S556 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

and TubS2 for PCR detection. The parameters for PCR were 94 ◦C for2 min, followed by 30 cycles of 98 ◦C for 10 sec, 59 ◦C for 30 sec and72 ◦C for 20 sec. Results: A 264 bp-PCR product was amplified fromthe suspicious isolates from the apples, positive control strain N.malicorticis, N. perennans and N. alba. In contrast, this PCR productwas not amplified from other strains. Based on the ˇ-tublin genesequences, we also designed universal primers TubC1 and TubC2to amplify 554-bp product for DNA sequencing. Discussion: Afterblasting in GenBank, Neofabraea spp can be differentiated on thespecies level. Based on these detection results, we concluded thatNeofabraea spp. captured in 2009 was N. perennans.

Uncited references

James, 2004; Sharon et al., 2001; Verkley, 1999.

References

James, H., 2004. Cunnington. Three Neofabraea species on pome fruit in Australia.Australia Plant Pathology 33, 453–454.

Sharon, N., de Jong, C., André Levesque, Gerard, J.M., Verkley, et al., June 2001.Phylogenetic relationships among Neofabraea species causing tree cankers andbull’s-eye rot of apple based on DNA sequencing of ITS nuclear rDNA, mitochon-drial rDNA, and the b-tubulin gene. Mycol. Res. 105 (6), 658–669.

Verkley, G.J.M., 1999. A monograph of the genus Pezicula and its anamorphs. Studiesin Mycology. 44, 1–180.

doi:10.1016/j.jbiotec.2010.10.013

Comparative analysis of pseudocowpoxvirus VEGF genes: evi-dence for interspecies recombination and different pattern ofreceptor activation

F. Vaccari 1, M. Battilani 1, G. Tasco 2, R. Casadio 2, A. Scagliarini 1,∗

1 Dipartimento di Sanità Pubblica Veterinaria e Patologia Animale2 Dipartimento di Biologia Evoluzionistica Sperimentale, Università diBologna, Italia

PCPV is a member of the parapoxvirus genus the type specis ofwhich is Orf virus (ORFV). PCPV is maintained in cattle, while ORFVis maintained in sheep and goats and both infect humans. Recently,a homolog of the vascular endothelial growth factor (VEGF) hasbeen identified in the VR634 pseudocowpoxvirus (PCPV genome).The relatedness between PCPV VR634 VEGF and ORFV NZ7 VEGFraised the possibility that ORFV NZ7 strain is a natural recombinantbetween strains of ORFV and PCPV and that the DNA segment span-ning the VEGF gene of PRFV NZ7 is derived from PCPV. In our study,the VEGF genes of two PCPV field strains (1303/05 and 380/06)recently isolated in Italy, were characterised. Phylogenetic analy-sis was carried out using the maximum likelihood approach anda variety of statistical analyses regarding genetic differentiationand gene flow were performed with DNASP version 4.10. To screenfor recombination, we employed two preliminary detection pro-grams: Single Breackpoint recombination and Genetic Algorithmsfor Recombination Detection, both implemented in Datamonkey.A breakpoint of recombination has been identified at nucleotideposition 237 of NZ-2 gene corresponding to a conserved codonencoding for CYS in all the viral variants indicating that recombina-tion may occur at this site while DNASP and statistical tests supportthe hypotesis that PCPV 1303 and 380 VEGFs differentiated fromNZ-2 variants. PCPV amino acid sequences were compared withother viral VEGFs using Clustal W. A 51% and 41,6% identity valueswere computed with the NZ-2 and VR634 variants, respectively.The secondary and tertiary structure analysis was performed withPSIPRED and Homology Modelling, indicating the conservation ofthe functional motifs and signature. The characterisation of the C-terminal has been performed to compare the amino acid residues

involved in the interaction with NP-1 and R1 receptors. C-terminalpeptides identified in the PCPV 1303 and 380 VEGF-variants sug-gest a low affinity with the receptor NP-1, while the presence offew glycosilation sites may lead to the activation of R1 which medi-ates monocyte migration. Taken together our results provide newinsights into interspecies recombination between bovine and sheepparapoxviruses and on the role of VEGF in normal and pathologicalconditions.

doi:10.1016/j.jbiotec.2010.10.014

Molecular markers linked to Salinity resistance genes in(Triticum aestivum L.). Wheat cultivars by QTL

Waqas Manzoor bhutta ∗, Naveed Ahmad

E-mail address: wmbhutta4055@yahoo.com (W.M. bhutta).

