Phage annealing proteins promote oligonucleotide-directed mutagenesis in <Emphasis Type="Italic">Escherichia coli</Emphasis> and mouse ES cells

Background

The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR) in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined.

Results

Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR) at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα) but only requires a phage annealing protein (RecT or Redβ). Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein.

Conclusion

Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

     

Growth and Endogenous Cytokinins of Juniper Shoots as Affected by High Metal Concentrations

The growth and the content of endogenous cytokinins (CKs) of current-year-old shoots from juniper plants (Juniperus communis L.) growing over and off ore site were compared. The juniper shoots from ore site (M plants) had higher metal content and exhibited delayed growth. Less bases and nucleosides of Z- and iP- type CK and more iP-conjugates were present in the M shoots. These changes were probably due to inhibited CK export from the roots and/or altered CK metabolism forming less biologically active CKs.     

Virion architecture unifies globally distributed pleolipoviruses infecting halophilic archaea

Our understanding of the third domain of life, Archaea, has greatly increased since its establishment some 20 years ago. The increasing information on archaea has also brought their viruses into the limelight. Today, about 100 archaeal viruses are known, which is a low number compared to the numbers of characterized bacterial or eukaryotic viruses. Here, we have performed a comparative biological and structural study of seven pleomorphic viruses infecting extremely halophilic archaea. The pleomorphic nature of this novel virion type was established by sedimentation analysis and cryo-electron microscopy. These nonlytic viruses form virions characterized by a lipid vesicle enclosing the genome, without any nucleoproteins. The viral lipids are unselectively acquired from host cell membranes. The virions contain two to three major structural proteins, which either are embedded in the membrane or form spikes distributed randomly on the external membrane surface. Thus, the most important step during virion assembly is most likely the interaction of the membrane proteins with the genome. The interaction can be driven by single-stranded or double-stranded DNA, resulting in the virions having similar architectures but different genome types. Based on our comparative study, these viruses probably form a novel group, which we define as pleolipoviruses.     

High Abundance of Neotropical Drosophilids (Diptera: Drosophilidae) in Four Cultivated Areas of Central Brazil

The drosophilid assemblages of four cultivated areas (soy, bean, corn, and orange plantations) grown in the core of the Neotropical region were analyzed by comparing their abundances and compositions. The collections, which were gathered using 38 banana traps, captured 12,560 drosophilids, including nine Neotropical and six exotic species. Most of the flies were collected in the bean (43%) and soy (42%) fields. The composition and relative abundance of species also varied among cultivated areas, with orange orchards presenting the highest relative abundance of exotics due to the dominance of the Afrotropical Zaprionus indianus (Gupta). Crop plantations were dominated by a Neotropical species, Drosophila cardini (Sturtevant), which has been shown to be well adapted to dry and disturbed environments. We discuss the drosophilid assemblages of the cultivated areas, comparing them with assemblages from neighbor urban and natural environments. The low drosophilid richness found in this study is similar to the richness found in urban environments and lower than the drosophilid richness of forests, supporting a pattern already known for other taxa. The high abundance of drosophilids in cultivated areas, as well as the dominance of a Neotropical species (D. cardini) in the crop assemblages, was a surprising result.     

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca²⁺) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca²⁺, DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca²⁺. In the absence of Ca²⁺, DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca²⁺-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μm as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional ¹H,¹⁵N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to ¹⁵N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca²⁺-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca²⁺ also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH₂-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.     

Phenotypic diversity of Erwinia amylovora in Bulgaria

Fifty-one strains of Erwinia amylovora isolated from nine host plants in Bulgaria were characterized phenotypically and identified by the API 20E and BIOLOG system. The identification was confirmed by PCR amplification of a specific region of the plasmid pEA29 and the genome ams region. The phenotypic diversity of the strains was studied on the basis of their API 20E and BIOLOG metabolic profiles, as well as of their SDS-PAGE protein profile. Metabolic diversity among the strains was established, but no connection with the origin of the strains was revealed. The Bulgarian strains showed API 20E metabolic profiles not found in previous studies of E. amylovora. The strains formed a homogenous group on the basis of their protein profiles. All the strains were sensitive to the antibiotics streptomycin, tetracycline and oxytetracycline. This study was an initial step towards an investigation of the diversity and evolution in the Bulgarian population of E. amylovora, and it was the first characterization of E. amylovora strains isolated from different host plants in the period 1995-2005 in Bulgaria.     

