Improved adsorption of an Enterococcus faecalis bacteriophage ΦEF24C with a spontaneous point mutation

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component.     

Isolation and Characterization of a Novel Bacteriophage φ4D Lytic Against Enterococcus faecalis Strains

In recent years, Enterococcus faecalis has emerged as an important opportunistic nosocomial pathogen capable of causing dangerous infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages have very effective bactericidal activity and several advantages over other antimicrobial agents and so far, no serious or irreversible side effects of phage therapy have been described. The objective of this study was to characterize a novel virulent bacteriophage φ4D isolated from sewage. Electron microscopy revealed its resemblance to Myoviridae, with an isometric head (74 ± 4 nm) and a long contractile tail (164 ± 4 nm). The φ4D phage genome was tested using pulsed-field gel electrophoresis and estimated to be 145 ± 2 kb. It exhibited short latent period (25 min) and a relatively small burst size (36 PFU/cell). Tests were conducted on the host range, multiplicities of infection (MOI), thermal stability, digestion of DNA by restriction enzymes, and proteomic analyses of this phage. The isolated phage was capable of infecting a wide spectrum of enterococcal strains. The results of these investigations indicate that φ4D is similar to other Myoviridae bacteriophages (for example φEF24C), which have been successfully used in phagotherapy.     

Identification of Nor-��-Lapachone Derivatives as Potential Antibacterial Compounds against Enterococcus faecalis Clinical Strain

A broad-spectrum antibiotic therapy has led to medical complications and emergence of multiresistant bacteria including Enterococcus faecalis. In this study, we designed, synthesized, and evaluated the antibacterial activity of 13 nor-β-lapachone derivatives against a drug resistant E. faecalis strain. Two triazole substituted compounds (1e = 8 μg/ml and 1c = 16 μg/ml) and the non-substituted derivative (1a = 8 μg/ml) were promising compared to chloramphenicol (12 μg/ml), an antibiotic currently available in the market. We also performed a structure-activity relationship analysis using a molecular modeling approach that pointed the low HOMO energy values; HOMO density concentrated on the nor-β-lapachone ring, lipophilicity, solubility and number HBA as important stereoelectronic features for the antibacterial profile. In addition the triazole compounds presented low theoretical toxicity profile, and drug-score higher than commercial antibiotics also fulfilling the Lipinski "Rule of Five", which pointed them as promising candidates for further studies in infections caused by multiresistant E. faecalis hospital strains.     

BRAF Mutation is Associated with an Improved Survival in Glioma—a Systematic Review and Meta-analysis

Newly emerged molecular markers in gliomas provide prognostic values beyond the capabilities of histologic classification. BRAF mutation, especially BRAF V600E, is common in a subset of gliomas and may represent a potential prognostic marker. The aim of our study is to investigate the potential use of BRAF mutations on prognosis of glioma patients. Four electronic databases were searched for potential articles, including PubMed, Scopus, ISI Web of Science, and Virtual Health Library (VHL). Data of hazard ratio (HR) for overall survival (OS) and progression-free survival (PFS) were directly obtained from original papers or indirectly estimated from Kaplan Meier curve (KMC). A random effect model weighted by inverse variance method was used to calculate the pooled HR. From 705 articles, we finally included 11 articles with 1308 glioma patients for the final analysis. The overall estimates showed that BRAF V600E was associated with an improved overall survival (OS) in glioma patients (HR = 0.60; 95% CI = 0.44–0.80). Results for progression-free survival (PFS), however, were not statistically significant (HR = 1.39; 95% CI = 0.82–2.34). In subgroup analyses, BRAF V600E showed its effect in improving survival in pediatric and young adult gliomas (under 35 years) but did not have prognostic value in old adult. Additionally, BRAF V600E was only associated with a favorable prognosis in lower grade glioma. Our meta-analysis provides evidence that BRAF mutation has a favorable prognostic impact in gliomas and its prognostic value might be dependent on patient age and tumor grade. This mutation can be used as a prognostic factor in glioma but additional studies are required to clarify its prognostic value taking into account other confounding factors.     

