Mutations identified in engineered Escherichia coli with a reduced genome.

Characterizing genes that regulate cell growth and survival in model organisms is important for understanding higher organisms. Construction of strains harboring large deletions in the genome can provide insights into the genetic basis of cell growth compared with only studying wild-type strains. We have constructed a series of genome-reduced strains with deletions spanning approximately 38.9% of the E. coli chromosome. Strains were constructed by combining large deletions in chromosomal regions encoding nonessential gene groups. We also isolated strains Δ33b and Δ37c, whose growth was partially restored by adaptive laboratory evolution (ALE). Genome sequencing of nine strains, including those selected following ALE, identified the presence of several Single Nucleotide Variants (SNVs), insertions, deletions, and inversions. In addition to multiple SNVs, two insertions were identified in ALE strain Δ33b. The first was an insertion at the promoter region of pntA, which increased cognate gene expression. The second was an insertion sequence (IS) present in sibE, encoding the antitoxin in a toxin-antitoxin system, which decreased expression of sibE. 5 strains of Δ37c independently isolated following ALE harboring multiple SNVs and genetic rearrangements. Interestingly, a SNV was identified in the promoter region of hcaT in all five strains, which increased hcaT expression and, we predict, rescued the attenuated Δ37b growth. Experiments using defined deletion mutants suggested that hcaT encodes a 3-phenylpropionate transporter protein and is involved in survival during stationary phase under oxidative stress. This study is the first to document accumulation of mutations during construction of genome-reduced strains. Furthermore, isolation and analysis of strains derived from ALE in which the growth defect mediated by large chromosomal deletions was rescued identified novel genes involved in cell survival.

Identification of a Formate-Dependent Uric Acid Degradation Pathway in Escherichia coli.

Purine is a nitrogen-containing compound that is abundant in nature. In organisms that utilize purine as a nitrogen source, purine is converted to uric acid, which is then converted to allantoin. Allantoin is then converted to ammonia. In Escherichia coli, neither urate-degrading activity nor a gene encoding an enzyme homologous to the known urate-degrading enzymes had previously been found. Here, we demonstrate urate-degrading activity in E. coli We first identified aegA as an E. coli gene involved in oxidative stress tolerance. An examination of gene expression revealed that both aegA and its paralog ygfT are expressed under both microaerobic and anaerobic conditions. The ygfT gene is localized within a chromosomal gene cluster presumably involved in purine catabolism. Accordingly, the expression of ygfT increased in the presence of exogenous uric acid, suggesting that ygfT is involved in urate degradation. Examination of the change of uric acid levels in the growth medium with time revealed urate-degrading activity under microaerobic and anaerobic conditions in the wild-type strain but not in the aegA ygfT double-deletion mutant. Furthermore, AegA- and YgfT-dependent urate-degrading activity was detected only in the presence of formate and formate dehydrogenase H. Collectively, these observations indicate the presence of urate-degrading activity in E. coli that is operational under microaerobic and anaerobic conditions. The activity requires formate, formate dehydrogenase H, and either aegA or ygfT We also identified other putative genes which are involved not only in formate-dependent but also in formate-independent urate degradation and may function in the regulation or cofactor synthesis in purine catabolism.IMPORTANCE The metabolic pathway of uric acid degradation to date has been elucidated only in aerobic environments and is not understood in anaerobic and microaerobic environments. In the current study, we showed that Escherichia coli, a facultative anaerobic organism, uses uric acid as a sole source of nitrogen under anaerobic and microaerobic conditions. We also showed that formate, formate dehydrogenase H, and either AegA or YgfT are involved in uric acid degradation. We propose that formate may act as an electron donor for a uric acid-degrading enzyme in this bacterium.

Subunit Composition of Ribosome in the yqgF Mutant Is Deficient in pre-16S rRNA Processing of Escherichia coli.

Escherichia coli 16S, 23S, and 5S ribosomal RNAs (rRNAs) are transcribed as a single primary transcript, which is subsequently processed into mature rRNAs by several RNases. Three RNases (RNase III, RNase E, and RNase G) were reported to function in processing the 5'-leader of precursor 16S rRNA (pre-16S rRNA). Previously, we showed that a novel essential YqgF is involved in that processing. Here we investigated the ribosome subunits of the yqgFts mutant by LC-MS/MS. The mutant ribosome had decreased copy numbers of ribosome protein S1, suggesting that the yqgF gene enables incorporation of ribosomal protein S1 into ribosome by processing of the 5'-end of pre-16S rRNA. The ribosome protein S1 is essential for translation in E. coli; therefore, our results suggest that YqgF converts the inactive form of newly synthesized ribosome into the active form at the final step of ribosome assembly.

