The identification of response regulator-specific binding sites reveals new roles of two-component systems in Bacillus cereus and closely related low-GC Gram-positives

ABSTRACT Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC50s) ranging from 1.9 to >500 μM and MICs ranging from 0.5 to >16 μg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4× MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC50s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 μg/ml). Fifteen of 24 compounds also caused hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.

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A survey of HK, HPt, and RR domains and their organization in two-component systems and phosphorelays proteins of organisms with fully sequenced genomes

10.7287/peerj.preprints.1267 ◽

2015 ◽

Author(s):

Baldiri Salvado ◽

Ester Vilaprinyo ◽

Albert Sorribas ◽

Rui Alves

Keyword(s):

Response Regulator ◽

Signal Transmission ◽

Pr Proteins ◽

Component Systems ◽

Two Component ◽

Domains Of Life ◽

Two Component Systems ◽

Operon Organization ◽

The Individual ◽

Selection Of

Two Component Systems and Phosphorelays (TCS/PR) are environmental signal transduction cascades in prokaryotes and, less frequently, in eukaryotes. The internal domain organization of proteins and the topology of TCS/PR cascades play an important role in shaping the responses of the circuits. It is thus important to maintain updated censuses of TCS/PR proteins in order to identify the various topologies used by nature and enable a systematic study of the dynamics associated with those topologies. To create such a census, we analyzed the proteomes of 7609 organisms from all domains of life with fully sequenced and annotated genomes. To begin, we survey each proteome searching for proteins containing domains that are associated with internal signal transmission within TCS/PR: Histidine Kinase (HK), Response Regulator (RR) and Histidine Phosphotranfer (HPt) domains, and analyze how these domains are arranged in the individual proteins. Then, we find all types of operon organization and calculate how much more likely are proteins that contain TCS/PR domains to be coded by neighboring genes than one would expect from the genome background of each organism. Finally, we analyze if the fusion of domains into single TCS/PR proteins is more frequently observed than one might expect from the background of each proteome. We find 50 alternative ways in which the HK, HPt, and RR domains are observed to organize into single proteins. In prokaryotes, TCS/PR coding genes tend to be clustered in operons. 90% of all proteins identified in this study contain just one of the three domains, while 8% of the remaining proteins combine one copy of an HK, a RR, and/or an HPt domain. In eukaryotes, 25% of all TCS/PR proteins have more than one domain. These results might have implications for how signals are internally transmitted within TCS/PR cascades. These implications could explain the selection of the various designs in alternative circumstances.

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The two-component system CopRS maintains femtomolar levels of free copper in the periplasm of Pseudomonas aeruginosa using a phosphatase-based mechanism

10.1101/2020.05.13.092544 ◽

2020 ◽

Author(s):

Lorena Novoa-Aponte ◽

Fernando C. Soncini ◽

José M. Argüello

Keyword(s):

Pseudomonas Aeruginosa ◽

Phosphatase Activity ◽

Response Regulator ◽

Redox Enzymes ◽

Two Component System ◽

Component System ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Systems Control

ABSTRACTTwo component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing domain of CusS, a cytoplasmic phosphotransfer domain phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains a non-phosphorylated CopR when the periplasmic Cu levels are below its activation threshold. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the ΔcopS strain showed constitutive high expression of the CopRS regulon, lower intracellular Cu+ levels, and larger Cu tolerance when compared to wild type cells. The invariant phospho-acceptor residue His235 of CopS was not required for the phosphatase activity itself, but necessary for its Cu-dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly explains the different binding stoichiometries in both systems. Interestingly, CopS binds Cu+/2+ with 30 × 10−15 M affinities, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm.IMPORTANCECopper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins, and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two component systems control the periplasmic response to metal overload. This manuscript shows that the copper sensing two component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of the response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the sensor is activated by copper levels compatible with the absence of free copper in the cell periplasm. This emphasizes the diversity of molecular mechanisms that have evolved in various bacteria to manage the copper cellular distribution.

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Molecular Characterization of Two-Component Systems of Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.182.8.2068-2076.2000 ◽

2000 ◽

Vol 182 (8) ◽

pp. 2068-2076 ◽

Cited By ~ 126

Author(s):

Dagmar Beier ◽

Rainer Frank

Keyword(s):

Helicobacter Pylori ◽

Histidine Kinase ◽

Response Regulator ◽

Response Regulators ◽

Reading Frame ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACT Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation. Here we report the characterization of members of the two-component systems of the gastric pathogenHelicobacter pylori deduced from the genome sequence of strain 26695. We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains. An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase-response regulator pairs. Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3′ end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165.

