Bacteriophages and their genomes.

The worrying rise of antibiotic resistance in pathogenic bacteria is leading to a renewed interest in bacteriophages as a treatment option. Novel sequencing technologies enable description of an increasing number of phage genomes, a critical piece of information to understand their life cycle, phage-host interactions, and evolution. In this work, we demonstrate how it is possible to recover more information from sequencing data than just the phage genome. We developed a theoretical and statistical framework to determine DNA termini and phage packaging mechanisms using NGS data. Our method relies on the detection of biases in the number of reads, which are observable at natural DNA termini compared with the rest of the phage genome. We implemented our method with the creation of the software PhageTerm and validated it using a set of phages with well-established packaging mechanisms representative of the termini diversity, i.e. 5′cos (Lambda), 3′cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of nine Clostridium difficile phages and six phages whose sequences were retrieved from the Sequence Read Archive. PhageTerm is freely available (https://sourceforge.net/projects/phageterm), as a Galaxy ToolShed and on a Galaxy-based server (https://galaxy.pasteur.fr).

https://hal-pasteur.archives-ouvertes.fr/pasteur-01613364/document

PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data.

Amino acid starvation in Escherichia coli results in a spectrum of changes in gene expression, including inhibition of rRNA and tRNA promoters and activation of certain promoters for amino acid biosynthesis and transport. The unusual nucleotide ppGpp plays an important role in both negative and positive regulation. Previously, we and others suggested that positive effects of ppGpp might be indirect, resulting from the inhibition of rRNA transcription and, thus, liberation of RNA polymerase for binding to other promoters. Recently, we showed that DksA binds to RNA polymerase and greatly enhances direct effects of ppGpp on the negative control of rRNA promoters. This conclusion prompted us to reevaluate whether ppGpp might also have a direct role in positive control. We show here that ppGpp greatly increases the rate of transcription initiation from amino acid promoters in a purified system but only when DksA is present. Activation occurs by stimulation of the rate of an isomerization step on the pathway to open complex formation. Consistent with the model that ppGpp/DksA stimulates amino acid promoters both directly and indirectly in vivo, cells lacking dksA fail to activate transcription from the hisG promoter after amino acid starvation. Our results illustrate how transcription factors can positively regulate transcription initiation without binding DNA, demonstrate that dksA directly affects promoters in addition to those for rRNA, and suggest that some of the pleiotropic effects previously associated with dksA might be ascribable to direct effects of dksA on promoters involved in a wide variety of cellular functions.

https://www.pnas.org/content/pnas/102/22/7823.full.pdf

DksA potentiates direct activation of amino acid promoters by ppGpp

Bacteriophage genomes have mosaic architectures and are replete with small open reading frames of unknown function, presenting challenges in their annotation, comparative analysis, and representation. We describe here a bioinformatic tool, Phamerator, that assorts protein-coding genes into phamilies of related sequences using pairwise comparisons to generate a database of gene relationships. This database is used to generate genome maps of multiple phages that incorporate nucleotide and amino acid sequence relationships, as well as genes containing conserved domains. Phamerator also generates phamily circle representations of gene phamilies, facilitating analysis of the different evolutionary histories of individual genes that migrate through phage populations by horizontal genetic exchange. Phamerator represents a useful tool for comparative genomic analysis and comparative representations of bacteriophage genomes.

/pdf/phamerator-a-bioinformatic-tool-for-comparative-15pyyikmxg.pdf

Phamerator: a bioinformatic tool for comparative bacteriophage genomics

Comparative genomic analysis of 60 Mycobacteriophage genomes: genome clustering, gene acquisition, and gene size.

The Genome of Bacillus Subtilis Bacteriophage SPO1.

http://www.bioc.rice.edu/~crs/pdf/Genes.pdf

Genes and Regulatory Sites of the ``Host-Takeover Module'' in the Terminal Redundancy of Bacillus subtilis Bacteriophage SPO1

Terminal redundancy of "high frequency of recombination" markers of Bacillus subtilis phage SPO1.

Some of the early genes of Bacillus subtilis bacteriophage SPO1 were hypothesized to function in the shutoff of host biosyntheses. Two of these genes, e3 and e22, were cloned and sequenced. E22 showed no similarity to any known protein, while E3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins. Each gene was immediately downstream of a very active early promoter. Each was expressed actively during the first few minutes of infection and was then rapidly shut off and its RNA rapidly degraded. An e3 nonsense mutation severely retarded the degradation of e3 RNA. Expression of a plasmid-borne e3 gene, in either B. subtilis or Escherichia coli, resulted in the inhibition of host DNA, RNA, and protein syntheses and prevented colony formation. However, the e3 nonsense mutation caused no measurable decrease in either burst size or host shutoff during infection and, in fact, caused an increased burst size at high multiplicities of infection. We suggest that e3 is one of several genes involved in host shutoff, that its function is dispensable both for host shutoff and for phage multiplication, and that its shutoff function is not entirely specific to host activities.

A cytotoxic early gene of Bacillus subtilis bacteriophage SPO1.

Mutants affected in cistrons 21 and 32 of bacteriophage SPO1 are defective specifically in the initiation of DNA replication. Mutations in cistron 32 also specifically affect the termination of replication.

Charles R. Stewart

Papers

The Genome of Bacillus Subtilis Bacteriophage SPO1.

Genes and Regulatory Sites of the ``Host-Takeover Module'' in the Terminal Redundancy of Bacillus subtilis Bacteriophage SPO1

Terminal redundancy of "high frequency of recombination" markers of Bacillus subtilis phage SPO1.

A cytotoxic early gene of Bacillus subtilis bacteriophage SPO1.

Initiation and Termination Mutants of Bacillus subtilis Bacteriophage SPO1