Starch synthase IIIa and starch branching enzyme IIb-deficient mutant rice line ameliorates pancreatic insulin secretion in rats: screening and evaluating mutant rice lines with antidiabetic functionalities.

Diabetes mellitus is a metabolic disease spreading worldwide that has been reported to worsen the development and progression of other diseases (cancer, vascular diseases and dementia). To establish functional rice lines with anti-postprandial hyperglycaemic effects, we developed mutant rice lines, which lack one or two gene(s) related to starch synthesis, and evaluated their effects. Powder of mutant rice lines or other grains was loaded to rats fasted overnight (oral grain powder loading test). Incremental area under time-concentration curves (iAUC) were calculated with monitored blood glucose levels. Rice lines with anti-postprandial hyperglycaemic effects were separated by cluster analysis with calculated iAUC. A double mutant rice #4019 (starch synthase IIIa (ss3a)/branching enzyme IIb (be2b)), one of the screened mutant rice lines, was fed to Goto-Kakizaki (GK) rats, an animal model for type 2 diabetes, for 5 weeks. Plasma levels of C-peptide, a marker of pancreatic insulin secretion, were measured with ELISA. For in vitro study, a rat pancreatic cell line was cultured with a medium containing rat serum which was sampled from rats fed #4019 diet for 2 d. After 24-h of incubation, an insulin secretion test was performed. Through the oral rice powder loading test, seven rice lines were identified as antidiabetic rice lines. The intake of #4019 diet increased plasma C-peptide levels of GK rats. This result was also observed in vitro. In rat serum added to cell medium, ornithine was significantly increased by the intake of #4019. In conclusion, the mutant rice #4019 promoted pancreatic insulin secretion via elevation of serum ornithine levels.

Involvement of the ytfK gene from the PhoB regulon in stationary-phase H2O2 stress tolerance in Escherichia coli.

The Escherichia coli PhoB-PhoR two-component system responds to phosphate starvation and induces the expression of many genes. Previous studies suggested that phosphate starvation induces oxidative stress, but the involvement of the PhoB regulon in oxidative stress tolerance has not been clarified. Here, we showed that ytfK, one of the PhoB regulon genes, is involved in cell tolerance to a redox-cycling drug, menadione, and H2O2 in stationary-phase cells. A ytfK deletion mutant was sensitive to H2O2 when the cells were grown anaerobically or micro-aerobically in the presence of nitrate. Genetic analysis suggested that the ytfK gene has a functional relationship with the oxyR and fur genes, among the oxyR regulon, at least, a catalase-encoding katG gene and peroxidase-encoding ahpCF genes. Overproduction of YtfK resulted in a KatG-dependent decrease of H2O2 concentration in the cell suspension, suggesting that katG is one of the targets of YtfK. Using a katG'-lacZ reporter fusion, we showed that YtfK enhances the transcription of katG although it was not clarified whether YtfK functions directly or not. We also showed that ytfK disruption results in reduced viability of stationary-phase cells under phosphate starvation. These results indicated that YtfK is involved in H2O2 tolerance by stimulating directly or indirectly the transcription of at least the catalase gene, and that this system plays an important role in cellular survival during phosphate starvation.

Involvement of formate dehydrogenases in stationary phase oxidative stress tolerance in Escherichia coli.

Previously, we constructed a series of reduced-genome strains of Escherichia coli by combining large-scale chromosome deletions and then tested the sensitivity of these strains to the redox-cycling drug menadione. In this study, we analyzed a deletion that increased menadione sensitivity and discovered that loss of selenocysteine synthase genes was responsible for the strain's reduced tolerance to oxidative stress. Mutants of formate dehydrogenases, which are selenocysteine-containing enzymes, were also sensitive to menadione, indicating that these enzymes are involved in oxidative stress during stationary phase, specifically under microaerobic conditions in the presence of glucose. Among three formate dehydrogenases encoded by the E. coli genome, two were responsible for the observed phenotypes: formate dehydrogenase-H and -O. In a mutant of fdhD, which encodes a sulfur transferase that is essential for formate dehydrogenase activity, formate dehydrogenase-O could still contribute to oxidative stress tolerance, revealing a novel role for this protein. Consistent with this, overproduction of the electron transfer subunits of this enzyme, FdoH and FdoI, increased menadione tolerance and supported survival in stationary phase. These results suggested that formate dehydrogenase-O serves as an electron transfer element in glucose metabolism to promote oxidative stress tolerance and survival in stationary phase.