A Novel Mitochondrial Heteroplasmic C13806A Point Mutation Associated with Iranian Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by decreased expression of the protein Frataxin. Frataxin deficiency leads to excessive free radical production and dysfunction of chain complexes. Mitochondrial DNA (mtDNA) could be considered a candidate modifier factor for FRDA disease, since mitochondrial oxidative stress is thought to be involved in the pathogenesis of this disease. It prompted us to focus on the mtDNA and monitor the nucleotide changes of genome which are probably the cause of respiratory chain defects and reduced ATP generation. We searched about 46% of the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE) and DNA fragments showing abnormal banding patterns were sequenced for the identification of exact mutations. In 18 patients, for the first time, we detected 26 mtDNA mutations; of which 5 (19.2%) was novel and 21 (80.8%) have been reported in other diseases. Heteroplasmic C13806A polymorphisms were associated with Iranian FRDA patients (55.5%). Our results showed that NADH dehydrogenase (ND) genes mutations in FRDA samples were higher than normal controls (P < 0.001) and we found statistically significant inverse correlation (r = −0.8) between number of mutation in ND genes and age of onset in FRDA patients. It is possible that mutations in ND genes could constitute a predisposing factor which in combination with environmental risk factors affects age of onset and disease progression.     

Improved Binding of Raf to Ras��GDP Is Correlated with Biological Activity

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras·Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras·GTP versus Ras·GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.     

Characterization of Phospholipase C�� Enzymes with Gain-of-Function Mutations

Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.Phosphoinositide-specific phospholipase C (PLC)² enzymes, comprising several families (PLCβ, γ, δ, ϵ, η, and ζ), have been established as crucial signaling molecules involved in regulation of a variety of cellular functions (1–4). PLC-catalyzed formation of the second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol, from phosphatidylinositol 4,5-bisphosphate (PIP₂), constitutes one of the major cell signaling responses. These second messengers provide a common link from highly specific receptors for hormones, neurotransmitters, antigens, and growth factors to downstream, intracellular targets; thus, they contribute to regulation of biological functions as diverse as cell motility, fertilization, and sensory transduction. Despite this central role for PLC enzymes in signaling networks, the molecular details of their regulation and possible subversion of these regulatory mechanisms in disease remain poorly understood.Of two PLCγ enzymes, PLCγ1 is ubiquitously expressed and appears to regulate a multitude of cellular functions in many tissues. Plcg1-null mice die by embryonic day 9, highlighting the widespread importance of this enzyme (5). PLCγ1 is activated in response to growth factor stimulation; in addition, its function in T-cell responses has been extensively documented (1). PLCγ2, in contrast, is most highly expressed in cells of the hematopoietic system and plays a key role in regulation of the immune response. Consistent with this, Plcg2-null mice display defects in the functioning of B cells, platelets, mast cells, and natural killer cells (6).Both PLCγ enzymes have also been implicated in signaling events underlying aberrant cellular responses. PLCγ1 is critically involved in the regulation of cancer cell motility (7–11) while PLCγ2 has been implicated in deregulation of the immune responses resembling Btk-dependent X-linked agammaglobulinaemia and SLE disease in humans (12–14). It has been suggested that, in cancer cells, PLCγ1 could function as a key, rate-limiting, common component involved in cell motility triggered by several growth factors and integrins (7). In some cancer cells, this increased motility could result from deregulation i.e. higher levels of expression of PLCγ1 (15, 16). The possibility that the activity of PLCγ could be up-regulated due to mutation has not yet been fully investigated in cancer. Previous studies of PLCγ2, however, have demonstrated the first gain-of-function mutation in a PLC molecule in the context of an organism, and shown that, in principle, PLC activity can be greatly enhanced by point mutations (13). Furthermore, this work has demonstrated that such a mutation is linked to a dramatic phenotypic disorder. By using a large scale ENU mutagenesis to discover new immune regulators, several mouse strains were generated with spontaneous autoimmune and inflammatory symptoms; two of these strains harbor a mutation in PLCγ2. In addition to the previously described ALI5 mutation (13) the ALI14 mutation has been identified very recently.³ Strikingly, the well-characterized ALI5 phenotype has shown that the mutation affects many cellular functions deregulated in Plcg2-null mice. Notably, while in Plcg2-null mice such responses are lacking, the ALI5 mutation resulted in their enhancement. In particular, further analyses of the ALI5 mutation in the context of signaling in B-cells have demonstrated that calcium responses to the crosslinking of the B-cell receptor were enhanced and prolonged resulting in enhanced deletion of B cells and autoreactivity (13).The domain organization of PLCγ enzymes is characterized by the insertion of a highly structured region (PLCγ-specific array, γSA) between the two halves of the TIM-barrel catalytic domain common to all PLCs. The γSA comprises a split PH (spPH) domain flanking two tandem SH2 domains and a SH3 domain (1). A distinct regulatory feature of PLCγ enzymes is that their activation is linked to an increase in phosphorylation of specific tyrosine residues (most notably within the γSA) by receptor and non-receptor tyrosine kinases (17, 18). Furthermore, multiple protein-protein interactions (mainly mediated by SH2 domains) also contribute to activation and have an important role in localizing PLCγ into protein complexes with different binding partners, depending on cell type and specific cellular compartments. One mode of activation that is specific for the PLCγ2 isozyme is direct binding to and activation by Rac. The interaction involves the spPH domain, and this activation mechanism does not require tyrosine phosphorylation (19, 20). In molecular terms, changes that lead to PLC activation in response to different input signals, or due to point mutations, are not well understood and require further studies.Here we describe further analysis of the two gain-of-function mutations, ALI5 and ALI14, obtained using ENU mutagenesis. These mutations map to different regions in PLCγ2, and we performed detailed analysis of these regions in both PLCγ isozymes. To characterize the molecular mechanism of gain-of-function, we combined studies in vitro and in different cellular signaling contexts. We have found that ALI5- and ALI14-type point mutations lead, by distinct mechanisms, to an enhancement of responses to a variety of input signals while their combination results in a constitutively active PLC enzyme.     

