Uptake System Research Articles

Structure-guided inhibitor design targeting CntL provides the first chemical validation of the staphylopine metallophore system in bacterial metal acquisition

To survive in the metal-scarce environment within the host, pathogens synthesize various small molecular metallophores to facilitate the acquisition of transition metals. The cobalt and nickel transporter (Cnt) system synthesizes and transports staphylopine, a nicotianamine-like metallophore, and serves as a primary transition metal uptake system in Gram-positive bacteria including the human pathogen Staphylococcus aureus. In this study, we report the design of the first inhibitor of the Cnt system by targeting the key aminobutanoyltransferase CntL which is involved in the biosynthesis of staphylopine. Through structure-guided fragment linking and optimization, a class of acceptor-adenosine dual-site inhibitors against S. aureus CntL (SaCntL) were designed and synthesized. The most potent inhibitor, compound 9, demonstrated a ΔTm value of 9.4 °C, a Kd value of 0.021 ± 0.004 μM, and an IC50 value of 0.06 μM against SaCntL. The detailed mechanism by which compound 9 inhibits SaCntL has been elucidated through a high-resolution co-crystal structure. Treatment with compound 9 resulted in a moderate downregulation of intracellular concentrations of iron, nickel, and cobalt ions in the S. aureus cells cultured in the metal-scarce medium, providing the first chemical validation of the important role of Cnt system in bacterial metal acquisition. Our findings pave the way for the development of CntL-based antibacterial agents in future.

Expanding the Substrate Selectivity of the Fimsbactin Biosynthetic Adenylation Domain, FbsH.

Nonribosomal peptide synthetases (NRPSs) produce diverse natural products including siderophores, chelating agents that many pathogenic bacteria produce to survive in low iron conditions. Engineering NRPSs to produce diverse siderophore analogs could lead to the generation of novel antibiotics and imaging agents that take advantage of this unique iron uptake system in bacteria. The highly pathogenic and antibiotic-resistant bacteria Acinetobacter baumannii produces fimsbactin, an unusual branched siderophore with iron-binding catechol groups bound to a serine or threonine side chain. To explore the substrate promiscuity of the assembly line enzymes, we report a structure-guided investigation of the stand-alone aryl adenylation enzyme FbsH. We report structures bound to its native substrate 2,3-dihydroxybenzoic acid (DHB) as well as an inhibitor that mimics the adenylate intermediate. We produced enzyme variants with an expanded binding pocket that are more tolerant for analogs containing a DHB C4 modification. Wild-type and mutant enzymes were then used in an in vitro reconstitution analysis to assess the production of analogs of the final product as well as several early stage intermediates. This analysis shows that some altered substrates progress down the fimsbactin assembly line to the downstream domains. However, analogs from alternate building blocks are produced at lower levels, indicating that selectivity exists in the downstream catalytic domains. These findings expand the substrate scope of producing condensation products between serine and aryl acids and identify the bottlenecks for chemoenzymatic production of fimsbactin analogs.

Genomic characterization of Pantoea dispersa A003 isolated from a clinical patient

