Identification of a sensor histidine kinase (BfcK) controlling biofilm formation in Clostridium acetobutylicum

Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been dissected so far.Here,to identify potential genes controlling C.acetobutylicum biofilm formation,over 40 gene candidates associated with extracellular matrix,cell surface,cell signaling or gene transcription,were tried to be disrupted to examine their individual impact.A total of 25 disruptants were finally obtained over years of attempts,for which biofilm and relevant phenotypes were characterized.Most of these disruptants formed robust biofilm still,or suffered both growth and biofilm defect.Only a strain with a disrupted histidine kinase gene(CA_C2730,designated bfcK in this study)abolished biofilm formation without impairing cell growth or solvent production.Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels.The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase(encoded by CA_C0404)that might negatively regulate biofilm formation.Based on these findings,possible bfcK-mediated mechanisms for biofilm formation were proposed.This is a big step toward understanding the biofilm formation in C.acetobutylicum and will help further engineering of its biofilm-based industrial processes.

Control of rice ratooning ability by a nucleoredoxin that inhibits histidine kinase dimerization to attenuate cytokinin signaling in axillary buds

Rice ratooning,the fast outgrowth of dormant buds on stubble,is an important cropping practice in rice production.However,the low ratooning ability(RA)of most rice varieties restricts the application of this cost-efficient system,and the genetic basis of RA remains unknown.In this study,we dissected the genetic architecture of RA by a genome-wide association study in a natural rice population.Rice ratooning ability 3(RRA3),encoding a hitherto not characterized nucleoredoxin involved in reduction of disulfide bonds,was identified as the causal gene of a major locus controlling RA.Overexpression of RRA3 in rice significantly accelerated leaf senescence and reduced RA,whereas knockout of RRA3 significantly delayed leaf senescence and increased RA and ratoon yield.We demonstrated that RRA3 interacts with Oryza sativa histidine kinase 4(OHK4),a cytokinin receptor,and inhibits the dimerization of OHK4 through disulfide bond reduction.This inhibition ultimately led to decreased cytokinin signaling and reduced RA.In addition,variations in the RRA3 promoter were identified to be associated with RA.Introgression of a superior haplotype with weak expression of RRA3 into the elite rice variety Guichao 2 significantly increased RA and ratoon yield by 23.8%.Collectively,this study not only uncovers an undocumented regulatory mechanism of cytokinin signaling through de-dimerization of a histidine kinase receptor-but also provides an eximious gene with promising value for ratoon rice breeding.

Structure-Function Analysis of Arabidopsis thaliana Histidine Kinase AHK5 Bound to Its Cognate Phosphotransfer Protein AHP1

The multi-step phosphorelay （MSP） system defines a key signal transduction pathway in plants and many eukaryotes. In this system, external stimuli first lead to the activation of a histidine kinase, followed by transfer of a phosphoryl group from the receiver domain of the kinase （HKRD） to downstream, cytosolic phosphotransfer proteins （HPs）. In order to establish the determinants of specificity for this signaling relay system, we have solved the first crystal structure of a plant HKRD, AHK5RD, in complex with one of its cognate HPs, AHP1. AHP1 binds AHK5RD via a prominent hydrogen bond docking ridge and a hydrophobic patch. These features are conserved among all AHP proteins, but differ significantly from other structurally characterized prokaryotic and eukaryotic HPs. Surface plasmon resonance experiments show that AHK5RD binds to AHP1-3 with similar, micromolar affinity, consistent with the transient nature of this signaling complex. Our correlation of structural and functional data provide the first insight, at the atomic level as well as with quantitative affinity data, into the molecular recognition events governing the MSP in plants.

Structural Aspects of Multistep Phosphorelay- Mediated Signaling in Plants

The multistep phosphorelay （MSP） is a central signaling pathway in plants integrating a wide spectrum of hormonal and environmental inputs and controlling numerous developmental adaptations. For the thor- ough comprehension of the molecular mechanisms underlying the MSP-mediated signal recognition and transduction, the detailed structural characterization of individual members of the pathway is critical. In this review we describe and discuss the recently known crystal and nuclear magnetic resonance structures of proteins acting in MSP signaling in higher plants, focusing particularly on cytokinin and ethylene signaling in Arabidopsis thaliana. We discuss the range of functional aspects of available structural infor- mation including determination of ligand specificity, activation of the receptor via its autophosphorylaUon, and downstream signal transduction through the phosphorelay. We compare the plant structures with their bacterial counterparts and show that although the overall similarity is high, the differences in structural de- tails are frequent and functionally important. Finally, we discuss emerging knowledge on molecular recog- nition mechanisms in the MSP, and mention the latest findings regarding structural determinants of signaling specificity in the Arabidopsis MSP that could serve as a general model of this pathway in all higher plants.

OsHK3 is a crucial regulator of abscisic acid signaling involved in antioxidant defense in rice

In this study, the role of the rice （Oryza sativa L.） histidine kinase OsHK3 in abscisic acid （ABA）-induced antioxidant defense was investigated. Treatments with ABA, H2O2, and polyethylene glycol （PEG） induced the expression of OsHK3 in rice leaves, and H2O2 is required for ABA-induced increase in the expression of OsHK3 under water stress. Subcellular localization analysis showed that OsHK3 is located in the cytoplasm and the plasma membrane. The transient expression analysis and the transient RNA interference test in rice protoplasts showed that OsHK3 is required for ABA-induced upreguiation in the expression of antioxidant enzymes genes and the activities of antioxidant enzymes. Further analysis showed that OsHK3 functions upstream of the calcium/ calmodulin-dependent protein kinase OsDMI3 and the mitogen-activated protein kinase OsMPK1 to regulate the activities of antioxidant enzymes in ABA signaling. Moreover, OsHK3 was also shown to regulate the expression of nicotinamide adenine dinucleotide phosphate oxidase genes, OsrbohB and OsrbohE, and the production of H2O2 in ABA signaling. Our data indicate that OsHK3 play an important role in the regulation of ABA-induced antioxidant defense and in the feedback regulation of H2O2 production in ABA signaling.

Engineering a fumaric acid-responsive two-component biosensor for dynamic range improvement in Escherichia coli

Due to the selective permeability of the cytomembrane,high-yield fumaric acid strains form a steep difference between intra-and extracellular concentrations.Intracellular biosensors cannot detect the real concentration change of extracellular fumaric acid.To overcome this limitation,a two-component biosensor(TCB)that could respond to extracellular fumaric acid was designed based on the DcuS-DcuR two-component system.The two-component system consists of a histidine kinase(SK)and response regulator.SK is a transmembrane histidine kinase sensor that can detect concentration changes in extracellular compounds.To improve the dynamic range of the constructed fumaric acid TCB,we optimized the expression ratio and expression intensity of dcuS and dcuR.We found that the optimum expression ratio of dcuS:dcuR was 46:54.Under this ratio,the higher was the expression level,the greater the dynamic range.In addition,we modified the ATP-binding site on the DcuS,and the final dynamic range of the TCB reached 6.6-fold.Overall,the obtained fumaric acid-responsive TCB with a high dynamic range is reported for the first time,providing a synthetic biology tool for high-throughput screening and dynamic metabolic regulation of fumaric acid cell factories.