A New Mechanism by Which Bacteriophage T5 Inhibits Growth of E. coli

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How can we better control pathogenic bacteria? Insights may come from studying bacteriophages, viruses that infect bacteria. There are a wide variety of bacteriophages, each of which is specialized to infect and replicate within a specific target bacteria. Learning how a bacteriophage takes over bacterial metabolism to direct resources towards generating more bacteriophage can both increase understanding of bacterial metabolism, and potentially provide ideas for new antibiotics or new means of controlling bacterial pathogens.

In recent work, Mahata et al from Tel Aviv University in Israel developed a high-throughput sequencing approach to identify functions for T5 proteins. The bacteria were mutagenized and then screened to identify bacterial mutants that were resistant to growth inhibition by the phage protein T5.015.

To demonstrate the DNA cleavage activity of T5.015, the Azure Sapphire™ Biomolecular Imager was used to detect cleavage products of Cy5.5-labeled oligomers separated by gel electrophoresis (Figure 1). High-throughput sequencing of the mutants characterized the DNA changes responsible for the resistance. The researchers found mutations in the ung gene made the bacteria resistant to the effects of T5.015.

Cy5.5 fluorescence of assay products from Azure Sapphire Biomolecular Imager resolved on denaturing urea polyacrylamide gel are shown.
Figure 1. 015 cleaves Ung-generated AP sites. (A) Schematic diagram of the Ung-generated AP site cleavage assay. (B and C) AP site cleavage assay performed in the presence of the Cy5.5-labeled dsDNA oligomer and the indicated purified proteins. In B, 33.4 nM (1 U) of Ung, 6.6 nM (2 U) of Ape1, 1.26 µM of 015, and 820 nM (2 U) of Ugi were used. In C, 315 nM of 015 and 6.6 nM of Ape1 were used. Cy5.5 fluorescence of assay products resolved on denaturing urea polyacrylamide gel are shown.

Ung, the protein encoded by the ung gene, is involved in uracil excision, removing uracils mistakenly incorporated into DNA. Normally the Ung protein removes the uracil, and the resulting abasic site in the DNA is repaired. However, the researchers found that in T5 infection, after Ung removes an uracil, T5.015 cleaves the DNA at the abasic site. DNA cleavage pauses DNA replication and inhibits bacterial growth. The authors hypothesize that halting DNA replication and cell division makes more resources available to the phage.

Conveniently,­ T5 encodes a dUTPase that reduces UTP levels in the bacteria after infection so newly synthesized phage DNA is much less likely to contain any uracil, and only bacterial DNA is targeted by T5.015. The mechanism identified by Mahata et al represents a previously unknown means of bacterial growth inhibition by a bacteriophage.

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Model of 015-mediated toxicity. T5 bacteriophage translocates its genetic material into the E. coli cell, which expresses 015 within several minutes (1). The 015 gene product forms a complex with the host Ung (2) and thus localizes to newly formed AP sites (3). 015 then attacks the AP site (4) and forms a nick in the chromosomal DNA (5), which leads to DNA replication arrest and ultimately to cell death (6). Licensed under CC BY 4.0

Bacteriophage T5 infects the bacteria Escherichia coli. Some strains of E. coli are found normally in the human gut, but other strains are pathogenic and are responsible for some cases of foodborne illness. T5 is an intriguing bacteriophage because of its large genome which encodes over 160 proteins, only about half of which have known, or proposed functions based on homology. Therefore, studying T5 holds the potential to reveal novel bacterial biological or biochemical mechanisms in addition to providing potential new avenues to controlling pathogens.

In addition to multichannel fluorescent imaging, the Sapphire Biomolecular Imager provides chemiluminescence, densitometry, phosphor, near-infrared and white light imaging of blots, gels, tissues, and more. Learn more about the Sapphire by clicking here and requesting a demo.

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