Analysis of the tf DNA ends.

<p>(A) Automated sequencing of the bacteriophage tf DNA at termini regions. Alternative end-nucleotides are indicated. (B) Results of the primer extension analysis of the 5′-ends in the tf DNA. Nucleotide positions corresponding to the stoppages at the phage DNA ends are indicated with arrows; black arrow for the position corresponding to the major band and gray arrows – position corresponding to minor bands at the left terminus only. (C) Analysis of the termini junction in the circularized tf DNA. Bacteriophage DNA was treated with T4 DNA polymerase and circularized using T4 DNA ligase. The junction region was analyzed by automated sequencing of the corresponding PCR product as described in the text. The junction identified is shown on the chromatogram by a vertical line and the schematic of the ligated genome ends is shown above the chromatogram. (D) Analysis of the 3′-ends in the tf DNA. Denatured tf DNA was treated with TdT enzyme in separate experiments in the presence of either dATP or dTTP and PCR products were obtained using resulting preparations and one phage specific and one poly-dT (poly-dA) specific primer. Chromatograms for all types of experiments are presented with the corresponding sequences shown at the bottom of the figure. Terminal sequences of the phage genome are shown by shading. (E) Structure of the tf DNA ends’ sequences.</p

Putative processing of the tf DNA from concatemer substrate.

<p>Regions on the concatemer containing terminase recognition sequences are boxed with a red box corresponding designating sequences found at the left end of the genome and a green one – at the right. A putative core recognition site for the terminase enzyme is shaded with grey and positions of cuts are indicated by vertical arrows. Palindrome sequences identified within these sites are shown by horizontal arrowed lines.</p

Identification and analysis of the localized nicks in tf DNA molecules.

<p>DNA preparation of bacteriophage tf was denatured in 0.1 M NaOH and subjected to electrophoresis in 0.9% agarose gel; in a control experiment a DNA from the same preparation was treated with T4 DNA ligase prior to denaturing in 0.1 M NaOH and electrophoresis.</p

Primer extension analysis of the sci-14.

<p>The schematic of the experiment is shown at the top of the Figure: non-ligated (1) and ligated (2) DNA preparations were digested with HaeIII restriction endonuclease and primer extension reaction were performed using the same [³²P] labeled primer. The results of primer extension with Klenow fragment (PE:K) and Taq DNA polymerases (PE:T) are presented at the bottom part of the Figure. Bands corresponding to the stoppages at the sci-14 and <i>Hae</i>III sites are indicated by arrows. Sequencing ladders (A, G, C, T) were generated by Sanger sequencing of the T4 DNA ligase treated tf DNA employing the same [³²P] labeled primer used in primer extension.</p

Genome organization of tf and its comparison with LUZ24 bacteriophage.

<p>Predicted genes are designated with arrows indicating their directions of transcription. Color and symbol codes adopted are shown at the bottom of the figure. The similarities between corresponding amino acid sequences of tf and LUZ24 are indicated by connecting lines.</p

Electron Microscopy of partially denatured tf DNA molecules.

<p>Nicks are visualized as partially denatured regions which are indicated by black arrows. Bacteriophage head attached to DNA molecule is indicated by white arrow.</p

Putative nickase recognition sites in the genomes of LUZ24-like phages.

*<p>,nucleotide sequences of the PaP3 (Accession number AY078382) and phiMR299-2 (JN254801) are reverse complemented.</p

Genomic Analysis of Pseudomonas putida Phage tf with Localized Single-Strand DNA Interruptions

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure--a blunt right end and a 4-nucleotide 3'-protruding left end--was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5'-TACT/RTGMC-3'. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages