Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated beta-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and (1)H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of beta-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.     

Role of (R)-specific enoyl coenzyme A hydratases of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp in the production of polyhydroxyalkanoates

Four (R)-specific enoyl CoA hydratases (PhaJ) interconnect the β-oxidation pathway with PHA biosynthesis in Pseudomonas aeruginosa. The use of antisense technique and over-expression to delineate the role of two of these enzymes, PhaJ1 and PhaJ4 forms the basis of this study. It has been observed that P. aeruginosa recombinant with phaJ1 antisense construct, fed with different fatty acids, produces PHA with less hydroxy octanoate (7–11% reduction) and a proportionate increase in other monomer fractions, compared to that of the control. Recombinants bearing phaJ4 antisense construct are found to contain less hydroxy decanoate (10–11% reduction) and more or less equal amount of hydroxy octanoate, compared to that of the control. P. aeruginosa has produced PHA with more hydroxy octanoate and decanoate (6–17% increase), respectively, when PhaJ1 and PhaJ4 have been over-expressed individually or along with PhaC1. PhaJ1 and PhaJ4 are found to be involved mainly in the production of hydroxy octanoyl CoA and hydroxy decanoyl CoA, respectively, in P. aeruginosa. The strongest accumulation of hydroxy octanoate and hydroxy decanoate has been observed along with hydroxy butyrate, in PHA, produced by E. coli, when PhaC1 has been co-expressed with PhaJ1 and PhaJ4, respectively. We have demonstrated, for the first time, the polymerization of hydroxy butyryl CoA monomers in recombinant E. coli by PhaC1 of P. aeruginosa.     

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background

The low pH environment of the human stomach is lethal for most microorganisms; but not Escherichia coli, which can tolerate extreme acid stress. Acid resistance in E. coli is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).

Results

In this study, we individually deleted seventy-nine sncRNA genes from the E. coli K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in E. coli. We demonstrate that GcvB enhances the ability of E. coli to survive low pH by upregulating the levels of the alternate sigma factor RpoS.

Conclusion

GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in E. coli.     

Extraction of polyhydroxyalkanoate from Sinorhizobium meliloti cells using Microbispora sp. culture and its enzymes

Sinorhizobium meliloti produced 50% polyhydroxyalkanoate (PHA) in the biomass in the presence of sucrose as carbon substrate. Isolation of the intracellular PHA was achieved through a secondary fermentation involving a cell lytic actinomycetes species namely Microbispora sp. without further supplementation of nutrients to the S. meliloti fermented broth, at 30 °C, 150 rpm up to 72 h. Microbispora sp. cells that showed pelleted growth was removed by filtration and the released polymer contained in the filtrate was extracted by chloroform or an admixture of Triton X 100 (0.6%) a surfactant and ethylene diamine tetra acetic acid (EDTA) a chelating agent. Yield of PHA obtained was 49, 41 and 7% of biomass weight after 24, 48 and 72 h of lytic culture fermentation, respectively. Corresponding recovery of the polymer was 94, 82 and 15% of 90% purity. Alternatively Microbispora sp. lytic enzyme was obtained by its cultivation in nutrient broth with S. meliloti cells as substrate and the supernatant was used for the hydrolysis of the PHA containing biomass to release PHA. A620 lytic activity value for the broth was 200 at 72 h. The enzyme showed optimized activity at 50 °C, pH 7 and this was used to hydrolyze 5 g/l of thermally inactivated biomass of S. meliloti to recover 94% of total PHA present in the cells and the polymer produced was 92% pure. Decreased cell lytic activity in the presence of soluble protein added in the form of bovine serum albumin indicated that the hydrolytic activity may be due to proteases. The polymer was characterized by GC, NMR and DSC and was found to be polyhydroxybutyrate-co-hydroxyvalerate (97:3 mol%) with a melt temperature of 169 °C.     

The Hoover's Sign of Pulmonary Disease: Molecular Basis and Clinical Relevance

Background

Mucosal-based immunotherapy has been already used as an alternative form of allergen delivery. In asthma, the poor success rate of immune modulation could be a consequence of inadequate immune modulation in the airways. Previously, we have found that subcutaneous (S.C) co-administration of a homemade allergenic extract from Chenopodium album (Ch.a) pollen and Guanine-Cytosine containing deoxynucleotides (CpG-ODNs) is effective to prevent the inflammatory responses in mouse. In this study we used CpG/Ch.a for immunotherapy of Ch.a-induced asthma and compared the intranasal (I.N) and S.C routes of administration concerning IFN-γ, IL-10 and total IgE responses.

Methods

Ch.a sensitized mice were treated intranasaly or subcutaneously using CpG and Ch.a. extract. IFN-γ, IL-10 and total IgE were measured in supernatant culture of splenocytes and bronchoalveolor lavage (BAL) fluids by ELISA. Student's t test was used in the analysis of the results obtained from the test and control mice.

