Joanna M. Łoś

Differential efficiency of induction of various lambdoid prophages responsible for production of Shiga toxins in response to different induction agents

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogenic strains responsible for bloody diarrhea and hemorrhagic colitis, with often severe complications. Shiga toxins are the main factors causing the phathogenicity of STEC. Production of these toxins depends on the presence of stx1 and stx2 genes, which are located on lambdoid prophages, and their expression is stimulated upon prophage induction. Therefore, a transition of the phage genome from the prophage state to an extrachromosomal genetic element, and its further propagation, is crucial for the pathogenic effects. However, our knowledge on specific conditions for induction of these prophages in bacteria occurring in human intestine is very limited. In this report we present results of our studies on five different phages, originally occurring in STEC strains, in comparison to bacteriophage lambda. We found that efficiencies of induction of prophages and their further development vary considerably in response to different induction agents. Moreover, efficiency of progeny phage production might be modulated by other factors, like temperature or bacterial growth rate. Therefore, it is likely that pathogenicity of different STEC strains may be significantly different under specific conditions in their natural habitats.

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.

Hydrogen peroxide-mediated induction of the Shiga toxin-converting lambdoid prophage ST2-8624 in Escherichia coli O157:H7

Shiga toxin-producing Escherichia coli (STEC) may cause bloody diarrhea and hemorrhagic colitis, with sometimes severe complications. Because genes coding for Shiga toxins are located on lambdoid prophages, effective toxin production occurs only after prophage induction. However, although agents that effectively induce prophage lambda (a paradigm of the family of lambdoid phages) under laboratory conditions, such as UV irradiation or DNA replication inhibitors, are well known, it is unlikely that such factors are present in human intestine infected with STEC. In this report, we demonstrate that induction of a Shiga toxin-converting prophage in its host (E. coli O157:H7) occurs not only in the presence of DNA-interfering antibiotics (mitomycin C and norfloxacin) but also under conditions of oxidative stress [following treatment with hydrogen peroxide (H(2)O(2))]. Under these conditions, we observed not only effective prophage induction but also expression of the reporter gene (replacing the original stx2 gene). In the light of previously published reports, indicating that oxidative stress conditions might occur during colonization of human intestine by enteric bacteria, and that neutrophil-produced H(2)O(2) can increase production of the Shiga toxin in a clinical isolate of STEC, these results suggest that oxidative stress may be one of the agents responsible for stimulating the pathogenicity determinants of STEC, leading to induction of Shiga toxin-converting prophages in these bacteria.

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.

Altruism of Shiga toxin-producing Escherichia coli: recent hypothesis versus experimental results.

Shiga toxin-producing Escherichia coli (STEC) may cause bloody diarrhea and hemorrhagic colitis (HC), with subsequent systemic disease. Since genes coding for Shiga toxins (stx genes) are located on lambdoid prophages, their effective production occurs only after prophage induction. Such induction and subsequent lytic development of Shiga toxin-converting bacteriophages results not only in production of toxic proteins, but also in the lysis (and thus, the death) of the host cell. Therefore, one may ask the question: what is the benefit for bacteria to produce the toxin if they die due to phage production and subsequent cell lysis? Recently, a hypothesis was proposed (simultaneously but independently by two research groups) that STEC may benefit from Shiga toxin production as a result of toxin-dependent killing of eukaryotic cells such as unicellular predators or human leukocytes. This hypothesis could make sense only if we assume that prophage induction (and production of the toxin) occurs only in a small fraction of bacterial cells, thus, a few members of the population are sacrificed for the benefit of the rest, providing an example of “bacterial altruism.” However, various reports indicating that the frequency of spontaneous induction of Shiga toxin-converting prophages is higher than that of other lambdoid prophages might seem to contradict the for-mentioned model. On the other hand, analysis of recently published results, discussed here, indicated that the efficiency of prophage excision under conditions that may likely occur in the natural habitat of STEC is sufficiently low to ensure survival of a large fraction of the bacterial host. A molecular mechanism by which partial prophage induction may occur is proposed. We conclude that the published data supports the proposed model of bacterial “altruism” where prophage induction occurs at a low enough frequency to render toxin production a positive selective force on the general STEC population.

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.

Plasmids derived from lambdoid bacteriophages as models for studying replication of mobile genetic elements responsible for the production of Shiga toxins by pathogenic Escherichia coli strains.

Background: Genes encoding Shiga toxins in pathogenic Escherichia coli strains (Shiga toxin-producing E. coli, STEC) are located on lambdoid prophages. However, studies on the replication of these phages were not reported to date. Methods: Plasmids derived from Shiga toxin-converting phages were constructed, introduced into wild-type E. coli strain and studied using methods of molecular genetics. Results: The studied replicons behaved similarly to plasmids derived from bacteriophage λ, and DNA sequence analysis revealed their high level of homology to the λ replication region. Nevertheless, contrary to λ and two of six tested plasmids, four tested replicons were able to replicate in the E. colidnaA46 mutant. Specific nucleotide differences, causing amino acid substitutions relative to wild-type λ O and P proteins, appeared to be responsible for this phenotype. Conclusions: We conclude that replicons of phages bearing Shiga toxin genes are functionally similar to that of bacteriophage λ, however, some of them have important differences which influence replication regulation. Furthermore, our results may suggest that there are newly discovered molecular interactions in the DNA replication regulation of λ and lambdoid phages. Hence, plasmids derived from lambdoid phages appear to be convenient models to study the replication of Shiga toxin-converting bacteriophages.

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 biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC50 = 20 μM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.

Influence of the Escherichia coli oxyR gene function on λ prophage maintenance

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of λ prophage. Here, we demonstrate that H2O2-mediated λ prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a λ lysogen in the presence of H2O2. On the other hand, stimulation of the pM promoter by cI857 overproduced from a multicopy plasmid was decreased in the ΔoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the pM promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the pM promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced λ prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating λ prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.