IntroductionPantoea dispersa is a Gram-negative bacterium generally considered as a plant pathogen and rarely causes human infections. To date, there have been 13 studies that have documented clinical infections linked to P. dispersa, with a primary emphasis on the initial identification of this pathogen. The genomic features and the mechanisms underlying the pathogenesis of P. dispersa remain largely uninvestigated. In the present study, we describe a clinical infection caused by P. dispersa and provide the first genomic analysis of this bacterium.MethodsThe bacterial strain designated A003 was isolated from blood samples. Preliminary identification of strain A003 was conducted using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) technology (VITEK® MS, bioMérieux, France). Biochemical and antimicrobial susceptibility assessments were carried out utilizing the VITEK-2 compact automated microbial analysis system (bioMérieux, France). Additionally, whole genome sequencing and subsequent analysis were performed to further elucidate the potential pathogenicity.ResultsThe bacterial isolate was cultured overnight at 35°C in a CO2-enriched environment on Columbia blood agar, resulting in the appearance of a white, smooth, Gram-negative rod-shaped bacterium with diameters ranging from 1 to 2 mm. Identification of strain A003 was achieved with a high confidence level of 99.9% as P. dispersa using MALDI-TOF mass spectrometry. The average nucleotide identity (ANI) value between strain A003 and the reference strain P. dispersa DSM 30073T was 98.08%, while the DNA–DNA hybridization (DDH) value was 84.10% (Formula 2) based on genome sequencing data, which further confirmed that A003 belonged to the P. dispersa species. Comprehensive analysis revealed the presence of 372 virulence factors, 15 antibiotic resistance genes, 222 carbohydrate-active enzymes, and 666 genes related to Type III secretion system (T3SS) effector proteins, as identified through the core databases of VFDB (Virulence Factors of Pathogenic Bacteria), CARD (Comprehensive Antibiotic Resistance Database), CAZY (Carbohydrate-Active enZYmes Database), and T3SS. The virulence factors included type IV pili (TFP), type VI secretion system, flagella, iron uptake system, and ompA, which are implicated in bacterial pathogenicity. The antibiotic resistance profile indicated resistance to fluoroquinolones, cephalosporins, penams, macrolides, and aminoglycosides, as annotated by the CARD database.ConclusionThe draft genome sequenced represents the inaugural genomic sequence of P. dispersa derived from clinical sources. This advancement may enhance clinical practitioners’ comprehension of the organism’s clinical attributes and serve as a foundational resource for future research into its virulence, antibiotic resistance, and host–pathogen interactions.

Coenzyme A biosynthesis in Bacillus subtilis: discovery of a novel precursor metabolite for salvage and its uptake system.

Vitamins are essential components of the diet of animals and humans. Vitamins are thus important targets for biotechnological production. While efficient fermentation processes have been developed for several vitamins, this is not the case for vitamin B5 (pantothenate), the precursor of coenzyme A. We have elucidated the complete pathway for coenzyme A biosynthesis in the biotechnological workhorse Bacillus subtilis. Moreover, a salvage pathway for coenzyme A synthesis was found in this study. Normally, this pathway depends on pantetheine; however, we observed activity of the salvage pathway on complex medium in mutants lacking the pantothenate biosynthesis pathway even in the absence of supplemented pantetheine. This required rewiring of metabolism by expressing a cystine transporter due to acquisition of mutations affecting the regulation of cysteine metabolism. This shows how the hidden "underground metabolism" can give rise to the rapid formation of novel metabolic pathways.

From grass to yeast; functional insights from heterologous expression of LfHKT2;1 in ion regulation

Saccharomyces cervisceae mutants lacking the major potassium trasnporters trk1 and trk2 show hypersensitivity to aminoglycoside antibiotic hygromycin (hyg). This study demonstrates that expression of the inward K+ transporter LfHKT2;1 can suppress this hygromycin sensitivity in the trk1, trk2 double mutant strain. Growth complementation was performed on solid yeast peptone dextrose (YPD) media supplemented with hygromycin B. Both, wild type and trk1 trk2 yeast strains exhibited growth inhibition in the presence of hygromycin. However the potassium uptake-deficient trk1 trk2 strain (control) showed complete growth arrest when exposed to hygromycin, while expression of LfHKT2;1 resulted in growth recovery. Increased sodium concentrations caused cellular toxicity in the trk1 trk2 strain, which was exacerbated by the addition of hygromycin to the media. The hypersensitivity of trk1 trk2 mutant yeast cells expressing LfHKT2;1 to sodium suggested the presence of an additional sodium uptake system on the membrane, which was further confirmed by transient GFP expression assays. These results provide conclusive evidence that heterologous expression of LfHKT2;1 confers both sodium and K+ uptake capabilities in hygromycin supplemented YPD media, thereby rescuing the growth of K+ transport-deficient S. cerevisiae mutants. This highlights the potential of plant gene expression in yeast as a valuable tool for studying ion transport mechanisms and gene function under stress conditions.

