Predisposing deleterious variants in the cancer-associated human kinases in the global populations.

Human kinases play essential and diverse roles in the cellular activities of maintaining homeostasis and growth. Genetic mutations in the genes encoding the kinases (or phosphotransferases) have been linked with various types of cancers. In this study, we cataloged mutations in 500 kinases genes in >65,000 individuals of global populations from the Human Genetic Diversity Project (HGDP) and ExAC databases, and assessed their potentially deleterious impact by using the in silico tools SIFT, Polyphen2, and CADD. The analysis highlighted 35 deleterious non-synonymous SNVs in the ExAC and 5 SNVs in the HGDP project. Notably, a higher number of deleterious mutations was observed in the Non-Finnish Europeans (26 SNVs), followed by the Africans (14 SNVs), East Asians (13 SNVs), and South Asians (12 SNVs). The gene set enrichment analysis highlighted NTRK1 and FGFR3 being most significantly enriched among the kinases. The gene expression analysis revealed over-expression of NTRK1 in liver cancer, whereas, FGFR3 was found over-expressed in lung, breast, and liver cancers compared to their expression in the respective normal tissues. Also, 13 potential drugs were identified that target the NTRK1 protein, whereas 6 potential drugs for the FGFR3 target were identified. Taken together, the study provides a framework for exploring the predisposing germline mutations in kinases to suggest the underlying pathogenic mechanisms in cancers. The potential drugs are also suggested for personalized cancer management.

Unveiling the fructose metabolism system in Staphylococcus aureus: insights into the regulatory role of FruR and the FruRKT operon in bacterial fitness.

BACKGROUND: The utilization of fructose as a carbon source and energy provider plays a crucial role in bacterial metabolism. Additionally, fructose metabolism directly impacts the pathogenicity and virulence of certain pathogenic microorganisms. RESULTS: In this study, we report the discovery of a fructose phosphotransferase system (PTS) in S. aureus. This system comprises three genes, namely fruR, fruK, and fruT, which are co-located in an operon that is indispensable for fructose utilization in S. aureus. Our findings confirm that these three genes are transcribed from a single promoter located upstream of the fruRKT operon. The fruR gene encodes a DeoR-type transcriptional regulator, designated as FruR, which represses the expression of the fruRKT operon by direct binding to its promoter region. Significantly, our experimental data demonstrate that the fruRKT operon can be induced by fructose, suggesting a potential regulatory mechanism involving intracellular fructose-1-phosphate as a direct inducer. Furthermore, we conducted RNA-seq analysis to investigate the specificity of FruR regulation in S. aureus, revealing that the fruRKT operon is predominantly regulated by FruR. CONCLUSIONS: In summary, this study has uncovered a fructose phosphotransferase system (PTS) in S. aureus, highlighting the essential role of the fruR, fruK, and fruT genes in fructose utilization. We confirmed their co-location within an operon and established FruR as a key regulator by binding to the operon's promoter. Importantly, we demonstrated that fructose can induce this operon, possibly through intracellular fructose-1-phosphate. Our identification of this PTS system represents the initial characterization of a fructose metabolism system in S. aureus.

Lactococcus lactis mutants resistant to lactococcin A and garvicin Q reveal missense mutations in the sugar transport domain of the mannose phosphotransferase system.

IMPORTANCE: Many bacteriocins target the sugar transporter mannose phosphotransferase system (man-PTS) to exert their antibacterial activity. The elucidation in recent years of the structure of man-PTS has facilitated our understanding of how bacteriocins might interact with the receptor and which domains of the transporter are involved in bacteriocin resistance. Here, we show that missense mutations in the sugar-binding domain of the man-PTS not only impede the uptake of sugars but also prevent the antibacterial activity of the bacteriocins lactococcin A and garvicin Q.

ArcB orchestrates the quorum-sensing system to regulate type III secretion system 1 in Vibrio parahaemolyticus.

