S444 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-M.47]

Isolation and characterisation of a new lytic bactriophagesagainst E. coli strains

Abdollah Ghasemian 1,∗, Zahra Moradpour 2

1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy,Shiraz University of Medical Sciences, Shiraz, Iran, Islamic Republic ofIran2 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy,Tabriz University of Medical Sciences, Tabriz, Iran, Islamic Republic ofIranKeywords: Bactriophage; E. coli; Phage therapy; Antibiotic resis-tance

Introduction: Treatment and prophylaxis of bacterial infectionsusing phage therapy is an old idea, but with regard to appearance ofmulti-drug resistance bacteria and the ineffectiveness of antibiotictherapy in case of E. coli infections, we looked for lytic bactriophagesto combat these photogenic bacteria.

Methods: The bacteriophage containing samples were collectedfrom different aquatic environments, soil and savage. Fallowingthe enrichment of phage from collected samples, the high titerof lytic phages was achieved. To test the lysates for the ability ofkilling E. coli stains, the top agar method was used. The purifiedphages obtained from resulting pluqes. In subsequent experiments,the number of pluqe forming units (PFU)/ ml of the purified sam-ples were calculated. Restriction patterns of total genomic DNA andelectron microscopy pictures of selected phages have provided.

Results: Several specific phages were isolated and characterisedfrom the natural samples. In vitro experiments demonstrateddifferent antimicrobial effect of phages on E. coli at different mul-tiplicity of infection -0.1, 1.0 and 10.0. The phage lysates were ableto significantly inhibit bacterial growth when incubated in Luria-Bertani media.

Discussion: It is believed that phage therapy may be consideredas an important alternative in post antibiotic era for treatment ofinfections. The new isolated phages increase the hope to cambatdrug resistant E. coli. The abundance and diversity of phages innature makes it necessary to expand investigations in the field ofisolation and charecterization of phages to exploit in phage therapy.

doi:10.1016/j.jbiotec.2010.09.635

[P-M.48]

The Mechanism of Bacterial Persistence

J.S. Kim ∗, P. Heo, Y.S. Yang, S.H. Kim, D.H. Kweon

Sungkyunkwan University, Republic of KoreaKeywords: Bacterial persistence; Hydroxyl radical; Alternativeelectorn acceptor; Anaerobic respiration

When a population of bacteria is treated with antibiotics aportion of bacterial subpopulation survives, which is called persis-tence. Persister cell are not genetically different from the dead cells(sensitive cell). The frequency of persistence formation is depen-dent on culture condition, aeration rate and biofilm formation. But,the mechanism of bacterial persistence is still elusive.

Recently, hydroxyl radical formation has been proposed to bethe common mechanism of cell death by bactericidal drugs. Wehypothised that persister cell would form if electron acceptorsother than oxygen are used because hydroxyl radicals cannot beformed in the absence of oxygen. In order to prove this hypothesis,persistence of bacteria to bactericidal drug was tested in aero-

bic and anaerobic condition. Different from aerobic condition, celldeath and hydroxyl radical formation did not occur in anaero-bic condition. Nitrate, fumarate, DMSO and TMAO may play asan electron acceptor in absence of oxygen. Addition of these elec-tron acceptors dramatically increased the frequency of persistenceand lowered hydroxyl radical formation, indicating that persistercells are these which does not use oxygen as the terminal electronacceptor. Various inhibitors against fumarate reductase and nitratereductase showed reduced persistence. Deletion mutation of thesegenes also reduced persistence. For conclusion, persister cells arethe sub population that does not use oxygen as electron acceptors.

doi:10.1016/j.jbiotec.2010.09.636

[P-M.49]

Enhanced production of recombinant staphylokinase inEscherichia coli carrying Vitreoscilla haemoglobin gene

A Jawed, K.L. Dikshit, D.K. Sahoo ∗

Institute of Microbial Technology, Chandigarh, IndiaKeywords: Staphylokinase; Escherichia coli; Vitreoscillahaemoglobin; Oxygen limitation

Escherichia coli is the host of choice for production of most het-erologous proteins. An important goal of an intracellular proteinproduction process is to produce high concentrations of desiredprotein, a parameter that is proportional to both cell density andspecific product yield, in active form. However, the replication andgene expression of a plasmid depend on a number of nutritional andmetabolic parameters. Oxygen is one such parameter and its lim-itation (dissolved oxygen concentration (DOC) level in cultivationmedium) following induction is known to adversely affect plas-mid maintenance and expression levels of recombinant proteinsin E. coli. Vitreoscilla haemoglobin (VHb), a bacterial haemoglobinmost likely binds to oxygen at low extracellular concentration anddeliver it to the terminal respiratory oxidase, thus enhancing respi-ration under these conditions. Thus, the purpose of this study wasto investigate the effect of co-expression of VHb gene with the geneof staphylokinase (a clot-dissolving protein) in E. coli BL-21 ( DE3)on its cell growth and staphylokinase expression at flask and biore-actor levels. A new plasmid construction was designed by ligation ofpUCVHB VHb along with its oxy promoter, with pSAK. In shake flaskstudies, the yield of SAK was 75.1 mg/L in comparison to 44.8 mg/Lwithout VHb expression, at agitation rate of 50 rpm where as at200 rpm, co-expression of VHb gene led to increased SAK expres-sion of 143.8 mg/L against 102 mg/L in culture without VHb. Inbioreactor studies, DOC was found to significantly affect cell growthand staphylokinase expression. The cells with VHb produced highercell density and SAK than those without VHb at correspondingDOC, co-expression of VHb gene enhancing cell growth and SAKexpression by 12-16% and 15-25%, respectively. Additional resultson implications of VHb co-expression on production process opti-mization and yield of SAK will be discussed.

doi:10.1016/j.jbiotec.2010.09.637