Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in Pseudomonas fluorescens.

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene (phaCPf) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (phaCPf+, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (phaCPf+) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaCPf, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

Oxidative stress response of Deinococcus geothermalis via a cystine importer.

A cystine-dependent anti-oxidative stress response is characterized in Deinococcus geothermalis for the first time. Nevertheless, the same transcriptional directed Δdgeo_1985F mutant strain was revealed to have an identical phenotype to the wild-type strain, while the reverse transcriptional directed Δdgeo_1985R mutant strain was more resistant to oxidative stress at a certain concentration of H2O2 than the wild-type strain. The wild-type and mutant strains expressed equal levels of superoxide dismutase and catalase under H2O2-induced stress. Although the expression levels of the general DNA-damage response-related genes recA, pprA, ddrA, and ddrB were up-regulated by more than five-fold in the wild-type strain relative to the Δdgeo_1985R mutant strain, the mutant strain had a higher survival rate than the wild-type under H2O2 stress. The Δdgeo_1985R mutant strain highly expressed a cystine-transporter gene (dgeo_1986), at levels 150-fold higher than the wild-type strain, leading to the conclusion that this cystine transporter might be involved in the defensive response to H2O2 stress. In this study, the cystine transporter was identified and characterized through membrane protein expression analysis, a cystine-binding assay, and assays of intracellular H2O2, cysteine, and thiol levels. The genedisrupted mutant strain of the cystine importer revealed high sensitivity to H2O2 and less absorbed cystine, resulting in low concentrations of total thiol. Thus, the absorbed cystine via this cystine-specific importer may be converted into cysteine, which acts as a primitive defense substrate that non-enzymatically scavenges oxidative stress agents in D. geothermalis.

Trials for the co-expression of the merozoite surface protein-1 and circumsporozoite protein genes of Plasmodium vivax.

Merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen, and circumsporozoite protein (CSP), a component of sporozoites that includes a Plasmodium vivax B-cell epitope, are strong candidates for use in a malaria vaccine. A chimeric recombinant gene containing portions of both msp-1 and csp from P. vivax separated by Pro-Gly linker motif was generated. The construct gene was named mlc (msp-1, linker, and csp). The MLC chimeric recombinant protein had a molecular weight of approximately 25 kDa when expressed in Escherichia coli, as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The purified chimeric protein reacted with the sera of patients infected with P. vivax but not with the sera of uninfected patients according to western blot analysis. The chimeric protein reacted well with sera of malaria patients (109/115, 94.78%) as assessed with enzyme-linked immunosorbent assay (ELISA). BALB/c mice that were orally immunized with the MLC chimeric recombinant protein successfully produced antigen-specific antibodies. Additionally, levels of the Th1-associated cytokines IL-12(p40), TNF-α, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Therefore, the E. coli-expressed MLC chimeric recombinant protein might be used as a valuable vaccine candidate for oral immunization against vivax malaria.

Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus.

BACKGROUND: To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope. METHODS: A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot. RESULTS: The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n=38) and a clinical specificity of 100% (n=24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. CONCLUSIONS: The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.

Study of the genetic discrimination between imported and autochthonous cases of malaria in South Korea.

There has been a great increase of Plasmodium vivax incidences in the Republic of Korea and the genetic diversity of the parasite became more complex with the rapid dissemination of newly introduced genotypes. Surveillance of imported malaria is very important, but there is no good way to determine imported vs. internal cases. In this study, we characterized imported vivax cases, analyzed the genetic sequence of three imported vivax malaria cases for the merozoite surface protein-1 (MSP-1) and circumsporozoite protein (CSP) genes, and clearly discriminated an imported vivax case that was misdiagnosed as indigenous by genetic analysis. PCR reaction for the merozoite surface protein-1 (MSP-1) and circumsporozoite protein (CSP) genes from three imported vivax cases were amplified and sequenced. The genetic variations were compared with a previously constructed database of South Korean isolates. The imported vivax cases showed various patterns on incubation period before onset. Most cases were from other parts of Asia. The MSP-1 gene sequence analysis of three imported cases showed that the imported cases had completely different sequences from any subtypes from Korean isolates. Case-1 and Case-2 exact match with an Indian isolate, and Case-3 had great similarity with isolates from countries neighboring Indonesia. CSP gene analysis based on the repeat patterns showed similar results that the sequences from the imported cases well matched with the patient's traveled countries and completely discriminated with indigenous cases. AMA-1 gene analysis also supported these results. We were able to clearly distinguish three imported vivax cases from indigenous by using a genetic database of Korean isolates and were able to suspect its origin by genotyping. This study demonstrated the usefulness of genetic survey on imported malaria cases.