Characterization of a bacteriocin-producing strain of Enterococcus faecalis from cow’s milk used in the production of Moroccan traditional dairy foods

A bacteriocin-producing lactic acid bacterium (strain 2.5) isolated from cow’s milk used in cheese production from Northern Morocco was selected for its strong anti-listerial activity. The producer strain was identified as Enterococcus faecalis by molecular methods. Strain 2.5 carried the genetic determinants for the two-peptide enterococcal bacteriocin enterocin 1071, and the active bacteriocin was purified to homogeneity by reversed-phase chromatography from culture broths of the producer strain. Strain 2.5 carried two plasmids (of ∼7 and 40 kb). Characterization of strain 2.5 at biosafety level indicated that this strain is non-haemolytic, and lacks the genetic determinants for most of the virulence factors described in enterococci (cylB, cylM, gelE, ace and agg) although it carried the genetic determinants cylA, efaAfs as well as determinants for the sex pheromone peptides cpd, cob, and ccf. Strain 2.5 was resistant to tetracycline, rifampicin, and ciprofloxacin, but it was sensitive to penicillin, ampicillin, vancomycin, and teicoplanin. Results from the present study support the potential role of strain 2.5 as an anti-listerial agent to be tested in traditional fermented foods.     

Characterization of Anti-Listeria monocytogenes Bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis Strains Isolated from Raïb, a Moroccan Traditional Fermented Milk

Seventy-four samples of raïb, a Moroccan traditional fermented milk, were screened for their anti-Listeria monocytogenes activity. Nine lactic acid bacteria with antilisterial activity were isolated and identified as Lactococcus lactis[⁴], Enterococcus faecium[⁴], and E. faecalis[¹]. Antibacterial spectra, determined against 45 target strains, led to the selection of four antibacterial-producing strains, which were further characterized. Their anti-microbial agents, inactivated by one or more proteases, were designed as bacteriocins. Lactococcin R9/2 and R10/1 showed the broadest range of inhibitory action. Anti-bacterial spectra and physico-chemical properties suggest that these bacteriocins were similar to nisin. Enterocin R69 had a specificity of action against Listeria spp., whereas Enterocin R18 had a broad spectrum of activity. Lc. lactis R9/2 and E. faecalis R18 were able to coagulate sterilised UHT milk at 30°C in 24 h and induced a 2 log reduction in L. monocytogenes ATCC 15313 population.     