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Unique Patterns and Biogeochemical Relevance of Two-Component Sensing in Marine Bacteria

mSystems ◽

10.1128/msystems.00317-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 7

Author(s):

Noelle A. Held ◽

Matthew R. McIlvin ◽

Dawn M. Moran ◽

Michael T. Laub ◽

Mak A. Saito

Keyword(s):

Marine Environment ◽

Marine Bacteria ◽

Response Regulator ◽

Bacterial Species ◽

South Pacific ◽

Response Regulators ◽

Orphan Genes ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACTTwo-component sensory (TCS) systems link microbial physiology to the environment and thus may play key roles in biogeochemical cycles. In this study, we surveyed the TCS systems of 328 diverse marine bacterial species. We identified lifestyle traits such as copiotrophy and diazotrophy that are associated with larger numbers of TCS system genes within the genome. We compared marine bacterial species with 1,152 reference bacterial species from a variety of habitats and found evidence of extra response regulators in marine genomes. Examining the location of TCS genes along the circular bacterial genome, we also found that marine bacteria have a large number of “orphan” genes, as well as many hybrid histidine kinases. The prevalence of “extra” response regulators, orphan genes, and hybrid TCS systems suggests that marine bacteria break with traditional understanding of how TCS systems operate. These trends suggest prevalent regulatory networking, which may allow coordinated physiological responses to multiple environmental signals and may represent a specific adaptation to the marine environment. We examine phylogenetic and lifestyle traits that influence the number and structure of two-component systems in the genome, finding, for example, that a lack of two-component systems is a hallmark of oligotrophy. Finally, in an effort to demonstrate the importance of TCS systems to marine biogeochemistry, we examined the distribution ofProchlorococcus/Synechococcusresponse regulator PMT9312_0717 in metaproteomes of the tropical South Pacific. We found that this protein’s abundance is related to phosphate concentrations, consistent with a putative role in phosphate regulation.IMPORTANCEMarine microbes must manage variation in their chemical, physical, and biological surroundings. Because they directly link bacterial physiology to environmental changes, TCS systems are crucial to the bacterial cell. This study surveyed TCS systems in a large number of marine bacteria and identified key phylogenetic and lifestyle patterns in environmental sensing. We found evidence that, in comparison with bacteria as a whole, marine organisms have irregular TCS system constructs which might represent an adaptation specific to the marine environment. Additionally, we demonstrate the biogeochemical relevance of TCS systems by correlating the presence of the PMT9312_0717 response regulator protein to phosphate concentrations in the South Pacific. We highlight that despite their potential ecological and biogeochemical relevance, TCS systems have been understudied in the marine ecosystem. This report expands our understanding of the breadth of bacterial TCS systems and how marine bacteria have adapted to survive in their unique environment.

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Insights into sRNA Genes Regulated by Two-Component Systems in the Bacillus cereus Group

Current Bioinformatics ◽

10.2174/157489361004150922151028 ◽

2015 ◽

Vol 10 (4) ◽

pp. 456-468 ◽

Cited By ~ 2

Author(s):

Han Mei ◽

Qing Tang ◽

Xinfeng Li ◽

Yaxi Wang ◽

Jieping Wang ◽

...

Keyword(s):

Bacillus Cereus ◽

Component Systems ◽

Two Component ◽

Two Component Systems

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Interactions between the YycFG and PhoPR two-component systems in Bacillus subtilis: the PhoR kinase phosphorylates the non-cognate YycF response regulator upon phosphate limitation

Molecular Microbiology ◽

10.1111/j.1365-2958.2006.05071.x ◽

2006 ◽

Vol 60 (2) ◽

pp. 535-535 ◽

Cited By ~ 2

Author(s):

Alistair Howell ◽

Sarah Dubrac ◽

David Noone ◽

Kottayil I. Varughese ◽

Kevin Devine

Keyword(s):

Bacillus Subtilis ◽

Response Regulator ◽

Phosphate Limitation ◽

Component Systems ◽

Two Component ◽

Two Component Systems

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Novel Two-Component System-Like Elements Reveal Functional Domains Associated with Restriction–Modification Systems and paraMORC ATPases in Bacteria

Genome Biology and Evolution ◽

10.1093/gbe/evab024 ◽

2021 ◽

Vol 13 (3) ◽

Author(s):

Daniel Bellieny-Rabelo ◽

Willem J S Pretorius ◽

Lucy N Moleleki

Keyword(s):

Response Regulator ◽

Structural Similarity ◽

Structural Features ◽

Bacterial Cells ◽

Bacterial Strains ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Genomic Regions ◽

