A T3 and T7 Recombinant Phage Acquires Efficient Adsorption and a Broader Host Range

It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress

Structures of four-way junctions in gene <i>17</i> of T3 and T7.

<p>(A) T3 nt 33324–33353 (B) T3 nt 33317–33347 (C) T7 nt 35109–35139 (D) T7 nt 35082–35117. One strand is highlighted in grey; the other is not. Arrows indicate Endo I cutting sites.</p

The mechanism of endonucleolytic cleavages at nonequivalent sites and strand annealing (CNSA).

<p>Colors and arrows are the same as those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030954#pone-0030954-g005" target="_blank">Figure 5</a>. The steps are: 1, production of DSBs by cutting at nonequivalent sites of T3 and T7 DNA; 2, 5′ resections of DSBs of T3 and T7; 3, annealing of the T3 DSBs with the T7 DSB; 4, removal of the nonhomologous nucleotides, filling the gap, and ligation.</p

Structures of four-way junctions in gene <i>18.5</i> of T3 and T7.

<p>(A) T3 nt 35315–35345 or equivalently T7 nt 37094–37124 (B) T7 nt 37163–37188. One of the strands is highlighted in grey. Endo I cutting sites are shown by arrows.</p

Adsorption efficiency (%) of phages on <i>E. coli</i> female and male strains.

<p>Adsorption efficiency (%) of phages on <i>E. coli</i> female and male strains.</p

Efficiency of plating of phages on <i>E. coli</i> female and male strains determined at 37°C.

<p>Efficiency of plating of phages on <i>E. coli</i> female and male strains determined at 37°C.</p

Alignment of T3 and T7 sequences near the crossover regions seen in the T3/7 phage.

<p>(A) Within gene <i>17</i>. (B) Within gene <i>18.5</i>. Sequences were aligned by ClustalW. The parent phages of the two sides of the crossover region (shown by arrows) are indicated on top of the alignment.</p

Mapping of the T3/7 and T7M DNA.

<p>The genomic DNA was digested by restriction endonucleases, and the fragments were observed by 1% (T3/7) or 0.8% (T7M) agarose gel electrophoresis. M: DNA marker. (A) Digestions of T3/7 DNA. Lane 1, <i>Mbo</i>I; lane 2, <i>Hpa</i>I; lane 3, <i>Nde</i>I; lane 4, <i>Stu</i>I; lane 5, <i>Avr</i>II. The markers are the same for the three slices of gels. (B) Digestion of T7M DNA. Lane 1, <i>Hpa</i>I. (C) Restriction sites in the T3/7 genome. Restriction sites are represented by vertical bars above the DNA. A: The T3 genome (black) and the region replaced by T7 DNA (in grey) in T3/7. The region of recombination is deduced from restriction mapping and sequencing. The two <i>Mbo</i>I sites in T7 that replace the single <i>Mbo</i>I site at T3 nt 34033 are shown by vertical bars. B to E show restriction sites in T3 genome (black); a dot above the bar indicates that the cutting site is present in T3 but not in T3/7. B: <i>Hpa</i>I sites, C: <i>Mbo</i>I sites, D: <i>Nde</i>I sites, E: <i>Avr</i>II cutting positions are indicated by bars, and the single <i>Stu</i>I site is shown by an arrow.</p