Determinants of Antibiotic Resistance and Virulence Factors in the Genome of Escherichia coli APEC 36 Strain Isolated from a Broiler Chicken with Generalized Colibacillosis.

Objective: Multidrug-resistant, highly pathogenic Escherichia coli strains are the primary causative agents of intestinal and extraintestinal human diseases. The extensive utilization of antibiotics for farm animals has been identified as a contributing factor to the emergence and dissemination of E. coli strains that exhibit multidrug resistance and possess high pathogenic potential. Consequently, a significant research objective is to examine the genetic diversity of pathogenic E. coli strains and to identify those that may pose a threat to human health. Methods: In this study, we present the results of genome sequencing and analysis, as well as the physiological characterization of E. coli strain APEC 36, which was isolated from the liver of a broiler chicken with generalized colibacillosis. Results: We found that APEC 36 possess a number of mechanisms of antibiotic resistance, including antibiotic efflux, antibiotic inactivation, and antibiotic target alteration/replacement/protection. The most widely represented group among these mechanisms was that of antibiotic efflux. This finding is consistent with the strain's documented resistance to multiple antibiotics. APEC 36 has an extremely rare variant of the beta-lactamase CTX-M-169. Notwithstanding the multitude of systems for interfering with foreign DNA present in the strain, seven plasmids have been identified, three of which may possess novel replication origins. Additionally, qnrS1, which confers resistance to fluoroquinolones, was found to be encoded in the genome rather than in the plasmid. This suggests that the determinants of antibiotic resistance may be captured in the genome and stably transmitted from generation to generation. Conclusions: The APEC 36 strain has genes for toxins, adhesins, protectins, and an iron uptake system. The obtained set of genetic and physiological characteristics allowed us to assume that this strain has a high pathogenic potential for humans.

The ammonium transporter AmtB is dispensable for the uptake of ammonium in the phototrophic diazotroph Rhodopseudomonas palustris

Ammonium (NH4+) transport across cell membranes plays an important role in assimilation or removal of the environmental nitrogen. The membrane protein AmtB has been considered to be the NH4+ transporter that is responsible for the NH4+ uptake. The phototrophic diazotroph Rhodopseudomonas palustris harboring two amtB genes has been widely used in wastewater treatment and bioremediation. However, the role of AmtB in NH4+ uptake remains unclear in R. palustris. Here, we employed an innovative approach combining stable isotope probing (SIP) with Raman spectroscopy to determine the physiological functions of AmtB1 and AmtB2 in R. palustris. This powerful technique allowed us to investigate NH4+ uptake at the single-cell level. The generated R. palustris ΔamtB1 ΔamtB2 mutant lacking AmtB1 and AmtB2 proteins was still capable of utilizing 15NH4+ even the 15NH4+ concentration was as low as 5 μM. These data demonstrate that both of the AmtB proteins are not essential for R. palustris to take up NH4+ regardless of environmental NH4+ levels. However, both AmtB1 and AmtB2 can contribute to NH4+ uptake under nitrogen-limiting conditions. Given that R. palustris primarily expresses AmtB2 in these conditions, AmtB2 plays a more important role in NH4+ uptake compared to AmtB1. In addition, transcriptomic analysis showed that the deletion of the amtB1 and amtB2 genes resulted in the upregulation of many transporter genes, providing potential targets for future investigation of alternative NH4+ uptake systems.

Whole cell affinity for 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) in the marine bacterium Candidatus Pelagibacter st. HTCC7211 explains marine dissolved HMP concentrations.

