(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in <named-content content-type="genus-species">Listeria monocytogenes</named-content>
ABSTRACT The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP...
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2020-08-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.01625-20 |
| _version_ | 1818931003928870912 |
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| author | Bret N. Peterson Megan K. M. Young Shukun Luo Jeffrey Wang Aaron T. Whiteley Joshua J. Woodward Liang Tong Jue D. Wang Daniel A. Portnoy |
| author_facet | Bret N. Peterson Megan K. M. Young Shukun Luo Jeffrey Wang Aaron T. Whiteley Joshua J. Woodward Liang Tong Jue D. Wang Daniel A. Portnoy |
| author_sort | Bret N. Peterson |
| collection | DOAJ |
| description | ABSTRACT The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenes. IMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes. Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation. |
| first_indexed | 2024-12-20T04:09:41Z |
| format | Article |
| id | doaj.art-fda165a1b7c14027812eb73e6e1bcba5 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-20T04:09:41Z |
| publishDate | 2020-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-fda165a1b7c14027812eb73e6e1bcba52022-12-21T19:53:56ZengAmerican Society for MicrobiologymBio2150-75112020-08-0111410.1128/mBio.01625-20(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in <named-content content-type="genus-species">Listeria monocytogenes</named-content>Bret N. Peterson0Megan K. M. Young1Shukun Luo2Jeffrey Wang3Aaron T. Whiteley4Joshua J. Woodward5Liang Tong6Jue D. Wang7Daniel A. Portnoy8Graduate Group in Microbiology, University of California, Berkeley, California, USADepartment of Bacteriology, University of Wisconsin, Madison, Wisconsin, USADepartment of Biological Sciences, Columbia University, New York, New York, USADepartment of Molecular and Cell Biology, University of California, Berkeley, California, USAGraduate Group in Infectious Diseases and Immunity, University of California, Berkeley, California, USADepartment of Microbiology, University of Washington, Seattle, Washington, USADepartment of Biological Sciences, Columbia University, New York, New York, USADepartment of Bacteriology, University of Wisconsin, Madison, Wisconsin, USADepartment of Molecular and Cell Biology, University of California, Berkeley, California, USAABSTRACT The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenes. IMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes. Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.https://journals.asm.org/doi/10.1128/mBio.01625-20bacteriacyclic dinucleotidestringent response |
| spellingShingle | Bret N. Peterson Megan K. M. Young Shukun Luo Jeffrey Wang Aaron T. Whiteley Joshua J. Woodward Liang Tong Jue D. Wang Daniel A. Portnoy (p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in <named-content content-type="genus-species">Listeria monocytogenes</named-content> mBio bacteria cyclic dinucleotide stringent response |
| title | (p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in <named-content content-type="genus-species">Listeria monocytogenes</named-content> |
| title_full | (p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in <named-content content-type="genus-species">Listeria monocytogenes</named-content> |
| title_fullStr | (p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in <named-content content-type="genus-species">Listeria monocytogenes</named-content> |
| title_full_unstemmed | (p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in <named-content content-type="genus-species">Listeria monocytogenes</named-content> |
| title_short | (p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in <named-content content-type="genus-species">Listeria monocytogenes</named-content> |
| title_sort | p ppgpp and c di amp homeostasis is controlled by cbpb in named content content type genus species listeria monocytogenes named content |
| topic | bacteria cyclic dinucleotide stringent response |
| url | https://journals.asm.org/doi/10.1128/mBio.01625-20 |
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