The pKO2 linear plasmid prophage of Klebsiella oxytoca

Abstract

Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage phiKO2. An analysis of the 64 predicted phiKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.

FIG. 1.

Electron micrograph of φKO2 virions. Virions were prepared, negatively stained, and examined by electron microscopy as described in Materials and Methods. Bar, 100 nm.

FIG. 2.

Map of the φKO2 genome. The genomes of phages φKO2, N15, and PY54 are shown with a scale of kilobase pairs for the φKO2 DNA. Predicted genes are indicated by rectangular boxes (those transcribed rightward are green and those transcribed leftward are red). Yellow and brown regions connect homologous regions of the two genomes. The predicted φKO2 transcription units are indicated below its genome. SOS refers to genes that may be involved in modulating and are controlled by the host SOS response. Double-headed arrows connect pairs of genes which have similarities to other genes of similar function but which have no discernible sequence similarity themselves. Asterisks denote putative φKO2 and N15 partitioning sites recognized by gene 27 and 28 proteins, and stem-loops denote predicted rho-independent transcription terminators (above the map for N15 and below the one for φKO2).

FIG. 3.

Separation of φKO2 proteins by SDS-polyacrylamide gel electrophoresis. The protein components of purified virions and heads, separated by sucrose gradient centrifugation, were displayed by SDS-10% polyacrylamide gel electrophoresis and were stained with Coomassie brilliant blue. The positions of standard proteins are indicated on the right, and the identities of φKO2 proteins are indicated on the left (gpX indicates the product of gene X).

FIG. 4.

Self-comparison of the putative φKO2 gene 21-encoded tail fiber. The gene 21 protein sequence was compared to itself by DNA Strider to produce a dot matrix plot in which each dot indicates seven or more identities in a 23-amino-acid scanning window. The repeat units are indicated below the matrix plot; the leftmost repeat is less conserved than the others and is barely recognizable at this stringency. Homology to the phage λ gene J protein is also indicated.

FIG. 5.

φKO2 gene 26 protein has protelomerase activity. Plasmid pBLSK DNA containing the cloned 56-bp sequence that includes the φKO2 telRL site between its HindIII and BamHI sites was first linearized with restriction endonuclease AluNI. It was then treated with 1 pmol of purified gene 26 protein (PtlK) at 37°C for 30 min in a solution containing 20 mM Tris-Cl (pH 7.5), 50 mM K-glutamate, 1 mM dithiothreitol, and 0.1 mM EDTA. The resulting DNA was separated in native and alkaline agarose gels and stained with ethidium bromide. Size standards (Std) are given in kilobase pairs to the left of each panel.

FIG. 6.

Region between φKO2 genes 22 and 23. The sequences of both strands of the region between genes 22 and 23 are shown, with a gray background marking the LexA-binding site, whose universally conserved base pairs are underlined. The −10 and −35 regions of putative promoters P₂₂ and P₂₃ are indicated by black and white rectangles, respectively, and arrows indicate the predicted mRNA start sites.

FIG. 7.

Phage-borne SOS genes and proteins. (A) Phage-, prophage-, and plasmid-borne dinI and umuD homologues are present in a number of different genetic contexts. Each line represents a section of the genome of the indicated genetic element (see text for details). The directional boxes represent genes and their transcriptional directions. Boxes of the same color are homologous genes, except for the orange boxes, which are all involved in tail fiber assembly but are not all homologues, and the black boxes, each of which has no homologue in the figure. (B) Comparison of 12 DinI family protein amino acid sequences. Seven phage-encoded DinI homologues are shown in the top group of sequences; the two DinI proteins from E. coli and Serratia marcescens, which are known by experimentation to down-regulate the SOS response, are shown in the middle;and two less similar phage homologues are shown below. Above the E. coli protein, asterisks indicate the seven negatively charged surface residues that make up its postulated DNA-mimic surface (78). These seven residues are indicated in red when homologous residues are present, and other highly conserved residues are shown in blue. From the top down, the proteins shown are as follows: (i) the phage 186 Tum protein; (ii) the phage Fels-2 gene STM2727 protein (we believe that a translation start site 25 codons 3′ of that reported by McClelland et al. [70] is the more likely true start for this gene, as STM2727 is identical to the retron 67-encoded Orf6 protein [50]); (iii) the phage Gifsy-2 gene STM1019 protein (identical to the phage Gifsy-1 gene STM2621 and prophage Sti1 STY1032 proteins); (iv) plasmid TP110-encoded ImpC protein; (v) phage PY54 gene 56 protein; (vi) E. coli prophage CP-933V gene Z3305 protein (the Shigella flexneri 301 prophage gene SF1879 protein only differs from Z3305 protein at its third amino acid); (vii) Salmonella defective prophage Stm6 gene STM2231 protein (16 predicted N-terminal amino acids are not shown; its poor Shine-Dalgarno and UUG start codon cast some doubt on the functionality of this gene); (viii) φKO2 gene 22 protein; (ix) E. coli K-12 dinI protein (gene b1061; identical proteins are encoded by the non-prophage-associated E. coli EDL933 gene Z1698 and Shigella flexneri 301 gene SF1067; S. enterica LT2 gene STM1162 and CT18 gene STY1200 proteins are 85 and 83% identical to the b1061 protein, respectively); (x) Serratia marcescens dinI protein; (xi) Gifsy-2 gene STM1056 protein; and (xi) Stm6 gene STM2244 protein.