Localization and phenotypic expression of a Tol marker in Citrobacter freundii

Among colicin-A-tolerant mutants of Citrobacter freundii we characterized some as Tol-2 mutants. The Tol-2 mutation results in insensitivity to bacteriocin S6 and an enhanced sensitivity to deoxycholate (DOC), ethylenediaminetetraacetic acid (EDTA) and to ampicillin. The Tol-2 mutation was mapped close near gal and the gene order pro-tol-gal was established in crosses between C. freundii Hfr tol ⁺ donor strains and C. freundii tol ^- acceptor strains. In these crosses a difference was observed in phenotypic expression of the pleiotropic properties of this tol ⁺ gene. Expression of resistance to DOC is substantially slower than the expression of EDTA-resistance. This phenomenon may play a disturbing role in those studies on cell envelope mutants, in which resistance to DOC is used as a selected marker. The differences in expression of DOC-and EDTA-resistance are discussed.     

Spontaneous frequency of a developmental mutant in Volvox

Two types of mutants, those resistant to the base analog 5-bromo-2′-deoxyuridine (BrdU) and somatic regenerator (SR) mutants, have been analyzed in Volvox carteri. In somatic regenerator mutants, the somatic cells which are normally terminally differentiated dedifferentiate and regenerate gonidia [Sessoms, A., and Huskey, R. J. (1973). Proc. Nat. Acad. Sci. USA70, 1335–1338; Starr, R. C. (1970). Develop. Biol. Suppl.4, 59–100]. The SR phenotype allows recovery of SR mutations arising in somatic cells, since such somatic cells would regenerate gonidia and give rise to mutant clones. Mutants of any phenotype other than SR can only be recovered if the mutation first appears in a gonidium. Since the somatic cells are 100-fold more numerous than reproductive cells (gonidia), we have determined the spontaneous frequency of both somatic regenerator mutants and mutations to BrdU resistance in order to determine if the SR mutation exerts its effect in the gonidium or in the somatic cell. The two frequencies were found to be nearly identical, suggesting that the SR mutation must first appear in a gonidium in order to be expressed.     

Synergistic anti-tumor efficacy of mutant isocitrate dehydrogenase 1 inhibitor SYC-435 with standard therapy in patient-derived xenograft mouse models of glioma

Clinical outcomes in patients with WHO grade II/III astrocytoma, oligodendroglioma or secondary glioblastoma remain poor. Isocitrate dehydrogenase 1 (IDH1) is mutated in > 70% of these tumors, making it an attractive therapeutic target. To determine the efficacy of our newly developed mutant IDH1 inhibitor, SYC-435 (1-hydroxypyridin-2-one), we treated orthotopic glioma xenograft model (IC-BT142AOA) carrying R132H mutation and our newly established orthotopic patient-derived xenograft (PDX) model of recurrent anaplastic oligoastrocytoma (IC-V0914AOA) bearing R132C mutation. In addition to suppressing IDH1 mutant cell proliferation in vitro, SYC-435 (15 mg/kg, daily x 28 days) synergistically prolonged animal survival times with standard therapies (Temozolomide + fractionated radiation) mediated by reduction of H3K4/H3K9 methylation and expression of mitochondrial DNA (mtDNA)-encoded molecules. Furthermore, RNA-seq of the remnant tumors identified genes (MYO1F, CTC1 and BCL9) and pathways (base excision repair, TCA cycle II, sirtuin signaling, protein kinase A, eukaryotic initiation factor 2 and α-adrenergic signaling) as mediators of therapy resistance. Our data demonstrated the efficacy SYC-435 in targeting IDH1 mutant gliomas when combined with standard therapy and identified a novel set of genes that should be prioritized for future studies to overcome SYC-435 resistance.     

Hfq variant with altered RNA binding functions

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS–lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq–RNA interaction but has probably no effect on the folding and the oligomerization of the protein.     

Sensing Cytosolic RpsL by Macrophages Induces Lysosomal Cell Death and Termination of Bacterial Infection

