Nonpermissive Research Articles

Structure of native photosystem II assembly intermediate from Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii Photosystem II (PSII) is a dimer consisting of at least 13 nuclear-encoded and four chloroplast-encoded protein subunits that collectively function as a sunlight-driven oxidoreductase. In this study, we present the inaugural structure of a green alga PSII assembly intermediate (pre-PSII-int). This intermediate was isolated from chloroplast membranes of the temperature-sensitive mutant TSP4, cultivated for 14 hours at a non-permissive temperature. The assembly state comprises a monomer containing subunits A, B, C, D, E, F, H, I, K, and two novel assembly factors, Psb1 and Psb2. Psb1 is identified as a novel transmembrane helix located adjacent to PsbE and PsbF (cytochrome b559). The absence of PsbJ, typically found in mature PSII close to this position, indicates that Psb1 functions as an assembly factor. Psb2 is an eukaryotic homolog of the cyanobacterial assembly factor Psb27. The presence of iron, coupled with the absence of QA, QB, and the manganese cluster, implies a protective mechanism against photodamage and provides insights into the intricate assembly process.

Structure-based identification of potential natural compound inhibitors targeting bacterial cytoskeleton protein FtsZ from Acinetobacter baumannii by computational studies

Acinetobacter baumannii is one of the multi-drug-resistant pathogens responsible for hospital-acquired infections reported worldwide. Clinically it is challenging to treat these pathogens as they have developed resistance against the existing class of antibiotics. Hence, there is an urgent need to develop a new class of antibiotics against these pathogens to prevent the spread of infections and mortality. In Acinetobacter baumannii, the filamentous temperature-sensitive mutant Z protein polymerizes at the imminent division site to form a Z-ring at the mid-point of the cell and act as a scaffold to recruit other cell division proteins involved in orchestrating septum synthesis in bacteria. Perturbation in the assembly of FtsZ affects bacterial cell dynamics and survival. Hence, FtsZ has emerged as a new drug target in antibiotic discovery to identify compounds that inhibit bacterial cell division. In this study, we have performed a virtual screening of 30,000 compounds from the ZINC Biogenic natural compound library targeting the nucleotide-binding site of FtsZ from Acinetobacter baumannii. We have identified 8 new natural compounds with binding energy in the range of −8.66 to −6.953 kcal/mol and analyzed them by 200 ns molecular dynamics simulations. Out of these eight compounds, ZINC14708526 showed the best binding with relatively optimal drug-likeness and medicinal chemistry as a potent inhibitor of abFtsZ. Thus, the identified FtsZ inhibitor ZINC14708526 is a promising lead compound to develop potent antimicrobial agents against Acinetobacter baumannii infections. Communicated by Ramaswamy H. Sarma

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a “molecular memory” of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3′-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

Antibacterial Activity and AbFtsZ Binding Properties of Fungal Metabolites Isolated from Mexican Mangroves

Antimicrobial resistance is emerging as a global health challenge that requires immediate and concerted attention. Accordingly, the WHO has issued alerts urging to continue developing antibiotics with novel mechanisms of action toward clinically important pathogens, including Acinetobacter baumannii. In this context, fungi have played a crucial role in the discovery and development of antibiotics. Therefore, in this work, three fungal strains were prioritized based on their metabolic profiles and antibacterial activity against a pan-resistant isolate of A. baumannii, to identify potential antibiotic molecules. Chemical investigation of the selected fungi (mangrove endophytes) led to the isolation of asperazine (1), aurasperone B (2), aurasperone F (3), TMC-256A1 (4), fonsecin B (5), dianhydroaurasperone C (6), aurasperone A (7), pyrophen (8), and penicillide (9). Moreover, an in vitro assay to detect ligands of the filamentous temperature-sensitive mutant Z enzyme of A. baumannii (AbFtsZ), a GTPase that plays a central role in bacterial division, was developed to correlate the antibacterial properties of the isolated molecules to a mechanism of action. Compounds 1–4 and 9 inhibited the growth of A. baumannii. Interestingly, compounds 2, 3, and 5–9 interacted with AbFtsZ1-412, increasing its GTPase activity. Conversely, compound 4 exhibited an outstanding ability to act as an inhibitor of both the enzymatic activity and the growth of the strain under study.Graphical

Cell type-specific regulation by different cytokinetic pathways in the early embryo.

