Infection of the culture at OD600 nm 0.5 only rarely resulted in cell lysis and the turbidity test showed no sensitivity to ΦBP. However, SCH727965 supplier the result of plaque assay indicated the sensitivity of P. polymyxa CCM 1465 to ΦBP. We observed the plaques on the plates where the culture of this strain with ΦBP had been plated. Phage particles examined by TEM (Fig. 1) were recovered from the cell-free supernatant of spontaneously lysed culture of P. polymyxa CCM 7400 and CsCl gradient purified. The phages had polyhedral heads with a diameter of 56±4 nm (mean±SD) (n=24) and tails with
a length of 144±8 nm (n=6) (n=number of measurements). The structural proteins of ΦBP were analyzed by SDS-PAGE (Fig. 2). At least 11 bands were revealed with molecular masses of putative proteins estimated at 13, 16, 22, 25, 26, 28, 35, 38, 51, 79 and 160 kDa. The most abundant protein bands were 28, 35, 38 and 51 kDa in size. We extracted nucleic acid from purified phage particles. The purified nucleic acid was sensitive to DNAse and resistant to RNAse treatment. To determine the genome size, ΦBP DNA was cut with restriction endonucleases HindIII,
EcoRV and XbaI. The length of the genome of about 43 kb was calculated as the sum of the Linsitinib chemical structure lengths of the restriction fragments (Fig. 3a). Restriction enzymes XhoI, PstI, BamHI and SalI did not cut ΦBP DNA. Analysis with four restriction enzymes (EcoRI, HindIII, XbaI, SpeI) showed an identical restriction pattern for DNA extracted from phage particles, which were recovered from both spontaneously lysed culture of P. polymyxa CCM 7400 and culture after external ΦBP infection (data not shown). Sequence homology analysis of eight DNA fragments from EcoRI-digested ΦBP DNA (Fig. 3b, Carnitine palmitoyltransferase II Table 1) revealed regions
with significant similarity to typical phage genes for two of them. Two regions within the 2.5-kbp fragment with predicted ORFs of 507 and 996 bp shared significant homology to phage holin and lysin genes, respectively. They represent a putative cassette of lytic genes, where the gene coding for predicted holin is closely followed by the lysin gene. We detected an overlap of both genes over a 23-bp region. The third gene of this cluster seems to be the second holin gene (555 bp). Two predicted ORFs with the length of 552 and 744 bp were identified within the 1.2-kbp fragment as putative small and large terminase subunit genes. These ORFs are incomplete due to the interruption caused by EcoRI digestion with the genes overlapping by 83 bp. Restriction and ORF maps of the 1.2- and 2.5-kbp fragments were constructed from the primary sequencing data (Fig. 4). The basic data of eight analyzed sequenced fragments, the sizes of the known sequences and results of the homology search are summarized in Table 1. Two pairs of specific oligonucleotide primers were derived from the proposed small terminase and holin gene sequences to detect the presence of ΦBP DNA sequences on P. polymyxa chromosome.