Three-piece-ligation PCR and application in disruption of chlorophyll synthesis genes in Synechocystis sp PCC 6803

Linear DNA, consisting of a drug-resistance marker and long flanking sequences, was synthesized by one-step polymerase chain reaction after a three-piece ligating reaction. Chlorophyll synthesis genes, chlH and chIL in Synechocystis sp. PCC 6803, were replaced by a kanamycin-resistance marker through double recombinations with flanking homology regions. Under LAHG conditions, the chIL but not chlH mutant stopped chlorophyll synthesis, while both synthesized chlorophyll in the light.

Microbial inactivation of Pseudomonas putida and Pichia pastoris using gene silencing.

Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.

Construction of bovine adenovirus type 3 E1 and E3, substitution plasmids and the sequencing analysis of DNA pol and pTP /

The relative ease to concentrate and purify adenoviruses, their well characterized mid-sized genome, and the ability to delete non-essential regions from their genome to accommodate foreign gene, made adenoviruses a suitable candidate for the construction of vectors. The use of adenoviral vectors in gene therapy, vaccination, and as a general vector system for expressing foreign genes have been documented for some time. In this study, the objective was to rescue a BAV3 E1 or E3 recombinant vector carrying the kanamycin resistant gene, a dominant selectable marker with useful applications in studying vectored gene expression in mammalian cells. To accomplish the objective of this study, more information about BAV3 DNA sequences was required in order to make the manipulation of the virus genome accessible. Therefore, sequencing of the BAV3 genome from 1 1 .7% to 30.8% was carried out. Analysis of the determined sequences revealed the primary structure of important viral gene products coded by E2 including BAV3 DNA pol and precursor to terminal protein. Comparative analysis of these proteins with their counterparts from human and non human adenoviruses revealed important insights as to the evolutionary lineage of BAV3. In order to insert the kanamycin resistance gene in either E1 or E3, it was necessary to delete BAV3 sequences to accommodate the foreign gene so as not to exceed the limit of the packaging capacity of the virus. To construct a recombinant BAV3 in which a foreign gene was inserted in the deleted E1 region, an E1 shuttle vector was constructed. This involved the deletion from the viral sequences a region between 1.3% to 9% and inserting the kanamycin resistance gene to replace the deletion. The E1 shuttle vector contained the left (0%- 53.9%) segment of the genome and was expected to generate BAV3 recombinants that can be grown and propagated in cells that can complement the missing E1 functions. To construct a similar shuttle vector for E3 deletion, DNA sequences extending from 78.9% to 82.5% (1281 bp) were deleted from within the E3 region that had been cloned into a plasmid vector. The deleted region corresponds to those that have been shown to be non-essential for viral replication in cell culture. The resulting plasmid was used to construct another recombinant plasmid with BAV3 DNA sequences extending from 37.1% to 100% and with a deletion of E3 sequences that were replaced by kanamycin resistance gene. This shuttle plasmid was used in cotransfections with digested viral DNA in an attempt to rescue a recombinant BAV3 carrying the kanamycin resistance gene to replace the deleted E3. In spite of repeated attempts of transfection, El or E3 recombinant BAV3 were not isolated. It seems that other approaches should be applied to make a final conclusion on BAV3 infectivity.

Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

Transformation von Osteospermum ecklonis mit Konstrukten zur Induktion von Virusresistenz und Blühverfrühung

Für die Etablierung einer Transformationsmethode züchterisch relevanter Sorten von Osteospermum ecklonis (Kapmargerite) wurde zunächst ein geeignetes Protokoll für die Regeneration adventiver Sprosse aus vegetativem Gewebe entwickelt. Anschließend wurden Transformationen von Markergenen durch Kokultur mit Agrobacterium tumefaciens durchgeführt. Hierzu wurden Konstrukte verwendet, die das Gen für ß-D-Glucuronidase (GUS) enthielten und deren Expression in transgenen Pflanzen histochemisch nachgewiesen werden konnte. Kanamycinresistenz erwies sich als geeigneter Selektionsmarker für die Transformation. Es konnten von verschiedenen O. ecklonis Sorten GUS-transgene, nicht-chimäre Pflanzen regeneriert werden.Zur Erzeugung transgener Pflanzen mit dem Ziel der Resistenz gegen LMV (lettuce mosaic potyvirus, Salat Mosaik Virus) wurden drei Konstrukte verwendet. Das erste enthält die kodierende Sequenz der Virusproteine VPg, Pro und 6K2. Durch PCR-Mutation wurde die Proteinase-Schnittstelle zwischen 6K2 und VPg zerstört, sowie Start- und Stopcodon eingeführt. Die anderen LMV-abgeleiteten Konstrukte enthalten nicht translatierbare Fragmente des coat protein Gens in sense und antisense Orientierung.Außerdem wurde O. ecklonis noch mit dem Gen des mutmaßlichen Transkriptionsfaktor SPL3 aus Arabidopsis thaliana unter der Kontrolle eines konstitutiven Promotors transformiert. SPL3 ist an der Regulierung der Blüteninduktion in A. thaliana beteiligt.Regenerierte O. ecklonis wurden durch PCR mit konstruktspezifischen Primern auf Anwesenheit des Transgens und Kontamination durch A. tumefaciens überprüft.

