The study of phage-host relationships is essential to understanding the dynamic of microbial systems. Here, we analyze genomewide interactions of Bacillus subtilis and its lytic phage 29 during the early stage of infection. Simultaneous high-resolution analysis of virus and host transcriptomes by deep RNA sequencing allowed us to identify differentially expressed bacterial genes. Phage 29 induces significant transcriptional changes in about 0.9% (38/4,242) and 1.8% (76/4,242) of the host protein-coding genes after 8 and 16 min of infection, respectively. Gene ontology enrichment analysis clustered upregulated genes into several functional categories, such as nucleic acid metabolism (including DNA replication) and protein metabolism (including translation). Surprisingly, most of the transcriptional repressed genes were involved in the utilization of specific carbon sources such as ribose and inositol, and many contained promoter binding-sites for the catabolite control protein A (CcpA). Another interesting finding is the presence of previously uncharacterized antisense transcripts complementary to the well-known phage 29 messenger RNAs that adds an additional layer to the viral transcriptome complexity. IMPORTANCEThe specific virus-host interactions that allow phages to redirect cellular machineries and energy resources to support the viral progeny production are poorly understood. This study provides, for the first time, an insight into the genome-wide transcriptional response of the Gram-positive model Bacillus subtilis to phage 29 infection. Due to their small dimension and limited size of genomes, bacteriophages have optimized the exploitation of host resources to increase the production of the viral progeny. A comprehensive understanding of these host-virus interactions requires the analysis of associated transcriptional changes in both organisms. Thus, we used the recently developed RNA sequencing (RNA-Seq) technology to monitor to a high level of accuracy and depth the genome-wide effect of the bacteriophage 29 on Bacillus subtilis transcription. The transcriptome profiles were analyzed at two early infection time points (8 and 16 min postinfection) so that the identification of the bacterial genes corresponding to these stages could allow the identification of potential phage targets.Phage 29 is a well-characterized lytic virus that belongs to the Podoviridae family. Over the years, it has been the subject of many extensive studies that have contributed to the understanding of several molecular mechanisms of biological processes, such as transcription regulation, viral DNA packaging, viral morphogenesis, and DNA replication (1). Phage 29 genome consists of a linear double-stranded DNA (dsDNA) molecule of 19,285 bp, which encodes 28 open reading frames (ORFs) transcribed from four early and one late promoters. The viral genes are expressed in a temporal sequence to ensure that DNA replication, and the production and assembly of viral components occur in an orderly fashion. Thus, bacterial cells inf...
5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.
Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.
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