Systematic identification of synthetic lethal mutations with reduced-genome Escherichia coli: synthetic genetic interactions among yoaA, xthA and holC related to survival from MMS exposure.

Reduced-genome Escherichia coli strains lacking up to 38.9% of the parental chromosome have been constructed by combining large-scale chromosome deletion mutations. Functionally redundant genes involved in essential processes can be systematically identified using these reduced-genome strains. One large-scale chromosome deletion mutation could be introduced into the wild-type strain but not into the largest reduced-genome strain, suggesting a synthetic lethal interaction between genes removed by the deletion and those already absent in the reduced-genome strain. Thus, introduction of the deletion mutation into a series of reduced-genome mutants could allow the identification of other chromosome deletion mutations responsible for the synthetic lethal phenotype. We identified a synthetic lethality caused by disruption of nfo and xthA, two genes encoding apurinic/apyrimidinic (AP) endonucleases involved in the DNA base excision repair pathway, and two other large-scale chromosome deletions. We constructed temperature-sensitive mutants harboring quadruple-deletion mutations in the affected genes/chromosome regions. Using these mutants, we identified two multi-copy suppressors: holC, encoding the chi subunit of DNA polymerase III, and yoaA, encoding a putative DNA helicase. Addition of the yoaA disruption increased the methyl methanesulfonate (MMS) sensitivity of xthA single-deletion or xthA nfo double-deletion mutants. This increased MMS sensitivity was not suppressed by the presence of multi-copy holC. These results indicate that yoaA is involved in MMS sensitivity and suggest that YoaA functions together with HolC.

Figure correction of a metallic ellipsoidal neutron focusing mirror.

An increasing number of neutron focusing mirrors is being adopted in neutron scattering experiments in order to provide high fluxes at sample positions, reduce measurement time, and/or increase statistical reliability. To realize a small focusing spot and high beam intensity, mirrors with both high form accuracy and low surface roughness are required. To achieve this, we propose a new figure correction technique to fabricate a two-dimensional neutron focusing mirror made with electroless nickel-phosphorus (NiP) by effectively combining ultraprecision shaper cutting and fine polishing. An arc envelope shaper cutting method is introduced to generate high form accuracy, while a fine polishing method, in which the material is removed effectively without losing profile accuracy, is developed to reduce the surface roughness of the mirror. High form accuracy in the minor-axis and the major-axis is obtained through tool profile error compensation and corrective polishing, respectively, and low surface roughness is acquired under a low polishing load. As a result, an ellipsoidal neutron focusing mirror is successfully fabricated with high form accuracy of 0.5 µm peak-to-valley and low surface roughness of 0.2 nm root-mean-square.

Novel essential gene Involved in 16S rRNA processing in Escherichia coli.

Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three-dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase-H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgF(ts)) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5' end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgF(ts) mutant cells showed that the expression of genes containing multiple Shine-Dalgarno-like sequences was observed to be lower than in wild type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation.

New fabrication method for an ellipsoidal neutron focusing mirror with a metal substrate.

We propose an ellipsoidal neutron focusing mirror using a metal substrate made with electroless nickel-phosphorus (NiP) plated material for the first time. Electroless NiP has great advantages for realizing an ellipsoidal neutron mirror because of its amorphous structure, good machinability and relatively large critical angle of total reflection for neutrons. We manufactured the mirror by combining ultrahigh precision cutting and fine polishing to generate high form accuracy and low surface roughness. The form accuracy of the mirror was estimated to be 5.3 µm P-V and 0.8 µm P-V for the minor-axis and major-axis direction respectively, while the surface roughness was reduced to 0.2 nm rms. The effect of form error on focusing spot size was evaluated by using a laser beam and the focusing performance of the mirror was verified by neutron experiments.

[A novel approach to techniques in genetic testing for cancer].

