scholarly journals Deciphering the activation and recognition mechanisms of Staphylococcus aureus response regulator ArlR

2019 ◽  
Vol 47 (21) ◽  
pp. 11418-11429 ◽  
Author(s):  
Zhenlin Ouyang ◽  
Fang Zheng ◽  
Jared Y Chew ◽  
Yingmei Pei ◽  
Jinhong Zhou ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Crystal Structures ◽  
Response Regulator ◽  
Regulatory System ◽  
Repeat Sequence ◽  
Effector Domain ◽  
Receiver Domain ◽  
Regulation Mechanisms ◽  
Significant Conformational Change

Abstract Staphylococcus aureus ArlRS is a key two-component regulatory system necessary for adhesion, biofilm formation, and virulence. The response regulator ArlR consists of a C-terminal DNA-binding effector domain and an N-terminal receiver domain that is phosphorylated by ArlS, the cognate transmembrane sensor histidine kinase. We demonstrate that the receiver domain of ArlR adopts the canonical α5β5 response regulator assembly, which dimerizes upon activation, using beryllium trifluoride as an aspartate phosphorylation mimic. Activated ArlR recognizes a 20-bp imperfect inverted repeat sequence in the ica operon, which is involved in intercellular adhesion polysaccharide production. Crystal structures of the inactive and activated forms reveal that activation induces a significant conformational change in the β4-α4 and β5-α5-connecting loops, in which the α4 and α5 helices constitute the homodimerization interface. Crystal structures of the DNA-binding ArlR effector domain indicate that it is able to dimerize via a non-canonical β1–β2 hairpin domain swapping, raising the possibility of a new mechanism for signal transduction from the receiver domain to effector domain. Taken together, the current study provides structural insights into the activation of ArlR and its recognition, adding to the diversity of response regulation mechanisms that may inspire novel antimicrobial strategies specifically targeting Staphylococcus.

scholarly journals In the Staphylococcus aureus Two-Component System sae, the Response Regulator SaeR Binds to a Direct Repeat Sequence and DNA Binding Requires Phosphorylation by the Sensor Kinase SaeS

2010 ◽  
Vol 192 (8) ◽  
pp. 2111-2127 ◽  
Author(s):  
Fei Sun ◽  
Chunling Li ◽  
Dowon Jeong ◽  
Changmo Sohn ◽  
Chuan He ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Response Regulator ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
Sensor Kinase ◽  
Two Component System ◽  
Component System ◽  
Direct Repeat Sequence ◽  
Two Component

ABSTRACT Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length SaeR could be restored by sensor kinase SaeS-induced phosphorylation. Phosphorylated SaeR is more resistant to digestion by trypsin, suggesting conformational changes. DNase I footprinting assays revealed that the SaeR protection region in the P1 promoter contains a direct repeat sequence (GTTAAN6GTTAA [where N is any nucleotide]). This sequence is critical to the binding of phosphorylated SaeR. Mutational changes in the repeat sequence greatly reduced both the in vitro binding of SaeR and the in vivo function of the P1 promoter. From these results, we concluded that SaeR recognizes the direct repeat sequence as a binding site and that binding requires phosphorylation by SaeS.

Structure of the Acinetobacter baumannii PmrA receiver domain and insights into clinical mutants affecting DNA-binding and promoting colistin resistance

2021 ◽  
Author(s):  
Samantha Palethorpe ◽  
Morgan E Milton ◽  
Everett C Pesci ◽  
John Cavanagh
Keyword(s):  
Dna Binding ◽  
Acinetobacter Baumannii ◽  
Structural Information ◽  
Response Regulator ◽  
Regulatory System ◽  
Nosocomial Pathogen ◽  
Colistin Resistance ◽  
X Ray ◽  
X Ray Crystallography ◽  
Receiver Domain

