Sequence analysis and interposon mutagenesis of a sensor-kinase (DctS) and response-regulator (DctR) controlling synthesis of the high-affinity C4-dicarboxylate transport system in Rhodobacter capsulatus

1993 ◽  
Vol 237-237 (1-2) ◽  
pp. 215-224 ◽  
Author(s):  
Mark J. Hamblin ◽  
Jonathan G. Shaw ◽  
David J. Kelly
Keyword(s):  
Sequence Analysis ◽  
Transport System ◽  
Rhodobacter Capsulatus ◽  
Response Regulator ◽  
Sensor Kinase ◽  
High Affinity ◽  
Dicarboxylate Transport ◽  
Interposon Mutagenesis

scholarly journals Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

2000 ◽  
Vol 66 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Lina M. Botero ◽  
Thamir S. Al-Niemi ◽  
Timothy R. McDermott
Keyword(s):  
Transport System ◽  
Response Regulator ◽  
Insertion Site ◽  
Dry Weight ◽  
Type Systems ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Atpase Inhibitor ◽  
Rhizobium Tropici ◽  
High Affinity

ABSTRACT Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropiciassociation is sensitive to the availability of phosphate (Pi). To better understand phosphorus movement between the bacteroid and the host plant, Pi transport was characterized in R. tropici. We observed two Pitransport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. TheKm and V max values for the low-affinity system were estimated to be 34 ± 3 μM Pi and 118 ± 8 nmol of Pi · min−1 · mg (dry weight) of cells−1, respectively, and the Km andV max values for the high-affinity system were 0.45 ± 0.01 μM Pi and 86 ± 5 nmol of Pi · min−1 · mg (dry weight) of cells−1, respectively. Both systems were inducible by Pi starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but Pi transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the Pi transport rate was correlated with the intracellular ATP concentration. Also, Pi movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both Pi transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology withkdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.

scholarly journals Identification and Characterization of a Two-Component Sensor-Kinase and Response-Regulator System (DcuS-DcuR) Controlling Gene Expression in Response to C4-Dicarboxylates in Escherichia coli

1999 ◽  
Vol 181 (4) ◽  
pp. 1238-1248 ◽  
Author(s):  
Paul Golby ◽  
Suzanne Davies ◽  
David J. Kelly ◽  
John R. Guest ◽  
Simon C. Andrews
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Carbon Sources ◽  
Receptor Protein ◽  
Growth Defect ◽  
Sensor Kinase ◽  
Triple Mutant ◽  
E Coli ◽  
Dicarboxylate Transport ◽  
Two Component

ABSTRACT The dcuB gene of Escherichia coli encodes an anaerobic C4-dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition,dcuB expression is strongly induced by C4-dicarboxylates, suggesting the presence of a novel C4-dicarboxylate-responsive regulator in E. coli. This paper describes the isolation of a Tn10mutant in which the 160-fold induction of dcuB expression by C4-dicarboxylates is absent. The corresponding Tn10 mutation resides in the yjdH gene, which is adjacent to the yjdG gene and close to thedcuB gene at ∼93.5 min in the E. colichromosome. The yjdHG genes (redesignateddcuSR) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the dcuSR operon restored the C4-dicarboxylate inducibility of dcuB expression in the dcuSmutant to levels exceeding those of the dcuS +strain by approximately 1.8-fold. The dcuS mutation affected the expression of other genes with roles in C4-dicarboxylate transport or metabolism. Expression of the fumarate reductase (frdABCD) operon and the aerobic C4-dicarboxylate transporter (dctA) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C4-dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the dcuS mutant was normal. However, under aerobic conditions with C4-dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a dctA mutant. Studies employing a dcuA dcuB dcuC triple mutant unable to transport C4-dicarboxylates anaerobically revealed that C4-dicarboxylate transport is not required for C4-dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a ∼140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C4-dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of Klebsiella pneumoniae. DcuS is not closely related to the C4-dicarboxylate-sensing DctS or DctB protein ofRhodobacter capsulatus or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.

scholarly journals Overlapping and Specialized Functions of the Molybdenum-Dependent Regulators MopA and MopB in Rhodobacter capsulatus

2006 ◽  
Vol 188 (24) ◽  
pp. 8441-8451 ◽  
Author(s):  
Jessica Wiethaus ◽  
Andrea Wirsing ◽  
Franz Narberhaus ◽  
Bernd Masepohl
Keyword(s):  
Transport System ◽  
Dna Sequences ◽  
Target Genes ◽  
Rhodobacter Capsulatus ◽  
Binding Protein ◽  
Transcriptional Unit ◽  
Purple Bacterium ◽  
Promoter Regions ◽  
Mobility Shift ◽  
High Affinity

ABSTRACT The phototrophic purple bacterium Rhodobacter capsulatus encodes two similar but functionally not identical molybdenum-dependent regulator proteins (MopA and MopB), which are known to replace each other in repression of the modABC genes (coding for an ABC-type high-affinity Mo transport system) and anfA (coding for the transcriptional activator of Fe-nitrogenase genes). We identified further Mo-regulated (mor) genes coding for a putative ABC-type transport system of unknown function (MorABC) and a putative Mo-binding protein (Mop). The genes coding for MopA and the ModABC transporter form part of a single transcriptional unit, mopA-modABCD, as shown by reverse transcriptase PCR. Immediately upstream of mopA and transcribed in the opposite direction is mopB. The genes coding for the putative MorABC transporter belong to two divergently transcribed operons, morAB and morC. Expression studies based on lacZ reporter gene fusions in mutant strains defective for either MopA, MopB, or both revealed that the regulators substitute for each other in Mo-dependent repression of morAB and morC. Specific Mo-dependent activation of the mop gene by MopA, but not MopB, was found to control the putative Mo-binding protein. Both MopA and MopB are thought to bind to conserved DNA sequences with dyad symmetry in the promoter regions of all target genes. The positions of these so-called Mo boxes relative to the transcription start sites (as determined by primer extension analyses) differed between Mo-repressed genes and the Mo-activated mop gene. DNA mobility shift assays showed that MopA and MopB require molybdenum to bind to their target sites with high affinity.

scholarly journals The KdpD Sensor Kinase of Escherichia coli Responds to Several Distinct Signals To Turn on Expression of the Kdp Transport System

2015 ◽  
Vol 198 (2) ◽  
pp. 212-220 ◽  
Author(s):  
Wolfgang Epstein
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Transport System ◽  
Kinase Activity ◽  
Response Regulator ◽  
Sensor Kinase ◽  
Monovalent Cations ◽  
Two Component System ◽  
Content Type ◽  
E Coli

ABSTRACTKdp, one of three saturable K+uptake systems inEscherichia coli, is the system with the highest affinity for K+and the only one whose expression is strongly controlled by medium K+concentration. Expression is controlled by a two-component system of KdpD, the sensor kinase, and KdpE, the response regulator. There is general agreement that expression occurs when the growth rate of cells begins to become limited by K+availability. How K+limitation results in expression has been controversial. Studying the roles of the major components of the growth medium shows that KdpD senses at least two distinct signals inside the cell, those of Na+and NH4+, and it probably senses other monovalent cations in the cell. KdpD does not sense turgor.IMPORTANCEThe expression of the Kdp K+transport system ofE. colioccurs when cells become limited in their growth rate by the availability of K+. Cells sense limited K+and try to compensate by taking up other monovalent cations, particularly Na+and NH4+. These cations are sensed in the cytoplasm by the KdpD response regulator, presumably to stimulate its kinase activity. It is shown that KdpD does not sense turgor, as was suggested earlier.

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