Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors

ABSTRACT Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-...

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Autores principales:	Henrik O’Brien, Joseph W. Alvin, Sanjay V. Menghani, Yamil Sanchez-Rosario, Koenraad Van Doorslaer, Michael D. L. Johnson
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	copper metal operator operon protein repressor Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1ef432d81d6945e6a0caeac264ac3ec9
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spelling	oai:doaj.org-article:1ef432d81d6945e6a0caeac264ac3ec92021-11-15T15:30:15ZRules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors10.1128/mSphere.00411-202379-5042https://doaj.org/article/1ef432d81d6945e6a0caeac264ac3ec92020-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00411-20https://doaj.org/toc/2379-5042ABSTRACT Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis. We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro. During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniae cop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs. IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.Henrik O’BrienJoseph W. AlvinSanjay V. MenghaniYamil Sanchez-RosarioKoenraad Van DoorslaerMichael D. L. JohnsonAmerican Society for MicrobiologyarticlecoppermetaloperatoroperonproteinrepressorMicrobiologyQR1-502ENmSphere, Vol 5, Iss 3 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	copper metal operator operon protein repressor Microbiology QR1-502
spellingShingle	copper metal operator operon protein repressor Microbiology QR1-502 Henrik O’Brien Joseph W. Alvin Sanjay V. Menghani Yamil Sanchez-Rosario Koenraad Van Doorslaer Michael D. L. Johnson Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors
description	ABSTRACT Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis. We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro. During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniae cop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs. IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.
format	article
author	Henrik O’Brien Joseph W. Alvin Sanjay V. Menghani Yamil Sanchez-Rosario Koenraad Van Doorslaer Michael D. L. Johnson
author_facet	Henrik O’Brien Joseph W. Alvin Sanjay V. Menghani Yamil Sanchez-Rosario Koenraad Van Doorslaer Michael D. L. Johnson
author_sort	Henrik O’Brien
title	Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors
title_short	Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors
title_full	Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors
title_fullStr	Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors
title_full_unstemmed	Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors
title_sort	rules of expansion: an updated consensus operator site for the copr-copy family of bacterial copper exporter system repressors
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/1ef432d81d6945e6a0caeac264ac3ec9
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Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors

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