Vitamin B1 is a universally required coenzyme in carbon metabolism. However, most marine microorganisms lack the complete biosynthetic pathway for this compound and must acquire thiamin, or precursor molecules, from the dissolved pool. The most common version of Vitamin B1 auxotrophy is for thiamin's pyrimidine precursor moiety, 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Frequent HMP auxotrophy in plankton and vanishingly low dissolved concentrations (approximately 0.1-50 pM) suggest that high-affinity HMP uptake systems are responsible for maintaining low ambient HMP concentrations. We used tritium-labelled HMP to investigate HMP uptake mechanisms and kinetics in cell cultures of Candidatus Pelagibacter st. HTCC7211, a representative of the globally distributed and highly abundant SAR11 clade. A single protein, the sodium solute symporter ThiV, which is conserved across SAR11 genomes, is the likely candidate for HMP transport. Experimental evidence indicated transport specificity for HMP and mechanistically complex, high-affinity HMP uptake kinetics. Km values ranged from 9.5 pM to 1.2 nM and were dramatically lower when cells were supplied with a carbon source. These results suggest that HMP uptake in HTCC7211 is subject to complex regulation and point to a strategy for high-affinity uptake of this essential growth factor that can explain natural HMP levels in seawater.

Structural basis for the ligand promiscuity of the hydroxamate siderophore binding protein FtsB from Streptococcus pyogenes.

Pathogenic bacteria must secure the uptake of nutritional metals such as iron for their growth, making their import systems attractive targets for the development of new antimicrobial modalities. In the pathogenic bacterium Streptococcus pyogenes, the iron uptake system FtsABCD transports iron encapsulated by siderophores of the hydroxamate class. However, the inability of S.pyogenes to produce these metabolites makes the biological and clinical relevance of this route unresolved. Herein, we demonstrated that the periplasmic binding protein FtsB recognizes not only the hydroxamate siderophore ferrichrome, as previously documented, but also ferrioxamine E (FOE), ferrioxamine B (FOB), and bisucaberin (BIS), each of them with high affinity (nM level). Up to seven aromatic residues in the binding pocket accommodate the variable backbones of the different siderophores through CH-π interactions, explaining ligand promiscuity. Collectively, our observations revealed how S.pyogenes exploits the diverse xenosiderophores produced by other microorganisms as iron sources to secure this precious nutrient.

Structural insights into polyamine spermidine uptake by the ABC transporter PotD-PotABC

Polyamines, characterized by their polycationic nature, are ubiquitously present in all organisms and play numerous cellular functions. Among polyamines, spermidine stands out as the predominant type in both prokaryotic and eukaryotic cells. The PotD-PotABC protein complex in Escherichia coli , belonging to the adenosine triphosphate–binding cassette transporter family, is a spermidine-preferential uptake system. Here, we report structural details of the polyamine uptake system PotD-PotABC in various states. Our analyses reveal distinct “inward-facing” and “outward-facing” conformations of the PotD-PotABC transporter, as well as conformational changes in the “gating” residues (F222, Y223, D226, and K241 in PotB; Y219 and K223 in PotC) controlling spermidine uptake. Therefore, our structural analysis provides insights into how the PotD-PotABC importer recognizes the substrate-binding protein PotD and elucidates molecular insights into the spermidine uptake mechanism of bacteria.

