Figure 1.
Replication-targeted inhibition of repλ phage plating.
A. Plasmid cloned λ DNA fragments used to map the sequence requirement(s) for an inhibition phenotype (IP). B. Genomic region spanning five contiguous and partially homologous genes of phages λ and P22 (see Fig. S2). Phage λ is naturally missing the orf48 gene between oop and O that is present between oop and 18 in P22 [37], [51]. C. Assay for EOP, defined as phage titer on strain 594 (with one of the indicated plasmids) / titer on 594 cells, where plating on 594 = EOP of 1.0. All of the plasmids shown were derived from pBR322. The oop+ oriλ+ plasmid used was p27. The DNA substitution of the “ice” [16] sequence of λ to make plasmid Δice oop+ oriλ+ ( = p50) is shown in Fig. S3A. Numbers in brackets represent standard error values.
Table 1.
E. coli K12 and Bacteriophage λ Strains.
Table 2.
Plasmidsa.
Table 3.
Primers used for plasmid modification.
Figure 2.
Thermal Induction of repλ or the repP22 –hybrid λcI857 prophages.
Lysogenic cultures of strain 594 were grown at 30° and each prophage was thermally induced by shifting the culture from 30° to 42° at time 0. A. Thermally induced repP22 prophage. B. Thermally induced repλ prophage. The results represent the averages for 2 independent assays. Plasmids within lysogenic cells: square, Po+ oop+ ori+ (results shown for p27R, but identical results were observed for p27); triangle, Po− oop+ ori+; inverted triangle, Δ (to-oop-Po) oriλ+ (ITN-AT+); diamond, cII-oop-Po+ Δoriλ; circle, none (no plasmid). The standard deviation is shown for all of the data points, but is too small for visualization in some data intervals.
Table 4.
Averaged EOP on host cells +/− plasmids with cloned O genea.
Figure 3.
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.
Figure 4.
Assay for prophage replication from oriλ.
This experiment was undertaken in parallel with the experiment shown in columns “e–g” of Fig. 3D. A. Map of λ fragment within Y836 cells. The thick solid line shows λ fragment within the E. coli chromosome (open boxes); the NdeI restriction sites within λ and chromosome are shown along with the DNA bands formed after cleavage; the λ region amplified to prepare a DNA probe is drawn. B. Map region of the λ DNA fragment cloned within plasmid p27R (2873 bp) (without indicating the small mutational changes within similar λ fragments in the other plasmids). C. Assay for replication initiation from oriλ after shifting Y836 culture cells from 30° to 42° to induce transcription and oriλ replication from the cryptic prophage. Cultures were grown to mid-log and aliquots were removed at time 0 as described in legend, Fig. 3. Thereupon, cultures were transiently swirled in a 60°C water bath and transferred to a 42°C shaking bath for one hour and aliquots were removed. Cell concentration of the 42°C aliquots was based upon the calculations for 30°C 0-time cultures, and DNA was prepared using Qiagen DNAeasy Kit from 2.0×109 cells. The concentration of extracted DNA was determined by spectrophotometer (A260nm x DNA dilution X 50 ng/ml). The Y836 cellular DNA (2.5 ug of ethanol precipitated and resuspended DNA) was digested 2 hrs with NdeI and digests were run on horizontal 0.7% agarose gel, followed by Southern transfer of DNA bands. The Southern blot bands for the 1774 bp chromosomal prophage fragments were each scanned 3X using GE Healthcare software program ImageQuant version 5.2 and the region under the peaks was integrated and averaged. The numbers below the bands compare the relative levels of 1774 bp fragment obtained for induced / noninduced sample pairs. Refer to Hayes et al.[18] for detailed hybridization methodology, and for comparing the effect of a cI+ repressor expressed from a plasmid on prophage induction, the influence of host recombination defects on replication initiation from oriλ from the prophage in Y836 cells, and the inhibition of replication initiation from oriλ by host mutations.
Table 5.
oriλ-dependent DNA replication inhibition is bypassed in multiply infected cells.
Figure 5.
Sequences of Sip and cro27 mutations.
For an alternative interpretation of the effect of Sip mutations on gene expression from pE refer to Fig. S7B. GeneBank Accession #'s for Sip mutants: 1 (DQ372057.1), 2 (DQ372058.1), 3 (DQ372059.1), 4 (DQ372060). Newer data for all Sip phages and for cro27 mutation in λcI857cro27 was submitted, BankIt1376628 : (12). Phage λcI[Ts]857cro27 was found to be WT between the end of cII and start of P, i.e., O+.