In molecular targeted drug therapy, genetic screening is carried out to identify the existence of target genes that are specifically expressed in cancer cells. Conventional methods for detecting the mutation of genes in cancer cells through the use of purified DNA is time consuming, especially in the case of the enzymatic treatment of pathological specimens, and it is difficult to finish all these protocols on the same day. Also, depending on the condition of the patients, it may be difficult to perform surgery or biopsy, and pathological specimens are not always obtainable. Thus, sometimes genetic screening using purified DNA and the enzymatic treatment of pathological specimens cannot be performed. We have successfully solved these problems using i-densy, a genetic analysis device, and two different methods of genetic testing for cancer. The first is a method which, without extracting DNA, uses simply pretreated pathological specimens for genetic screening. Using deparaffinized specimens that have only been heat-treated for a short period of time, we were able to obtain the exact same results as if we had extracted DNA. The second is the highly specific genetic screening technique, the MBP-QP method. Using this method, we were able to confirm the detection of genetic mutation from the DNA of blood plasma. It is now possible to screen for the mutation of genes in cancer cells using just a blood sample from patients without using tissue or cells, which also has little burden on the patient.

Initial reduction of oxidative stress by angiotensin receptor blocker contributes long term outcomes after percutaneous coronary intervention.

BACKGROUND: It remains unclear whether administration of ARB with reactive oxygen species (ROS) scavenging effects improves the prognosis of patients undergoing PCI. OBJECTIVES: This study investigated whether the pre-intervention antioxidant effect of angiotensin receptor blocker (ARB) affects long-term outcomes in patients after successful percutaneous coronary intervention (PCI) without early adverse events. METHODS: Fifty-two patients who underwent elective PCI were randomly assigned for treatment with or without ARB, which was administered within 48 hours before PCI. ROS levels in mononuclear cells (MNCs) and serum superoxide dismutase (SOD) activity were measured pre-PCI and 6 months post-PCI. After exclusion of unexpected early adverse events during angiographic follow-up period, the long-term outcome (major adverse cerebro-cardiovascular event; MACCE) was assessed in eligible patients. RESULTS: Forty-three patients (non-ARB n = 22, ARB n = 21) were followed up in this study. During angiographic follow-up period, ROS formation in MNCs was significantly increased in the non-ARB group (from 29.4 [21.6-35.2] to 37.2 [30.7-45.1] arbitrary units; p = 0.031) compared to that in the ARB group. Meanwhile, SOD activity was significantly impaired in the non-ARB group alone (from 24.0 ± 17.0 to 16.3 ± 13.8%, p = 0.004). During the follow-up period (median, 63.3 months), MACCEs were observed in 6 patients. The cumulative event ratio of MACCE was significantly higher in the non-ARB group than in the ARB group (p = 0.018). CONCLUSIONS: Concomitant administration of ARB effectively reduced ROS production of PCI patients during angiographic follow-up period. Initial ROS inhibition following ARB administration may contribute to improvement of worse outcomes in patients who have undergone successful PCI.

Yucasin is a potent inhibitor of YUCCA, a key enzyme in auxin biosynthesis.

Indole-3-acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole-3-pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC-expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1-His suggested that yucasin strongly inhibited YUC1-His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over-expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss-of-function mutant of TAA1, sav3-2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l-kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin-treated sav3-2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.

Color selectivity of surface-plasmon holograms illuminated with white light.

By using the optical frequency dependence of surface-plasmon polaritons, color images can be reconstructed from holograms illuminated with white light. We report details on the color selectivity of the color holograms. The selectivity is tuned by the thickness of a dielectric film covering a plasmonic metal film. When the dielectric is SiO(2) and the metal is silver, the appropriate thicknesses are 25 and 55 nm, respectively. In terms of spatial color uniformity, holograms made of silver-film corrugations are better than holograms recorded on photographic film on a flat silver surface.

Effects on IS1 transposition frequency of a mutation in the ygjD gene involved in an essential tRNA modification in Escherichia coli.

The YgjD protein is essential for the synthesis of the universal tRNA modification, N(6) -threonylcarbamoyladenosine (t(6) A), which is necessary for the decoding of ANN codons. We isolated a suppressor (ygjDsup ) of the ygjD(ts) mutant by its permissive growth at high temperature in Escherichia coli. Resequencing of the ygjDsup mutant genome showed the presence of a complicated chromosome rearrangement, an inverse insertion of a large duplicated region (c. 450 kb) into a small deleted region. The temperature-resistant growth associated with ygjDsup was due to the presence of multicopy suppressor genes, yjeE and groL, of the ygjD(ts) mutation in the duplicated region. This DNA rearrangement was not simply mediated by IS1 transposition, but the duplicated region was flanked by IS1. We showed that the frequency of IS1 transposition was increased in ygjD(ts) mutants. The transposase of IS1 is coded for by the insB gene, and its translation occurs through a frameshift of a ribosome translating upstream of the insA gene. We showed that this frameshifting frequency was increased by the ygjD(ts) mutation. These results indicated that the mutation of the gene for tRNA modification, t(6) A, affected IS1 transposition.