In many Vibrio species, virulence is regulated by quorum sensing, which is regulated by a complex, multichannel, two-component phosphorelay circuit. Through this circuit, sensor kinases transmit sensory information to the phosphotransferase LuxU via a phosphotransfer mechanism, which in turn transmits the signal to the response regulator LuxO. For Vibrio parahaemolyticus, type III secretion system 1 (T3SS1) is required for cytotoxicity, but it is unclear how quorum sensing regulates T3SS1 expression. Herein, we report that a hybrid histidine kinase, ArcB, instead of LuxU, and sensor kinase LuxQ and response regulator LuxO, collectively orchestrate T3SS1 expression in V. parahaemolyticus. Under high oxygen conditions, LuxQ can interact with ArcB directly and phosphorylates the Hpt domain of ArcB. The Hpt domain of ArcB phosphorylates the downstream response regulator LuxO instead of ArcA. LuxO then activates transcription of the T3SS1 gene cluster. Under hypoxic conditions, ArcB autophosphorylates and phosphorylates ArcA, whereas ArcA does not participate in regulating the expression of T3SS1. Our data provides evidence of an alternative regulatory path involving the quorum sensing phosphorelay and adds another layer of understanding about the environmental regulation of gene expression in V. parahaemolyticus.

A putative lipase affects Pseudomonas aeruginosa biofilm matrix production.

Pseudomonas aeruginosa is an opportunistic pathogen that is widely known for infecting patients with underlying conditions. This species often survives antibiotic therapy by forming biofilms, in which the cells produce a protective extracellular matrix. P. aeruginosa also produces virulence factors that enhance its ability to cause disease. One signaling pathway that influences virulence is the nitrogen-related phosphotransferase system (Nitro-PTS), which consists of an initial phosphotransferase, PtsP, a phosphocarrier, PtsO, and a terminal phosphate receptor, PtsN. The physiological role of the Nitro-PTS in P. aeruginosa is poorly understood. However, PtsN, when deprived of its upstream phosphotransfer proteins, has an antagonistic effect on biofilm formation. We thus conducted a transposon mutagenesis screen in an unphosphorylated-PtsN (i.e., ∆ptsP) background to identify downstream proteins with unacknowledged roles in PtsN-mediated biofilm suppression. We found an unstudied gene, PA14_04030, whose disruption restored biofilm production. This gene encodes a predicted phospholipase with signature alpha/beta hydrolase folds and a lipase signature motif with an active-site Ser residue. Hence, we renamed the gene bipL, for biofilm-impacting phospholipase. Deletion of bipL in a ∆ptsP background increased biofilm formation, supporting the idea that BipL is responsible for reducing biofilm formation in strains with unphosphorylated PtsN. Moreover, substituting the putative catalytic Ser for Ala phenocopied bipL deletion, indicating that this residue is important for the biofilm-suppressive activity of BipL in vivo. As our preliminary data suggest that BipL is a lipase, we performed lipidomics to detect changes in the lipid profile due to bipL deletion and found changes in some lipid species. IMPORTANCE Biofilm formation by bacteria occurs when cells secrete an extracellular matrix that holds them together and shields them from environmental insults. Biofilms of bacterial opportunistic human pathogens such as Pseudomonas aeruginosa pose a substantial challenge to clinical antimicrobial therapy. Hence, a more complete knowledge about the bacterial factors that influence and regulate production of the biofilm matrix is one key to formulate more effective therapeutic strategies. In this study, we screen for factors that are important for reducing biofilm matrix production in certain genetic backgrounds. We unexpectedly found a gene encoding a putative lipase enzyme and showed that its predicted catalytic site is important for its ability to reduce biofilm formation. Our findings suggest that lipase enzymes have previously uncharacterized functions in biofilm matrix regulation.

PerTurboID, a targeted in situ method reveals the impact of kinase deletion on its local protein environment in the cytoadhesion complex of malaria-causing parasites.

Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the Plasmodium falciparum-exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria-causing parasite, PfEMP1. We generated independent TurboID fusions of two proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

Enzymes known as protein kinases regulate a huge variety of biological processes inside cells by attaching small tags known as phosphate groups onto specific locations on certain proteins. For example, the parasite that causes malaria infections in humans and great apes, injects a protein kinase called FIKK4.1 into certain cells in its host. This enzyme then adds phosphate groups to various parasite and host proteins that, in turn, causes them to form a large group of proteins (known as the cytoadhesion complex) to protect the parasite from being cleared by the hosts' immune defences. However, it remains unclear how and where the complex forms, and how the parasite regulates it. Proximity labelling is a well-established method that allows researchers to label and identify proteins that are near to a protein of interest. To investigate how the FIKK4.1 enzyme alters host cells to make the cytoadhesion complex, Davies et al. combined proximity labelling with methods that disturb the normal state of cells at a specific timepoint during development. The team used this new approach ­ named PerTurboID ­ to identify the proteins surrounding three components in the cytoadhesion complex. This made it possible to create a map of proteins that FIKK4.1 is likely to modify to build and control the cytoadhesion complex. Further experiments examined what happened to these surrounding proteins when FIKK4.1 was inactivated. This revealed that some protein targets of FIKK4.1 become either more or less accessible to other enzymes that attach a molecule known as biotin to proteins. This could be a result of structural changes in the cytoadhesion complex that are normally regulated by the FIKK4.1 kinase. In the future, PerTurboID may be useful to study how genetics or environmental changes affect other groups of proteins within specific environments inside cells, such as protein complexes required for DNA replication or cell division, or assembly of temporal structures required for cell movement.

