Environmental potassium regulates bacterial flotation, antibiotic production and turgor pressure in Serratia through the TrkH transporter.
ABSTRACT: Serratia sp. strain ATCC 39006 (S39006) can float in aqueous environments due to natural production of gas vesicles (GVs). Expression of genes for GV morphogenesis is stimulated in low oxygen conditions, thereby enabling migration to the air-liquid interface. Quorum sensing (via SmaI and SmaR) and transcriptional and post-transcriptional regulators, including RbsR and RsmA, respectively, connect the control of cell buoyancy, motility and secondary metabolism. Here, we define a new pleiotropic regulator found in screens of GV mutants. A mutation in the gene trkH, encoding a potassium transporter, caused upregulation of GV formation, flotation, and the prodigiosin antibiotic, and downregulation of flagellar motility. Pressure nephelometry revealed that the mutation in trkH affected cell turgor pressure. Our results show that osmotic change is an important physiological parameter modulating cell buoyancy and antimicrobial production in S39006, in response to environmental potassium levels.
Project description:Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. During its infectious cycle, F. tularensis is not only exposed to the intracellular environment of macrophages but also resides transiently in extracellular compartments, in particular during its systemic dissemination. The screening of a bank of F. tularensis LVS transposon insertion mutants on chemically defined medium (CDM) led us to identify a gene, designated trkH, encoding a homolog of the potassium uptake permease TrkH. Inactivation of trkH impaired bacterial growth in CDM. Normal growth of the mutant was only restored when CDM was supplemented with potassium at high concentration. Strikingly, although not required for intracellular survival in cell culture models, TrkH appeared to be essential for bacterial virulence in the mouse. In vivo kinetics of bacterial dissemination revealed a severe defect of multiplication of the trkH mutant in the blood of infected animals. The trkH mutant also showed impaired growth in blood ex vivo. Genome sequence analyses suggest that the Trk system constitutes the unique functional active potassium transporter in both tularensis and holarctica subspecies. Hence, the impaired survival of the trkH mutant in vivo is likely to be due to its inability to survive in the low potassium environment (1-5 mM range) of the blood. This work unravels thus the importance of potassium acquisition in the extracellular phase of the F. tularensis infectious cycle. More generally, potassium could constitute an important mineral nutrient involved in other diseases linked to systemic dissemination of bacterial pathogens.
Project description:Gas vesicles (GVs) are proteinaceous, gas-filled organelles used by some bacteria to enable upward movement into favorable air/liquid interfaces in aquatic environments. <i>Serratia</i> sp. ATCC39006 (S39006) was the first enterobacterium discovered to produce GVs naturally. The regulation of GV assembly in this host is complex and part of a wider regulatory network affecting various phenotypes, including antibiotic biosynthesis. To identify new regulators of GVs, a comprehensive mutant library containing 71,000 insertion mutants was generated by random transposon mutagenesis and 311 putative GV-defective mutants identified. Three of these mutants were found to have a transposon inserted in a LacI family transcription regulator gene (<i>rbsR</i>) of the putative ribose operon. Each of these <i>rbsR</i> mutants was GV-defective; no GVs were visible by phase contrast microscopy (PCM) or transmission electron microscopy (TEM). GV deficiency was caused by the reduction of <i>gvpA1</i> and <i>gvrA</i> transcription (the first genes of the two contiguous operons in the GV gene locus). Our results also showed that a mutation in <i>rbsR</i> was highly pleiotropic; the production of two secondary metabolites (carbapenem and prodigiosin antibiotics) was abolished. Interestingly, the intrinsic resistance to the carbapenem antibiotic was not affected by the <i>rbsR</i> mutation. In addition, the production of a siderophore, cellulase and plant virulence was reduced in the mutant, whereas it exhibited increased swimming and swarming motility. The RbsR protein was predicted to bind to regions upstream of at least 18 genes in S39006 including <i>rbsD</i> (the first gene of the ribose operon) and <i>gvrA</i>. Electrophoretic mobility shift assays (EMSA) confirmed that RbsR bound to DNA sequences upstream of <i>rbsD</i>, but not <i>gvrA</i>. The results of this study indicate that RbsR is a global regulator that affects the modulation of GV biogenesis, but also with complex pleiotropic physiological impacts in S39006.
Project description:<h4>Background</h4>Secondary metabolism in Serratia sp. ATCC 39006 (Serratia 39006) is controlled via a complex network of regulators, including a LuxIR-type (SmaIR) quorum sensing (QS) system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in Serratia 39006.<h4>Results</h4>We demonstrate that a mutation in the high affinity phosphate transporter pstSCAB-phoU, believed to mimic low phosphate conditions, causes upregulation of secondary metabolism and QS in Serratia 39006, via the PhoBR two-component system. Phosphate limitation also activated secondary metabolism and QS in Serratia 39006. In addition, a pstS mutation resulted in upregulation of rap. Rap, a putative SlyA/MarR-family transcriptional regulator, shares similarity with the global regulator RovA (regulator of virulence) from Yersina spp. and is an activator of secondary metabolism in Serratia 39006. We demonstrate that expression of rap, pigA-O (encoding the prodigiosin biosynthetic operon) and smaI are controlled via PhoBR in Serratia 39006.<h4>Conclusion</h4>Phosphate limitation regulates secondary metabolism in Serratia 39006 via multiple inter-linked pathways, incorporating transcriptional control mediated by three important global regulators, PhoB, SmaR and Rap.
