Systematic screening of Escherichia coli single-gene knockout mutants for improving recombinant whole-cell biocatalysts

Systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) was performed to select mutants that could enhance the deethylation of 7-ethoxycoumarin catalyzed by CYP154A1. After 96-well plate high-throughput screening followed by test tube assays, four mutants (ΔcpxA, ΔgcvR, ΔglnL, and an unknown-gene-deleted one (Δuk)) were able to increase the CYP154A1 activity by approximately 1.4–1.7 times compared with that of the control strain. When new mutants were constructed by disrupting individually the cpxA, gcvR, glnL, and uk genes in E. coli BW25113, three of them (ΔcpxA, ΔgcvR, and ΔglnL) showed high levels of CYP154A1 activity. However, the uk-disruptant failed to enhance the CYP154A1 activity, suggesting that the high CYP154A1 activity of the Δuk mutant in the Keio collection was due to a spontaneous mutation in the chromosome. In-frame deletion mutants of ΔcpxA, ΔgcvR, and ΔglnL also exhibited high enzyme activity, and complementation of these mutations could decrease CYP154A1 activity. These results indicated that the enhancement of the enzyme activity was not caused by polar effects on their neighbor genes. To our knowledge, this is the first report on a genome-wide screening of the genes for deletion to improve the activity of a recombinant whole-cell biocatalyst.     

Phosphorylation of human DNMT1: implication of cyclin-dependent kinases

DNA methylation plays a central role in the epigenetic regulation of gene expression during development and progression of cancer diseases. The inheritance of specific DNA methylation patterns are acquired in the early embryo and are specifically maintained after cellular replication via the DNA methyltransferase 1 (DNMT1). Recent studies have suggested that the enzymatic activity of DNMT1 is possibly modulated by phosphorylation of serine/threonine residues located in the N-terminal domain of the enzyme. In the present work, we report that cyclin-dependent kinases (CDKs) 1, 2 and 5 can phosphorylate Ser154 of human DNMT1 in vitro. Further evidence of phosphorylation of endogenous DNMT1 at position 154 by CDKs is also found in 293 cells treated with roscovitine, a specific inhibitor of CDK1, 2 and 5. To determine the importance of Ser154 phosphorylation, a mutant of DNMT1 encoding a single-point mutation at position 154 (S154A) was generated. This mutation induced a severe loss of enzymatic activity when compared to wild type DNMT1. Moreover, after treatment with 5-Aza-2′-Deoxycytidine (5-aza-dC), a faster decline in DNMT1 protein level was observed for HEK-293 cells expressing DNMT1(S154A) as compared to cells expressing wild type DNMT1. Our data suggest that phosphorylation of DNMT1 at Ser154 by CDKs is important for enzymatic activity and protein stability of DNMT1. Considering that tumour-associated cell cycle defects are often mediated by alterations in CDK activity, our results suggest that dysregulation of cell cycle via CDKs could induce abnormal phosphorylation of DNMT1 and lead to DNA hypermethylation often observed in cancer cells.     

A new approach to the production of the recombinant SOD protein by methylotrophic <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The gene for the copper, zinc–superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl–cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity.     

Plant regeneration in a jute species (C. capsularis) and its possible relationship with glyoxalase-I

Summary The addition of 3 mg/l of nordihydroguaiaretic acid (NDGA) to BAP and tyrosine fortified MS medium was essential to obtain organogenic callus from the hypocotyl segments of two varieties (D-154 and CVL-1) of Corchorus capsularis — one of the two jute species. When the organogenic callus, which is rich in large starch granules, was transferred to MS basal medium, it differentiated into single or multiple shoots usually in the first subculture and sometimes in the second. The activity of glyoxalase-I of the organogenic callus was found to be significantly lower than that observed in the nonorganogenic callus initiated on MS medium supplemented with 2,4-D, tyrosine, BAP or just BAP and tyrosine. This suggests an inverse relationship between differentiation and the level of glyoxalase-I activity in the two varieties of C. capsularis jute.Abbreviations BAP 6-benzylaminopurine - CVL 1 Corchorus capsularis var. CVL-1 - D-154 C. capsularis var. D 154 - O-4 C. olitorius var. O-4 - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NDGA nordihydroguaiaretic acid - tyr tyrosine     

Degradation of cyclin A does not require its phosphorylation by CDC2 and cyclin-dependent kinase 2

Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27(KIP1) were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E.     