323 Advances in using membrane vesicles for studying intestinal functions

Abstract Enterocytes are the major cell lineage of the small intestinal epithelia along the crypt-villus axis in gut mucosa. Enterocytes participate in the gut terminal nutrient digestion and absorption; and have essential roles in nutrient and metabolite recycling and efflux, nutrient and hormonal sensing, interactions with symbiotic microbiome and binding to infectious viruses, thus influencing the host homeostasis and whole-body physiological status. Intestinal membrane vesicles have been widely used for studying various gut functions in the past several decades. Villus enterocytic brush border membrane and the apical membrane vesicles along the crypt-villus axis in gut mucosa can be prepared and isolated by Mg2+- and Ca2+-precipitation and differential centrifugation procedures. Whereas, gut mucosal basolateral membrane vesicles can be prepared, fractionated and differentiated from other intracellular organelles such as the mitochondrial inner membrane by the Percoll gradient ultracentrifugation. Nutrient transport and exchange activities in the membrane vesicles in vitro are conducted often by rapid hand filtration, and in excess, in an automated fast sampling apparatus. Nutrient transport and exchange activity kinetics are derived according to the well-established classic Michaelis-Menten and the tracer inhibitory kinetic models with a simple linear diffusional component. Physiological and biochemical properties associated with various nutrient uptake systems such as pH optimum, requirements for co-transported ions (e.g., Na+, K+, Cl-), transporter affinity and substrate specificity can be effectively investigated in vitro with the membrane vesicles by altering the compositions of uptake buffers and intra-vesicular pre-loading buffers. Some of the fundamental gut functions are established via using the membrane vesicles in different mammalian species. And these include H+-peptide cotransport, Na+-cotransport and Na+-independent systems of hexose uptake across the apical membrane and the low-affinity Na+-independent hexose bidirectional uptake across the basolateral membrane that were characterized in rodents; Na+-dependent cotransport and Na+-independent systems of amino acid (AA) transport across the apical membrane that were revealed in rodents and pigs; as well as the Na+-hexose and AA cotransport and the co-expression of the coronavirus SARS-CoV-2 host epithelial apical membrane binding receptor, i.e., the angiotensin-converting enzyme-2 (ACE2), along the gut crypt-villus axis that were identified in liquid formula-fed piglets. In the post genomic and metagenomic sequencing era, further research efforts need to pursue understanding of expression and various functionality of the gut terminal hydrolases, nutrient transporters, nutrient exchangers, nutrient sensors, substrate and particle receptors, taste receptors, viral binding receptors such as ACE2 and hormonal receptors and their interactions with luminal factors such as nutrient, metabolites, symbiotic bacterial outer membrane vesicles etc., along the longitudinal and crypt-villus axes, through using the mammalian primary enterocytic apical and the basolateral membrane vesicles under various research settings.

Biophysical and Solution Structure Analysis of Critical Residues Involved in the Interaction between the PupB N-Terminal Signaling Domain and PupR C-Terminal Cell Surface Signaling Domain from Pseudomonas capeferrum.

Cell surface signaling (CSS) is a means of rapidly adjusting transcription in response to extracellular stimuli in Gram-negative bacteria. The pseudobactin BN7/8 uptake (Pup) system not only imports iron but also upregulates its own transcription through CSS in Pseudomonas capeferrum. In the absence of ferric pseudobactin BN7/8, the signaling components are maintained in a resting state via the formation of a periplasmic complex between the N-terminal signaling domain (NTSD) of the outer membrane iron-transporter, PupB, and the C-terminal CSS domain (CCSSD) of the sigma regulator, PupR. The previously determined 1.6 Å crystal structure of this periplasmic complex has allowed us to probe the structural and thermodynamic consequences of mutating key interfacial residues. In this report, we describe the solution structure of the PupB NTSD and use Nuclear Magnetic Resonance spectroscopy, Isothermal Titration Calorimetry, and Circular Dichroism spectroscopy together with thermal denaturation to investigate whether three PupB point mutations, Q69K, H72D, and L74A, influence the interaction merely due to the chemical nature of the amino acid substitution or also cause changes in overall protein structure. Our results demonstrate that binding to the PupR CCSSD does not alter the structure of PupB NTSD and that the individual mutations have only minor effects on structure. The mutations generally lower thermodynamic stability of the NTSD and weaken binding to the CCSSD. These findings validate the X-ray crystal structure interface, emphasizing the importance of amino acid chemical nature at the interface.

A fungal endophyte increases plant resilience to low nutrient availabilities: a case of Fe acquisition in legumes.

