Search Results (5)

Background/Objectives: The ShiLan domain was previously identified as an insertion sequence in a phage DNA methylase gene that exhibited similar evolutionary patterns to that of an active intein or self-splicing intron but could not be identified as either. It produces no internal stop codons when read in frame with its host methylase gene, leading to the thought that it may not be an intron and rather be an abnormal type of intein. However, the sequence has no detectable self-splicing domains, which are essential for intein persistence, as preventing an intein from successfully splicing is often detrimental to proper host protein function. Methods: The analysis of alternate open reading frames for the full nucleotide sequence of this insertion element revealed the insertion to be an out-of-frame histidine-asparagine-histidine (HNH) endonuclease. A GTG start codon is located 18 bp into the insertion, and a TAA stop codon within the last four bases of the insertion (TAAC). When this frame is read, an HNH endonuclease is revealed. In-depth computational analysis could not retrieve support for this element being any known type of self-splicing element, neither intein nor intron. When read in-frame with the methylase gene, this insertion is predicted to take on a looping structure that may be able to avoid interference with the DNA methylase activity. We performed searches for sequences similar in nature to the inserted out-of-frame HNH and found several in other phages and prokaryotes. We present our survey of these out-of-frame endonuclease insertion elements as well as some speculation on how these endonucleases are getting translated to facilitate their homing activity. Conclusions: These findings expand our understanding of the possible arrangements for and prevalence of unorthodox mobile genetic elements and overlapping open reading frames in phages. Full article

The last thirty years have seen a meteoric rise in the number of sequenced bacteriophage genomes, spurred on by both the rise and success of groups working to isolate and characterize phages, and the rapid and significant technological improvements and reduced costs associated with sequencing their genomes. Over the course of these decades, the tools used to glean evolutionary insights from these sequences have grown more complex and sophisticated, and we describe here the suite of computational and bioinformatic tools used extensively by the integrated research–education communities such as SEA-PHAGES and PHIRE, which are jointly responsible for 25% of all complete phage genomes in the RefSeq database. These tools are used to integrate and analyze phage genome data from different sources, for identification and precise extraction of prophages from bacterial genomes, computing “phamilies” of related genes, and displaying the complex nucleotide and amino acid level mosaicism of these genomes. While over 50,000 SEA-PHAGES students have primarily benefitted from these tools, they are freely available for the phage community at large. Full article

Inteins, often referred to as protein introns, are highly mobile genetic elements that invade conserved genes throughout the tree of life. Inteins have been found to invade a wide variety of key genes within actinophages. While in the process of conducting a survey of these inteins in actinophages, we discovered that one protein family of methylases contained a putative intein, and two other unique insertion elements. These methylases are known to occur commonly in phages as orphan methylases (possibly as a form of resistance to restriction–modification systems). We found that the methylase family is not conserved within phage clusters and has a disparate distribution across divergent phage groups. We determined that two of the three insertion elements have a patchy distribution within the methylase protein family. Additionally, we found that the third insertion element is likely a second homing endonuclease, and that all three elements (the intein, the homing endonuclease, and what we refer to as the ShiLan domain) have different insertion sites that are conserved in the methylase gene family. Furthermore, we find strong evidence that both the intein and ShiLan domain are partaking in long-distance horizontal gene transfer events between divergent methylases in disparate phage hosts within the already dispersed methylase distribution. The reticulate evolutionary history of methylases and their insertion elements reveals high rates of gene transfer and within-gene recombination in actinophages. Full article

Streptomyces are well-known antibiotic producers, also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing and life style predictions suggested that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces. Full article

Here, we describe the structure of three actinobacteriophage capsids that infect Mycobacterium smegmatis. The capsid structures were resolved to approximately six angstroms, which allowed confirmation that each bacteriophage uses the HK97-fold to form their capsid. One bacteriophage, Rosebush, may have a novel variation of the HK97-fold. Four novel accessory proteins that form the capsid head along with the major capsid protein were identified. Two of the accessory proteins were minor capsid proteins and showed some homology, based on bioinformatic analysis, to the TW1 bacteriophage. The remaining two accessory proteins are decoration proteins that are located on the outside of the capsid and do not resemble any previously described bacteriophage decoration protein. SDS-PAGE and mass spectrometry was used to identify the accessory proteins and bioinformatic analysis of the accessory proteins suggest they are used in many actinobacteriophage capsids. Full article