Mutational analysis of OGG1, MYH, MTH1 in FAP, HNPCC and sporadic colorectal cancer patients: R154H OGG1 polymorphism is associated with sporadic colorectal cancer patients

MYH, OGG1 and MTH1 are members of base excision repair (BER) families, and MYH germline mutations were recently identified in patients with multiple adenomas or familial adenomatous polyposis (FAP). A total of 20 APC-negative Korean FAP patients were analyzed for OGG1, MYH and MTH1 germline mutations. A total of 19 hereditary nonpolyposis colorectal cancer (HNPCC), 86 suspected HNPCC, and 246 sporadic colorectal cancer cases were investigated for OGG1 and MYH mutations. A total of 14 R154H OGG1 polymorphisms were identified in hereditary, sporadic colorectal cancers, and normal controls. For the case-control analysis of OGG1 R154H, a total of 625 hereditary or sporadic colorectal cancer patients and 527 normal controls were screened. R154H was a rare polymorphism associated with sporadic colorectal cancer patents (OR: 3.586, P= 0.053). R154H does not segregate with cancer phenotypes. Upon examining the possibility of recessive inheritance of R154H, we could not identify any complementary mutations in OGG1, MYH or MTH1. Samples with R154H were further screened for mutations of K-ras, -catenin, APC, p53, BRAF and the microsatellite instability (MSI) status. Eight somatic mutations were identified in these genes and G:C to T:A transversion mutations were not dominant in samples harboring R154H. This result raises the possibility that OGG1 R154H may function as a low/moderate-penetrance modifier for colorectal cancer development.I.-J. Kim and J.-L. Ku contributed equally to this work     

Plantacin B, a bacteriocin produced by Lactobacillus plantarum NCDO 1193

Abstract Strains of Lactobacillus plantarum and Leuconostoc mesenteroides were tested for bacteriocin production against each other and a range of closely related bacteria. L. plantarum 1193 was found to produce an inhibitory substance active against L. plantarum 340 and 1752, L. mesenteroides 8015 and Pediococcus damnosus 1832. This substance is a potential bacteriocin and has been named plantacin B.     

Cyclic‐di‐AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis

The second messenger cyclic‐di‐adenosine monophosphate (c‐di‐AMP) plays important roles in growth, virulence, cell wall homeostasis, potassium transport and affects resistance to antibiotics, heat and osmotic stress. Most Firmicutes contain only one c‐di‐AMP synthesizing diadenylate cyclase (CdaA); however, little is known about signals and effectors controlling CdaA activity and c‐di‐AMP levels. In this study, a genetic screen was employed to identify components which affect the c‐di‐AMP level in Lactococcus. We characterized suppressor mutations that restored osmoresistance to spontaneous c‐di‐AMP phosphodiesterase gdpP mutants, which contain high c‐di‐AMP levels. Loss‐of‐function and gain‐of‐function mutations were identified in the cdaA and gdpP genes, respectively, which led to lower c‐di‐AMP levels. A mutation was also identified in the phosphoglucosamine mutase gene glmM, which is commonly located within the cdaA operon in bacteria. The glmM I154F mutation resulted in a lowering of the c‐di‐AMP level and a reduction in the key peptidoglycan precursor UDP‐N‐acetylglucosamine in L. lactis. C‐di‐AMP synthesis by CdaA was shown to be inhibited by GlmM^I154F more than GlmM and GlmM^I154F was found to bind more strongly to CdaA than GlmM. These findings identify GlmM as a c‐di‐AMP level modulating protein and provide a direct connection between c‐di‐AMP synthesis and peptidoglycan biosynthesis.     

Overexpression of a novel osteopetrosis-related gene CCDC154 suppresses cell proliferation by inducing G2/M arrest

Osteopetrosis, a disorder of skeletal bone, can cause death during childhood. We previously described a new spontaneous autosomal recessive osteopetrosis mouse mutant, “new toothless” (ntl). In this study, we reported for the first time the identification, cloning and characterization of the coiled-coil domain-containing 154 (CCDC154), a novel gene whose deletion of ~5 kb sequence including exons 1–6 was completely linked to the ntl mutant. The CCDC154 was conserved between mouse and human and is wildly expressed in mouse tissues. The cellular localization of CCDC154 was in the early endosomes. Overexpression of CCDC154 inhibited cell proliferation of HEK293 cells by inducing G₂/M arrest. CCDC154 also inhibited tumor cell growth, and the soft agar assay revealed a significant decrease of the colony size of Hela cells upon transfection of CCDC154. Our results indicate that CCDC154 is a novel osteopetrosis-related gene involved in cell cycle regulation and tumor suppression growth.     

Alcohol fermentation of sweet potato. I. Acid hydrolysis and factors involved

Factors affecting acid hydrolysis of sweet potato powder (SSP) to fermentable sugars were examined. These include HCl concentration, temperature, time, and levels of SPP. Maximum reducing sugar, reported as dextrose equivalent (DE), was detected after 24 min hydrolysis (1% SPP) in 0.034N HCl heated at 154°C. These samples also had 3.43% droxymethylfurfural (HMF) based on dry weight. A high level of HMF (9.2%) was detected in 1% SPP heated at 154° in 0.10N HCl for 18 min. The lowest concentration of HMF formed (1.8%), at maximal DE of 61%, was established in samples containing 5% SPP and heated at 154° in 0.034N HCl for 48 min. Aqueous extracts of uncured SPP, examined by HPLC, contained glucose, fructose and sucrose, but degraded SPP had only glucose and fructose. Products of degraded SPP, under appropriate conditions, could be used for alcohol fermentation.     