Improved Virus Neutralization by Plant-produced Anti-HIV Antibodies with a Homogeneous ��1,4-Galactosylated N-Glycan Profile

It is well established that proper N-glycosylation significantly influences the efficacy of monoclonal antibodies (mAbs). However, the specific immunological relevance of individual mAb-associated N-glycan structures is currently largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. Here we report on the generation of a plant-based expression platform allowing the efficient production of mAbs with a homogeneous β1,4-galactosylated N-glycosylation structure, the major N-glycan species present on serum IgG. This was achieved by the expression of a highly active modified version of the human β1,4-galactosyltransferase in glycoengineered plants lacking plant-specific glycosylation. Moreover, we demonstrate that two anti-human immunodeficiency virus mAbs with fully β1,4-galactosylated N-glycans display improved virus neutralization potency when compared with other glycoforms produced in plants and Chinese hamster ovary cells. These findings indicate that mAbs containing such homogeneous N-glycan structures should display improved in vivo activities. Our system, using expression of mAbs in tobacco plants engineered for post-translational protein processing, provides a new means of overcoming the two hurdles that limit the therapeutic use of anti-human immunodeficiency virus mAbs in global health initiatives, low biological potency and high production costs.About 40 million people are estimated to be infected with HIV-1,² and the HIV-1/AIDS epidemic continues to escalate, with the most devastating consequences seen in the most impoverished nations (1). Two strategies that have been pursued over the past 2 decades for stopping the AIDS pandemic/epidemic are the generation of vaccines to prevent HIV infection and the development of microbicides to prevent HIV transmission. Highly effective monoclonal antibodies (mAbs) are suitable to be used in both modalities. To date, only a handful of anti-HIV mAbs with neutralizing activities has been explored in more detail (2). In a recent clinical study, it has been demonstrated that a combination of three broadly neutralizing anti-HIV antibodies (2G12, 2F5, and 4E10) shows promise as AIDS treatment (3). However, despite effective in vitro neutralization activities, relatively modest in vivo effects were obtained, suggesting that the in vivo properties of these antibodies require further improvement (2). Noteworthy, these antibodies bind to HIV envelope proteins thus inhibiting viral entry into target cells (2, 4, 5). In addition to their potential use in therapeutic modalities, this renders them as promising candidates for microbicide development. However, high production costs using mammalian-cell technologies and insufficient efficacy of anti-HIV antibodies are remaining hurdles for their effective use. Among recent advances in generating antibodies with enhanced activities, glyco-engineering has been proven to be a powerful tool (6). It is well established that proper N-glycosylation significantly influences the efficacy of mAbs. Nevertheless, the specific immunological relevance of individual mAb-associated N-glycan structures is largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. A series of studies emphasize the critical role of IgG glycoforms lacking core α1,6-fucose for cell-mediated immunological activities (6). However, the immunological significance of N-glycans with terminal β1,4-galactose residues, the major N-glycan species present on serum IgG, has not yet been established.During the last 2 decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. This resulted in a significant enhancement of expression levels (up to 100-fold) and a reduction of production time (7, 8), which makes the system economically interesting. Another important achievement was the generation of plant glycosylation mutants, which allows a controlled human-type glycosylation of recombinant glycoproteins (9, 10). Recently, we have generated different glycoforms of anti-HIV mAb 2G12 in the tobacco-related plant species Nicotiana benthamiana (9). All of them were functionally active, and HIV neutralization potency was comparable with CHO-derived 2G12. This process involved the generation of a plant glycosylation mutant (ΔXT/FT), which was found to produce mAbs carrying homogeneous N-glycans with terminal N-acetylglucosamine (Gn) residues (i.e. GnGn structures) lacking unwanted plant-specific β1,2-xylose and core α1,3-fucose residues. These glycans are devoid of any β1,4-linked galactose residues; thus in this study, we set out to glyco-engineer ΔXT/FT plants for quantitative β1,4-galactosylation. A highly active modified version of human β1,4-galactosyltransferase was used to transform ΔXT/FT and progeny screened for efficient protein β1,4-galactosylation. In total four glycoforms from the two anti-HIV mAbs 2G12 and 4E10 (plant- and CHO-derived) were generated and compared toward antigen binding and virus neutralization capacities.     

Hepatitis C Virus Impairs p53 via Persistent Overexpression of 3��-Hydroxysterol ��24-Reductase

Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3β-hydroxysterol Δ24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.     