Defective immunogenic cell death of HMGB1-deficient tumors: compensatory therapy with TLR4 agonists

Immunogenic cell death induced by anticancer chemotherapy is characterized by a series of molecular hallmarks that include the exodus of high-mobility group box 1 protein (HMGB1) from dying cells. HMGB1 is a nuclear nonhistone chromatin-binding protein. It is secreted at the late stages of cellular demise and engages Toll-like receptor4 (TLR4) on dendritic cells (DCs) to accelerate the processing of phagocytic cargo in the DC and to facilitate antigen presentation by DC to T cells. The absence of HMGB1 expression by dying tumor cells exposed to anthracyclines or oxaliplatin compromises DC-dependent T-cell priming by tumor-associated antigens. Here, we show that transplantable tumors exhibiting weak expression of nuclear HMGB1 respond to chemotherapy more effectively if the treatment is combined with the local or systemic administration of a highly purified and physiochemically defined and standardized lipopolysaccharide solution, which acts as a high-potency and exclusive TLR4 agonist, called Dendrophilin (DEN). The synergistic antitumor effects mediated by the combination of chemotherapy and immunotherapy relied upon the presence of the MyD88 (myeloid differentiation primary response gene) adapter of TLR4 (but not that of the TIR-domain-containing adapter-inducing interferon-β adapter), in line with the well-characterized action of DEN on the MyD88 signaling pathway. DEN and anthracyclines synergized to induce intratumoral accumulation of interferon-γ-producing CD4⁺ and CD8⁺ T lymphocytes. Moreover, DEN could restore the immunogenicity of dying tumor cells from which HMGB1 had been depleted by RNA interference. These findings underscore the potential clinical utility of combination regimens involving immunogenic chemotherapy and certain TLR4 agonists in advanced HMGB1-deficient cancers.     

Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3 inflammasome‐dependent secretion of IL‐1β and the danger signals ASC and HMGB1

Fusobacterium nucleatum is an invasive anaerobic bacterium that is associated with periodontal disease. Previous studies have focused on virulence factors produced by F. nucleatum, but early recognition of the pathogen by the immune system remains poorly understood. Although an inflammasome in gingival epithelial cells (GECs) can be stimulated by danger‐associated molecular patterns (DAMPs) (also known as danger signals) such as ATP, inflammasome activation by this periodontal pathogen has yet to be described in these cells. This study therefore examines the effects of F. nucleatum infection on pro‐inflammatory cytokine expression and inflammasome activation in GECs. Our results indicate that infection induces translocation of NF‐κB into the nucleus, resulting in cytokine gene expression. In addition, infection activates the NLRP3 inflammasome, which in turn activates caspase‐1 and stimulates secretion of mature IL‐1β. Unlike other pathogens studied until now, F. nucleatum activates the inflammasome in GECs in the absence of exogenous DAMPs such as ATP. Finally, infection promotes release of other DAMPs that mediate inflammation, such as high‐mobility group box 1 protein and apoptosis‐associated speck‐like protein, with a similar time‐course as caspase‐1 activation. Thus, F. nucleatum expresses the pathogen‐associated molecular patterns necessary to activate NF‐κB and also provides an endogenous DAMP to stimulate the inflammasome and further amplify inflammation through secretion of secondary DAMPs.     

Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli

Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.     

Modified coat protein forms the flexible spindle‐shaped virion of haloarchaeal virus His1

Extremophiles are found in all three domains of cellular life. However, hyperthermic and hypersaline environments are typically dominated by archaeal cells which also hold the records for the highest growth temperature and are able to grow even at saturated salinity. Hypersaline environments are rich of virus‐like particles, and spindle‐shaped virions resembling lemons are one of the most abundant virus morphotypes. Spindle‐shaped viruses are archaea‐specific as all the about 15 such virus isolates infect either hyperthermophilic or halophilic archaea. In the present work, we studied spindle‐shaped virus His1 infecting an extremely halophilic euryarchaeon, Haloarcula hispanica. We demonstrate that His1 tolerates a variety of salinities, even lower than that of seawater. The detailed analysis of the structural constituents showed that the His1 virion is composed of only one major and a few minor structural proteins. There is no lipid bilayer in the His1 virion but the major structural protein VP21 is most likely lipid modified. VP21 forms the virion capsid, and the lipid modification probably enables hydrophobic interactions leading to the flexible nature of the virion. Furthermore, we propose that euryarchaeal virus His1 may be related to crenarchaeal fuselloviruses, and that the short‐tailed spindle‐shaped viruses could form a structure‐based viral lineage.