Controlling chronic Pseudomonas aeruginosa infections by strategically interfering with the sensory function of SagS

The hybrid sensor SagS plays a central role in the formation of Pseudomonas aeruginosa biofilms, by enabling the switch from the planktonic to the biofilm mode of growth and by facilitating the transition of biofilm cells to a highly tolerant state. In this study, we examined the importance of the SagS key amino acid residues associated with biofilm formation (L154) and antibiotic tolerance (D105) in P. aeruginosa virulence. Recombinant P. aeruginosa ΔsagS and ΔsagS chromosomally expressing wild‐type sagS, or its two variants D105A and L154A, were tested for their potential to form biofilms and cause virulence in plants and mouse models of acute and chronic pneumonia. Although mutation of sagS did not alter P. aeruginosa virulence during acute infections, a significant difference in pathogenicity of sagS mutants was observed during chronic infections, with the L154A variant showing reduced bacterial loads in the chronic pneumonia model, while interference with the D105 residue enhanced the susceptibility of P. aeruginosa biofilms during tobramycin treatment. Our findings suggest that interference with the biofilm or tolerance regulatory circuits of SagS affects P. aeruginosa pathogenicity in chronic but not acute infections, and reveal SagS to be a promising new target to treat P. aeruginosa biofilm infections.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine thein vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5–6 times more than the basal control. And the uptake of D-[3-³H]-glucose is linear as the logarithm of insulin concentration from 0.2 ώg/L to 1.0 ώg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-³H]-glucose uptake into adipocytes. By this method, thein vitro biological activity of [B₂-Lys]-insulin and [B₃-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

Geographic Origins of Korean and Chinese Kimchi Determined by Multiple Elements

By use of the promoter probe transposon Tn5-B21, two promoter fragments were isolated. One promoter (822 bp) (GC=63.5%) isolated from P. putida loses all of its promoter activity by exo-nuclease or restriction enzyme deletion. Another promoter (264 bp) (GC=49.2%) isolated from P. fluorescens could be shortened to 154 bp by exonuclease deletion without any effect on its promoter activity in several Pseudomonas species.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine the in vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5-6 times more than the basal control. And the uptake of D-[3-3H]-glucose is linear as the logarithm of insulin concentration from 0.2 μg/L to 1.0 μg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-3H]-glucose uptake into adipocytes. By this method, the in vitro biological activity of [B2-Lys]-insulin and [B3-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

Production of TMC-151, TMC-154 and TMC-171, a new class of antibiotics, is specific to ‘Gliocladium roseum’ group

A novel class of fungal metabolites, TMC-151, TMC-154, and TMC-171 series compounds, was found exclusively inGliocladium catenulatum, Clonostachys rosea and closely related strains. These compounds were not detected in any other fungi examined. The production spectrum of each component was correlated to the morphology of the secondary conidiophores and the conidia. TMC-151 was limited toClonostachys rosea (formerlyG. roseum) forming navicular or reniform conidia orG. catenulatum with gray-green conidial masses, whereas TMC-154 and 171 were limited to the strains closely related toGliocladium roseum, which grew more slowly and formed more symmetrical conidia.     

Glycoproteins in rabbit uterus during implantation

Summary The synthesis of glycoproteins in rabbit uterine epithelium during the late preimplantation period was studied using tritiated N-acetylglucosamine. In vivo labelling was achieved by the intra-uterine implantation of agar gel columns containing the precursor. Autoradiography showed the radioactivity to be predominantly localized in the apical cell surfaces, with single cells exhibiting an accumulation of silver grains in their supranuclear cytoplasm. After gel electrophoresis of uterine flushings, activity was mainly found in the -glycoprotein fraction. Fluorescein isothiocyanate (FITC)-conjugated wheat-germ agglutinin reacted with the apical cytoplasm and surfaces of the endometrial cells. However, FITC-conjugated concanavalin A exhibited a different binding pattern, reacting first with the basal cytoplasm, and later with the apical cytoplasm. After concanavalin-A staining, single cells exhibited positive vesicles in their lateral and apical parts. These cells may be released into the uterine lumen until 210 h post coitum. Neither of the lectins reacted with ciliated cells. Concanavalin A showed an affinity for the -glycoprotein fraction of the uterine secretion. The results indicate that, although all endometrial cells contain glycoproteins, only a few of these seem to be involved in the synthesis of secretory products.Supported by grants Ki 154/9-3 and 154/10-1 from the Deutsche Forschungsgemeinschaft     

Proteolysis upon dehydration of yeasts saccharomyces cerevisiae

The effect of dehydration on proteolysis and activity of proteases A, B and C in the cells of baker's yeast Saccharomyces cerevisiae was investigated. It can be concluded, that under investigated conditions of yeast Saccharomyces cerevisiae drying a decrease of proteases activity takes place. In cells a limited proteolysis takes place which is indicated by an increase in amino nitrogen content and a decrease of tryptophane synthase activity. Adding the protease inhibitor to yeast suspension prevents decrease of tryptophane synthase activity upon dehydration.