Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

ABSTRACT Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) fro...

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Autores principales:	Daniel Woods, Sweta Vangaveti, Ikechukwu Egbanum, Allison M. Sweeney, Zhong Li, Valjean Bacot-Davis, Danielle S. LeSassier, Matthew Stanger, Gabrielle E. Hardison, Hongmin Li, Marlene Belfort, Christopher W. Lennon
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	conditional protein splicing DNA helicase intein mycobacteria Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b2f33535d95e4d2088e994a5737fc469
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spelling	oai:doaj.org-article:b2f33535d95e4d2088e994a5737fc4692021-11-15T15:56:44ZConditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria10.1128/mBio.01403-202150-7511https://doaj.org/article/b2f33535d95e4d2088e994a5737fc4692020-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01403-20https://doaj.org/toc/2150-7511ABSTRACT Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis. We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis. Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae. Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc. IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.Daniel WoodsSweta VangavetiIkechukwu EgbanumAllison M. SweeneyZhong LiValjean Bacot-DavisDanielle S. LeSassierMatthew StangerGabrielle E. HardisonHongmin LiMarlene BelfortChristopher W. LennonAmerican Society for Microbiologyarticleconditional protein splicingDNA helicaseinteinmycobacteriaMicrobiologyQR1-502ENmBio, Vol 11, Iss 4 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	conditional protein splicing DNA helicase intein mycobacteria Microbiology QR1-502
spellingShingle	conditional protein splicing DNA helicase intein mycobacteria Microbiology QR1-502 Daniel Woods Sweta Vangaveti Ikechukwu Egbanum Allison M. Sweeney Zhong Li Valjean Bacot-Davis Danielle S. LeSassier Matthew Stanger Gabrielle E. Hardison Hongmin Li Marlene Belfort Christopher W. Lennon Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria
description	ABSTRACT Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis. We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis. Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae. Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc. IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.
format	article
author	Daniel Woods Sweta Vangaveti Ikechukwu Egbanum Allison M. Sweeney Zhong Li Valjean Bacot-Davis Danielle S. LeSassier Matthew Stanger Gabrielle E. Hardison Hongmin Li Marlene Belfort Christopher W. Lennon
author_facet	Daniel Woods Sweta Vangaveti Ikechukwu Egbanum Allison M. Sweeney Zhong Li Valjean Bacot-Davis Danielle S. LeSassier Matthew Stanger Gabrielle E. Hardison Hongmin Li Marlene Belfort Christopher W. Lennon
author_sort	Daniel Woods
title	Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria
title_short	Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria
title_full	Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria
title_fullStr	Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria
title_full_unstemmed	Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria
title_sort	conditional dnab protein splicing is reversibly inhibited by zinc in mycobacteria
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/b2f33535d95e4d2088e994a5737fc469
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Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

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