Catalytic process of anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa.

The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the pairing of hydrolytic ring opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM, and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the ß-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

Functional characterization of the phosphotransferase system in Parageobacillus thermoglucosidasius.

Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis.

PtsN in Pseudomonas aeruginosa Is Phosphorylated by Redundant Upstream Proteins and Impacts Virulence-Related Genes.

The bacterial nitrogen-related phosphotransfer (PTSNtr; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and phosphorylation. The Nitro-PTS comprises an enzyme I (EI), PtsP; an intermediate phosphate carrier, PtsO; and a terminal acceptor, PtsN, which is thought to exert regulatory effects that depend on its phosphostate. For instance, biofilm formation by Pseudomonas aeruginosa can be impacted by the Nitro-PTS, as deletion of either ptsP or ptsO suppresses Pel exopolysaccharide production and additional deletion of ptsN elevates Pel production. However, the phosphorylation state of PtsN in the presence and absence of its upstream phosphotransferases has not been directly assessed, and other targets of PtsN have not been well defined in P. aeruginosa. We show that PtsN phosphorylation via PtsP requires the GAF domain of PtsP and that PtsN is phosphorylated on histidine 68, as in Pseudomonas putida. We also find that FruB, the fructose EI, can substitute for PtsP in PtsN phosphorylation but only in the absence of PtsO, implicating PtsO as a specificity factor. Unphosphorylatable PtsN had a minimal effect on biofilm formation, suggesting that it is necessary but not sufficient for the reduction of Pel in a ptsP deletion. Finally, we use transcriptomics to show that the phosphostate and the presence of PtsN do not appear to alter the transcription of biofilm-related genes but do influence genes involved in type III secretion, potassium transport, and pyoverdine biosynthesis. Thus, the Nitro-PTS influences several P. aeruginosa behaviors, including the production of its signature virulence factors. IMPORTANCE The PtsN protein impacts the physiology of a number of bacterial species, and its control over downstream targets can be altered by its phosphorylation state. Neither its upstream phosphotransferases nor its downstream targets are well understood in Pseudomonas aeruginosa. Here, we examine PtsN phosphorylation and find that the immediate upstream phosphotransferase acts as a gatekeeper, allowing phosphorylation by only one of two potential upstream proteins. We use transcriptomics to discover that PtsN regulates the expression of gene families that are implicated in virulence. One emerging pattern is a repression hierarchy by different forms of PtsN: its phosphorylated state is more repressive than its unphosphorylated state, but the expression of its targets is even higher in its complete absence.

The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling.

Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations.

Functional and structural diversification of incomplete phosphotransferase system in cellulose-degrading clostridia.

Carbohydrate utilization is critical to microbial survival. The phosphotransferase system (PTS) is a well-documented microbial system with a prominent role in carbohydrate metabolism, which can transport carbohydrates through forming a phosphorylation cascade and regulate metabolism by protein phosphorylation or interactions in model strains. However, those PTS-mediated regulated mechanisms have been underexplored in non-model prokaryotes. Here, we performed massive genome mining for PTS components in nearly 15,000 prokaryotic genomes from 4,293 species and revealed a high prevalence of incomplete PTSs in prokaryotes with no association to microbial phylogeny. Among these incomplete PTS carriers, a group of lignocellulose degrading clostridia was identified to have lost PTS sugar transporters and carry a substitution of the conserved histidine residue in the core PTS component, HPr (histidine-phosphorylatable phosphocarrier). Ruminiclostridium cellulolyticum was then selected as a representative to interrogate the function of incomplete PTS components in carbohydrate metabolism. Inactivation of the HPr homolog reduced rather than increased carbohydrate utilization as previously indicated. In addition to regulating distinct transcriptional profiles, PTS associated CcpA (Catabolite Control Protein A) homologs diverged from previously described CcpA with varied metabolic relevance and distinct DNA binding motifs. Furthermore, the DNA binding of CcpA homologs is independent of HPr homolog, which is determined by structural changes at the interface of CcpA homologs, rather than in HPr homolog. These data concordantly support functional and structural diversification of PTS components in metabolic regulation and bring novel understanding of regulatory mechanisms of incomplete PTSs in cellulose-degrading clostridia.

