Yellow stars indicate these data points are in agreement with RB-TnSeq, CRISPRi, and Dub-seq data for E. To investigate BL21 genes essential for phage growth, we constructed an RB-TnSeq library made up of approximately 97,000 mutants . For fitness assays, we used the same set of phages that were assayed with K-12 library except phages N4 and 186.
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In addition, we also did not identify strong effects for bacterial defense mechanisms (for example, R-M and CRISPR systems). coli K-12 BW25113 and BL21, CRISPR and type I R-M systems are disrupted but other R-M systems such as McrA, McrBC, and Mrr are intact . Other than strains with high-scoring mcrB in the presence of T2 phage, we did not see high fitness scores for other restriction systems in our Dub-seq fitness screens. We speculate that the expression of restriction enzyme subunit has to be at optimum level to counter the phage infectivity cycle and also to compete with high fitness strains in the pooled fitness assays. One way to improve the detection of overexpression gene phenotypes is to use phage mutants that lack anti-R-M systems and nonessential regions.
Both N4 and 186 phages do not infect BL21 because of a lack of N4 phage receptors and truncated LPS that probably limits 186 binding. We performed 53 pooled fitness experiments using the BL21 RB-TnSeq library in both liquid and solid plate assay format at varying MOI and nine no-phage control experiments. In total, we identified 115 high-scoring hits, made up of 50 unique phage-gene combinations and representing 32 unique genes . Largely, the BL21 LOF data are in agreement with our K-12 results for T3, T5, T6, T7, CEV2, LZ4, λ, P1, and P2 phages, especially the high-scoring genes that either code the phage receptor or its regulators .
Red lines represent fragments covering highlighted genes completely , whereas gray-colored fragments either cover the highlighted gene partially or do not cover the highlighted gene completely. Additional Dub-seq viewer plots are provided in S2–S4 Figs. dsDNA, double-stranded DNA; Dub-seq, dual-barcoded shotgun expression library sequencing; MOI, multiplicity of infection. Compared to assays performed in planktonic cultures, a few additional gene mutants showed stronger fitness effects on plate assays. For example, trxA, encoding thioredoxin 1, is known to be essential for T7 phage propagation and scored high in the solid plate assays but not in our planktonic growth assays .
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Finally, to investigate whether increased gene copy number of host factors interferes with phage growth in BL21, we constructed a BL21 Dub-seq library. This library is made up of randomly sheared BL21 genomic DNA cloned into a dual-barcoded vector and is made up of a total 65,788 unique 3-kb fragments . We then screened the BL21 Dub-seq library in a variant of BL21 as the host . From 24 pooled fitness experiments in planktonic cultures and on solid media in the presence of 12 phages, we identified 39 high-scoring candidates (fitness score ≥ 4, FDR of 0.74, Methods). Other than a few top-scoring candidates in the presence of λ phage, the BL21 dataset was considerably different from K-12 dataset .
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Heatmap of Dub-seq data for 13 dsDNA phages at different MOI and 30 genes with large fitness benefits. Overexpression or higher dosage of these genes interferes with the phage infectivity cycle and impart fitness benefits to the host. Only genes with high-confidence effects and gene fitness score of ≥4 in at least one phage assay are shown.
Yellow boxes highlight genes that are known to show resistance when overexpressed. The pooled fitness assays performed on solid plate agar are marked with stars. Dub-seq viewer plots for high-scoring mlc- , pdeL- , and ygbE-containing fragments in the presence of λ, N4, and T4 phages, respectively.
Heatmap of BL21 LOF RB-TnSeq data for 12 dsDNA phages at a single MOI, and selected genes with high-confidence fitness benefits are shown. Heatmap of GOF BL21 Dub-seq data for 12 dsDNA phages with high-confidence fitness benefit. Fitness scores of ≥4 in at least one phage assay are shown.