The intracellular bacterial pathogen Legionella pneumophila provokes strong host responses and has proven to be a valuable model for the discovery of novel immunosurveillance pathways. Our previous work revealed that an environmental isolate of L. pneumophila induces a noncanonical form of cell death, leading to restriction of bacterial replication in primary mouse macrophages. Here we show that such restriction also occurs in infections with wild type clinical isolates. Importantly, we found that a lysine to arginine mutation at residue 88 (K88R) in the ribosome protein RpsL that not only confers bacterial resistance to streptomycin, but more importantly, severely attenuated the induction of host cell death and enabled L. pneumophila to replicate in primary mouse macrophages. Although conferring similar resistance to streptomycin, a K43N mutation in RpsL does not allow productive intracellular bacterial replication. Further analysis indicated that RpsL is capable of effectively inducing macrophage death via a pathway involved in lysosomal membrane permeabilization; the K88R mutant elicits similar responses but is less potent. Moreover, cathepsin B, a lysosomal protease that causes cell death after being released into the cytosol upon the loss of membrane integrity, is required for efficient RpsL-induced macrophage death. Furthermore, despite the critical role of cathepsin B in delaying RpsL-induced cell death, macrophages lacking cathepsin B do not support productive intracellular replication of L. pneumophila harboring wild type RpsL. This suggests the involvement of other yet unidentified components in the restriction of bacterial replication. Our results identified RpsL as a regulator in the interactions between bacteria such as L. pneumophila and primary mouse macrophages by triggering unique cellular pathways that restrict intracellular bacterial replication.     

Development of Antibiotic-Overproducing Strains by Site-Directed Mutagenesis of the rpsL Gene in Streptomyces lividans

Certain rpsL (which encodes the ribosomal protein S12) mutations that confer resistance to streptomycin markedly activate the production of antibiotics in Streptomyces spp. These rpsL mutations are known to be located in the two conserved regions within the S12 protein. To understand the roles of these two regions in the activation of silent genes, we used site-directed mutagenesis to generate eight novel mutations in addition to an already known (K88E) mutation that is capable of activating antibiotic production in Streptomyces lividans. Of these mutants, two (L90K and R94G) activated antibiotic production much more than the K88E mutant. Neither the L90K nor the R94G mutation conferred an increase in the level of resistance to streptomycin and paromomycin. Our results demonstrate the efficacy of the site-directed mutagenesis technique for strain improvement.     

Vinculin transmits high-level integrin tensions that are dispensable for focal adhesion formation

Focal adhesions (FAs) transmit force and mediate mechanotransduction between cells and the matrix. Previous studies revealed that integrin-transmitted force is critical to regulate FA formation. As vinculin is a prominent FA protein implicated in integrin tension transmission, this work studies the relation among integrin tensions (force), vinculin (protein), and FA formation (structure) by integrin tension manipulation, force visualization and vinculin knockout (KO). Two DNA-based integrin tension tools are adopted: tension gauge tether (TGT) and integrative tension sensor (ITS), with TGT restricting integrin tensions under a designed T_tol (tension tolerance) value and ITS visualizing integrin tensions above the T_tol value by fluorescence. Results show that large FAs (area >1 μm²) were formed on the TGT surface with T_tol of 54 pN but not on those with lower T_tol values. Time-series analysis of FA formation shows that focal complexes (area <0.5 μm²) appeared on all TGT surfaces 20 min after cell plating, but only matured to large FAs on TGT with T_tol of 54 pN. Next, we tested FA formation in vinculin KO cells on TGT surfaces. Surprisingly, the T_tol value of TGT required for large FA formation is drastically decreased to 23 pN. To explore the cause, we visualized integrin tensions in both wild-type and vinculin KO cells using ITS. The results showed that integrin tensions in FAs of wild-type cells frequently activate ITS with T_tol of 54 pN. With vinculin KO, however, integrin tensions in FAs became lower and unable to activate 54 pN ITS. Force signal intensities of integrin tensions reported by 33 and 43 pN ITS were also significantly reduced with vinculin KO, suggesting that vinculin is essential to transmit high-level integrin tensions and involved in transmitting intermediate-level integrin tensions in FAs. However, the high-level integrin tensions transmitted by vinculin are not required by FA formation.     

A Missense Mutation in CASK Causes FG Syndrome in an Italian Family

First described in 1974, FG syndrome (FGS) is an X-linked multiple congenital anomaly/mental retardation (MCA/MR) disorder, characterized by high clinical variability and genetic heterogeneity. Five loci (FGS1-5) have so far been linked to this phenotype on the X chromosome, but only one gene, MED12, has been identified to date. Mutations in this gene account for a restricted number of FGS patients with a more distinctive phenotype, referred to as the Opitz-Kaveggia phenotype. We report here that a p.R28L (c.83G→T) missense mutation in CASK causes FGS phenotype in an Italian family previously mapped to Xp11.4-p11.3 (FGS4). The identified missense mutation cosegregates with the phenotype in this family and is absent in 1000 control X chromosomes of the same ethnic origin. An extensive analysis of CASK protein functions as well as structural and dynamic studies performed by molecular dynamics (MD) simulation did not reveal significant alterations induced by the p.R28L substitution. However, we observed a partial skipping of the exon 2 of CASK, presumably a consequence of improper recognition of exonic splicing enhancers (ESEs) induced by the c.83G→T transversion. CASK is a multidomain scaffold protein highly expressed in the central nervous system (CNS) with specific localization to the synapses, where it forms large signaling complexes regulating neurotransmission. We suggest that the observed phenotype is most likely a consequence of an altered CASK expression profile during embryogenesis, brain development, and differentiation.     

