2.4 Materials and Methods
3.1.4 T7 bacteriophage
T7 bacteriophage is an efficient bacterial killing machine. It is fast-replicating (~17 minutes), lytic, and carries its own transcription and replication systems, making it largely independent from the host machinery (Molineux, 2006). For these reasons, it could be an attractive chassis for Phage Assisted Continuous Evolution (PACE) or PACE-inspired techniques that involve continuous phage growth. Fast replication mean more rounds per time unit, its lytic nature reduce or eliminate biofilm which complicate bioreactor operation, and the independent replication machinery might allow the use of T7 in other hosts. Unfortunately a major drawback to its use is the absence of an easy, robust cloning method for engineering the T7 genome. However, homologous recombination followed by selection has been used to delete portions
of the T7 genome. Selection for mutants is achieved by incorporating the E. coli genescmkortrxA, that are important for T7 replication, between homology arms. Recombinant phage is then selected by plating on E. colideficient in such genes (Qimron et al., 2006; Tridgett, 2015).
T7 bacteriophage life cycle
The T7 genome is 39,937 bp long, coding for 56 genes (Dunn et al., 1983), encapsulated in a 60 nm icosahedral capsid, with a 23 nm tail (Rontó et al., 1983; Molineux, 2006). T7 can infectE. colias well as someSalmonellaandShigellastrains, and some mutants can infectY. pestisstrains (Molineux, 2006). T7 infection starts when the tail fibre interacts with the outer membrane lipopolysaccharide (LPS), the first step of phage adsorption. After binding, the inner core of the phage is ejected from the virion capsid into the outer membrane and periplasm, where it degrades the peptidoglycan layer, presumably forming a channel for DNA translocation, although this has not been well characterised (Molineux, 2006). The first ~850 bp of the genome then enter the cell, allowing initiation of transcription by theE. coliRNA polymerase that recognises the promoters present at the start of the genome. Transcription of the genome by the endogenous polymerases pull the rest of the phage genome into the cell (Garcia and Molineux, 1995). This gradual entry of the genome into the host cell forms a first layer of gene regulation. Genes transcribed from the phage can be divided into three classes (see fig. 3.2). Class I genes are expressed early in the infection stage – up to 8 minutes – from three strongE. colipromoters (Moak and Molineux, 2000) situated at the start of the genome. They consist of 9 genes, from gene 0.3 to 1.3, expressed from mRNAs transcribed from different promoters but that terminate at the TE terminator that followsgp1.3. Two important proteins are gene 0.7 and gene 1. The product ofgp0.7inactivates host transcription and phosphorylates many host proteins, including ribosomal subunits, RNase III and RNase E, andE. coli polymerase subunits (McAllister and Barrett, 1977; Molineux, 2006). Gene 1 codes for T7 RNA polymerase, which goes on to initiate transcription of late genes 60
3.1. Introduction
from T7 promoters. Late genes are divided into class II and class III. Class II genes are expressed from 6 to 15 minutes after infection at 30°C, and consist of enzymes required for T7 replication. gp2also inhibits theE. coliRNA polymerase (Nechaev and Severinov, 1999), switching off transcription of early genes. gp3 and gp6 are responsible for degrading the host chromosome. These nucleotides are then recycled by endogenousE. coliproteins, and make up 80% of the progeny phage DNA. The Cmk protein is responsible for the recycling of cytidine monophosphate (CMP) nucleotides. Its deletion greatly reduces phage replication, allowing its use as a selection gene. gp5is the T7 DNA polymerase which replicates the phage chromosome, in complex with theE. coliprotein thioredoxin (TrxA). TrxA acts as a processivity factor for the phage DNA polymerase, increasing the speed of DNA polymerization 1000 fold, and is essential for T7 replication. Class III genes are responsible for DNA packaging, viron assembly and the eventual lysis of the host which releases the virons (Molineux, 2006). Class III gene mRNAs are highly expressed, which may reduce class II gene expression by ribosome sequestering.
T7 targets F-E. coli Initial attachment of phage legs to cell surface via LPS
Adsorption
DNA penetration
Insertion of inner core into membrane Degradation of peptidoglycan Entry of first part of genome T7 RNAP, regulatory proteins Transcription by E. coli RNAP T7 genome pulled into cell by RNAP
Class I genes
DNA pol Nucleases Regulators E. coli RNAP inhibited by Gp2 Transcription by T7 RNAPClass II genes
Capsid proteins T7 genome replication by Gp5 Nucleotide recycling by CmkClass III genes
Packaging of new phages
Cell lysis and virion release
Burst and release
E. coli RNAP E. colichromosome T7 RNAP Gp5 TrxA dNTPs Gp5 mRNA
Figure 3.2 – The life cycle of T7. Adsorption starts with attachment of phage legs to LPS. The inner core enters the cell membrane, 850 bp of the genome enter the cytoplasm, where theE. coliRNAP expresses Class I genes, simultaneously pulling in the rest of the genome. T7 RNAP expresses Class II then III genes. The T7 genome is replicated by the Gp5-TrxA complex. Nucleases degrade theE. coligenome, and nucleotides are recycled.
3.1. Introduction