-f8d78df25cb7400cb30879aff810c7c3 |
| institution | Directory Open Access Journal |
| issn | 1741-7007 |
| language | English |
| last_indexed | 2024-03-10T17:02:27Z |
| publishDate | 2023-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biology |
| spelling | doaj.art-f8d78df25cb7400cb30879aff810c7c32023-11-20T10:55:44ZengBMCBMC Biology1741-70072023-10-0121112210.1186/s12915-023-01684-1Protein secretion zones during overexpression of amylase within the Gram-positive cell wallManuel Strach0Felicitas Koch1Svenja Fiedler2Klaus Liebeton3Peter L. Graumann4Centre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität MarburgCentre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität MarburgCentre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität MarburgBRAIN Biotech AGCentre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität MarburgAbstract Background Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis. Results We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term “secretion zones”. Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system. Conclusions Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus.https://doi.org/10.1186/s12915-023-01684-1Protein secretionGram-positive cell wallAmylaseSecABacterial cell biology |
| spellingShingle | Manuel Strach Felicitas Koch Svenja Fiedler Klaus Liebeton Peter L. Graumann Protein secretion zones during overexpression of amylase within the Gram-positive cell wall BMC Biology Protein secretion Gram-positive cell wall Amylase SecA Bacterial cell biology |
| title | Protein secretion zones during overexpression of amylase within the Gram-positive cell wall |
| title_full | Protein secretion zones during overexpression of amylase within the Gram-positive cell wall |
| title_fullStr | Protein secretion zones during overexpression of amylase within the Gram-positive cell wall |
| title_full_unstemmed | Protein secretion zones during overexpression of amylase within the Gram-positive cell wall |
| title_short | Protein secretion zones during overexpression of amylase within the Gram-positive cell wall |
| title_sort | protein secretion zones during overexpression of amylase within the gram positive cell wall |
| topic | Protein secretion Gram-positive cell wall Amylase SecA Bacterial cell biology |
| url | https://doi.org/10.1186/s12915-023-01684-1 |
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