Analysing the possible influence of transposon TCl4.7 insertion on the function of the genome of Cydia pomonella granulovirus

CpGV-MCp5 is a natural mutant of the Cydia pomonella Granulovirus (Mexican isolate) (CpGV-M) that harbors an insect host transposon termed TCl4.7 in its genome. TCl4.7 is located between the open reading frames Cp15 and Cp16 and separates two homologous regions hr3 and hr4, which have been recently shown to be origins of replication of CpGV-M. The MCp5 has a significant replication disadvantage in the presence of the wild-type CpGV-M. In this study, the possible effects of TCl4.7 transposon insertion on the genome function of its insertion site has been analysed. The role of Cp15 and Cp16 in the context of the virus infection cycle was examined by generating a CpGV-Bacmid (CpBAC) and Cp15 knock-out (CpBACCp15KO) and Cp16 knock-out (CpBACCp16KO) mutants. The mutant CpBACCp15KO was not able to replicate in CM larvae suggesting that Cp15 was essential for virus replication. In contrast, the mutant CpBACCp16KO infected CM larvae and produced viable occlusion bodies (OBs) demonstrating that Cp16 is a non-essential gene for virus in vivo infection of C. pomonella. The temporal transcription of Cp15 and Cp16, as well as of Cp31 (F protein) as a control, was analysed using RT-PCR and quantitative real-time PCR. It suggested a general delay or reduction of gene transcription of MCp5 compared to the parental CpGV-M. Western blot analyses using anti-Cp15 and anti-Cp16 polyclonal antibodies, however, did not show any immuno-reactive response. Thus, a direct influence of TCl4.7 on the expression of Cp15 and Cp16 could not be substantiated. To investigate whether the interruption of hr3 and hr4 palindromes affects the virus replication, two mutant bacmids with a deletion of hr3 and hr4 (CpBAChr3/hr4-KO) and another with an insertion of a Kanamycin resistance cassette between hr3 and hr4 (CpBAChr3-kan-hr4) were generated. Both mutant bacmids replicated and produced infectious virus OBs, which did not significantly differ in their median lethal concentration (LC50) and median survival time (ST50) compared to the parental CpBAC. Interestingly, the mutant CpBAChr3-kan-hr4 was very effectively out-competed by parental CpBAC, when CM larvae were co-infected with known ratios of OBs of CpBAC and the mutant CpBAChr3-kan-hr4. These observations suggested a functional co-operation between hr3 and hr4 which was interrupted by the KanR insertion in CpBAChr3-kan-hr4 and possibly by TCl4.7 transposon insertion in the mutant MCp5. This hypothesis may explain the observed replication disadvantage of the mutants MCp5 and CpBAChr3-kan-hr4 in the presence of the parental viruses CpGV-M and CpBAC, respectively.

Role of FbpA and FbpB in the pathogenesis and mycolyltransferase activity of Mycobacterium tuberculosis

Mycobacterium tuberculosis infects more people worldwide each year than any other single organism. The Antigen 85 Complex, a family of fibronectin-binding proteins (Fbps) found in several species of mycobacteria and possibly involved in host interaction, is considered among the putative virulence factors of M. tuberculosis. These proteins are implicated in the production of trehalose dimycolate (TDM) and arabinogalactan-mycolate (AG-M), two prominent components of the mycobacterium cell wall and potent modulators of the immune system during infection. For these reasons, the principal members of the complex, FbpA and FbpB, were the focus of these studies. The genes encoding these proteins, fbpA and fbpB, were each disrupted by insertion of a kanamycin resistance cassette in a pathogenic strain of M. tuberculosis, H37Rv. Neither mutation affected growth in routine broth culture. Thin layer chromatography analysis of TDM and AG-M showed no difference in content between the parent strain H37Rv and the FbpA- and FbpB-deficient mutants grown under two different culture conditions. However, metabolic radiolabeling of the strains showed that the production of TDM (but not its precursor TMM) was delayed in the FbpA- and FbpB-deficient mutants compared to the parent H37Rv. During this same labeling period, FbpA-deficient mutant LAa1 failed to produce AG-M and in the FpbB-deficient mutant LAb1 production was decreased. In macrophage tissue culture assay, LAa1 failed to multiply when bacteria in early log phase were used to infect monolayers while LAb1 grew like the parent strain. The growth deficiency of LAa1 as well as the deficiencies in TDM and AG-M production were restored by complementing LAa1 with a functional fbpA gene. These results suggest that the FbpA and FbpB proteins are involved in synthesis of TDM (but not its precursor TMM) as well as AG-M. Other members of the complex appear to compensate for defects in synthesis caused by mutation of single genes in the complex over time. Mutation of the FbpA gene causes greater in vivo effect than mutation of the FbpB gene despite very similar deficiencies in the rate of production of mycolate containing molecules on the cell surface. ^

Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus

The α C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from the bca gene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), which contain large fragments of genomic DNA, have been successfully used as transgenes to create mouse models of dose-dependent diseases. They are also potentially valuable as transgenes for dominant diseases given that point mutations and/or small rearrangements can be accurately introduced. Here, we describe a new method to introduce small alterations in BACs, which results in the generation of point mutations with high frequency. The method involves homologous recombination between the original BAC and a shuttle vector providing the mutation. Each recombination step is monitored using positive and negative selection markers, which are the Kanamycin-resistance gene, the sacB gene and temperature-sensitive replication, all conferred by the shuttle plasmid. We have used this method to introduce four different point mutations and the insertion of the β-galactosidase gene in a BAC, which has subsequently been used for transgenic animal production.

Pollen selection: a transgenic reconstruction approach.

A transgenic reconstruction experiment has been performed to determine the feasibility of male gametophytic selection to enhance transmission of genes to the next sporophytic generation. For tobacco pollen from a transgenic plant containing a single hygromycin-resistance (hygromycin phosphotransferase, hpt-) gene under control of the dc3 promoter, which is active in both sporophytic and gametophytic tissues, 3 days of in vitro maturation in hygromycin-containing medium was sufficient to result in a 50% reduction of germinating pollen, as expected for meiotic segregation of a single locus insert. Pollination of wild-type plants with the selected pollen yielded 100% transgenic offspring, as determined by the activity of the linked kanamycin-resistance gene--present within the same transferred T-DNA borders--under control of the nos promoter. This is direct proof that selection acting on male gametophytes can be a means to alter the frequency of genes in the progeny.

The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

A conjugative plasmid carrying the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacter baumannii isolate

OBJECTIVES: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate. METHODS: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted. RESULTS: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus. CONCLUSIONS: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4.

Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant Staphylococcus aureus Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.

Antibiotic resistance of Staphylococcus aureus strains isolated from fish processing factory workers

One hundred and twenty two strains of Staphylococcus aureus isolated from throats and palms of 39 workers from 6 fish processing factories situated in and around Cochin were tested for their sensitivity to nine commonly used antibiotics-ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, penicillin, polymyxin-B, streptomycin and tetracycline. Highest percentage of resistance was observed towards ampicillin followed by penicillin i.e. 64.75% and 59.84%. Resistance towards other antibiotics like tetracycline, polymyxin-B, erythromycin, kanamycin, neomycin, chloramphenicol and streptomycin were shown by 22.95, 16.39, 7.38, 5.74, 3.28 and 1.64% of the isolates respectively.

Genetic mechanisms of multi-antimicrobial resistance in a pathogenic Edwardsiella tarda strain

TX01, a pathogenic Edwardsiella tarda strain isolated from diseased fish at an epidemic-inflicted fish farm in China, exhibits resistance to multiple classes of antimicrobial agents. The genes (kn(R). catA3, and tet(A), respectively) encoding resistance to kanamycin, chloramphenicol, and tetracycline were cloned and found to be 99-100% identical to the corresponding genes carried by known plasmids and transposons of human, animal, and environmental isolates. Further study demonstrated that TX01 harbors a plasmid, pETX, which proved to be (i) the carrier of the tet and cut operons; (ii) a mobile genetic element that is capable of transferring between bacteria of different genera. These results, which, to our knowledge, documented for the first time the co-existence of chloramphenicol and tetracycline resistance determinants on a conjugative plasmid in a pathogenic E tarda strain, indicated that gene acquisition via horizontal transferring of pETX-like mobile genetic entities may have played an important part in the dissemination of antimicrobial resistance and that there have existed for some time widespread genetic exchanges between bacteria of human, animal/fish, and environmental origins. (C) 2008 Elsevier B.V. All rights reserved.