Dimer Asymmetry and Light Activation Mechanism in Brucella Blue-Light Sensor Histidine Kinase

ABSTRACT The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus. Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain. IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.

Download Full-text

HAMP Domain Rotation and Tilting Movements Associated with Signal Transduction in the PhoQ Sensor Kinase

mBio ◽

10.1128/mbio.00616-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 29

Author(s):

Susana Matamouros ◽

Kyle R. Hager ◽

Samuel I. Miller

Keyword(s):

Histidine Kinase ◽

Kinase Activity ◽

Catalytic Domain ◽

Structural Information ◽

Mechanistic Model ◽

Full Length ◽

Physical Interaction ◽

Content Type ◽

Serovar Typhimurium ◽

Α Helix

ABSTRACTHAMP domains are α-helical coiled coils that often transduce signals from extracytoplasmic sensing domains to cytoplasmic domains. Limited structural information has resulted in hypotheses that specific HAMP helix movement changes downstream enzymatic activity. These hypotheses were tested by mutagenesis and cysteine cross-linking analysis of the PhoQ histidine kinase, essential for resistance to antimicrobial peptides in a variety of enteric pathogens. These results support a mechanistic model in which periplasmic signals which induce an activation state generate a rotational movement accompanied by a tilt in α-helix 1 which activates kinase activity. Biochemical data and a high-confidence model of the PhoQ cytoplasmic domain indicate a possible physical interaction of the HAMP domain with the catalytic domain as necessary for kinase repression. These results support a model of PhoQ activation in which changes in the periplasmic domain lead to conformational movements in the HAMP domain helices which disrupt interaction between the HAMP and the catalytic domains, thus promoting increased kinase activity.IMPORTANCEMost studies on the HAMP domain signaling states have been performed with chemoreceptors or the HAMP domain of Af1503. Full-length structures of the HAMP-containing histidine kinases VicK and CpxA or a hybrid between the HAMP domain of Af1503 and the EnvZ histidine kinase agree with the parallel four-helix bundle structure identified in Af1503 and provide snapshots of structural conformations experienced by HAMP domains. We took advantage of the fact that we can easily regulate the activation state of PhoQ histidine kinase to study its HAMP domain in the context of the full-length protein in living cells and provide biochemical evidence for different conformational states experienced bySalmonella entericaserovar Typhimurium PhoQ HAMP domain upon signaling.

Download Full-text

Full-Length Structure of a Sensor Histidine Kinase Pinpoints Coaxial Coiled Coils as Signal Transducers and Modulators

Structure ◽

10.1016/j.str.2013.04.024 ◽

2013 ◽

Vol 21 (7) ◽

pp. 1127-1136 ◽

Cited By ~ 121

Author(s):

Ralph P. Diensthuber ◽

Martin Bommer ◽

Tobias Gleichmann ◽

Andreas Möglich

Keyword(s):

Histidine Kinase ◽

Coiled Coils ◽

Full Length ◽

Signal Transducers ◽

Sensor Histidine Kinase

Download Full-text

Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.00995-15 ◽

2016 ◽

Vol 198 (8) ◽

pp. 1326-1336 ◽

Cited By ~ 16

Author(s):

Stephanie L. Kellogg ◽

Christopher J. Kristich

Keyword(s):

Cell Wall ◽

Enterococcus Faecalis ◽

Histidine Kinase ◽

Response Regulator ◽

Wall Stress ◽

Opportunistic Pathogen ◽

Content Type ◽

Cell Wall Stress ◽

Cephalosporin Resistance ◽

Sense And Respond

ABSTRACTBacteria use two-component signal transduction systems (TCSs) to sense and respond to environmental changes via a conserved phosphorelay between a sensor histidine kinase and its cognate response regulator. The opportunistic pathogenEnterococcus faecalisutilizes a TCS comprised of the histidine kinase CroS and the response regulator CroR to mediate resistance to cell wall stresses such as cephalosporin antibiotics, but the molecular details by which CroRS promotes cephalosporin resistance have not been elucidated. Here, we analyzed mutants ofE. faecaliscarrying substitutions in CroR and CroS to demonstrate that phosphorylated CroR drives resistance to cephalosporins, and that CroS exhibits kinase and phosphatase activities to control the level of CroR phosphorylationin vivo. Deletion ofcroSin various lineages ofE. faecalisrevealed a CroS-independent mechanism for CroR phosphorylation and led to the identification of a noncognate histidine kinase capable of influencing CroR (encoded byOG1RF_12162; here calledcisS). Further analysis of this TCS network revealed that both systems respond to cell wall stress.IMPORTANCETCSs allow bacteria to sense and respond to many different environmental conditions. The opportunistic pathogenEnterococcus faecalisutilizes the CroRS TCS to mediate resistance to cell wall stresses, including clinically relevant antibiotics such as cephalosporins and glycopeptides. In this study, we use genetic and biochemical means to investigate the relationship between CroRS signaling and cephalosporin resistance inE. faecaliscells. Through this, we uncovered a signaling network formed between the CroRS TCS and a previously uncharacterized TCS that also responds to cell wall stress. This study provides mechanistic insights into CroRS signaling and cephalosporin resistance inE. faecalis.

