Analysis of diverse mobile genetic elements infecting Shewanella oneidensis MR-1

Abstract

Mobile genetic elements (MGEs) are autonomous genetic agents that utilise host cells for selfish reproduction. MGEs are considered to be drivers of microbial evolution, as they largely control horizontal gene transfer. Bacteriophages (Phages), the viruses that infect bacteria, are classified as MGEs. Phages are known to inject their genome into a host cell and then hijack the host cell, turning it into a virion-producing factory. Interestingly, not only host cells are exploited by MGEs, some MGEs also exploit other MGEs for their selfish horizontal spread. So-called phage satellites are specialised in the hijacking of phages. The majority of phage satellites are integrative elements present in bacterial chromosomes, which contain some, but not enough, structural genes to generate virions. They therefor are dependent on phage structural proteins for horizontal transfer via transduction. The resulting satellite-dependent exploitation of a phage often leads to a reduction in the virion output of the hijacked phage. In this work, a new Shewanella phage, named Dolos, was characterised. Dolos belongs to the viral family Inoviridae and thus is a phage that carries out a chronic-productive infection. Consequently, Dolos-mediated virion release occurs independently of host cell lysis. Phage Dolos has been found to be exploited by a plasmid called pDolos. pDolos appears to be a cryptic, non-mobilizable plasmid that has been shown to be stable in cells during the absence of phage Dolos. In the presence of Dolos, pDolos can be transferred to other cells by transduction. Like other phage satellites, pDolos drastically reduces the virion output of the helper phage Dolos. Furthermore, a higher number of pDolos virions was detected in a supernatant containing both pDolos and Dolos virions. Interestingly, in contrast to all other phage satellites, pDolos has no recognisable phage genes. Thus, this study shows that phage satellites are much more diverse than previously assumed and that pDolos can be regarded as the first member of a new family of phage satellites, characterised by autonomously replicating plasmids without phage genes that hijack inoviruses. Considering the relatively simple requirements for a plasmid to hijack an inovirus, it is likely that such satellite systems are widespread. In addition, this study demonstrates a new transmission strategy for non-mobilizable plasmids by acting as phage satellites. As an additional part of this study the mechanism of host acquisition by the virulent Shewanella phage Thanatos was investigated. Thanatos encodes two ADP-ribosyltransferases (ARTs), which are enzymes known to be host acquisition factors of Escherichia phage T4. These Thanatos ARTs are homologues of the phage T4 Alt protein and were consequently named Alt1 and Alt2. Phage particle proteomics demonstrated that both enzymes are highly abundant proteins of the Thanatos particle. In addition, both Thanatos ARTs are injected into the host cell during the phage DNA injection process and thus immediately ADP-ribosylate host proteins. Alt1 and Alt2 have been shown to be autocatalytic and have different ADP-ribosylation profiles. A protein interaction study revealed interactions with host proteins that are likely involved in anti-phage defence. Furthermore, it was shown that a functional loss of Alt2 drastically reduces the virion output of the phage Thanatos. This work therefor provides first insights into the function of ARTs for Thanatos-mediated host acquisition.