Search

T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ 70 , by two T7 proteins: Gp2 and Gp5.7. Although the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit Eσ S , the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growing E. coli as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth., Competing Interests: The authors declare no conflict of interest.

Over the past six decades, there has been a decline in novel therapies to treat tuberculosis, while the causative agent of this disease has become increasingly resistant to current treatment regimens. Bacteriophages (phages) are able to kill bacterial cells and understanding this process could lead to novel insights for the treatment of mycobacterial infections. Phages inhibit bacterial gene transcription through phage-encoded proteins which bind to RNA polymerase (RNAP), thereby preventing bacterial transcription. Gp2, a T7 phage protein which binds to the beta prime (β') subunit of RNAP in Escherichia coli, has been well characterized in this regard. Here, we aimed to determine whether Gp2 is able to inhibit RNAP in Mycobacterium tuberculosis as this may provide new possibilities for inhibiting the growth of this deadly pathogen. Results from an electrophoretic mobility shift assay and in vitro transcription assay revealed that Gp2 binds to mycobacterial RNAP and inhibits transcription; however to a much lesser degree than in E. coli. To further understand the molecular basis of these results, a series of in silico techniques were used to assess the interaction between mycobacterial RNAP and Gp2, providing valuable insight into the characteristics of this protein-protein interaction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The σ factor is a functionally obligatory subunit of the bacterial transcription machinery, the RNA polymerase. Bacteriophage-encoded small proteins that either modulate or inhibit the bacterial RNAP to allow the temporal regulation of bacteriophage gene expression often target the activity of the major bacterial σ factor, σ 70 . Previously, we showed that during Xanthomonas oryzae phage Xp10 infection, the phage protein P7 inhibits the host RNAP by preventing the productive engagement with the promoter and simultaneously displaces the σ 70 factor from the RNAP. In this study, we demonstrate that P7 also inhibits the productive engagement of the bacterial RNAP containing the major variant bacterial σ factor, σ 54 , with its cognate promoter. The results suggest for the first time that the major variant form of the host RNAP can also be targeted by bacteriophage-encoded transcription regulatory proteins. Since the major and major variant σ factor interacting surfaces in the RNAP substantially overlap, but different regions of σ 70 and σ 54 are used for binding to the RNAP, our results further underscore the importance of the σ-RNAP interface in bacterial RNAP function and regulation and potentially for intervention by antibacterials., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Most DNA-binding bacterial transcription factors contact DNA through a recognition α-helix in their DNA-binding domains. An emerging class of DNA-binding transcription factors, predominantly found in pathogenic bacteria interact with the DNA via a relatively novel type of DNA-binding domain, called the LytTR domain, which mainly comprises β strands. Even though the crystal structure of the LytTR domain of the virulence gene transcription factor AgrA from Staphylococcus aureus bound to its cognate DNA sequence is available, the contribution of specific amino acid residues in the LytTR domain of AgrA to transcription activation remains elusive. Here, for the first time, we have systematically investigated the role of amino acid residues in transcription activation in a LytTR domain-containing transcription factor. Our analysis, which involves in vivo and in vitro analyses and molecular dynamics simulations of S. aureus AgrA identifies a highly conserved tyrosine residue, Y229, as a major amino acid determinant for maximal activation of transcription by AgrA and provides novel insights into structure-function relationships in S. aureus AgrA., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)