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, location of the genomic deletion. (z) represented the genome size of mutant SNJ1 724 viruses and the smaller genomes were noted in bold
, Late exponential 729 phase culture of CJ7 was infected with SNJ1 orf4 mut and SNJ1 ?orf4 viruses (generated 730 from CJ7/pYC-S-4M and CJ7/pYC-S D1-575 culture respectively), and only clear 731 plaques were observed. b. Expression of ORF4 in trans restored turbid plaque 732 formation of clear-plaque SNJ1 mutant viruses and inhibited plaque formation 733 dramatically. Ten-fold serial dilutions of SNJ1 viruses or its clear plaque mutants were 734 spotted onto the lawns of CJ7-F/pFJ6-MCS, or CJ7-F/pFJ6-1-656 (with orf4), Fig 3. Identification of orf4 as a critical factor for SNJ1 lysis-lysogeny switch. a. 728 orf4 disrupted viruses formed only clear plaques on lawns of CJ7
, Cells 746 were collected by centrifugation and washed twice in same volume of 18% MGM to 747 remove MMC. Cell pellets were resuspended in 18% MGM + Mev medium and 748 cultured for 24 h. Samples were taken for qPCR analysis using primer pairs 749 vector-F/vector-R, orf14-F/orf14-R and radA-F/radA-R to determine the relative 750 plasmid copy number to chromosome. Three independent experiments were 751 performed, and error bars indicated standard deviations. b. Plasmid stability of 752 pYC-SHS and pYC-SHS
, 4M+pFJ6-1-656 was 754 inoculated into 5 mL of Halo-2 medium every day. Samples were taken and measured 755 by qPCR using primer pairs vector-F/vector-R, orf14-F/orf14-R and radA-F/radA-R 756 for five days. Three independent experiments were performed
, and SpoVT (residues 1-55, PDB code 2W1T)(35) are aligned with ORF4 768 by Clustal Omega. The secondary-structure of ORF4 was predicted by PSIPRED in
, The same color is used for other proteins. c. The 771 N-terminal 33 amino acids of SNJ1 were necessary and sufficient for immunity to 772 against SNJ1. Ten-fold serial dilutions of SNJ1 were spotted onto lawns of CJ7-F 773 harboring plasmid pFJ6-MCS or its derivatives carrying different portion of ORF4, 774 superscript denotes the amino acid residues of ORF4. Hpro stands for the promoter of 775 heat-shock protein 70 from Haloferax volcanii DS52, MPI Bioinformatics Toolkit. ORF4 contains 5 b-strands (light-grey arrows) and one 770 a-helix (dark-grey cylinder)
, ORF4 blocks SNJ1 infection by inhibiting viral gDNA replication. a. ORF4 779 did not affect SNJ1 adsorption to host cells CJ7. SNJ1 was incubated with 780 early-exponential phase cultures of CJ7, J7-1, CJ7-F/pFJ6-MCS (-orf4) and
/pFJ6-Hpro-orf4 (+ orf4) at different MOI (0.1, 1 and 10) for about 1 h at 45? ,