Rapid dissemination of newly introduced Plasmodium vivax genotypes in South Korea.

Reemerged Plasmodium vivax malaria in South Korea has not yet been eradicated despite continuous governmental efforts. It has rather become an endemic disease. Our study aimed to determine the genetic diversity in P. vivax merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) genes over an extended period after its reemergence to its current status. Sequence analysis of PvMSP-1 gene sequences from the 632 P. vivax isolates during 1996-2007 indicates that most isolates recently obtained were different from isolates obtained in the initial reemergence period. There was initially only one subtype (recombinant) present but its subtypes have varied since 2000; six MSP-1 subtypes were recently found. A similar variation was observed by CSP gene analysis; a new CSP subtype was found. Understanding genetic variation patterns of the parasite may help to analyze trends and assess extent of endemic malaria in South Korea.

Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4.

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.

Genotype of Toxoplasma gondii from blood of stray cats in Gyeonggi-do, Korea.

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5' and 3' ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.

Prevalence of Toxoplasma gondii in stray cats of Gyeonggi-do, Korea.

Toxoplasma gondii is an obligate intracellular zoonotic protozoan with a worldwide distribution. It infects humans as well as a broad spectrum of vertebrate hosts. Cats and wild felidae play crucial roles in the epidemiology of toxoplasmosis. This study was performed to survey the prevalence of T. gondii infection among stray cats in the Gyeonggi-do, Republic of Korea. A total of 174 stray cat blood samples were collected from Gwacheon-si (n=20), Bucheon-si (82), and Yangju-si (72). Positive sera for T. gondii were identified in 14 samples (8.1%) exclusively via the latex agglutination test, 28 (16.1%) via ELISA, and 23 (13.2%) via PCR analysis. The overall infection rate of female stray cats (29.2%) presented as higher than that of male cats (24.0%). This study suggests that T. gondii is widespread in the stray cat population of Gyeonggi-do, Korea. It is urgently needed to control urban stray cat population and to reduce the risk of zoonotic transmission of toxoplasmosis to other animal hosts and humans.

Characterization of a chromosomal nickel resistance determinant from Klebsiella oxytoca CCUG 15788.

Klebsiella oxytoca CCUG 15788 is resistant to Ni2+ at a concentration of 10 mM and grows in an inducible manner when exposed to lower concentrations of Ni2+. The complete genomic sequence of a 4.2-kb HindIII-digested fragment of this strain was determined from genomic DNA. It was shown to contain four nickel resistance genes (nirA, nirB, nirC, and nirD) encoding transporter and transmembrane proteins for nickel resistance. When the plasmid pKOHI4, encoding nirABCD, was transformed into Escherichia coli JM109, the cells were able to grow in Tris-buffered mineral medium containing 3 mM nickel. TnphoA'-1 insertion mutants in the four nickel genes nirA, nirB, nirC, and nirD showed nickel sensitivity. The nir genes were heterogeneously expressed in E. coli, suggesting functional roles of these genes in nickel resistance.

Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0A resolution.

A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M.tuberculosis ThyX (MtbThyX) enzyme complements the E.coli chi2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.

Nucleotide sequence and expression of the ncr nickel and cobalt resistance in Hafnia alvei 5-5.

The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced. The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX. The predicted amino acid sequences of ncrA were homologous to the amino acid sequences of nreB of Achromobacter xylosoxidans 31A. Expression of ncr with the T7 RNA polymerase-promoter system allowed Escherichia coli BL21 (DE3) to overexpress NcrA, NcrB, and NcrC but not NcrY, and NcrX. The apparent molecular masses of NcrA, NcrB, and NcrC were 30, 33, and 17 kDa, respectively. Primer-extension analysis showed that ncr mRNA started at nucleotide position 23 upstream from ncrA. The promoter region of the ncr operon possessed a strong, putative -35 element of sigma(32)-type promoter sequence, and transcriptional 'lacZ fusion studies indicated that the -35 element influenced sigma(32)-specific transcription.