OsSYMRK Plays an Essential Role in AM Symbiosis in Rice (Oryza sativa).

Arbuscular mycorrhizal (AM) fungi establish mutualistic symbiosis with a wide range of terrestrial plants, including rice. However, the mechanisms underlying the initiation of AM symbiosis are yet to be elucidated, particularly in nonleguminous plants. We previously demonstrated that chitin elicitor receptor kinase 1 (OsCERK1), a lysin motif receptor-like kinase essential for chitin-triggered immunity, also plays a key role in AM symbiosis in rice. However, the mechanisms underlying the regulation of switching between immunity and symbiosis by OsCERK1 are yet to be fully elucidated. SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK)/DOES NOT MAKE INFECTIONS 2 (DMI2) is a leucine-rich repeat receptor-like kinase associated with both root nodule symbiosis and AM symbiosis in legumes. The homolog of SYMRK in rice, OsSYMRK, has a shorter form than that in legumes because OsSYMRK lacks a malectin-like domain (MLD). The MLD reportedly contributes to symbiosis in Lotus japonicus; however, the contribution of OsSYMRK to AM symbiosis in rice remains unclear. Phylogenetic analyses indicated that the MLD of SYMRK/DMI2 is widely conserved even in mosses and ferns but absent in commelinids, including rice. To understand the function of OsSYMRK, we produced an Ossymrk knockout mutant using genome editing technology. AM colonization was mostly abolished in Ossymrk with a more severe phenotype than Oscerk1. Ca2+ spiking against chitin tetramer was also diminished in Ossymrk. In contrast, comparable defense responses against chitin heptamer to the wild type were observed in Ossymrk. Bimolecular fluorescence complementation studies demonstrating an interaction between OsSYMRK and OsCERK1 indicate that OsSYMRK may play an important role in switching from immunity to symbiosis through the interaction with OsCERK1 in rice.

BarA/UvrY differentially regulates prodigiosin biosynthesis and swarming motility in Serratia marcescens FS14.

BarA/UvrY, a two-component system and global regulator that controls expression of more than a hundred of genes involved in virulence, motility, biofilm formation, and central carbon metabolism under various stress conditions. In this study, we investigated the function of BarA/UvrY system in Serratia marcescens FS14. The disruption of barA or/and uvrY results in the yield increase of secondary metabolite prodigiosin. We further demonstrated that BarA/UvrY system represses prodigiosin production by inhibiting the transcription level of pig gene cluster with direct binding to the pigA promoter. In addition, deletion of barA or/and uvrY abolished the swarming motility of FS14, but not the swimming motility. We revealed that BarA/UvrY activates swarming through directly upregulating the expression of the biosurfactant synthesis gene swrW rather than flagella system. We also observed that BarA/UvrY positively regulates the resistance to H2O2 same as in Escherichia coli highlighting the importance of BarA/UvrY on hydrogen peroxide resistance. Our results demonstrated that the BarA/UvrY system differentially regulates the biosynthesis of the secondary metabolite prodigiosin and swarming motility in S. marcescens FS14. Comparison of our results with those observed for Serratia sp. 39006 suggests that BarA/UvrY's role in regulation of secondary metabolite production is different among Serratia species.

CK2 promotes jasmonic acid signaling response by phosphorylating MYC2 in Arabidopsis.