Cytokinesis, the physical division of one cell into two, is typically assumed to use the same molecular process across animal cells. However, regulation of cell division can vary significantly among different cell types, even within the same multicellular organism. Using six fast-acting temperature-sensitive (ts) cytokinesis-defective mutants, we found that each had unique cell type-specific profiles in the early 2-cell through 8-cell C. elegans embryo. Certain cell types were more sensitive than others to actomyosin and spindle signaling disruptions, disrupting two members of the same complex could result in different phenotypes, and protection against actomyosin inhibition did not always protect against spindle signaling inhibition.

Characterization of temperature-sensitive alleles of Schizosaccharomyces pombe septation initiation network components.

The Schizosaccharomyces pombe septation initiation network (SIN) promotes cytokinesis and septation. Comprised of a protein kinase cascade triggered by activation of a small GTPase and inhibited by a two-component GAP that localize to the spindle pole bodies in a cell cycle specific manner. Here, we characterized temperature-sensitive mutants isolated in the 1990s in four SIN components. We determined the mutations within each cdc14 , cdc16 , sid1 , and sid2 mutant allele and analyzed their growth at different temperatures compared with known mutant alleles. The new mutants described here expand the toolkit for studying SIN signaling.

Investigating postsynaptic effects of a Drosophila neuropeptide on muscle contraction.

The Drosophila neuropeptide, DPKQDFMRFamide, was previously shown to enhance excitatory junctional potentials (EJPs) and muscle contraction by both presynaptic and postsynaptic actions. Since the peptide acts on both sides of the synaptic cleft, it has been difficult to examine postsynaptic modulatory mechanisms, particularly when contractions are elicited by nerve stimulation. Here, postsynaptic actions are examined in 3rd instar larvae by applying peptide and the excitatory neurotransmitter, l-glutamate, in the bathing solution to elicit contractions after silencing motor output by removing the central nervous system (CNS). DPKQDFMRFamide enhanced glutamate-evoked contractions at low concentrations (EC50 1.3 nM), consistent with its role as a neurohormone, and the combined effect of both substances was supra-additive. Glutamate-evoked contractions were also enhanced when transmitter release was blocked in temperature-sensitive (Shibire) mutants, confirming the peptide's postsynaptic action. The peptide increased membrane depolarization in muscle when co-applied with glutamate, and its effects were blocked by nifedipine, an L-type channel blocker, indicating effects at the plasma membrane involving calcium influx. DPKQDFMRFamide also enhanced contractions induced by caffeine in the absence of extracellular calcium, suggesting increased calcium release from the sarcoplasmic reticulum (SR) or effects downstream of calcium release from the SR. The peptide's effects do not appear to involve calcium/calmodulin-dependent protein kinase II (CaMKII), previously shown to mediate presynaptic effects. The approach used here might be useful for examining postsynaptic effects of neurohormones and cotransmitters in other systems.NEW & NOTEWORTHY Distinguishing presynaptic and postsynaptic effects of neurohormones is a long-standing challenge in many model organisms. Here, postsynaptic actions of DPKQDFMRFamide are demonstrated by assessing its ability to potentiate contractions elicited by direct application of the neurotransmitter, glutamate, when axons are silent and when transmitter release is blocked. The peptide acts at multiple sites to increase contraction, increasing glutamate-induced depolarization at the cell membrane, acting on L-type channels, and acting downstream of calcium release from the sarcoplasmic reticulum.

T5S1607 identified as a antibacterial FtsZ inhibitor：Virtual screening combined with bioactivity evaluation for the drug discovery

Due to antibiotic overuse, many bacteria have developed resistance, creating an urgent need for novel antimicrobial agents. It has been established that the filamentous temperature-sensitive mutant Z (FtsZ) of the bacterial cell division protein is an effective and promising antibacterial target. In this study, the optimal proteins were assessed by early recognition ability and the processed compound libraries were virtually screened using Vina. This effort resulted in the identification of 14 potentially active antimicrobial compounds. Among them, the compound T5S1607 demonstrated remarkable antibacterial efficacy against Bacillus subtilis ATCC9732 (MIC = 1 μg/mL) and Staphylococcus aureus ATC5C6538 (MIC = 4 μg/mL). Furthermore, in vitro experiments demonstrated that the selected compound T5S1607 rapidly killed bacteria and induced FtsZ protein aggregation, preventing bacterial division and leading to bacterial death. Additionally, cell toxicity and hemolysis experiments indicate that compound T5S1607 exhibits minimal toxicity to LO2 cells and shows no significant hemolytic effects on mammalian cells in vitro at the MIC concentration range. All the results indicate that compound T5S1607 is a promising antibacterial agent and a potential FtsZ inhibitor. In conclusion, this work successfully discovered FtsZ inhibitors with good activity through the virtual screening drug discovery process.