AbstractWheat is an important staple food and cash crop of the world. To

obtain better yield from saline soils and saline waters, it is imper-ative that salt tolerant wheat varieties should be developed. Soilsalinity is one of the greatest problems faced by agriculture today.There are many mechanisms by which plants tolerant the problemsassociated with salinity all of which are genetically controlled. Salttolerance is a mixture of different morpho-physiological processeswhich are controlled by many genes across the wheat genome. DNAmarkers enhance the recovery rate of isogenic recurrent genomeafter hybridization and facilitate the introgression of multiple geneswith additive effects necessary to increase stress tolerance. Molec-ular markers are used to transfer alleles of interest from wildrelatives into cultivated genotypes. SSR primer pairs were used tofind DNA marker linked with salinity resistance. A total 200 SSRprimer pairs used. Out of which four SSR primers were polymor-phic with the parents. These four polymorphic primers applied onF2 population. Development of microsarellite markers provided anew approach to MAS in breeding practice. Based on these criteriamicrosatellite markers are preferable to RFLP markers. Once devel-oped microsatellite markers are easy to use and less expensive toassay because the procedure for microsatellite analysis virtuallyinvolves only amplification and electrophoresis.

doi:10.1016/j.jbiotec.2010.10.015

Fluorescence staining of asbestos using asbestos-binding pro-teins and its application to rapid and sensitive asbestosdetection

Akio Kuroda, Takenori Ishida, Maxym Alexandrov, TomokiNishimura, Kenji Minagawa, Ryuichi Hirota

Department of Molecular Biotechnology, Graduate School of AdvancedSciences of Matter, Hiroshima University, Higashi-Hiroshima,Hiroshima 739-8530, Japan

Asbestos is fibrous silicate minerals that had been used as anacoustic insulator, thermal insulation, fire proofing, and many otherbuilding materials due to chemical and thermal stability. Asbestosstill remains common in most of the world where the incidence ofasbestos-linked pleural mesothelioma and other lung cancers con-tinues to rise. Of the several types of asbestos, the serpentine fiberchrysotile accounts for more than 90% of industrial asbestos, whileamosite and crocidolite are rod-like, durable amphibole fibers asso-ciated with a greater risk of pleural mesothelioma. Having accuratetechniques for measuring asbestos levels is critical in determin-ing the extent of asbestos contamination and the health risks forhumans.

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S557

Fluorescence microscopy (FM) is one of the most important ana-lytical tools in modern life sciences, sufficiently sensitive to allowthe detection of single molecules. Despite its superior sensitivity,utility of FM for micro- and nanoscale inorganic materials has beenrather limited due to the lack of specific probes. Recently, we dis-covered that E. coli protein DksA can specifically bind to serpentineasbestos, chrysotile that can cause asbestosis, mesothelioma andlung cancer. Fluorescent-labeled DksA allows detection of very thinchrysotile fibers. We also screened E. coli lysate for proteins thatbind to amphibole asbestos such as amosite and crocidolite, andfound the novel ability of GatZ to bind to the amphibole asbestos.We conjugated GatZ with a different fluorescent dye and demon-strated selective and highly sensitive detection of two asbestostypes using FM.

doi:10.1016/j.jbiotec.2010.10.016

Metabolic engineering of Clostridium acetobutylicum ATCC 824for increased butanol production

S.Y. Lee 1,2,∗, Y.-S. Jang 1, J.Y. Lee 1, J. Lee 1

1 Department of Chemical and Biomolecular Engineering (BK21 pro-gram) and BioProcess Engineering Research Center, Institute for theBioCentury, KAIST, Republic of Korea2 Department of Bio and Brain and Bioinformatics Research Center,KAIST, Republic of Korea

E-mail address: leesy@kaist.ac.kr (S.Y. Lee).