Restriction Modification

Abstract Two-component systems (TCS) are important types of machinery allowing for efficient signal recognition and transmission in bacterial cells. The majority of TCSs utilized by bacteria is composed of a sensor histidine kinase (HK) and a cognate response regulator (RR). In the present study, we report two newly predicted protein domains—both to be included in the next release of the Pfam database: Response_reg_2 (PF19192) and HEF_HK (PF19191)—in bacteria which exhibit high structural similarity, respectively, with typical domains of RRs and HKs. Additionally, the genes encoding for the novel predicted domains exhibit a 91.6% linkage observed across 644 genomic regions recovered from 628 different bacterial strains. The remarkable adjacent colocalization between genes carrying Response_reg_2 and HEF_HK in addition to their conserved structural features, which are highly similar to those from well-known HKs and RRs, raises the possibility of Response_reg_2 and HEF_HK constituting a new TCS in bacteria. The genomic regions in which these predicted two-component systems-like are located additionally exhibit an overrepresented presence of restriction–modification (R–M) systems especially the type II R–M. Among these, there is a conspicuous presence of C-5 cytosine-specific DNA methylases which may indicate a functional association with the newly discovered domains. The solid presence of R–M systems and the presence of the GHKL family domain HATPase_c_3 across most of the HEF_HK-containing genes are also indicative that these genes are evolutionarily related to the paraMORC family of ATPases.

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Accurate prediction of bacterial two-component signaling with a deep recurrent neural network ORAKLE

10.1101/532721 ◽

2019 ◽

Author(s):

Jan Balewski ◽

Zachary F. Hallberg

Keyword(s):

Neural Network ◽

Amino Acid ◽

Response Regulator ◽

Amino Acid Sequences ◽

Histidine Kinases ◽

Deep Recurrent Neural Network ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Recommender Algorithm

AbstractTwo-component systems (2CS) are a primary method that bacteria use to detect and respond to environmental stimuli. Receptor histidine kinases (HK) detect an environmental signal, activating the appropriate response regulator (RR). Genes for such cognate HK-RR pairs are often located proximally on the chromosome, allowing easier identification of the target for a particular signal. However, almost half of all HK and RR proteins are orphans, with no nearby partner, complicating identification of the proteins that respond to a particular signal. To address this problem, we trained a neural network on the amino acid sequences of known 2CS pairs. Next, we developed a recommender algorithm that ranks a set of HKs for an arbitrary fixed RR and arbitrary species whose amino acid sequences are known. The recommender strongly favors known 2CS pairs, and correctly selects orphan pairs in Escherichia coli. We expect that use of these results will permit rapid discovery of orphan HK-RR pairs.

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Dynamics and activation in response regulators: the β4-α4 loop

BioMolecular Concepts ◽

10.1515/bmc-2011-0063 ◽

2012 ◽

Vol 3 (2) ◽

pp. 175-182 ◽

Cited By ~ 3

Author(s):

Benjamin G. Bobay ◽

James A. Hoch ◽

John Cavanagh

Keyword(s):

Response Regulator ◽

Short Review ◽

Phosphoryl Group ◽

Sensor Kinase ◽

Response Regulators ◽

Component Systems ◽

Two Component ◽

Phosphorylation Event ◽

Primary Means ◽

Two Component Systems

AbstractTwo-component signal transduction systems of microbes are a primary means to respond to signals emanating from environmental and metabolic fluctuations as well as to signals coordinating the cell cycle with macromolecular syntheses, among a large variety of other essential roles. Signals are recognized by a sensor domain of a histidine kinase which serves to convert signal binding to an active transmissible phosphoryl group through a signal-induced ATP-dependent autophosphorylation reaction directed to histidine residue. The sensor kinase is specifically mated to a response regulator, to which it transfers the phosphoryl group that activates the response regulator’s function, most commonly gene repression or activation but also interaction with other regulatory proteins. Two-component systems have been genetically amplified to control a wide variety of cellular processes; for example, both Escherichia coli and Pseudomonas aeruginosa have 60 plus confirmed and putative two-component systems. Bacillus subtilis has 30 plus and Nostoc punctiformis over 100. As genetic amplification does not result in changes in the basic structural folds of the catalytic domains of the sensor kinase or response regulators, each sensor kinase must recognize its partner through subtle changes in residues at the interaction surface between the two proteins. Additionally, the response regulator must prepare itself for efficient activation by the phosphorylation event. In this short review, we discuss the contributions of the critical β4-α4 recognition loop in response regulators to their function. In particular, we focus on this region’s microsecond-millisecond timescale dynamics propensities and discuss how these motions play a major role in response regulator recognition and activation.

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