Abstract Acinetobacter baumannii is an insidious emerging nosocomial pathogen that has developed resistance to all available antimicrobials, including the last resort antibiotic, colistin. Colistin resistance often occurs due to mutations in the PmrAB two component regulatory system. To better understand the regulatory mechanisms contributing to colistin resistance, we have biochemically characterized the A. baumannii PmrA response regulator. Initial DNA-binding analysis shows that A. baumannii PmrA bound to the Klebsiella pneumoniae PmrA box motif. This prompted analysis of the putative A. baumannii PmrAB regulon which indicated that the A. baumannii PmrA consensus box is 5′- HTTAAD N5 HTTAAD. Additionally, we provide the first structural information for the A. baumannii PmrA N-terminal domain through X-ray crystallography, and we present a full-length model using molecular modeling. From these studies, we were able to infer the effects of two critical PmrA mutations, PmrA::I13M and PmrA::P102R, both of which confer increased colistin resistance. Based on these data, we suggest structural and dynamic reasons for how these mutations can affect PmrA function and hence encourage resistive traits. Understanding these mechanisms will aid in the development of new targeted antimicrobial therapies.

scholarly journals Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

2005 ◽  
Vol 187 (5) ◽  
pp. 1833-1844 ◽  
Author(s):  
Martin K. Safo ◽  
Qixun Zhao ◽  
Tzu-Ping Ko ◽  
Faik N. Musayev ◽  
Howard Robinson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Crystal Structures ◽  
Double Helix ◽  
Free Form ◽  
Binding Motif ◽  
Dimerization Domain ◽  
Gene Encoding ◽  
Dna Binding Sites ◽  
Dna Double Helix

ABSTRACT The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding β-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to β-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when β-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0- and 2.7-Å resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix α3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.

scholarly journals The DNA-binding domain of the Escherichia coli CpxR two-component response regulator is constitutively active and cannot be fully attenuated by fused adjacent heterologous regulatory domains

Microbiology ◽  
2006 ◽  
Vol 152 (2) ◽  
pp. 431-441 ◽  
Author(s):  
Caroline Tapparel ◽  
Antoinette Monod ◽  
William L. Kelley
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Environmental Changes ◽  
Response Regulator ◽  
Effector Domain ◽  
Regulatory Domains ◽  
Rapid Responses ◽  
Two Component ◽  
Two Component Systems ◽  
Constitutively Active

Two-component systems (TCS) based on a sensor histidine kinase and a phosphorylated cognate target regulator allow rapid responses to environmental changes. TCS are highly evolutionarily conserved, though in only a few cases are the inducing signals understood. This study focuses on the Escherichia coli CpxR response regulator that responds to periplasmic and outer-membrane stress. N-terminal deletion mutations have been isolated that render the transcription factor constitutively active, indicating that the N terminus functions, in part, to keep the C-terminal winged-helix DNA-binding effector domain in an inactive state. Analysis of truncations spanning the CpxR interdomain region revealed that mutants retaining the α5 helix significantly augment activation. Hybrid proteins obtained by fusing the CpxR effector domain to structurally similar heterologous N-terminal regulatory domains, or even GFP, failed to restore repression to the C-terminal domain. These findings shed light on the mechanism of CpxR effector domain activation and on the investigation of constitutive mutants obtained by truncation in other TCS.

Structure of the DNA-binding domain of the response regulator SaeR fromStaphylococcus aureus

2015 ◽  
Vol 71 (8) ◽  
pp. 1768-1776 ◽  
Author(s):  
Xiaojiao Fan ◽  
Xu Zhang ◽  
Yuwei Zhu ◽  
Liwen Niu ◽  
Maikun Teng ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Response Regulator ◽  
Regulatory System ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Promoter Regions ◽  
Winged Helix

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors inStaphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeRDBD) was solved at 2.5 Å resolution. The structure reveals that SaeRDBDexists as a monomer and has the canonical winged helix–turn–helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix–turn–helix module were verified to be important for binding to the P1 promoterin vitroand for the physiological function of SaeRin vivo.

scholarly journals The Syringate O-Demethylase Gene of Sphingobium sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate Catabolism Genes Are Regulated by DesR