Butanol is a primary alcohol with a C4-structure (C4H10O).Recent studies have shown that butanol can be used as an alterna-tive renewable energy source. In recent years, interest in butanolas potential fuel has grown up due to its advantages over ethanolsuch as high energy density, phase stability on blending with otherfuels, and non-corrosiveness. Clostridium acetobutylicum ATCC 824is the most frequently used organism for butanol production by ABEfermentation. The present work was carried out to develop a strainof C. acetobutylicum ATCC 824 with improved butanol productioncapabilities. To achieve this objective, a gene knockout systemwas constructed by using an L1.LtrB group II intron from Lactococ-cus lactis. The gene knockout plasmid pCACYS3 was constructedby cloning the L1.LtrB group II intron into the pIMPH, whichwas generated by removing both HindIII restriction sites from C.acetobutylicum-Escherichia coli shuttle vector pIMP1. The transcrip-tion of the L1.LtrB group II intron on pCACYS3 was controlled by athl promoter. Based on this knockout system, mutants on aceticand butyric acids pathways have been constructed from C. aceto-butylicum ATCC 824 and characterized. [This work was supportedby the Korea–Australia Collaborative Research Project on the Devel-opment of Sucrose-Based Bioprocess Platform (#10030795) fromthe Korean Ministry of Knowledge Economy. Further support byGS Caltex, BioFuelChem, Microsoft, and the World Class UniversityProgram (R32-2008-000-10142-0) of the MEST are appreciated.].

doi:10.1016/j.jbiotec.2010.10.017

Genome-scale metabolic network analysis of multi-drug resis-tant pathogen Acinetobacter baumannii AYE for drug targeting

S.Y. Lee 1,2,∗, H.U. Kim 1, T.Y. Kim 1

1 Department of Chemical and Biomolecular Engineering (BK21 pro-gram) and BioProcess Engineering Research Center, Institute for theBioCentury, KAIST, Republic of Korea2 Department of Bio and Brain and Bioinformatics Research Center,KAIST, Republic of Korea

E-mail address: leesy@kaist.ac.kr (S.Y. Lee).

Acinetobacter baumannii has emerged as a new clinical threat tohuman health, particularly to the immunocompromised patients inhospital environment. Rapidly increasing number of drug-resistantstrains of this organism urges us to conduct systems-level studiesthat could contribute to the development of an effective therapy.In this study, we reconstructed the genome-scale metabolic net-work of this pathogen, and used it for subsequent drug targeting.The genome-scale metabolic network of A. baumannii AYE wasfirst reconstructed based on its genome annotation data and bio-chemical knowledge from literatures and databases. The in silicomodel was simulated using constraints-based flux analysis withappropriate constraints according to the specific known conditions.Essential gene/reaction analysis and essential metabolite analysiswere performed using this simulation method, each of which iden-tifies essential genes/reactions and metabolites critical to the cellgrowth. The gene/reaction essentiality was investigated to validatethe in silico cell in comparison with available literature data. Themetabolite essentiality approach was undertaken to predict essen-tial metabolites that are critical to the cell growth. The EMFilter,a framework that filters initially predicted essential metabolitesto find the most effective ones as drug targets, was also devel-oped. EMFilter considers metabolite types, number of total andconsuming reaction linkage with essential metabolites, and pres-ence of essential metabolites and their relevant enzymes in humanmetabolism. Final drug target candidates obtained by this systemframework are presented along with implications of this approach.

[This work was supported by the Korean Systems BiologyResearch Project (20090065571) of the Ministry of Education,Science and Technology (MEST) through the National ResearchFoundation (NRF) of Korea. Further supports by the World ClassUniversity Program (R32-2008-000-10142-0) of the MEST, LGChem Chair Professorship, IBM SUR program, and Microsoft areappreciated.]

doi:10.1016/j.jbiotec.2010.10.018

Degradation of ipso-substitution products in Sphingomonas sp.strain TTNP3: are further novel enzymes involved in alkylphenolmetabolism?

B.A. Kolvenbach 1,2,∗, M. Lenz 1, F.L.P. Gabriel 3, H.-P.E. Kohler 3, A.Schäffer 2, P.F.X. Corvini 1

1 Institute for Ecopreneurship, School of Life Sciences, University ofApplied Sciences Northwestern Switzerland, Muttenz, Switzerland2 Institute for Environmental Research, Rheinisch-Westfälische Tech-nische Hochschule, Aachen, Germany3 Swiss Federal Institute of Aquatic Science and Technology, Dübendorf,Switzerland

E-mail address: Boris.kolvenbach@fhnw.ch (B.A. Kolvenbach).

A number of pollutants of environmental concern, e.g. p-nitrophenol, pentachlorophenol, hexachlorocyclohexane, certainnonylphenols, and bisphenol A, are microbially degraded via hydro-quinone (HQ) as a key metabolite. Oxidative degradation of HQ