Biological Characterization and Evolution of Bacteriophage T7-△holin During the Serial Passage Process

Abstract

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5, which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process.

Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences

“…Thus, the C-terminal Api801 and Api805 sequences reduced sfGFP expression to around one-third and two-thirds, respectively. Assuming a burst size of 180 T7Select phages per E. coli cell [ 30 ] and a copy number of 415 proteins per phage [ 31 ], these numbers are close to the theoretically expected yield of 74,700 molecules per cell. Thus, sfGFP was expressed at the normal cellular capacity.…”

Single-cell imaging of the lytic phage life cycle in bacteria

“…Larger production rates in larger cells would be consistent with the presence of more ribosomes, which is likely the limiting factor in phage protein production. The reason behind the positive correlation between production time and cell size is less clear: lysis, which marks the end of production, is thought to be controlled by the accumulation of holins in the cell up to a critical concentration 30,31 , thus requiring a larger number of holins in larger cells. However, since larger cells produce proteins exponentially more rapidly, the time to lysis is expected overall to decrease with increasing cell size.…”

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

“…Lysis time courses and EOP assays were used to compare the relative T7 fitness. Here, lysis onset time is defined as the time post-T7 infection at which optical density begins to decrease as previously described . Log-phase BL21 carrying CRISPRi vectors (OD = 0.3) were infected with T7 to a multiplicity of infection (MOI) = 3 and then OD kinetics were tracked (Figures a and S2).…”

“…Log-phase BL21 carrying CRISPRi vectors (OD = 0.3) were infected with T7 to a multiplicity of infection (MOI) = 3 and then OD kinetics were tracked (Figures a and S2). Overall, in cultures without CRISPRi induction, the lysis onset was ∼25 min, later than the typical T7 lysis onset of ∼15 min . We attribute this to the metabolic burden of plasmid maintenance and chloramphenicol in selective media.…”

“…coli BL21 (non-Rosetta) as the TXTL donor. We choose T7 as it is perhaps the most well characterized model phage and has featured in most CFBS peer-reviewed literature. ,,− Previous genome-wide investigations of T7-host interactions identified at least 11 genes essential to T7 replication in vivo, but most of these are related to lipopolysaccharide (LPS) biosynthesis. , LPS presented on the outer membrane is the receptor by which T7 recognized its hosts, without which infection cycles cannot initiate . For CFBS, phage infection is not a relevant process as phages are synthesized from naked DNA.…”