Use of a Dominant rpsL Allele Conferring Streptomycin Dependence for Positive and Negative Selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

The effects of traT insertion mutations on detergent sensitivity and serum resistance of Escherichia coli and Salmonella typhimurium

The SS-A mutation of Salmonella typhimurium, which probably causes the production of a mutated TraT-like protein, sensitizes the bacteria to hydrophobic antibiotics. A similar phenotype is caused by insertion mutations in the cloned traT gene of R6-5. While the SS-A mutants are resistant to detergents and have unaltered serum resistance, the insertion mutations sensitize both S. typhimurium and Escherichia coli to detergents and abolish the increase in serum resistance caused by the wild-type traT gene product in E. coli.     

Studies on the cross-resistance of Mycobacterium tuberculosis, strain H37Rv, to aminoglycoside- and peptide-antibiotics

Various phenotypes of the resistance to aminoglycoside- and peptide-antibodies of Mycobacterium tuberculosis strain H37Rv were produced by single- and/or two-step selection of the parent strain. Mutants obtained by single-step selection with antibiotics were classified into ten phenotypes; one of single resistance, two of triple resistance, three of quadruple resistance, and four of sextuple resistance. There were two kinds of sextuple resistance (high resistance to enviomycin, viomycin, capreomycin, kanamycin, lividomycin). One was isolated from the parent strain by single-step selection and could be eliminated by mutation to isoniazid resistance, the other was obtained by two-step selections and was not eliminated by mutation to isoniazid resistance. Interaction between mutation to streptomycin resistance and mutation to quadruple resistance (4R phenotype) was observed. Streptomycin resistance interfered with the formation of the 4R phenotype and produced a different phenotype, KR instead of the 4R phenotype. The existence of mutation of the 4R phenotype did not usually interfere with mutation to streptomycin resistance, but a small portion of the mutants with the 4R phenotype were altered in their phenotype from 4R to KR after addition of the mutation to streptomycin resistance. This effect of the mutation to streptomycin resistance was not observed in mutants which already had a mutation to klR phenotype (mutation to low concentrations of kanamycin only).     

Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential.     

Control of plasmid R6K copy numbers in isogenic rep+ and rep Escherichia coli strains

Summary We have analysed as a function of cell doubling times the control of R6K plasmid replication in rep ⁺ and rep strains of Escherichia coli. The rep mutation results in an alteration or loss of an enzyme that unwinds helical DNA. We found in rep ⁺ bacteria that R6K relative dosage (plasmids per genome equivalent) remained nearly constant as growth rates increased. From this we concluded that the average plasmid concentration (plasmids per unit cell mass or volume) fell relative to the average concentration of chromosome origins when growth rates increased. In this context, the control of R6K replication is similar to that of other plasmids as seen by different workers. We also found that the relative dosage of R6K in rep mutants is greater than in rep ⁺ bacteria when both strains were grown at fast growth rates. This finding was expected since at fast growth rates the number of genome equivalents per unit mass is expected to be lower in rep mutants. Unexpectedly, however, we found the effect of the rep mutation on R6K relative dosage had occurred in a step-like manner at a slow growth rate of about 120 min per generation. This implies that both the relative dosage and concentration of R6K had increased in a step-like manner. We also found that the effect of the rep mutation on R6K concentration was lost at fast growth rates while the effect of the mutation on R6K relative dosage was not lost.     

Comparative genomic analysis of Erwinia amylovora reveals novel insights in phylogenetic arrangement,plasmid diversity,and streptomycin resistance

Erwinia amylovora is a destructive pathogen of Rosaceous plants and an economic concern worldwide. Herein, we report 93 new E. amylovora genomes from North America, Europe, the Mediterranean, and New Zealand. This new genomic information demonstrates the existence of three primary clades of Amygdaloideae (apple and pear) infecting E. amylovora and suggests all three independently originate from North America. The comprehensive sequencing also identified and confirmed the presence of 7 novel plasmids ranging in size from 2.9 to 34.7 kbp. While the function of the novel plasmids is unknown, the plasmids pEAR27, pEAR28, and pEAR35 encoded for type IV secretion systems. The strA-strB gene pair and the K43R point mutation at codon 43 of the rpsL gene have been previously documented to confer streptomycin resistance. Of the sequenced isolates, rpsL-based streptomycin resistance was more common and was found with the highest frequency in the Western North American clade.     