Microbial inocula are considered a promising and effective alternative solution to the use of chemical fertilizers to support plant growth and productivity since they play a key role in the availability and uptake of nutrients. Here, the effect of a beneficial of a fungal root endophyte, Fusarium solani strain K (FsK), on nutrient acquisition efficiency of the legume Lotus japonicus was studied, and putative mode-of-action of the endophyte at a molecular level was determined. Plant colonization with the endophyte resulted in increased shoot and root fresh weight under Fe deficiency compared to control nutrient conditions. Plant inoculation with FsK was associated with a significant increase in macro- and micronutrient concentration in leaves at an early stage of endophyte inoculation and a replenishment of Fe content under prolonged iron starvation. The mechanistic basis of the plant growth promotion capabilities of the endophyte is exerted at the transcriptional level since we recorded changes in the expression levels of genes related to iron uptake in FsK-colonized plants under stress conditions compared to uninoculated plants. In addition, the observed changes in the ethylene biosynthesis-related genes suggest a possible implication of ethylene in the mode of action used by FsK to enhance plant response to nutrient stress conditions. Finally, we demonstrated that the endophyte possesses a reductive high-affinity Fe uptake system and identified a ferric reductase that was induced in planta under Fe deficiency conditions, indicating that this fungal Fe homeostasis mechanism may result in a benefit in nutrient acquisition for the plant as well.

Proteomic characterization of Tenacibaculum dicentrarchi under iron limitation reveals an upregulation of proteins related to iron oxidation and reduction metabolism, iron uptake systems and gliding motility.

A strategy for vaccine design involves identifying proteins that could be involved in pathogen-host interactions. The aim of this proteomic study was to determine how iron limitation affects the protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron-limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB-dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.

Genomic analysis of two penicillin- and rifampin-resistant Corynebacterium rouxii strains isolated from cutaneous infections in dogs

Although diphtheria is a vaccine-preventable disease, numerous cases are still reported around the world, as well as outbreaks in countries, including European ones. Species of the Corynebacterium diphtheriae complex are potentially toxigenic and, therefore, must be considered given the possible consequences, such as the circulation of clones and transmission of antimicrobial resistance and virulence genes. Recently, Corynebacterium rouxii was characterized and included among the valid species of the complex. Therefore, two cases of C. rouxii infection arising from infections in domestic animals are presented here. We provide molecular characterization, phylogenetic analyses, genome sequencing, and CRISPR-Cas analyses to contribute to a better understanding of the molecular bases, pathogenesis, and epidemiological monitoring of this species, which is still little studied. We confirmed its taxonomic position with genome sequencing and in silico analysis and identified the ST-918 for both strains. The clinical isolates were sensitive resistance to benzylpenicillin and rifampin. Antimicrobial resistance genes, including tetB, rpoB2, and rbpA genes, were predicted. The bla and ampC genes were not found. Several virulence factors were also detected, including adhesion, iron uptake systems, gene regulation (dtxR), and post-translational modification (MdbA). Finally, one prophage and the Type I-E CRISPR-Cas system were identified.

Regulation of potassium uptake in Caulobacter crescentus.

Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Here, we describe the regulation of potassium uptake systems in the oligotrophic α-proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by mainly using two K+ transporters to maintain potassium homeostasis, the low-affinity Kup and the high-affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by increasing the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABCDE expression, suggest that the inner membrane sensor regulatory component KdpD mainly works as a phosphatase to limit the growth when cells reach late exponential phase. Our data therefore suggest that KdpE is phosphorylated by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability.IMPORTANCEPotassium (K+) is a key metal ion involved in many essential cellular processes. Here, we show that the oligotroph Caulobacter crescentus can support growth at micromolar concentrations of K+ by mainly using two K+ uptake systems, the low-affinity Kup and the high-affinity Kdp. Using genome-wide approaches, we also determined the entire set of genes required for C. crescentus to survive at low K+ concentration as well as the full K+-dependent regulon. Finally, we found that the transcriptional regulation mediated by the KdpDE two-component system is unconventional since unlike Escherichia coli, the inner membrane sensor regulatory component KdpD seems to work rather as a phosphatase on the phosphorylated response regulator KdpE~P.

