scholarly journals Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

2019 ◽  
Author(s):  
Henrik O’Brien ◽  
Joseph W. Alvin ◽  
Sanjay V. Menghani ◽  
Koenraad Van Doorslaer ◽  
Michael D. L. Johnson
Keyword(s):  
Gene Regulation ◽  
Streptococcus Pneumoniae ◽  
Dna Binding ◽  
Consensus Sequence ◽  
Copper Chaperone ◽  
Enterococcus Hirae ◽  
Bacterial Gene ◽  
Operator Sequence ◽  
Operator Site

ABSTRACTCopper 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 repression. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, these homologs have been characterized to bind a 10-base consensus sequence T/GACAnnTGTA. Here, we bioinformatically and empirically characterize these operator sites across species using S. pneumoniae CopY as a guide for binding. By examining the 21-base repeat operators for the pneumococcal cop operon and comparing binding of recombinant CopY to this, and the operator sites found in Enterococcus hirae, we show using biolayer interferometry that the T/GACAnnTGTA sequence is essential to binding, but it is not sufficient. We determine a more comprehensive S. pneumoniae CopY operator sequence to be RnYKACAAATGTARnY (where “R” is purine, “Y” is pyrimidine, and “K” is either G or T) binding with an affinity of 28 nM. We further propose that the cop operon operator consensus site of pneumococcal homologs be RnYKACAnnYGTARnY. This study illustrates the necessity to explore bacterial operator sites further to better understand bacterial gene regulation.

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

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Henrik O’Brien ◽  
Joseph W. Alvin ◽  
Sanjay V. Menghani ◽  
Yamil Sanchez-Rosario ◽  
Koenraad Van Doorslaer ◽  
...  
Keyword(s):  
Dna Binding ◽  
Consensus Sequence ◽  
Species Interaction ◽  
Copper Stress ◽  
Copper Chaperone ◽  
Binding Studies ◽  
Content Type ◽  
Operator Sequence ◽  
Export System

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.

scholarly journals Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding

2011 ◽  
Vol 13 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Trevor Siggers ◽  
Abraham B Chang ◽  
Ana Teixeira ◽  
Daniel Wong ◽  
Kevin J Williams ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Dna Binding ◽  
Specific Gene ◽  
Comprehensive Characterization

scholarly journals CodY of Streptococcus pneumoniae: Link between Nutritional Gene Regulation and Colonization

2007 ◽  
Vol 190 (2) ◽  
pp. 590-601 ◽  
Author(s):  
Wouter T. Hendriksen ◽  
Hester J. Bootsma ◽  
Silvia Estevão ◽  
Theo Hoogenboezem ◽  
Anne de Jong ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Nutritional Regulation ◽  
Mobility Shift ◽  
Gene And Protein Expression

ABSTRACT CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.

scholarly journals Characterization of the Alternative Sigma Factor σ54 and the Transcriptional Regulator FleQ of Legionella pneumophila, Which Are Both Involved in the Regulation Cascade of Flagellar Gene Expression

2004 ◽  
Vol 186 (9) ◽  
pp. 2540-2547 ◽  
Author(s):  
Sebastian Jacobi ◽  
Rüdiger Schade ◽  
Klaus Heuner
Keyword(s):  
Gene Regulation ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Consensus Sequence ◽  
Transcriptional Regulator ◽  
Alternative Sigma Factor ◽  
Flagellar Gene ◽  
Wild Type ◽  
Interaction Domain

ABSTRACT We cloned and analyzed Legionella pneumophila Corby homologs of rpoN (encoding σ54) and fleQ (encoding σ54 activator protein). Two other genes (fleR and pilR) whose products have a σ54 interaction domain were identified in the genome sequence of L. pneumophila. An rpoN mutant strain was nonflagellated and expressed very small amounts of the FlaA (flagellin) protein. Like the rpoN mutant, the fleQ mutant strain of L. pneumophila was also nonflagellated and expressed only small amounts of FlaA protein compared to the amounts expressed by the wild type. In this paper we show that the σ54 factor and the FleQ protein are involved in regulation of flagellar gene operons in L. pneumophila. RpoN and FleQ positively regulate the transcription of FliM and FleN, both of which have a σ54-dependent promoter consensus sequence. However, they seemed to be dispensable for transcription of flaA, fliA, or icmR. Our results confirmed a recently described model of the flagellar gene regulation cascade in L. pneumophila (K. Heuner and M. Steinert, Int. J. Med. Microbiol. 293:133-145, 2003). Flagellar gene regulation was found to be different from that of Enterobacteriaceae but seems to be comparable to that described for Pseudomonas or Vibrio spp.

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