The toxicity of an ��artificial�� amyloid is related to how it interacts with membranes

Despite intensive research into how amyloid structures can impair cellular viability, the molecular nature of these toxic species and the cellular mechanisms involved are not clearly defined and may differ from one disease to another. We systematically analyzed, in Saccharomyces cerevisiae, genes that increase the toxicity of an amyloid (M8), previously selected in yeast on the sole basis of its cellular toxicity (and consequently qualified as “artificial”). This genomic screening identified the Vps-C HOPS (homotypic vacuole fusion and protein sorting) complex as a key-player in amyloid toxicity. This finding led us to analyze further the phenotype induced by M8 expression. M8-expressing cells displayed an identical phenotype to vps mutants in terms of endocytosis, vacuolar morphology and salt sensitivity. The direct and specific interaction between M8 and lipids reinforces the role of membrane formation in toxicity due to M8. Together these findings suggest a model in which amyloid toxicity results from membrane fission.Key words: aggregates, amyloid, yeast, euroscarf     

Replication of Bacteriophage φX174 DNA in a Temperature-Sensitive dnaE Mutant of Escherichia coli C

Bacteriophage phiX174 cannot grow in a temperature-sensitive dnaE (DNA polymerase III) mutant of Escherichia coli C at the nonpermissive temperature. The inability to grow is the result of inhibition of virus DNA synthesis. The synthesis of the parental replicative form is unaffected, but the replication of the replicative form and the synthesis of the single-stranded virus DNA are inhibited.     

Restoration of Growth Phenotypes of Escherichia coli DH5α in Minimal Media through Reversal of a Point Mutation in purB