SlCIPK9 regulates pollen tube elongation in tomato plants via a K+-independent mechanism

Potassium (K+) is an essential macronutrient which contributes to osmotic- and turgor-related processes in plants. Calcineurin-B like Interacting Protein Kinases (CIPKs) play crucial roles in plants under low-K+ supply since they activate root K+ uptake transport systems such as AKT1 and AtHAK5. In Arabidopsis, AtCIPK9 is important for low-K+ tolerance since atcipk9 plants exhibited poor growth and leaf chlorosis when K+ was scarce. Part of these phenotypes could be ascribed to the activation of AtHAK5 by AtCIPK9. It has been reported that important differences exist between Arabidopsis and other plant species such as tomato with respect to the regulation of K+ uptake systems. Thus, our aim was to evaluate the contribution of SlCIPK9, the homologous protein of AtCIPK9 in tomato, to K+ nutrition. Unexpectedly, phenotyping experiments carried out with slcipk9 loss-of-function mutants revealed that SlCIPK9 did not play a clear role in tomato K+ homeostasis. By contrast, it was found that SlCIPK9 contributed to pollen tube elongation, but not to pollen germination, via a K+-independent mechanism. Therefore, our results highlight the remarkable differences that exist in Ca2+ signaling pathways between plant species and encourage the realization of more comparative studies as the one presented here.

Whole-genome sequencing of multidrug-resistant Klebsiella pneumoniae with capsular serotype K2 isolates from mink in China

BackgroundKlebsiella pneumoniae is a zoonotic opportunistic pathogen, and also one of the common pathogenic bacteria causing mink pneumonia. The aim of this study was to get a better understanding of the whole-genome of multi-drug resistant Klebsiella pneumoniae with K2 serotype in China. This study for the first time to analyze Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, resistance and virulence genes of Klebsiella pneumoniae in mink.ResultsThe isolate was Klebsiella pneumoniae with serotype K2 and ST6189 by PCR method. The string test was positive and showed high mucus phenotype. There was one plasmid with IncFIB replicons in the genome. The virulence factors including capsule, lipopolysaccharide, adhesin, iron uptake system, urease, secretory system, regulatory gene (rcsA, rcsB), determinants of pili adhesion, enolase and magnesium ion absorption related genes. The strain was multi-drug resistant. A total of 26 resistance genes, including beta-lactam, aminoglycosides, tetracycline, fluoroquinolones, sulfonamides, amide alcohols, macrolides, rifampicin, fosfomycin, vancomycin, diaminopyrimidines and polymyxin. Multidrug-resistant efflux protein AcrA, AcrB, TolC, were predicted in the strain.ConclusionIt was the first to identify that serotype K2 K. pneumonia with ST6189 isolated from mink in China. The finding indicated that hypervirulent and multi-drug resistant K. pneumoniae was exist in Chinese mink. The whole-genome of K. pneumoniae isolates have importance in mink farming practice.

Crystal structure of pectocin M1 reveals diverse conformations and interactions during its initial step via the ferredoxin uptake system.

Pectocin M1 (PM1), the bacteriocin from phytopathogenic Pectobacterium carotovorum which causes soft rot disease, has a unique ferredoxin domain that allows it to use FusA of the plant ferredoxin uptake system. To probe the structure-based mechanism of PM1 uptake, we determined the X-ray structure of full-length PM1, containing an N-terminal ferredoxin and C-terminal catalytic domain connected by helical linker, at 2.04 Å resolution. Based on published FusA structure and NMR data for PM1 ferredoxin domain titrated with FusA, we modeled docking of the ferredoxin domain with FusA. Combining the docking models with the X-ray structures of PM1 and FusA enables us to propose the mechanism by which PM1 undergoes dynamic domain rearrangement to translocate across the target cell outer membrane.

Functional plasticity of HCO3– uptake and CO2 fixation in Cupriavidus necator H16

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of β-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3– uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing.