Piotr Golec

Simple Method for Plating Escherichia coli Bacteriophages Forming Very Small Plaques or No Plaques under Standard Conditions

ABSTRACT The use of low concentrations (optimally 2.5 to 3.5 μg/ml, depending on top agar thickness) of ampicillin in the bottom agar of the plate allows for formation of highly visible plaques of bacteriophages which otherwise form extremely small plaques or no plaques on Escherichia coli lawns. Using this method, we were able to obtain plaques of newly isolated bacteriophages, propagated after induction of prophages present in six E. coli O157:H− strains which did not form plaques when standard plating procedures were employed.

Specific detection of Salmonella enterica and Escherichia coli strains by using ELISA with bacteriophages as recognition agents

The use of bacteriophages, instead of antibodies, in the ELISA-based detection of bacterial strains was tested. This procedure appeared to be efficient, and specific strains of Salmonella enterica and Escherichia coli could be detected. The sensitivity of the assay was about 105 bacterial cells/well (106/ml), which is comparable with or outperforms other ELISA tests detecting intact bacterial cells without an enrichment step. The specificity of the assay depends on the kind of bacteriophage used. We conclude that the use of bacteriophages in the detection and identification of bacteria by an ELISA-based method can be an alternative to the use of specific antibodies. The advantages of the use of bacteriophages are their environmental abundance (and, thus, a possibility to isolate various phages with different specificities) and the availability of methods for obtaining large amounts of phage lysates, which are simple, rapid, cheap, and easy.

Effective inhibition of lytic development of bacteriophages λ, P1 and T4 by starvation of their host, Escherichia coli

BackgroundBacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful.ResultsHere, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, λ, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats.ConclusionBacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively.

A reliable method for storage of tailed phages

A universal and effective method for long-term storage of bacteriophages has not yet been described. We show that randomly selected tailed phages could be stored inside the infected cells at -80°C without a major loss of phage and host viability. Our results suggest the suitability of this method as a standard for phage preservation.

Inhibition of development of Shiga toxin-converting bacteriophages by either treatment with citrate or amino acid starvation.

OBJECTIVES Shiga toxin-producing Escherichia coli (STEC) are pathogenic strains, whose virulence depends on induction of Shiga toxin-converting prophages and their subsequent lytic development. We explored which factors or conditions could inhibit development of these phages, potentially decreasing virulence of STEC. MATERIALS AND METHODS Lytic development of Shiga toxin-converting bacteriophages was monitored after mitomycin C-provoked prophage induction under various conditions. Phage DNA replication efficiency was assessed by measurement of DNA amount in cells using quantitative polymerase chain reaction. RESULTS We demonstrated that the use of citrate delayed Shiga toxin-converting phage development after prophage induction. This effect was independent on efficiency of prophage induction and phage DNA replication. However, an excess of glucose reversed the effect of citrate. Amino acid starvation prevented the phage development in bacteria both able and unable to induce the stringent response. CONCLUSIONS Lytic development of Shiga toxin-converting bacteriophages can be inhibited by either the presence of citrate or amino acid starvation. We suggest that the inhibition caused by the latter condition may be due to a block in prophage induction or phage DNA replication or both. APPLICATIONS Our findings may facilitate development of procedures for treatment of STEC-infected patients.

Persistence of bacteriophage T4 in a starved Escherichia coli culture: evidence for the presence of phage subpopulations

Bacteriophage T4 is able to adjust its development to the growth parameters of the host cell. Here, we present evidence for the production of two different subpopulations of phage particles, which differ in their ability to infect starved Escherichia coli cells. The ability of phage T4 to produce a fraction of virions unable to infect starved cells is linked to the functions of genes rI and rIII, as well as rIIA. This may represent the adaptation of phage T4 in order to persist in unfavourable environmental conditions.

