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Open AccePoster PresentationA simple emergency procedure to be used if biotechnological protein production is endangered by bacteriophage infection of Escherichia coli cultures: effective inhibition of bacteriophage lytic development in infected cultures by removing a carbon source from the mediumGrzegorz Wegrzyn*1, Marcin Los1 and Peter Neubauer2

Address: 1Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland and 2Biocenter Oulu and Department of Process and Environmental Engineering, University of Oulu, Finland

* Corresponding author

BackgroundBacteriophage infections cause serious problems in bothresearch laboratories and large biotechnological compa-nies. Once infected by bacteriophages, bacterial culturesare usually completely destroyed, as phage lytic develop-ment in a bacterium ends up with cell lysis and liberationof progeny phages that infect neighbor bacterial cells. Apossibility of spreading of bacteriophages throughout alaboratory is even more dangerous than a loss of a singleculture. Namely, subsequent cultures may be infected,which can lead to cultivation problems lasting even sev-eral months or longer. Therefore, a method for inhibitionof bacteriophage lytic development in infected cultureswould be useful. Perhaps it is not difficult in small cul-tures (e.g. flask cultures), when simple sterilization of thewhole material and a flask should be sufficient. However,phage contamination in bioreactors is a serious technicalproblem indeed.

Escherichia coli is one of the most widely used bacteriumin genetic engineering and biotechnology. This bacteriumis, however, a host for many bacteriophages and thus, it isendangered by phage infections. Bacteriophages havebeen considered as models in genetic and biochemicalstudies for a long time. However, many physiologicalaspects of bacteriophages' growth were not sufficiently

investigated relative to extensive molecular biology stud-ies. On the other hand, recent reports indicated that devel-opment of bacteriophages largely depends on thephysiology of the host cells. In laboratories, the physio-logical status of a cell depends, in turn, on cultivation con-ditions. Therefore, we aimed to find cultivationconditions that may result in inhibition of bacteriophagedevelopment and are not deleterious for bacterial cells.Previous studies indicated that development of phages T4and λ is significantly less effective in slowly growing hostcells than in rapidly growing bacteria. Thus, we aimed totest whether induction of starvation, caused be depletionof a carbon source from the culture medium, may inhibitphage development effectively. Growth of bacteriophagesin bacterial cells cultured on solid (agar) media support-ing various growth rates and at different temperatures wasalso investigated.

ResultsWe found that a decrease in temperature of an infectedbacterial culture might impair development of some bac-teriophages (e.g. λ) but not others (e.g. T4). Therefore,usefulness of a method of inhibition of phage develop-ment in infected bacterial cultures based on changes oftemperature would be limited.

from The 4th Recombinant Protein Production Meeting: a comparative view on host physiologyBarcelona, Spain. 21–23 September 2006

Published: 10 October 2006

Microbial Cell Factories 2006, 5(Suppl 1):P81 doi:10.1186/1475-2859-5-S1-P81

<supplement> <title> <p>The 4th Recombinant Protein Production Meeting: a comparative view on host physiology</p> </title> <sponsor> <note>The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting.</note> </sponsor> <note>Meeting abstracts – A single PDF containing all abstracts in this supplement is available <a href="http://www.biomedcentral.com/content/files/pdf/1475-2859-5-S1-full.pdf">here</a></note> <url>http://www.biomedcentral.com/content/pdf/1475-2859-5-S1-info.pdf</url> </supplement>

© 2006 Wegrzyn et al; licensee BioMed Central Ltd.

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Microbial Cell Factories 2006, 5(Suppl 1):P81

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The presence of various carbon sources in the same kindof medium results in different bacterial growth rates. Weinvestigated phage plaque formation on lawns of bacteriagrowing in media containing various carbon sources (glu-cose, glycerol, succinate or acetate) at the same tempera-ture. The changes in plaque morphology of both phageswere significant, though more pronounced in λ, as thisphage was not able to form plaques when host grew onthe medium with acetate as a carbon source, i.e. at thelowest growth rate. Removing the carbon source inducesstarvation conditions and minimal bacterial growth rate.Therefore, we asked whether removing the carbon sourcecan lead to inhibition of formation of progeny phages ininfected bacterial cultures.

We found that formation of phage progeny was com-pletely inhibited in infected cultures devoid of the carbonsource. This was true for all tested bacteriophages (λ, P1and T4). Addition of glucose to infected cultures ofstarved bacteria resulted in restoration of phage progenyproduction, indicating that depletion of the carbon sourcewas the sole reason for inhibition of development ofphages λ, P1 and T4.

ConclusionDevelopment of bacteriophages λ, P1 and T4 is com-pletely inhibited after removing a carbon source frominfected E. coli cultures. Therefore, to minimize deleteri-ous effects of phage contamination, especially in high-celldensity and/or fed-batch cultivations, it may be recom-mended to stop feeding bacteria immediately after obser-vation of first signs of phage infection. Such a procedureshould lead to starvation of bacteria and inhibition ofproduction of phage progeny. Although unambiguousdetection of phage contamination at early stages of infec-tion may be difficult using traditional methods, a newlydeveloped technology of electric DNA chips allows forearly detection of phages in bacterial cultures, even a fewgenerations before they cause visible lysis of host cells.

An interesting side aspect of the performed study is thereduced ability of T4 and the inability of λ to form plaqueson medium with acetate as the only carbon source.Although it remains unclear whether this effect is due tothe very low growth rate of E. coli under these conditionsor to the specific effect of acetate on the ΔpH and conse-quently on the proton motive force, or osmotic pressureof the host cell, this effect is interesting in connection tothe proposed strategy to avoid phage propagation by stopof the feeding. Most E. coli fed-batch cultivations, kept atglucose limitation, would immediately stop phagegrowth, even if acetate is still available.

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