Production of Shiga toxins by enterohemorrhagic Escherichia coli (EHEC) which is responsible for the pathogenicity of these strains, is strictly correlated with induction of lambdoid bacteriophages present in the host’s genome, replication of phage DNA and expression of stx genes. Antibiotic treatment of EHEC infection may lead to induction of prophage into a lytic development, thus increasing the risk of severe complications. This, together with the spread of multi-drug resistance, increases the need for novel antimicrobial agents. We report here that isothiocyanates (ITC), plant secondary metabolites, such as sulforaphane (SFN), allyl isothiocyanate (AITC), benzyl isothiocynanate (BITC), phenyl isothiocyanate (PITC) and isopropyl isothiocyanate (IPRITC), inhibit bacterial growth and lytic development of stx-harboring prophages. The mechanism underlying the antimicrobial effect of ITCs involves the induction of global bacterial stress regulatory system, the stringent response. Its alarmone, guanosine penta/tetraphosphate ((p)ppGpp) affects major cellular processes, including nucleic acids synthesis, which leads to the efficient inhibition of both, prophage induction and toxin synthesis, abolishing in this way EHEC virulence for human and simian cells. Thus, ITCs could be considered as potential therapeutic agents in EHEC infections.

BackgroundUntil now, the direct link between central carbon metabolism and DNA replication has been demonstrated only in Bacillus. subtilis. Therefore, we asked if this is a specific phenomenon, characteristic for this bacterium and perhaps for its close relatives, or a more general biological rule.ResultsWe found that temperature-sensitivity of mutants in particular genes coding for replication proteins could be suppressed by deletions of certain genes coding for enzymes of the central carbon metabolism. Namely, the effects of dnaA46(ts) mutation could be suppressed by dysfunction of pta or ackA, effects of dnaB(ts) by dysfunction of pgi or pta, effects of dnaE486(ts) by dysfunction of tktB, effects of dnaG(ts) by dysfunction of gpmA, pta or ackA, and effects of dnaN159(ts) by dysfunction of pta or ackA. The observed suppression effects were not caused by a decrease in bacterial growth rate.ConclusionsThe genetic correlation exists between central carbon metabolism and DNA replication in the model Gram-negative bacterium, E. coli. This link exists at the steps of initiation and elongation of DNA replication, indicating the important global correlation between metabolic status of the cell and the events leading to cell reproduction.

bThe pathogenicity of enterohemorrhagic Escherichia coli (EHEC) depends on production of Shiga toxins, which are encoded by stx genes located in the genomes of lambdoid prophages. Efficient expression of these genes requires prophage induction and lytic development of phages. Treatment of EHEC infections is problematic due to not only the resistance of various strains to antibiotics but also the fact that many antibiotics cause prophage induction, thus resulting in high-level expression of stx genes. Here we report that E. coli growth, Shiga toxin-converting phage development, and production of the toxin by EHEC are strongly inhibited by phenethyl isothiocyanate (PEITC). We demonstrate that PEITC induces the stringent response in E. coli that is mediated by massive production of a global regulator, guanosine tetraphosphate (ppGpp). The stringent response induction arises most probably from interactions of PEITC with amino acids and from amino acid deprivation-mediated activation of ppGpp synthesis. In mutants unable to synthesize ppGpp, development of Shiga toxin-converting phages and production of Shiga toxin are significantly enhanced. Therefore, ppGpp, which appears at high levels in bacterial cells after stimulation of its production by PEITC, is a negative regulator of EHEC virulence and at the same time efficiently inhibits bacterial growth. This is in contrast to stimulation of virulence of different bacteria by this nucleotide reported previously by others.

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bThe pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains depends on the production of Shiga toxins that are encoded on lambdoid prophages. Effective production of these toxins requires prophage induction and subsequent phage replication. Previous reports indicated that lytic development of Shiga toxin-converting bacteriophages is inhibited in amino acidstarved bacteria. However, those studies demonstrated that inhibition of both phage-derived plasmid replication and production of progeny virions occurred during the stringent as well as the relaxed response to amino acid starvation, i.e., in the presence as well as the absence of high levels of ppGpp, an alarmone of the stringent response. Therefore, we asked whether ppGpp influences DNA replication and lytic development of Shiga toxin-converting bacteriophages. Lytic development of 5 such bacteriophages was tested in an E. coli wild-type strain and an isogenic mutant that does not produce ppGpp (ppGpp 0 ). In the absence of ppGpp, production of progeny phages was significantly (in the range of an order of magnitude) more efficient than in wild-type cells. Such effects were observed in infected bacteria as well as after prophage induction. All tested bacteriophages formed considerably larger plaques on lawns formed by ppGpp 0 bacteria than on those formed by wild-type E. coli. The efficiency of synthesis of phage DNA and relative amount of lambdoid plasmid DNA were increased in cells devoid of ppGpp relative to bacteria containing a basal level of this nucleotide. We conclude that ppGpp negatively influences the lytic development of Shiga toxin-converting bacteriophages and that phage DNA replication efficiency is limited by the stringent control alarmone.