Download Full-text

Light Regulation of Swarming Motility in Pseudomonas syringae Integrates Signaling Pathways Mediated by a Bacteriophytochrome and a LOV Protein

mBio ◽

10.1128/mbio.00334-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 34

Author(s):

Liang Wu ◽

Regina S. McGrane ◽

Gwyn A. Beattie

Keyword(s):

Signaling Pathways ◽

Blue Light ◽

Pseudomonas Syringae ◽

Cross Talk ◽

Histidine Kinase ◽

Kinase Activity ◽

Red Light ◽

Swarming Motility ◽

Content Type ◽

Light Sensing

ABSTRACT The biological and regulatory roles of photosensory proteins are poorly understood for nonphotosynthetic bacteria. The foliar bacterial pathogen Pseudomonas syringae has three photosensory protein-encoding genes that are predicted to encode the blue-light-sensing LOV (light, oxygen, or voltage) histidine kinase (LOV-HK) and two red/far-red-light-sensing bacteriophytochromes, BphP1 and BphP2. We provide evidence that LOV-HK and BphP1 form an integrated network that regulates swarming motility in response to multiple light wavelengths. The swarming motility of P. syringae B728a deletion mutants indicated that LOV-HK positively regulates swarming motility in response to blue light and BphP1 negatively regulates swarming motility in response to red and far-red light. BphP2 does not detectably regulate swarming motility. The histidine kinase activity of each LOV-HK and BphP1 is required for this regulation based on the loss of complementation upon mutation of residues key to their kinase activity. Surprisingly, mutants lacking both lov and bphP1 were similar in motility to a bphP1 single mutant in blue light, indicating that the loss of bphP1 is epistatic to the loss of lov and also that BphP1 unexpectedly responds to blue light. Moreover, whereas expression of bphP1 did not alter motility under blue light in a bphP1 mutant, it reduced motility in a mutant lacking lov and bphP1, demonstrating that LOV-HK positively regulates motility by suppressing negative regulation by BphP1. These results are the first to show cross talk between the LOV protein and phytochrome signaling pathways in bacteria, and the similarity of this regulatory network to that of photoreceptors in plants suggests a possible common ancestry. IMPORTANCE Photosensory proteins enable organisms to perceive and respond to light. The biological and ecological roles of these proteins in nonphotosynthetic bacteria are largely unknown. This study discovered that a blue-light-sensing LOV (light, oxygen, or voltage) protein and a red/far-red-light-sensing bacteriophytochrome both regulate swarming motility in the foliar pathogen Pseudomonas syringae. These proteins form an integrated signaling network in which the bacteriophytochrome represses swarming motility in response to red, far-red, and blue light, and LOV positively regulates swarming motility by suppressing bacteriophytochrome-mediated blue-light signaling. This is the first example of cross talk between LOV and phytochrome signaling pathways in bacteria, which shows unexpected similarity to photoreceptor signaling in plants.

Download Full-text

The Sensor Histidine Kinase RgfC Affects Group B Streptococcal Virulence Factor Expression Independent of Its Response Regulator RgfA

Infection and Immunity ◽

10.1128/iai.02738-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1078-1088 ◽

Cited By ~ 7

Author(s):

Claire Gendrin ◽

Annalisa Lembo ◽

Christopher Whidbey ◽

Kellie Burnside ◽

Jessica Berry ◽

...

Keyword(s):

Histidine Kinase ◽

Response Regulator ◽

Group B Streptococci ◽

Altered Expression ◽

Content Type ◽

Sensor Histidine Kinase ◽

Opportunistic Bacteria ◽

Extracellular Matrix Components ◽

Group B

Group B streptococci (GBS;Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation inrgfAresults in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion ofrgfCin GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with thergfCmutant increased induction of proinflammatory signaling pathwaysin vivo. Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in thergfCmutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of thergfCmutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis.

Download Full-text

Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

mBio ◽

10.1128/mbio.02105-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 30

Author(s):

Felipe Trajtenberg ◽

Daniela Albanesi ◽

Natalia Ruétalo ◽

Horacio Botti ◽

Ariel E. Mechaly ◽

...

Keyword(s):

Signaling Pathways ◽

Histidine Kinase ◽

Response Regulator ◽

Activation Mechanism ◽

Response Regulators ◽

Content Type ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. IMPORTANCE The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways.