Jasmonic acid (JA) signaling plays a pivotal role in plant development and defense. MYC2 is a master transcription factor in JA signaling, and was found to be phosphorylated and negatively regulated by MAP kinase and receptor-like kinase. However, the kinases that positively regulate MYC2 through phosphorylation and promote MYC2-mediated activation of JA response have not been identified. Here, we identified CK2 as a kinase that phosphorylates MYC2 and thus regulates the JA signaling. CK2 holoenzyme can interact with MYC2 using its regulatory subunits and phosphorylate MYC2 at multiple sites with its catalytic subunits. Inhibition of CK2 activity in a dominant-negative plant line, CK2mut, repressed JA response. On the other hand, increasing CK2 activity by overexpression of CKB4, a regulatory subunit gene of CK2, enhanced JA response in a MYC2-dependent manner. Substitution of the Ser and Thr residues at phosphorylation sites of MYC2 by CK2 with Ala impaired MYC2 function in activating JA response. Further investigations evidenced that CK2 facilitated the JA-induced increase of MYC2 binding to the promoters of JA-responsive genes in vivo. Our study demonstrated that CK2 plays a positive role in JA signaling, and reveals a previously undiscovered mechanism that regulates MYC2 function.

Rare multiple congenital anomalies-hypotonia-seizures syndrome type 1 (MCAHS1) - the clinical and molecular summary.

Multiple congenital anomalies-hypotonia-seizures syndrome type 1 (MCAHS1) is a rare autosomal recessive genetic disease belonging to glycosylphosphatidylinositols biosynthesis defects (GPIBD), a group of recessive disorders characterized by intellectual disability, hypotonia, and seizures. Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor and remodel cell proteins. These processes are highly conserved and fundamental in the metabolism of all eukaryotes, including humans. Here, we have reported a male patient presenting with hypotonia, intellectual disability, and epilepsy, who underwent whole exome sequencing (WES). The analysis revealed the presence of two deleterious variants in PIGN that encodes GPI ethanolamine phosphate transferase-1 - one novel (c.1247_1251delAAGTG; p.Glu416Glyfs*22), and one that has been previously reported in the medical literature (c.1434+5G>A) resulting in MCAHS1. The detailed clinical assessment followed by the medical literature review also pointed out transient macrosomia and unreported in MCAHS1 advanced bone age and postnatal tall stature. These symptoms suggest that MCAHS1 shares a phenotypic overlap with disorders associated with overgrowth. To conclude, our case report and summary of the medical literature may be helpful for clinicians and geneticists who diagnose patients presenting with hypotonia accompanied by tall stature, advanced bone age, and transient macrosomia.

A screening of inhibitors targeting the receptor kinase FERONIA reveals small molecules that enhance plant root immunity.

Receptor-like kinases (RLKs) constitute the largest receptor family involved in the regulation of plant immunity and growth, but small-molecule inhibitors that target RLKs to improve agronomic traits remain unexplored. The RLK member FERONIA (FER) negatively regulates plant resistance to certain soil-borne diseases that are difficult to control and cause huge losses in crop yields and economy. Here, we identified 33 highly effective FER kinase inhibitors from 1494 small molecules by monitoring FER autophosphorylation in vitro. Four representative inhibitors (reversine, cenisertib, staurosporine and lavendustin A) inhibited the kinase activity of FER and its homologues in several crops by targeting the conserved ATP pocket in the kinase structure. FER contributes to the physiological impact of representative inhibitors in plants. The treatment of roots with reversine, staurosporine and lavendustin A enhanced innate immunity in plant roots and thus alleviated soil-borne diseases in tobacco, tomato and rice without growth penalties. Consistently, RNA sequencing assays showed that lavendustin A and reversine exert profound impacts on immunity-related gene expression. Our results will set a new milestone in the development of the plant RLK kinase regulation theory and provide a novel strategy for the prevention and control of plant soil-borne diseases without growth penalties.

PIGN-Related Disease in Two Lithuanian Families: A Report of Two Novel Pathogenic Variants, Molecular and Clinical Characterisation.

Background and Objectives: Pathogenic variants of PIGN are a known cause of multiple congenital anomalies-hypotonia-seizures syndrome 1 (MCAHS1). Many affected individuals have clinical features overlapping with Fryns syndrome and are mainly characterised by developmental delay, congenital anomalies, hypotonia, seizures, and specific minor facial anomalies. This study investigates the clinical and molecular data of three individuals from two unrelated families, the clinical features of which were consistent with a diagnosis of MCAHS1. Materials and Methods: Next-generation sequencing (NGS) technology was used to identify the changes in the DNA sequence. Sanger sequencing of gDNA of probands and their parents was used for validation and segregation analysis. Bioinformatics tools were used to investigate the consequences of pathogenic or likely pathogenic PIGN variants at the protein sequence and structure level. Results: The analysis of NGS data and segregation analysis revealed a compound heterozygous NM_176787.5:c.[1942G>T];[1247_1251del] PIGN genotype in family 1 and NG_033144.1(NM_176787.5):c.[932T>G];[1674+1G>C] PIGN genotype in family 2. In silico, c.1942G>T (p.(Glu648Ter)), c.1247_1251del (p.(Glu416GlyfsTer22)), and c.1674+1G>C (p.(Glu525AspfsTer68)) variants are predicted to result in a premature termination codon that leads to truncated and functionally disrupted protein causing the phenotype of MCAHS1 in the affected individuals. Conclusions: PIGN-related disease represents a wide spectrum of phenotypic features, making clinical diagnosis inaccurate and complicated. The genetic testing of every individual with this phenotype provides new insights into the origin and development of the disease.