A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae), resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).     

Molecular Analysis of RNF213 Gene for Moyamoya Disease in the Chinese Han Population

Background

Moyamoya disease (MMD) is an uncommon cerebrovascular disorder characterized by progressive occlusion of the internal carotid artery causing cerebral ischemia and hemorrhage. Genetic factors in the etiology and pathogenesis of MMD are being increasingly recognized. Previous studies have shown that the RNF213 gene was related to MMD susceptibility in the Japanese population. However, there is no large scale study of the association between this gene and MMD in the Chinese Han population. Thus we designed this case-control study to validate the R4810K mutation and to define the further spectrum of RNF213 mutations in Han Chinese.

Methodology/Principal Findings

Genotyping of the R4810K mutation in the RNF213 gene was performed in 170 MMD cases and 507 controls from a Chinese Han population. The R4810K mutation was identified in 22 of 170 MMD cases (13%), including 21 heterozygotes and a single familial homozygote. Two of the 507 controls (0.4%) were heterozygous R4810K carriers. The R4810K mutation greatly increased the risk for MMD (OR = 36.7, 95% CI: 8.6∼156.6, P = 6.1 E-15). The allele frequency of R4810K was significantly different between patients with ischemia and hemorrhage (OR = 5.4, 95% CI: 1.8∼16.1, P = 0.001). Genomic sequencing covering RNF213 exon 40 to exon 68 also identified eight other non-R4810K variants; P4007R, Q4367L, A4399T, T4586P, L4631V, E4950D, A5021V and M5136I. Among them A4399T polymorphism was found in 28/170 cases (16.5%) and 45/507 controls (8.9%) and was associated with MMD (OR = 2.0, 95% CI: 1.2∼3.3, P = 0.004), especially with hemorrhage (OR = 2.8, 95% CI: 1.2∼6.5, P = 0.014).

Conclusions

RNF213 mutations are associated with MMD susceptibility in Han Chinese. The ischemic type MMD is particularly related to the R4810K mutation. However, A4399T is also a susceptible variant for MMD, primarily associated with hemorrhage. Identification of novel variants in the RNF213 gene further highlights the genetic heterogeneity of MMD.     

Somatic cell mutation. Detection and quantification of x-ray-induced mutation in cultured, diploid human fibroblasts

We describe a system for detecting somatic cell mutation to 8-azaguanine (8AG) resistance in cultured, diploid human fibroblasts. Hypoxanthine-guanine phosphoribosyltransferase (HG-PRT)-deficient, AG-resistant fibroblasts from boys with the X-chromosomal, Lesch-Nyhan (L-N) mutation served as one type of prototype mutant cells. Both spontaneous and X-ray-induced mutation were studied. Recovery of L-N cells was a function both of density of normal cells and of the AG concentration used for selection. Optimum recovery was achieved at an initial inoculum of 2·10⁴ normal cells per 60 mm diameter culture dish and an AG concentration of 8·10^?6M. Efficiency of recovery was between 39 and 90% and controls to determine this efficiency were included in mutagenesis experiments.Attempts to free normal cell populations of pre-existing AG-resistant mutant cells by pregrowth in HAT medium failed because, unlike L-N mutants, most spontaneous AG-resistant mutants can grow in HAT medium. Although pre-existing mutants probably caused overestimation, the average spontaneous mutation rate derived from our experiments was 4.5·10^?6 per cell generation. Eliminating one large-yieldv experiment reduced this estimate to 1.9·10^?6. Clonal survival of cultured human fibroblasts as a function of X-ray dose was studied. X-Irradiation increased the mutation rate above spontaneous background. Minimum estimates of the increases were 1.13·10^?9 per R per cell at 75 R, 7.49·10^?8 per R per cell at 125 R, 6.87·10^?8 per R per cell at 150 R and 2.16·10^?7 per R per cell at 250 R. The total mutagenic effect and the induced mutation rate appeared to be dose-dependent. Normal parental cell strains and their derived AG-resistant mutants had similar X-ray sensitivities indicating that X-rays induced mutations rather than selected for pre-existing mutants.Because of the realism of the cultured diploid, human fibroblast model vis-a-vis in vivohuman cellular events, the mutation detection system described herein is proposed as being potentially useful for environmental monitoring.     

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase

Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40°C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line.