A point mutation (E115K) resulting in slower growth of Escherichia coli DH5α and XL1-Blue in minimal media was identified in the purB gene, coding for adenylosuccinate lyase (ASL), through complementation with an E. coli K-12 genomic library and serial subcultures. Chromosomal modification reversing the mutation to the wild type restored growth phenotypes in minimal media.The Escherichia coli DH5α strain possesses many beneficial genotypes (recA, deoR, gyrA, and endA1) and has been widely used for many purposes, such as gene cloning and protein production (5). However, E. coli DH5α also exhibits inferior growth phenotypes, especially in minimal media, compared to other E. coli strains. As such, the utilization of this bacterium has been limited to the laboratory despite its numerous advantages. We can assume that these inferior growth phenotypes have resulted from unknown accumulated mutations during the strain development process (5). Some of those mutations, which might impact growth in minimal media, have been characterized, including the phenotypes for thiamine requirement and relaxed amino acid synthesis (5). Still, there may be other uncharacterized mutations whose interactions hamper the growth of E. coli DH5α in minimal media.Based on successful identifications (6, 7) of gene targets for metabolic engineering (3), we performed serial subcultures of E. coli DH5α transformants with an E. coli K-12 genomic library based on a multicopy plasmid (9) to isolate genes that improve growth phenotypes in minimal media. The M9 minimal medium and R medium (11) were chosen for enrichment experiments because of their popular use in metabolic engineering (1, 2, 7) and in high-cell-density fermentation (8, 10, 11). After 11 serial transfers of the transformants in the M9 medium, and 27 transfers in the R medium, cultured cells were diluted and plated onto LB agar for single-colony isolation. Although more than 10 colonies were picked, only three distinctive plasmids, containing different inserts, were isolated from the transformants enriched in M9 medium. In the case of R medium enrichment, all isolated plasmids were identical. Sequencing of the isolated plasmids revealed the exact genome coordinates of each insert. A diagram of the inserts in the context of the E. coli genome sequence is shown in Fig. ​Fig.1.1. Interestingly, all of the isolated plasmids contained similar regions of genomic DNA. mnmA (tRNA 5-methylaminomethyl-2-thiouridylate-methyltransferase), purB (adenylosuccinate lyase), and hflD (lysogenization regulator) were the annotated genes in the overlapping region among distinctive isolated fragments. However, since the N-terminal portions of mnmA and hflD were truncated in some of the inserts, we selected only the M3 and R1 plasmids for further experimentation. These two plasmids were retransformed into E. coli DH5α for confirmation of their beneficial effects on growth of E. coli in minimal media. The newly transformed strains showed growth phenotypes almost identical to those of the previously isolated transformants. When cultured in flasks, the specific growth rate of E. coli DH5α with the R1 plasmid was 1.5-fold higher (0.53 versus 0.36 h⁻¹) than the rate of cells transformed with a control plasmid (pZE). The R1 transformant reached the stationary phase much earlier, arriving at an optical density at 600 nm (OD₆₀₀) of 10 within 16 h, whereas the control transformant reached this cell density after 24 h. However, the final cell densities were almost equivalent. Acetate accumulation, as well as glucose consumption, by the R1 transformant was much higher than that of the control transformant (2.2 versus 0.3 g acetate/liter). The increased accumulation of acetate could be the result of increased cell density. These findings confirm that the enhanced growth phenotypes of the isolated transformants were conferred not by accumulated spontaneous mutations in the genome during enrichment but by the introduced plasmids.Open in a separate windowFIG. 1.Diagram of open reading frames in the identified genomic DNA fragments. M1, M2, and M3 were isolated from the serial subculture using M9 medium. R1 was isolated from the serial subculture using R medium.The open reading frame (ORF) of purB was amplified and cloned into a multicopy plasmid under the control of a strong promoter (rrnB). Transformation of the resulting plasmid (pZE-purB) into E. coli DH5α resulted in a growth phenotype almost identical to that of the R1 transformant. This result suggested that overexpression of purB is a specific genetic perturbation improving growth phenotypes of E. coli DH5α in minimal media. We also performed 1-liter batch fermentation experiments with three DH5α transformants: one containing the control plasmid (pZE), one with the isolated plasmid (R1), and a third with the purB overexpression plasmid (pZE-purB). Growth phenotypes of these strains were very similar to results obtained from shaker flask experiments (Fig. ​(Fig.2).2). Next, we tested whether the overexpression of purB is beneficial to the growth of other E. coli strains by introducing the R1 and pZE-purB plasmids into various other strains (K-12, BL21, and XL1-Blue) that are commonly used in biotechnological research. Among the four strains tested in our various experiments, the positive effects of purB overexpression on growth phenotypes were observed only in DH5α and XL1-Blue, both of which have been favored in molecular cloning. These results suggest that an uncharacterized mutation might have been introduced into both strains during strain development. This unknown mutation might cause growth inhibition, which can be suppressed by the overexpression of purB. Therefore, we concluded that expression of an exogenous, K-12-derived copy of the purB gene under a constitutive promoter can enhance growth phenotypes of E. coli DH5α and XL1-Blue strains in minimal media.Open in a separate windowFIG. 2.Comparison of levels of cell growth (♦) (OD₆₀₀), glucose consumption (▪) (g/liter), and acetate production (▴) (g/liter) by E. coli DH5α transformants with a control plasmid (A), the isolated R1 plasmid (B), and the pZE-purB plasmid (C) in R medium with glucose in a bioreactor.However, it is plausible that a mutation is located in the purB locus of DH5α and XL1-Blue that decreases the activity of the encoded enzyme. In order to identify a putative mutation in purB, we sequenced the chromosomal purB gene of DH5α and XL1-Blue. A point mutation resulting in the transition of nucleotide 343 of purB from guanine (G) to adenine (A) was identified in the genomes of both strains. This mutation causes a change of the 115th residue of adenylosuccinate lyase from glutamate to lysine (E115K). This finding explains why the expression of exogenous, K-12-derived purB in DH5α and XL1-Blue strains enhances growth phenotypes in minimal media. The E115K mutation of purB was named purB20 for simple notation.Chromosomal modification of the mutant allele in E. coli DH5α or XL1-Blue might be desirable for practical applications. To this end, the purB20 mutant allele was replaced by purB amplified from E. coli K-12 through recombination based on phage lambda Red recombinase (4). The resulting strain (SC1) showed growth phenotypes similar to those of E. coli DH5α strains harboring the pZE-purB or R1 plasmid. The specific growth rate of SC1 in M9 medium was 40% higher than that of DH5α (0.50 versus 0.36 h⁻¹). These results show that what we had originally interpreted as overexpression of the purB gene was actually complementation of the mutant purB20 allele with wild-type purB. We also tested whether the modification from purB20 to wild-type purB elicits a change in the transformation efficiency. Chemically induced competent SC1 cells exhibited approximately 2.5-fold lower transformation efficiency than E. coli DH5α cells did when induced under identical conditions (1.8 ± 0.1 × 10⁶ versus 4.6 ± 0.3 × 10⁶ CFU/μg pUC19 DNA). Still, the transformation efficiency of the SC1 strain was of the same order of magnitude as that of E. coli DH5α, suggesting that the SC1 strain would be useful for many biotechnological applications, such as the mass production of DNA vectors and recombinant proteins.     

