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. 2018 Apr 5;7(2):31.
doi: 10.3390/antibiotics7020031.

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes

Sidney Hayes  1 , Karthic Rajamanickam  2 , Connie Hayes  3
Affiliations

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes

Sidney Hayes et al. Antibiotics (Basel). .

Abstract

λ genes O and P are required for replication initiation from the bacteriophage λ origin site, oriλ, located within gene O. Questions have persisted for years about whether O-defects can indeed be complemented in trans. We show the effect of original null mutations in O and the influence of four origin mutations (three are in-frame deletions and one is a point mutation) on complementation. This is the first demonstration that O proteins with internal deletions can complement for O activity, and that expression of the N-terminal portion of gene P can completely prevent O complementation. We show that O-P co-expression can limit the lethal effect of P on cell growth. We explore the influence of the contiguous small RNA OOP on O complementation and P-lethality.

Keywords: O and P initiator proteins; O complementation by oriλ-defective alleles; bacteriophage lambda (λ); bi-directional replication initiation from oriλ; influence of O:P interactions on cell growth and O activity; oriλ interaction site.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Replicative Killing, RK+ phenotype and selection for RK− mutants. (A) Defective prophage strains were made where the int-kil or int-ral genes of λ were substituted with the bio275 or bio10 regions of specialized transducing phage to remove a phenotype termed “killing to the left”, dependent on kil [23]. The starting cells included the chlA deletion Δ434 that removed all of the late genes, i.e., cell lysis, head and tail for λ [11]. These constructs include (i) an active immλ region with gene cI[Ts]857 encoding a repressor that blocks transcription from promoters pL and pR along with the cro repressor just right of pR, and (ii) the repλ region that includes genes O and P and the oriλ target for replication initiation from the λ genome. The genome for strain Y836, shown, has the bio+ operon to the left of the λ fragment and Δ431deletion to the right; (B) As long as strain Y836 maintains CI repressor activity the cells can grow normally without gene expression from the repressed λ fragment; (C) When the cells are shifted to growth conditions where the CI[Ts] repressor loses its ability to block transcription from pL and pR the remaining λ genes become derepressed, the phage replication initiation genes O and P are expressed and rounds of replication initiation arise from oriλ. The λ replication forks extend bidirectionally into the adjacent regions of the E. coli genome, likely colliding with E. coli replication forks. The event is highly lethal to the cell because the λ fragment has no mechanism for excision from the genome and was termed Replicative Killing [18]; (D) When cells with a conditionally repressible defective λ prophage are shifted from growth at 30 °C to 42 °C the Replicative Killing, RK+, phenotype is triggered, resulting in cell death. Rare mutations that suppress the loss of λ replication control are selected as RK− clones capable of colony formation at 42 °C. These survivor CFU have lost the capacity for λ replication. This strategy is based on the PDS selection [15], where an intact prophage is made N-defective, so that expression of int-xis and late/cell lysis gene expression is limited without N-antitermination of pL and pR transcription upon prophage induction. There are many possibilities for RK− mutants; (E) Cells acquiring defects in host genes participating λ replication are termed RK− Hd−. For example, the GrpD55 mutation in dnaB is of this type, though not isolated as shown [24,25]; (F) A marker rescue recombination assay is used to determine if the immλ regions genes and target sites remain functional (i.e., FI+) when substituted for the imm434 region of a hybrid phage. The FI assay scores for the activity of the pR promoter, but in practice it is a good indication of whether the λ fragment in Y836 cells was partially or fully deleted. An example of the deletion endpoints of RK− FI− mutants from Y836 is shown [26,27,28]; (G) It was found that brief pretreatment RK+ of cells held at 30 °C with a mutagenic substance, prior to shifting them to 42 °C increases the frequency of RK− mutants. This assay, termed the RK Mutatest, proved very sensitive due to the rather large target potential for RK− mutants [29,30,31].
Figure 2
Figure 2
RK− ilr mutations characterized within λ genes O-P. The minimal oriλ size was suggested to include a HIGH-AT-rich region to the right of four iteron sequences, ITN’s1-4 [43,44], which each contain an 18 bp inverted repeat of hyphenated symmetry, joined by adenine residues that can cause oriλ to assume a bent structure [45]. The mutants shown designated ori95, ori96 and ori98 were obtained from WD from prophage with r-mutants r-95, r-96, and r-98. Note that Denniston-Thompson, et al., [46] sequenced the r-mutants r-99, r-96 and r93 which represent Δ12 bp (39120–39131), Δ15 bp (39138–39152) and Δ24 bp (39092–39115) [42]. Our sequence localization for ori96 (r96) differs by one bp from that assigned in [42].
Figure 3
Figure 3
Expression vector pcIpR-(O alleles)-timm. Expression of gene O or an allele occurs upon inactivation of the CI repressor by raising cells grown at 30 °C to 42 °C. Immediately following the 299 codons of O is an ochre stop codon, where the last base in TAA represents the first base of the ClaI restriction site ATCGAT.
Figure 4
Figure 4
Influence of induced O, P gene expression on cell growth at 42 °C. All the strains were made by transforming hosts 594 or 594 dnaB-GrpD55 with pcIpR-(‥)-timm plasmids that included the cloned O, P DNA fragment, and selecting the transformants on LBAmp50 agar (medium composition is described in footnote to Table 3). The plasmid inserts in each CFU employed were verified by DNA sequence analysis. Cells were inoculated from overnight cultures grown up overnight in LBAmp50 broth and then 0.4 mL of culture was added to triplicate 20 mL fresh LB cultures that were incubated at 30 °C for ~30 min to reach an A575 = 0.1. Upon reaching an absorbance of 0.1 the cultures were transferred to a shaking 42 °C water bath. Aliquots were sampled every 30 min for 3 h. The average absorbance is shown, with a standard error for each culture time of less than 5% the averaged absorbance value.
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