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Takuma Araki ◽  
Kenta Tanatani ◽  
Naofumi Kamimura ◽  
Yuichiro Otsuka ◽  
Muneyoshi Yamaguchi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Aromatic Compounds ◽  
Regulatory System ◽  
Value Added ◽  
Transcriptional Regulator ◽  
Inducible Promoter ◽  
Repeat Sequence ◽  
Chemical Degradation ◽  
Lignin Biodegradation ◽  
Rt Pcr

ABSTRACT Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while those of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here, we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA. Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicated that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB. The ferulate catabolism genes (ferBA), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of desA. RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX. IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (encoding a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6.

scholarly journals Mutated Response Regulator graR Is Responsible for Phenotypic Conversion of Staphylococcus aureus from Heterogeneous Vancomycin-Intermediate Resistance to Vancomycin-Intermediate Resistance

2007 ◽  
Vol 52 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Hui-min Neoh ◽  
Longzhu Cui ◽  
Harumi Yuzawa ◽  
Fumihiko Takeuchi ◽  
Miki Matsuo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Response Regulator ◽  
Regulatory System ◽  
Vancomycin Resistance ◽  
Whole Genome ◽  
Two Component ◽  
Intermediate Resistance ◽  
Mrsa Strains

ABSTRACT Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR, designated graR*, but not intact graR, designated graRn, could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR* expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR* increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.

Dimerization and DNA binding of the Salmonella enterica PhoP response regulator are phosphorylation independent

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 3979-3987 ◽  
Author(s):  
Philippe Perron-Savard ◽  
Gregory De Crescenzo ◽  
Hervé Le Moual
Keyword(s):  
Dna Binding ◽  
Salmonella Enterica ◽  
Response Regulator ◽  
Gel Filtration ◽  
Regulatory System ◽  
Tryptophan Fluorescence ◽  
Biochemical Properties ◽  
Wild Type ◽  
His Tag ◽  
Target Dna

In Salmonella enterica, PhoP is the response regulator of the PhoP/PhoQ two-component regulatory system that controls the expression of various virulence factors in response to external Mg2+. Previous studies have shown that phosphorylation of a PhoP variant with a C-terminal His tag (PhoPHis) enhances dimerization and binding to target DNA. Here, the effect of phosphorylation on the oligomerization and DNA binding properties of both wild-type PhoP (PhoP) and PhoPHis are compared. Gel filtration chromatography showed that PhoP exists as a mixture of monomer and dimer regardless of its phosphorylation state. In contrast, unphosphorylated PhoPHis was mostly monomeric, whereas PhoPHis∼P existed as a mixture of monomer and dimer. By monitoring the tryptophan fluorescence of the proteins and the fluorescence of the probe 1-anilinonaphthalene-8-sulfonic acid bound to them, it was found that PhoP and PhoPHis exhibited different spectral properties. The interaction between PhoP or PhoPHis and the PhoP box of the mgtA promoter was monitored by surface plasmon resonance. Binding of PhoP to the PhoP box was barely influenced by phosphorylation. In contrast, phosphorylation of PhoPHis clearly increased the interaction of PhoPHis with target DNA. Altogether, these data show that a His tag at the C-terminus of PhoP affects its biochemical properties, most likely by affecting its conformation and/or its oligomerization state. More importantly, these results show that wild-type PhoP dimerization and interaction with target DNA are independent of phosphorylation, which is in contrast to the previously proposed model.

scholarly journals Crystal Structures of the Receiver Domain of the Response Regulator PhoP from Escherichia coli in the Absence and Presence of the Phosphoryl Analog Beryllofluoride

2007 ◽  
Vol 189 (16) ◽  
pp. 5987-5995 ◽  
Author(s):  
Priti Bachhawat ◽  
Ann M. Stock
Keyword(s):  
Escherichia Coli ◽  
Crystal Structures ◽  
Response Regulator ◽  
Active State ◽  
Two Component System ◽  
Component System ◽  
Regulatory Domains ◽  
Receiver Domain ◽  
Two Component ◽  
High Concentrations

ABSTRACT The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg2+. Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation.

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