A role for accessory genes rI.-1 and rI.1 in the regulation of lysis inhibition by bacteriophage T4

Lysis inhibition (LIN) is a known feature of the T-even family of bacteriophages. Despite its historical role in the development of modern molecular genetics, many aspects of this phenomenon remain mostly unexplained. The key element of LIN is an interaction between two phage-encoded proteins, the T holin and the RI antiholin. This interaction is stabilized by RIII. In this report, we demonstrate the results of genetic experiments which suggest a synergistic action of two accessory proteins of bacteriophage T4, RI.-1, and RI.1 with RIII in the regulation of LIN.

Proteomic profiles and kinetics of development of bacteriophage T4 and its rI and rIII mutants in slowly growing Escherichia coli

Bacteriophage T4 survival in its natural environment requires adjustment of phage development to the slow bacterial growth rate or the initiation of mechanisms of pseudolysogeny or lysis inhibition (LIN). While phage-encoded RI and probably RIII proteins seem to be crucial players in pseudolysogeny and LIN phenomena, the identity of proteins involved in the regulation of T4 development in slowly growing bacteria has remained unknown. In this work, using a chemostat system, we studied the development of wild-type T4 (T4wt) and its rI (T4rI) and rIII (T4rIII) mutants in slowly growing bacteria, where T4 did not initiate LIN or pseudolysogeny. We determined eclipse periods, phage propagation times, latent periods and burst sizes of T4wt, T4rI and T4rIII. We also compared intracellular proteomes of slowly growing Escherichia coli infected with either T4wt or the mutants. Using two-dimensional PAGE analyses we found 18 differentially expressed proteins from lysates of infected cells. Proteins whose amounts were different in cells harbouring T4wt and the mutants are involved in processes of replication, phage-host interactions or they constitute virion components. Our data indicate that functional RI and RIII proteins - apart from their already known roles in LIN and pseudolysogeny - are also necessary for the regulation of phage T4 development in slowly growing bacteria. This regulation may be more complicated than previously anticipated, with many factors influencing T4 development in its natural habitat.

Phage-Directed Synthesis of Photoluminescent Zinc Oxide Nanoparticles under Benign Conditions

Biological systems, especially bacteriophages and peptides, are an attractive green alternative to other known methods of nanoparticle synthesis. In this work, for the first time, bacteriophages were employed to synthesize a specific peptide, capable of producing nanoparticles (NPs). Derivatives of M13 bacteriophage exposing a ZnO-binding peptide (TMGANLGLKWPV) on either pIII or pVIII phage coat protein were constructed and used as a biotemplate. The exposition of the ZnO-binding peptide, synthesized by phages during their propagation in bacteria, on M13 virions provided a groundwork for growing ZnO nanostructures. Depending on the recombinant phage type used (M13-pIII-ZnO or M13-pVIII-ZnO), well separated ZnO NPs or complex 3D structures of ZnO NPs of ca. 20-40 nm were synthesized at room temperature. The synthesized ZnO nanoparticles served as a luminescent material that emitted light near the short wavelength end of the visible region (at ca. 400 nm). The next very low intensity emission band at 530 nm demonstrated that the ZnO material obtained is characterized by a low concentration of surface defects.

Methods for Bacteriophage Preservation

In a view of growing interest in bacteriophages as the most abundant members of microbial communities and as antibacterial agents, reliable methods for bacteriophage long-term preservation, that warrant the access to original or mutant stocks of unchanged properties, have become of crucial importance. A storage method that retains the infectivity of any kind of bacteriophage virions, either in a cell lysate or in a purified suspension, does not exist, due to the enormous diversity of bacteriophages and hence the differentiation of their sensitivity to various storage conditions. Here, we describe a method of long-term bacteriophage preservation, which is based on freezing of freshly infected susceptible bacteria at early stages of bacteriophage development. The infected bacteria release mature bacteriophages upon melting enabling the recovery of bacteriophage virions with high efficiency. The only limitation of this method is the sensitivity of bacteriophage host to deep-freezing, and thus it can be used for the long-term preservation of the vast majority of bacteriophages.