Download Full-text

Systems mapping for technology development in CBRN response

International Journal of Emergency Services ◽

10.1108/ijes-08-2017-0044 ◽

2018 ◽

Vol 7 (2) ◽

pp. 111-119 ◽

Cited By ~ 4

Author(s):

Graham Hancox ◽

Sue Hignett ◽

Hilary Pillin ◽

Spyros Kintzios ◽

Jyri Silmäri ◽

...

Keyword(s):

Blue Light ◽

Large Scale ◽

New Technologies ◽

Technology Development ◽

End Users ◽

Future Research ◽

Content Type ◽

Development Platform ◽

Gold Silver ◽

The Uk

Purpose The purpose of this paper is to develop an EU sociotechnical systems (STSs) map to represent a harmonised concept of operations (CONOPS) as a future development platform for technologies used in multi-services emergency responses to chemical, biological, radiological and nuclear (CBRN) incidents. Design/methodology/approach AcciMaps were developed to locate where technologies are currently used, and opportunities for new technologies. The AcciMaps were iteratively co-designed with end users (fire, ambulance, police and military) across three EU countries (the UK, Finland and Greece). Data were collected using document analysis and interviews with senior ranking (Gold or Silver Command level) representatives of the participating end users. Findings Despite differences in terminology and between service sectors, consensus was achieved for the command structures (Gold, Silver and Bronze), and Hot Zone responders (specialist blue light responders and blue light responders (BLR)). A control room was included as the communication spine. BLR activities were limited by their scope of practice and available equipment, for example, breathing apparatus. The harmonised EU AcciMap offers a high-level STSs map of CBRN response. Critical segments have been identified which offer opportunities for technology developments that can add value in terms of response capabilities (e.g. tag and trace). Originality/value A large scale major CBRN incident may need cross-border and cross-professional engagement where efficient interoperability is vital. This research is the first EU consensus of a STS map for CONOPS. It supports future research for technology development, e.g., detection and decontamination equipment design and use, communication, diagnosis and response technologies.

Download Full-text

Financial distress determinants among SMEs: empirical evidence from Sweden

Journal of Economic Studies ◽

10.1108/jes-01-2019-0030 ◽

2020 ◽

Vol 47 (3) ◽

pp. 547-560 ◽

Cited By ~ 1

Author(s):

Darush Yazdanfar ◽

Peter Öhman

Keyword(s):

Financial Crisis ◽

Financial Distress ◽

Large Scale ◽

Global Financial Crisis ◽

Binary Logistic Regression ◽

Data Availability ◽

Cross Sectional ◽

Data Set ◽

Content Type ◽

The Global Financial Crisis

PurposeThe purpose of this study is to empirically investigate determinants of financial distress among small and medium-sized enterprises (SMEs) during the global financial crisis and post-crisis periods.Design/methodology/approachSeveral statistical methods, including multiple binary logistic regression, were used to analyse a longitudinal cross-sectional panel data set of 3,865 Swedish SMEs operating in five industries over the 2008–2015 period.FindingsThe results suggest that financial distress is influenced by macroeconomic conditions (i.e. the global financial crisis) and, in particular, by various firm-specific characteristics (i.e. performance, financial leverage and financial distress in previous year). However, firm size and industry affiliation have no significant relationship with financial distress.Research limitationsDue to data availability, this study is limited to a sample of Swedish SMEs in five industries covering eight years. Further research could examine the generalizability of these findings by investigating other firms operating in other industries and other countries.Originality/valueThis study is the first to examine determinants of financial distress among SMEs operating in Sweden using data from a large-scale longitudinal cross-sectional database.

Download Full-text

FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants

Genome Biology ◽

10.1186/s13059-021-02288-0 ◽

2021 ◽

Vol 22 (1) ◽

Cited By ~ 2

Author(s):

Yanping Long ◽

Zhijian Liu ◽

Jinbu Jia ◽

Weipeng Mo ◽

Liang Fang ◽

...

Keyword(s):

Single Cell ◽

Cell Walls ◽

Large Scale ◽

Full Length ◽

Cell Level ◽

Root Cells ◽

Rna Profiling ◽

Different Types ◽

Long Read ◽

Single Nucleus

AbstractThe broad application of single-cell RNA profiling in plants has been hindered by the prerequisite of protoplasting that requires digesting the cell walls from different types of plant tissues. Here, we present a protoplasting-free approach, flsnRNA-seq, for large-scale full-length RNA profiling at a single-nucleus level in plants using isolated nuclei. Combined with 10x Genomics and Nanopore long-read sequencing, we validate the robustness of this approach in Arabidopsis root cells and the developing endosperm. Sequencing results demonstrate that it allows for uncovering alternative splicing and polyadenylation-related RNA isoform information at the single-cell level, which facilitates characterizing cell identities.

Download Full-text