Replication of Bacteriophage φX174 DNA in a Temperature-Sensitive dnaC Mutant of Escherichia coli C

Bacteriophage phiX174 cannot grow in a temperature-sensitive dnaC mutant of Escherichia coli C at the nonpermissive temperature. The inability to grow is the result of inhibition of virus DNA synthesis. Parental replicative form synthesis is not temperature sensitive. Single-stranded virus DNA continues to be synthesized for at least 45 min after shifting to the nonpermissive temperature late in infection. In contrast, the replication of the replicative form terminates within 5 min after shifting to the nonpermissive temperature.     

Mechanism of Adsorption and Eclipse of Bacteriophage φX174 II. Attachment and Eclipse with Isolated Escherichia coli Cell Wall Lipopolysaccharide

A mixture of aqueous phenol, choloroform, and ether extracts the lipopolysaccharides (LPS) from the phiX174-sensitive strain, Escherichia coli C/1, and resistant strains, C/phiX and K12. Interaction of the C/1 LPS with phiX in a starvation buffer containing 10(-3) M CaCl(2) at 37 C, but not at 15 C, results in a first-order inactivation that is specific for C/1 LPS. After interaction for 60 min at 15 C, followed by centrifugation, 37 and 20% of a (14)C-phiX preparation are bound to the C/1 and C/phiX LPS pellets, respectively. The results for intact cells are 75 and 10%. Supporting the conclusion that this represents specific attachment of phiX to its receptor site in the LPS is the fact that EDTA-borate buffer is required to elute 85% of the (14)C-phiX from the C/1 LPS, whereas starvation buffer elutes the same amount from C/phiX LPS. Moreover, 95% of the PFU are found in the C/1 LPS pellets as compared with 50% in the resistant strain LPS pellets. When the products of interaction between phiX and LPS at 37 C are examined by sucrose density gradients in EDTA-borate, a single 60 to 90S peak is observed in the C/1 sample, and the single peak cosediments with the 120S marker phiX in the C/phiX sample. This change in S(20, w) is very similar to that reported for the eclipse of phiX in vivo. If the inactivation at 37 C is carried out on phiX-LPS complexes first formed at 15 C, the first-order kinetics are biphasic and nearly identical to that observed for the eclipse kinetics of phiX attached to intact cells. Thus, the phiX-LPS system is suitable for in vitro studies on the early events in phiX infection.     

Modeling Effect of a ��-Secretase Inhibitor on Amyloid-�� Dynamics Reveals Significant Role of an Amyloid Clearance Mechanism

Aggregation of the small peptide amyloid beta (A??) into oligomers and fibrils in the brain is believed to be a precursor to Alzheimer??s disease. A?? is produced via multiple proteolytic cleavages of amyloid precursor protein (APP), mediated by the enzymes ??- and ??-secretase. In this study, we examine the temporal dynamics of soluble (unaggregated) A?? in the plasma and cerebral-spinal fluid (CSF) of rhesus monkeys treated with different oral doses of a ??-secretase inhibitor. A dose-dependent reduction of A?? concentration was observed within hours of drug ingestion, for all doses tested. A?? concentration in the CSF returned to its predrug level over the monitoring period. In contrast, A?? concentration in the plasma exhibited an unexpected overshoot to as high as 200% of the predrug concentration, and this overshoot persisted as late as 72 hours post-drug ingestion. To account for these observations, we proposed and analyzed a minimal physiological model for A?? dynamics that could fit the data. Our analysis suggests that the overshoot arises from the attenuation of an A?? clearance mechanism, possibly due to the inhibitor. Our model predicts that the efficacy of A?? clearance recovers to its basal (pretreatment) value with a characteristic time of >48 hours, matching the time-scale of the overshoot. These results point to the need for a more detailed investigation of soluble A?? clearance mechanisms and their interaction with A??-reducing drugs.