Fission Yeast TORC1 Promotes Cell Proliferation through Sfp1, a Transcription Factor Involved in Ribosome Biogenesis.

Target of rapamycin complex 1 (TORC1) is activated in response to nutrient availability and growth factors, promoting cellular anabolism and proliferation. To explore the mechanism of TORC1-mediated proliferation control, we performed a genetic screen in fission yeast and identified Sfp1, a zinc-finger transcription factor, as a multicopy suppressor of temperature-sensitive TORC1 mutants. Our observations suggest that TORC1 phosphorylates Sfp1 and protects Sfp1 from proteasomal degradation. Transcription analysis revealed that Sfp1 positively regulates genes involved in ribosome production together with two additional transcription factors, Ifh1/Crf1 and Fhl1. Ifh1 physically interacts with Fhl1, and the nuclear localization of Ifh1 is regulated in response to nutrient levels in a manner dependent on TORC1 and Sfp1. Taken together, our data suggest that the transcriptional regulation of the genes involved in ribosome biosynthesis by Sfp1, Ifh1, and Fhl1 is one of the key pathways through which nutrient-activated TORC1 promotes cell proliferation.

Naturally occurring PAE206K point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures

The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century. In this study, we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients. Sequence comparison of the three main protein subunits (PB2, PB1, and PA) of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution (E206K) was responsible for the observed impaired replication phenotype. Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/WSN/33 virus in mammalian cells and a reduction in the virus’s pathogenicity in vivo. Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1–PA complex to the nucleus at high temperature (39.5 ​°C). Hence, this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines.

Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast.

Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.

FRI014 Exploring The Epigenetic Fingerprint Of Sedentary Behavior

Abstract Disclosure: A. Brener: None. D. Lorber: None. A. Reuveny: None. Y. Lebenthal: None. T. Volk: None. Eating habits and physical activity play a central role among the environmental factors that modulate biological aging. Sedentary lifestyle, characterized by increased waking time spent in minimal energy expenditure activity, is closely associated with increased cardiometabolic risk, a principal hallmark of aging. The biological mechanisms linking a sedentary lifestyle with metabolic derangements are incompletely understood. Exercise was reported as a stimulator of epigenetic modifications of the DNA in human muscle cell nuclei (myonuclei). Exploring the specific impact of sedentary behavior on epigenetics, especially among younger individuals, requires the exclusion of the effects of inflammatory and nutritional contributors to medical conditions. We developed an experimental animal model that imitates sedentary behavior in which a chromatin epigenetic landscape could be explored. We used third-instar Drosophila larvae carrying a temperature-sensitive mutation in the shibire gene coding for GTPase involved in neuronal synaptic vesicle transport. Shibire homozygous mutant larvae are paralyzed at restrictive temperature (>∼29°C) due to inhibition of neuromuscular junction vesicular transport, while muscle contraction is abolished without affecting other systems. Shibire and control (y,w) larvae were kept at 18°C or 30°C without food for six hours. They were then dissected, fixed and double-labeled with antibodies specific for epigenetic marks of active (H3K9ac) and repressive (H3K27me3) histone modifications. An anti-lamin fluorescent antibody served for nuclear membrane demarcation. Immunofluorescence images of epigenetic marks were acquired on a confocal microscope Zeiss LSM 800, after which a quantitative analysis of epigenetics marks was performed. At the restrictive temperature (30°C), the crawling motion of shibire mutant larvae ceased in contrast to control (shibire at 18°C). The crawling motion of the y,w larvae was similar at 18°C and 30°C. The mean repressive mark H3K27me3 decreased (p<0.001), while the mean active H3KPac increased (p<0.001) in the myonuclei of sedentary shibire larvae (30°C). The mean myonuclear active/repressed ratio of epigenetic marks (H3K9ac/H3K27me) was similar in both active shibire (18°C) and y,w at 18°C, while it increased significantly in sedentary shibire (30°C) (p<0.001). In contrast, this ratio decreased in the control larvae (y,w at 30°C) (p<0.001). The findings of this model, which was aimed at reflecting sedentary behavior in youth, revealed that muscle inactivity changed the epigenetic fingerprint in myonuclei, with a balance shift towards decreased chromatin methylation relative to acetylation. Our finding suggests a decline in the epigenetic control of gene transcription, reminiscent of decreased epigenetic repression described with aging. Presentation: Friday, June 16, 2023

Detection and Characterisation of Endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in Elaeidobius kamerunicus (Coleoptera: Curculionoidea), Pollinating Agent of Oil Palm, and Its Relationships between Populations.