Nucleotide sequence and expression of the ncr nickel and cobalt resistance in Hafnia alvei 5-5 / Secuencia nucleotídica y expresión del determinante ncr de resistencia a níquel y cobalto en Hafnia alvei 5-5

The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced. The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX. The predicted amino acid sequences of ncrA were homologous to the amino acid sequences of nreB of Achromobacter xylosoxidans 31A. Expression of ncr with the T7 RNA polymerase-promoter system allowed Escherichia coli BL21 (DE3) to overexpress NcrA, NcrB, and NcrC but not NcrY, and NcrX. The apparent molecular masses of NcrA, NcrB, and NcrC were 30, 33, and 17 kDa, respectively. Primer-extension analysis showed that ncr mRNA started at nucleotide position 23 upstream from ncrA. The promoter region of the ncr operon possessed a strong, putative -35 element of sigma(32)-type promoter sequence, and transcriptional 'lacZ fusion studies indicated that the -35 element influenced sigma(32)-specific transcription (AU)

Los genes estructurales de la resistencia a níquel y cobalto del plásmido conjugativo pEJH 501 de Hafnia alvei 5-5, contenido en un fragmento SalI-EcoRI de 4,8 kb, fueron clonados y secuenciados. La secuencia de DNA incluye cinco genes en el siguiente orden: ncrA, ncrB, ncrC, ncrY, y ncrX. Las secuencias de aminoácidos equivalentes a ncrA fueron homólogas a las secuencias de aminoácidos codificadas por nreB en Achromobacter xylosoxidans 31A. La expresión de los genes ncr mediante el sistema promotor de la RNA polimerasa T7 permite a Escherichia coli BL21 (DE3) sobreexpresar NcrA, NcrB, y NcrC, pero no NcrY ni NcrX. Los pesos moleculares aparentes de NcrA, NcrB y NcrC fueron 30, 33, y 17 kDa, respectivamente. El análisis de extensión de los cebadores mostró que el mRNA de ncr se iniciaba a una distancia de 23 nucleótidos corriente arriba del ncrA.La región promotora del operón ncr posee una fuerte secuencia promotora de tipo sigma32 en la posición -35, y estudios transcripcionales de fusión con ´lacZ indicaron que el elemento situado en -35 influye sobre la transcripción específica de sigma32 (AU)

Conjugative plasmid mediated inducible nickel resistance in Hafnia alvei 5-5.

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni(2+) or 2 mM Co(2+). The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni(2+), and cobalt resistance to 2 mM Co(2+). A 4.8-kb Sal- EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni(2+)) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni(2+). By random Tn phoA'-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A.

Conjugative plasmid pEJH501-mediated inducible nickel resistance in Hafnia alvei 5-5 / Resistencia al níquel en Hafnia alvei 5-5 mediada por el plásmido conjugativo pEJH501

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni(2+) or 2 mM Co(2+). The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni(2+), and cobalt resistance to 2 mM Co(2+). A 4.8-kb Sal- EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni(2+)) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni(2+). By random Tn phoA'-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A (AU)

Hafnia alvei 5-5, aislada en un vertedero bajo la copa del árbol hiperacumulante de níquel Sebertia acuminata (Sapoteacea) en Nueva Caledonia, resultó ser resistente a 30 mM Ni2+ y 2 mM Co2+. El plásmido de 70 kilopares de bases (kb), pEJH501 se transfirió por conjugación a Escherichia coli, Serratia marcescens y Klebsiella oxytoca. Las cepas transconjugantes expresaron resistencia inducible a entre 5 y 17 mM Ni2+, y a 2 mM Co2+. Se subclonó un fragmento Sal-EcoRI que contenía el determinante de resistencia al níquel, y el plásmido híbrido se descubrió que confería un nivel de resistencia moderado al níquel (7 mM Ni2+), incluso en E. coli. La expresión de la resistencia al níquel era inducible por exposición a concentraciones de cloruro de níquel de como mínimo 0,5 mM Ni2+. Mediante mutagénesis por inserción aleatoria de TnphoA'-1, se encontraron en el fragmento tanto genes estructurales como regiones reguladoras para la resistencia al níquel. Estudios de hibridación southern mostraron que el determinante de la resistencia al níquel del plásmido pEJH501 en H. alvei 5-5 era homólogo al de pTOM9 de Alcaligenes xylosoxydans 31A (AU)