Mutations in the essential Escherichia coli gene, yqgF, and their effects on transcription.

The Escherichia coli yqgF gene is highly conserved across a broad spectrum of bacterial genomes. The gene was first identified as being essential for cell growth during screening for targets for broad-spectrum antibiotics. YqgF is structurally similar to RuvC, a Holliday junction resolvase, but its function has not been established. This study describes the isolation of a temperature-sensitive yqgF mutant, the growth of which was inhibited by rho or nusA multicopy plasmids, indicating that YqgF is involved in transcription. Rho is a global transcription termination factor that acts at Rho-dependent terminator sites, which exist not only at the ends of genes but also within genes. The transcription of genes possessing intragenic, or upstream, Rho-dependent terminators was reduced in temperature-sensitive yqgF mutants. This transcription inhibition was sensitive to the Rho inhibitor, bicyclomycin. In addition, the transcription of mutant tnaA genes defective for upstream Rho-dependent termination was not significantly affected by the yqgF mutation. Taken together, these results suggest that YqgF is involved in anti-termination at Rho-dependent terminators in vivo.

Diffuse skeletal muscles uptake of [18F] fluorodeoxyglucose on positron emission tomography in primary muscle peripheral T-cell lymphoma.

A 40-year-old man presented with weakness of neck extensor muscles. Cervical magnetic resonance imaging showed high-intensity areas in muscles of the left lateral cervical region on T2-weighted images. Fluorodeoxyglucose-positron emission tomography scan demonstrated striking fluorodeoxyglucose uptake by multiple skeletal muscles of the neck, chest, and abdominal region. Muscle biopsy demonstrated peripheral T-cell lymphoma, unspecified. The diagnosis was primary skeletal muscle peripheral T-cell lymphoma. Primary skeletal muscle non-Hodgkin's lymphoma of T-cell immunophenotype is extremely rare and fluorodeoxyglucose-positron emission tomography demonstrated striking fluorodeoxyglucose uptake in multiple skeletal muscles and served as a quite useful modality for the diagnosis of this patient.

Effects on transcription of mutations in ygjD, yeaZ, and yjeE genes, which are involved in a universal tRNA modification in Escherichia coli.

The Escherichia coli ygjD gene is critical for the universal tRNA modification N(6)-threonylcarbamoyladenosine, together with two other essential genes, yeaZ and yjeE. This study showed that the transcription of the thr and ilv operons in ygjD mutants was increased through the inhibition of transcription attenuation and that dnaG transcription was reduced.

Oxidative stress sensitivity of engineered Escherichia coli cells with a reduced genome.

The construction of engineered bacterial cells with a reduced genome allows the investigation of molecular mechanisms that may be cryptic in wild-type strains and derivatives. Previously, a large-scale combined deletion mutant of Escherichia coli that lacked 29.7% of the parental chromosome was constructed by combining large chromosome deletions. In this work, we improved the system for making markerless-chromosomal deletions and obtained mutants with a genome that lacked up to 38.9% of the parental chromosome. Although the large-scale deletion mutants possessed genes needed for resistance to oxidative stress, including superoxide dismutase, catalase, and RpoS, they were sensitive to menadione, which induces reactive oxygen species during stationary phase. Small genome size did not necessarily correlate with greater sensitivity to menadione as several mutants with large deletions were more resistant to menadione. The sensitivity to menadione depended on whether the mutants were grown aerobically or anaerobically, suggesting that the mechanism governing menadione resistance depended on the oxygen tension of the growth medium. Further analysis of the large-scale deletion mutants should help identify the regulatory networks that are important for cellular defense against oxidative stress.

Surface-plasmon holography with white-light illumination.

The recently emerging three-dimensional (3D) displays in the electronic shops imitate depth illusion by overlapping two parallax 2D images through either polarized glasses that viewers are required to wear or lenticular lenses fixed directly on the display. Holography, on the other hand, provides real 3D imaging, although usually limiting colors to monochrome. The so-called rainbow holograms--mounted, for example, on credit cards--are also produced from parallax images that change color with viewing angle. We report on a holographic technique based on surface plasmons that can reconstruct true 3D color images, where the colors are reconstructed by satisfying resonance conditions of surface plasmon polaritons for individual wavelengths. Such real 3D color images can be viewed from any angle, just like the original object.