Results

We found that I.N administration of CpG/Ch.a in sensitized mice significantly increased the production of systemic and mucosal IFN-γ and IL-10 compared to phosphate buffered saline (PBS), Ch.a alone and control ODNs treated sensitized mice (P ≤ 0.001). On the other hand, S.C. route induced the systemic and mucosal IFN-γ in the lower levels than in I.N one, and failed to increase systemic IL-10 induction (P = 0.06). Total serum IgE in CpG/Ch.a treated mice in both routes showed significant decreases compared to three control groups (P ≤ 0.01). The amounts of IgE in BAL fluids were not measurable in all groups.

Conclusion

According to the results of this experiment we concluded that immunotherapy via the I.N co-administration of CpG/Ch.a in comparison with S.C route is more effective to stimulate the mucosal and regulatory responses in Ch.a induced asthma.     

An in vitro investigation of Malaysian Phytophthora palmivora isolates and pathogenicity study on oil palm

Bud rot disease affecting oil palm in South American countries is reported to be caused by Phytophthora palmivora. P. palmivora is a local pathogen affecting various crops in Malaysia, and this finding caused an alarm, which prompted an investigation of pathogenicity using Malaysian P. palmivora to assess the potentials of this Oomycete to infect oil palm in Malaysia. A total of 11 P. palmivora isolates were obtained from cocoa and durian for the study. Leaf bioassays via artificial inoculation using 50,000 zoospores/ml and mycelial agar disc showed severe necrotic lesions on the infection spot of oil palm (DxP) spear leaves. Mild infection was observed in oil palm spear leaves of OxG hybrids indicating lower susceptibility against P. palmivora infection. Phylogenetic analysis using internal transcribed spacer (ITS) data revealed that Malaysian isolates were genetically similar to Colombian isolates supported by significant bootstrap values. The leaf bioassay results revealed that Malaysian oil palm materials are susceptible towards local P. palmivora infection. The Colombian P. palmivora isolates causing bud rot incidence may have evolved over a long period of time, undergone sequential genetic shift to become more virulent towards Colombian oil palm planting materials.     

Crystal structures of diketopiperazines containing α-aminoisobutyric acid: Cyclo(Aib-Aib) and cyclo(Aib-L-Ile)

The crystal and molecular structures of two α-aminoisobutyric acid (Aib)-containing diketopiperazines, cyclo(Aib-Aib) 1 and cyclo(Aib-L -Ile) 2 , are reported. Cyclo(Aib-Aib) crystallizes in the space group P1 with a = 5.649(3), b = 5.865(2), c = 8.363(1), α = 69.89(6), β = 113.04(8), γ = 116.0(3), and Z = 1, while 2 occurs in the space group P2₁2₁2₁ with a = 6.177(1), b = 10.791(1), c = 16.676(1), and Z = 4. The structures of 1 and 2 have been refined to final R factors of 0.085 and 0.086, respectively. In both structures the diketopiperazine ring shows small but significant deviation from planarity. A very flat chair conformation is adopted by 1, in which the C^α atoms are displaced by 0.07 Å on each side of the mean plane, passing through the other four atoms of the ring. Cyclo(Aib-Ile) favors a slight boat conformation, with Aib C^α and Ile C^α atoms displaced by 0.11 and 0.05 Å on the same side of the mean plane formed by the other ring atoms. Structural features in these two molecules are compared with other related diketopiperazines.     

Induced potential for male sex expression inRicinus communis L. by ethyl hydrogen-1-propylphosphonate

Summary Spray application of ethyl hydrogen-1-propylphosphonate (NIA 10637) to juvenile plants ofRicinus communis L. at concentrations ranging from 4,000 to 16,000 ppm caused a change in the normal pattern of sex expression resulting in the transformation of the female into male flowers. Intergrades of sex expression were observed in plants treated with 4,000 and 8,000 ppm of the chemical and the plants were wholly male in those that had received 16,000 ppm, but showing delay in flowering. Pollen sterility was observed in the treated plants.Abbreviations CEPA (2-Chloroethyl)phosphonic acid - TIBA 2,3,5-triiodobenzoic acid - CCC (2-chloroethyl)trimethylammonium chloride - GA₃ gibberellic acid - Kn kinetin     

A new domain family in the superfamily of alkaline phosphatases

During the course of our large-scale genome analysis a conserved domain, currently detectable only in the genomes of Drosophila melanogaster, Caenorhabditis elegans and Anopheles gambiae, has been identified. The function of this domain is currently unknown and no function annotation is provided for this domain in the publicly available genomic, protein family and sequence databases. The search for the homologues of this domain in the non-redundant sequence database using PSI-BLAST, resulted in identification of distant relationship between this family and the alkaline phosphatase-like superfamily, which includes families of aryl sulfatase, N-acetylgalactosomine-4-sulfatase, alkaline phosphatase and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM). The fold recognition procedures showed that this new domain could adopt a similar 3-D fold as for this superfamily. Most of the phosphatases and sulfatases of this superfamily are characterized by functional residues Ser and Cys respectively in the topologically equivalent positions. This functionally important site aligns with Ser/Thr in the members of the new family. Additionally, set of residues responsible for a metal binding site in phosphatases and sulphtases are conserved in the new family. The in-depth analysis suggests that the new family could possess phosphatase activity.