Replication silencing of repλ phages requires oop, and iterons

(ITN) from oriλ. A. The non-excisable cryptic λ fragment (short arrow) inserted within the E. coli chromosome in strain Y836 [13], [35] remains repressed at 30° where the prophage repressor is active. Shifting cells to about 39° inactivates the CI857 repressor that prevents λ prophage transcription and replication initiation from oriλ. Multiple λ bidirectional replication initiation events from oriλ generate the onion-skin replication structure drawn at right. B. Map showing oop-oriλ region. The DNA sequence for oriλ, shown as a rectangle around ITN-AT within gene O has four repeated 18 bp iteron sequences (ITN1 to ITN4), each separated by short spacer, and joined by a 38 bp high AT-rich sequence. The genes cII and O are each shown truncated and are transcribed rightward from pR. The oop sequence, which overlaps cII is transcribed leftward from pO. C. Illustrated mutations within the λ DNA region in plasmids numbered 1–6 (Table 2). Plasmid p27R (shown as #1) carries with WT sequence from which other plasmids were derived. In each plasmid the rop gene was deleted to provide higher plasmid copy number per cell to test the stringency of introduced mutations. The “X” in #2 inactivates the pO promoter for oop gene; the filled rectangle in #3 (mutation oopR45) substitutes random 45 bp for 45 bp within oop providing a 77nt RNA without internal secondary structure (Fig. S3B); and the gaps in #'s 4–6 are deletions (Table 2). D. Columns (left ‘a,’ to right ‘g’): Lane ‘a’ shows the plasmid number and common name (Table 2), with plasmid genotype indicated in part C. Lanes ‘b’ and ‘c’: EOP of repP22 and repλ phages on 594 host cells with indicated plasmid; ‘d’ summary of the inhibitory effect of a plasmid in 594 cells to the plating of repP22 or repλ phages, where NONE is essentially no inhibition of plating and FULL indicates that plaque formation was prevented by the presence of the plasmid. Lanes “e” through “g” indicate the results of a separate experiment to determine if plasmids #1–5, transformed into strain Y836, can suppress Replicative Killing, which occurs upon prophage induction when the Y836 cells are raised above 38°C. Prophage induction leads to replication initiation from oriλ within the chromosome, as shown in part A, which is very lethal to cell. Lane ‘e’ shows the level of cell survival upon shifting the cells to 42°C. The survival of Y836 cells that were diluted and spread on plates incubated at 42°C requires plasmid suppression / interference of replication initiation and cell killing upon de-repression of the prophage in Y836 cells. Two single colonies of each transformant of Y836 cells were inoculated into 20 ml TB +50 ug/ml ampicillin and grown overnight at 30°C. The following day the cultures were subcultured (2.5 ml overnight culture +17.5 ml TB and grown to mid-log (∼0.35 A575nm), whereupon, cells were diluted into buffer and spread on TB plates that were incubated for 24 hr at 30°C, and onto TB and TBamp50 plates that were incubated at 42°C for 24 hr. Survival to Replicative Killing was assessed by dividing the average cfu/ml at 42°C incubation (the cell titers on both TB and TBamp50 plates were equivalent) by average titer for cell dilutions incubated at 30°C. Lane ‘f’ is a summary of the plasmid's effect on Replicative Killing of induced Y836 cells, where NONE indicates the cells were killed upon induction, and FULL reflects high cell survival as determined by colony formation at 42°. The values in parentheses show standard error for at least two independent determinations. Lane “g” shows the level of each plasmid present in the cells at 30°C (noninduced), immediately prior to shifting cells to 42°C (see legend, Fig. 4). The duplicate cultures processed at time 0 were extracted for DNA using Qiagen DNAeasy Kit, estimating 1.0×108 cells per 0.1 A575nm and calculating the amount of cell culture needed for 2.0×109 cells per DNA preparation. All DNA samples were prepared in duplicate. The gel purified bands for the plasmid DNA present in the 0 time cultures was assessed by hybridization as described in Fig. 4.



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