Project description:TrkH belongs to a superfamily of K(+) transport proteins required for growth of bacteria in low external K(+) concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K(+) channel and may have a gating mechanism substantially different from K(+) channels. TrkH assembles with TrkA, a cytosolic protein comprising two RCK (regulate the conductance of K(+)) domains, which are found in certain K(+) channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two markedly different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA.
Project description:The TrkH/TrkG/KtrB proteins mediate K(+) uptake in bacteria and probably evolved from simple K(+) channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K(+) channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K(+) and Rb(+) over smaller ions such as Na(+) or Li(+). Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K(+) flux. These results reveal the molecular basis of K(+) selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.
Project description:TrkH is a bacterial ion channel implicated in K<sup>+</sup> uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.
Project description:The corrected sequence of the trkH gene of Escherichia coli predicts that the TrkH protein is a hydrophobic membrane protein of 483 amino acid residues, of which 41% are identical to those of the homologous and functionally analogous TrkG protein. These two proteins form the transmembrane component of the Trk system for the uptake of K+. Each protein alone is sufficient for high-level Trk activity. When Trk is assembled with the TrkG protein, Rb+ and K+ are transported with a Km near or below 1 mM; however, the Vmax for Rb+ is only about 7% of that for K+. When Trk is formed with TrkH, the affinities for both for K+ and Rb+ are somewhat lower, and the Vmax for Rb+ is only 1% of that for K+ transport. The kinetics of transport in strains with wild-type alleles at trkG and at trkH suggest that both products participate in transport.
Project description:The halophilic bacterium Halomonas elongata accumulates K+, glutamate, and the compatible solute ectoine as osmoprotectants. By functional complementation of Escherichia coli mutants defective in K+ uptake, we cloned three genes that are required for K+ uptake in H. elongata. Two adjacent genes, named trkA (1,374 bp) and trkH (1,449 bp), were identified on an 8.5-kb DNA fragment, while a third gene, called trkI (1,479 bp), located at a different site in the H. elongata chromosome, was found on a second 8.5-kb fragment. The potential protein expressed by trkA is similar to the cytoplasmic NAD+/NADH binding protein TrkA from E. coli, which is required for the activity of the Trk K+ uptake system. The deduced amino acid sequences of trkH and trkI showed significant identity to the transmembrane protein of Trk transporters. K+ transport experiments with DeltatrkH and DeltatrkI mutants of H. elongata revealed that TrkI exhibits a Km value of 1.12 mM, while the TrkH system has a half-saturation constant of 3.36 mM. Strain KB12, relying on TrkH alone, accumulated K+ with a lower Vmax and required a higher K+ concentration for growth in highly saline medium than the wild type. Strain KB15, expressing only TrkI, showed the same phenotype and the same K+ transport kinetics as the wild type, proving that TrkI is the main K+ transport system in H. elongata. In the absence of both transporters TrkH and TrkI, K+ accumulation was not detectable. K+ transport was also abolished in a trkA deletion mutant, indicating that TrkI and TrkH depend on one type of TrkA protein. Reverse transcriptase PCR experiments and Northern hybridization analyses of the trkAH locus revealed cotranscription of trkAH as well as a monocistronic transcript with only trkA.
Project description:Members of the Superfamily of K(+) Transporters (SKT) are integral membrane proteins that mediate the uptake of ions into non-animal cells. Although these proteins are homologous to the well-characterized K(+) channel family, relatively little was known about their transport and gating mechanisms until the recent determination of crystal structures for two SKT proteins, TrkH and KtrB. These structures reveal that the SKT proteins are channels, containing a flexible loop in the middle of the permeation pathway that may act as a gate. Two different conformational changes have been observed for the associated gating rings, suggesting different mechanisms of regulation by the binding of nucleotides.
Project description:The gene for the Thermotoga maritima Trk potassium transporter component TrkA was originally thought to be a frameshift mutation and not to encode a functional protein. However, expression from this gene yielded a complex consisting of two distinct proteins designated TM1088A and -B. Genetic complementation of Escherichia coli mutants unable to transport potassium suggests that TM1088A/B is part of a functional Trk potassium transporter complex with the membrane protein TM1089. The protein structure for TM1088A shows a characteristic Rossmann fold indicating an NAD+ binding site and has structural similarity to potassium channel-related proteins. Ligand binding studies indicated that ATP, ADP, and AMP stabilized TM1088A to a much greater degree than NADH and NAD, consistent with the crystal structure of TM1088A, which contains a bound AMP natural ligand at the characteristic GXGXXG nucleotide binding site. Mutation of single and all glycines at this nucleotide binding site eliminated in vitro protein stabilization by the ligand, yet these mutated proteins could still functionally complement the E. coli potassium uptake mutants. We predict that this new two-subunit class of TrkA proteins is present in a number of organisms. A further subclass of the predicted two-subunit TrkA proteins lack an identifiable membrane-spanning subunit of the Trk K+ transporter. This class, as exemplified by Mycobacterium tuberculosis, did not complement E. coli potassium transport with the native E. coli TrkH; thus, it may require a novel TrkH-like protein for activity or provide an alternate function in vivo.