Elaeidobius kamerunicus is the most efficient pollinator of oil palm. Wolbachia is an endosymbiotic bacteria associated with E. kamerunicus that has a potential to affect the fecundity and fitness of the E. kamerunicus. Despite their importance, no studies have been conducted to investigate its prevalence in E. kamerunicus. The objectives of this study were to detect and characterise Wolbachia in E. kamerunicus and determine the phylogenetic relationship of Wolbachia strains that infect E. kamerunicus by using three genetic markers namely Filamenting temperature-sensitive mutant Z (ftsZ), Chaperonin folding protein (groEL), and Citrate Synthase Coding Gene (gltA). DNA was extracted from 210 individuals of E. kamerunicus and the Wolbachia infections were detected using the wsp marker. The infected samples (n = 25, 11.9%) were then sequenced using ftsZ, gltA and groEL markers for strain characterization. In this study, a combination of four markers was used to construct the phylogeny of Wolbachia. Similar topologies were shown in all trees; Neighbour-Joining (NJ), Maximum Parsimony (MP), and Bayesian Inference (BI), which showed the mixing of individuals that harbor Wolbachia between populations. Interestingly, Wolbachia on E. kamerunicus was claded together with the species Drosophila simulans under supergroup B. This is the first report of Wolbachia infecting E. kamerunicus which is very valuable and significant as one of the parameters to evaluate the quality of the E. kamerunicus population for sustaining its function as a great pollinator for oil palm.

Where is the EXIT? Phenotypic screens for new egress factors in apicomplexan parasites.

Apicomplexans, such as Plasmodium and Toxoplasma are obligate intracellular parasites that invade, replicate and finally EXIT their host cell. During replication within a parasitophorous vacuole (PV), the parasites establish an extensive F-actin-containing network that connects individual parasites and is required for material exchange, recycling and the final steps of daughter cell assembly. After multiple rounds of replication, the parasites exit the host cell involving multiple signalling cascades, disassembly of the network, secretion of microneme proteins and activation of the acto-myosin motor. Blocking the host cell EXIT process leads to the formation of large PVs, making the screening for genes involved in exiting the cell relatively straightforward. Given that apicomplexans are highly diverse from other eukaryotes, approximately 30% of all genes are annotated as hypothetical, some apicomplexan-specific factors are likely to be critical during EXIT. This motivated several labs to design and perform forward genetic and phenotypic screens using various approaches, such as random insertion mutagenesis, temperature-sensitive mutants and, more recently, CRISPR/Cas9-mediated targeted editing and conditional mutagenesis. Here we will provide an overview of the technological developments over recent years and the most successful stories that led to the identification of new critical factors in Toxoplasma gondii.

Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae.

The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. Our findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating our findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases.

Animal Models for Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular protozoan parasite that commonly infects mammals and birds throughout the world. This protocol describes murine models of acute T. gondii infection, toxoplasmic encephalitis and toxoplasma retinochoroiditis. T. gondii infection in severe combined immunodeficient (SCID) mice, deficient in T and B cells, has allowed for the study of T cell-independent mechanisms of defense against intracellular organisms, as described here. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii induce protection against challenge with virulent strains of the parasite. They have allowed studies of immunization and adoptive-transfer experiments. A protocol is provided for infection with these mutant strains. The EGS strain of T. gondii has the unique feature of spontaneously forming tissue cysts in cell culture. Dual fluorescent reporter stains of this strain have allowed the study of tachyzoite to bradyzoite transitions in vitro and in vivo. A protocol for in vitro and in vivo growth of this strain and tissue cyst isolation is provided. Genetic manipulation of T. gondii and mice has led to the development of parasites that express fluorescent proteins as well as mice with fluorescently labeled leukocytes. This together with the use of T. gondii that express model antigens and transgenic mice that express the appropriate T cell receptor have facilitated the in vivo study of parasite host-interaction. In addition, parasites that express bioluminescent markers have made it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite that includes these newer methodologies. In addition, support protocols address the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of acute T. gondii infection in mice Basic Protocol 2: Model of toxoplasmic encephalitis and toxoplasma retinochoroiditis in chronically infected mice Basic Protocol 3: Assessment of T. gondii invasion into neural tissue Basic Protocol 4: T. gondii infection in scid/scid (SCID) mice Basic Protocol 5: Infection with the uracil auxotroph strain CPS1-1 or the temperature-sensitive TS-4 strain of T. gondii Basic Protocol 6: In vivo and in vitro maintenance of the EGS strain of T. gondii Support Protocol 1: Assessment of progression of infection and immune response to T. gondii Support Protocol 2: Maintenance of a bank of T. gondii cysts of the ME49 strain Support Protocol 3: Maintenance of T. gondii tachyzoites using human foreskin fibroblasts Support Protocol 4: Maintenance of T. gondii tachyzoites in mice Support Protocol 5: Preparation of T. gondii lysate antigens Support Protocol 6: Isolation of T. gondii tissue cysts from brain.

Targeting Filamenting temperature-sensitive mutant Z (FtsZ) with bioactive phytoconstituents: An emerging strategy for antibacterial therapy.

The rise and widespread occurrence of bacterial resistance has created an evident need for novel antibacterial drugs. Filamenting temperature-sensitive mutant Z (FtsZ) is a crucial bacterial protein that forms a ring-like structure known as the Z-ring, playing a significant role in cell division. Targeting FtsZ is an effective approach for developing antibiotics that disrupt bacterial cell division and halt growth. This study aimed to use a virtual screening approach to search for bioactive phytoconstituents with the potential to inhibit FtsZ. The screening process proceeded with the filtering compounds from the IMPPAT library of phytochemicals based on their physicochemical properties using the Lipinski rule of five. This was followed by molecular docking, Pan-assay interference compounds (PAINS) filter, absorption, distribution, metabolism, excretion, and toxicity (ADMET), prediction of activity spectra for biologically active substances (PASS), and molecular dynamics (MD) simulations. These filters ensured that any adverse effects that could impede the identification of potential inhibitors of FtsZ were eliminated. Following this, two phytocompounds, Withaperuvin C and Trifolirhizin, were selected after the screening, demonstrating noteworthy binding potential with FtsZ's GTP binding pocket, acting as potent GTP-competitive inhibitors of FtsZ. The study suggested that these compounds could be further investigated for developing a novel class of antibiotics after required studies.

Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.

Synthesis of epitope-targeting nanobody based on native protein-protein interactions for FtsZ filamentation suppressor.

Phage display and biopanning are powerful tools for generating binding molecules for a specific target. However, the selection process based only on binding affinity provides no assurance for the antibody's affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein-protein interactions to generate epitope-targeting antibodies. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the nanobody, and a nonrelated CDR loop (in the grafted nanobody) was randomized to create a phage display library. In this construction of nanobodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence added functionality to the nanobody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein-protein interactions. To apply for biological tool with growth screening, model nanobodies with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) from Staphylococcus aureus were constructed and completely inhibited the polymerization of FtsZ as a function. Consequently, the expression of these nanobodies drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein-protein interactions for steady epitope-specific evolutionary engineering.

Functional analysis of 3-Oxacyl-Acyl carrier protein reductase homolog (FabG3) in Xanthomonas arboricola pv. juglandis

Most of the pathogenic microorganisms that cause serious production and economic losses for the agricultural industry are members of the genus Xanthomonas. The main bacterial pathogen causing walnut black spot is Xanthomonas arboricola pv. juglandis (Xaj). Members of the genus Xanthomonas produce a yellow pigment called xanthomonadin, which is membrane-bound and protects the bacteria against photobiological and peroxidative damage. Here, we report the characterization of a gene named XajfabG3 (a homologous gene of fabG3 in Xanthomonas campestris pv. campestris), demonstrating that it is responsible for xanthomonadin synthesis. Expression of XajfabG3 in Escherichia coli fabG temperature-sensitive mutant CL104 restored the growth of CL104 under nonpermissive temperature, although XajFabG3 had a lower activity than XajFabG1 or FabG from Escherichia coli. In vitro assays demonstrated that XajFabG3 could reduce 3-oxoacyl-acyl carrier proteins (-ACPs) of fatty acid synthesis reactions and showed that although deletion of XajfabG3 did not affect bacterial growth and fatty acid composition, xanthomonadin production was abolished in a XajfabG3-deficient strain. Xanthomonadin production could be restored only by wild-type fabG3 and not by XajfabG1, another 3-oxoacyl-ACP reductase-encoding gene in Xaj, indicating that XajFabG3 specifically participates in the biosynthesis of xanthomonadin. Moreover, our study showed that the XajfabG3 knockout mutant affected virulence in the host Juglans regia L. Our elucidation of the biological function of the fabG3 gene in Xaj provides a target for the development of prevention and treatment agents for walnut blight.