Escherichia coli genes that are required for the Ff phage lifecycle : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Manawatū, New Zealand
Ff phages have remarkably few genes yet are able to perform the complex task of infecting Escherichia coli via a process involving F pilus binding and retraction at the start of its life cycle, and releasing progeny from a host cell by a secretion-like process without killing it. The Ff phage-encoded assembly-secretion (egress) machinery operates across all layers of E. coli envelope – inner membrane, periplasm, peptidoglycan and outer membrane. Nevertheless, phage encodes only three proteins that build a functional assembly-secretion machine. In contrast, analogous trans-envelope systems in bacteria, that mediate pilus assembly, conjugation, DNA transfer and protein secretion require tens of proteins. To date, only a few host proteins required for Ff phages infection and DNA replication have been identified, of which only one was found to be involved in assembly, and only in one of three known Ff phages. Because the Ff phages are able to be assembled and secreted using a strategy similar to those that complex transenvelope secretion-assembly systems do yet only use 3 phage-encoded proteins for this, a hypothesis can be made that the Ff phage is using the host (E. coli)-encoded proteins to complete its lifecycle. The hypothesis that many host proteins are involved in the Ff lifecycle was tested by systematic screening of 212 E. coli single knockout mutants, which were mated to become F positive, in a Ff phage plaque and phagemid transduction assays. The screen found 66 E. coli proteins which were required for the phage lifecycle, most of which had impaired the infection step, which is dependent on the F-pilus as the primary receptor. Of the mutants that failed to be infected by Ff phage, there is some overlap with previously identified mutants that were defective in F pilus conjugation donor function, which indicates that there is an overlap between the F-pilus Ff receptor and conjugation function. Some mutants identified in this thesis were not functional as donors in conjugation, hence they added to the tally of E. coli chromosomal genes that cannot conjugate using the F pilus. Majority of the chromosomal mutants required for the Ff lifecycle found in this screen encode proteins that play a role in stress response pathways or control the envelope morphology. Many proteins encoded by mutated genes that did not fall into these two broad categories were also found. Due to the large number of mutants found that are implicated in the Ff phage infection and conjugation, from just a small systematic screen in this study, a further genetic screen is warranted to find the true extent of E. coli genes that are required for filamentous phage infection and conjugation. Phage infection and conjugation play major roles in horizontal gene transfer between bacteria. Thus finding E. coli genes that are vital for these processes is crucial to fully understand the mechanisms of inter-bacterial horizontal gene transfer. Having a complete list of genes involved in gene transfer mediated by filamentous phage infection and conjugation would give more targets to control the spread of antibiotic resistance during antibiotic treatment, or to develop other biotechnology applications.
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