Phosphotransferase system sugars immediately induce mutations of Cra in an Escherichia coli ptsH mutant.

Most bacteria use the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) to catalyse coupled transport and phosphorylation of sugars. The PTS consists of several sugar-specific components (enzyme IIs) and two general components: enzyme I, encoded by ptsI, and HPr, encoded by ptsH, which are common to most PTS carbohydrates. Although both enzyme I and HPr are believed to be required to utilize these PTS sugars, an E. coli ptsH mutant has been reported to exhibit a leaky growth phenotype on these sugars. Here, we show that this phenomenon occurs because the ptsH mutant undergoes adaptive mutations in the presence of PTS sugars within a few generation times. The ptsH mutant cells once exposed to a PTS sugar showed a growth rate similar to that of the wild-type strain when transferred to fresh medium supplemented with the same PTS sugar, suggesting the acquisition of additional genetic variations. Genome sequencing revealed that the PTS sugar-adapted variants harboured loss-of-function mutations in cra, which increased expression of the fruBKA operon. Our results suggest that the presence of a PTS sugar can exert a strong selective pressure when a general PTS component is defective.

Escherichia coli BarA-UvrY regulates the pks island and kills Staphylococci via the genotoxin colibactin during interspecies competition.

Wound infections are often polymicrobial in nature, biofilm associated and therefore tolerant to antibiotic therapy, and associated with delayed healing. Escherichia coli and Staphylococcus aureus are among the most frequently cultured pathogens from wound infections. However, little is known about the frequency or consequence of E. coli and S. aureus polymicrobial interactions during wound infections. Here we show that E. coli kills Staphylococci, including S. aureus, both in vitro and in a mouse excisional wound model via the genotoxin, colibactin. Colibactin biosynthesis is encoded by the pks locus, which we identified in nearly 30% of human E. coli wound infection isolates. While it is not clear how colibactin is released from E. coli or how it penetrates target cells, we found that the colibactin intermediate N-myristoyl-D-Asn (NMDA) disrupts the S. aureus membrane. We also show that the BarA-UvrY two component system (TCS) senses the environment created during E. coli and S. aureus mixed species interaction, leading to upregulation of pks island genes. Further, we show that BarA-UvrY acts via the carbon storage global regulatory (Csr) system to control pks expression. Together, our data demonstrate the role of colibactin in interspecies competition and show that it is regulated by BarA-UvrY TCS during interspecies competition.

Controlling catabolite repression for isobutanol production using glucose and xylose by overexpressing the xylose regulator.

Using lignocellulosic biomass is immensely beneficial for the economical production of biochemicals. However, utilizing mixed sugars from lignocellulosic biomass is challenging because of bacterial preference for specific sugar such as glucose. Although previous studies have attempted to overcome this challenge, no studies have been reported on isobutanol production from mixed sugars in the Escherichia coli strain. To overcome catabolite repression of xylose and produce isobutanol using mixed sugars, we applied the combination of three strategies: (1) deletion of the gene for the glucose-specific transporter of the phosphotransferase system (ptsG); (2) overexpression of glucose kinase (glk) and glucose facilitator protein (glf); and (3) overexpression of the xylose regulator (xylR). xylR gene overexpression resulted in 100% of glucose and 82.5% of xylose consumption in the glucose-xylose mixture (1:1). Moreover, isobutanol production increased by 192% in the 1:1 medium, equivalent to the amount of isobutanol produced using only glucose. These results indicate the effectiveness of xylR overexpression in isobutanol production. Our findings demonstrated various strategies to overcome catabolite repression for a specific product, isobutanol. The present study suggests that the selected strategy in E. coli could overcome the major challenge using lignocellulosic biomass to produce isobutanol.