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 KO2. An analysis of the 64 predicted KO2 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 KO2 virion is unusual in that its phage -like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The KO2 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.Temperate bacteriophages usually physically integrate their prophage DNAs into the host bacterium's chromosome when they establish lysogeny. However, a few prophages, such as those of phages P1, N15, LE1, 20, and BB-1, are not integrated but replicate in the lysogen as low-copy-number plasmids (32,38,52,53,84). Of these, the Escherichia coli doublestranded DNA (dsDNA) tailed-phage N15 has an unusual linear prophage plasmid that has covalently closed hairpin telomeres (67, 68). Although N15 has been studied in some detail (83,85,87), few other phages with similar linear DNA prophages have been described. While this paper was being prepared, another linear plasmid prophage, PY54, was reported for Yersinia enterocolitica (48). Furthermore, the linear, hairpin-ended plasmids Lp28-2 and Lp54 in the spirochete Borrelia burgdorferi B31-MI carry sequence similarities to dsDNA phage virion assembly genes (19, 33); however, they have not yet been shown to be bone fide prophages. Linear plasmids are very rare in the ␥-Proteobacteria, but it has been reported that Klebsiella oxytoca strain CCUG 15788 (a nickelresistant bacterium isolated from the mineral oil-water coolant-lubricant emulsion of a metal working machine in Göte-borg, Sweden, in 1989 [69]) harbors a linear plasmid, pKO2, of about 50 kbp (95). Since this is close to the size of the N15 linear plasmid prophage, we were prompted to investigate the possibility that this plasmid could be a prophage. We report here the complete nucleotide sequence of pKO2, a linear plasmid prophage that encodes the synthesis of a tailed-phage particle, and the analysis of some of its properties. MATERIALS AND METHODSBacterial strains and plasmid construction. The bacterial st...

In a particular genetic system, selection stimulates reversion of a lac mutation and causes genome-wide mutagenesis (adaptive mutation). Selection allows rare plated cells with a duplication of the leaky lac allele to initiate clones within which further lac amplification improves growth rate. Growth and amplification add mutational targets to each clone and thereby increase the likelihood of reversion. We suggest that general mutagenesis occurs only in clones whose lac amplification includes the nearby dinB ؉ gene (for error-prone DNA polymerase IV). Thus mutagenesis is not a programmed response to stress but a side effect of amplification in a few clones; it is not central to the effect of selection on reversion.W hen a particular lac mutant of Escherichia coli is plated on selective medium, revertant colonies accumulate over several days (1, 2). Two models assume that mutations arise in the nongrowing population (3). Directed mutation proposes that stress preferentially induces beneficial (i.e., Lac ϩ ) mutations (1, 2). The hypermutable state proposes that stress induces general (genome-wide, undirected) mutagenesis in a subset of cells (Ϸ0.1%), and this mutagenesis produces the Lac ϩ revertants (4-6).An alternative model, amplification mutagenesis, proposes that reversion occurs in cells growing under selection and requires no change in mutability. On selective medium, rare preexisting cells with a lac duplication initiate slow-growing clones within which the growth rate increases progressively as amplification increases the copy number of the partially functional mutant lac allele (7,8). The probability of reversion within each clone increases with the number of target lac copies. After reversion, selection holds the revertant lac ϩ allele and favors cells that stabilize this allele by loss of mutant copies. This model is a specific form of a more general hypothesis proposed by Lenski et al. (9).Genomewide mutagenesis (an Ϸ100-fold increase in rate) is experienced by some revertants. This mutagenesis depends on the error-prone DNA polymerase, DinB (10-12), which may be induced when the SOS regulon is activated by DNA fragments released during segregation of the amplified lac region (8). Three problems complicate understanding how selection might cause general mutagenesis:(i) Induction of SOS does not mutagenize strains with a single wild-type dinB ϩ gene (13-15).(ii) Only 10% of Lac ϩ revertants arising under selection experience general mutagenesis (16,17).(iii) Selection causes mutagenesis only when lac is near the dinB gene (16,(18)(19)(20)(21).Evidence is presented that general mutagenesis occurs only in those developing clones whose amplified lac region includes the nearby dinB ϩ gene. Thus general mutagenesis is not a programmed response to stress in stationary phase but rather a side effect in a subset of developing clones growing under strong selection. Materials and MethodsSupporting Information. More detailed descriptions of methods and complete genotypes of all strains are published as supportin...

The multidrug resistance plasmid pBWH301 was shown to contain a sulI-associated integron with five inserted gene cassettes, aacA7-catB3-aadB-oxa2-orfD, all of which can be mobilized by the integron-encoded DNA integrase. The aadB, oxa2, and orfD cassettes are identical to known cassettes. The aacA7 gene encodes a protein that is a member of one of the three known families of aminoglycoside acetyltransferases classified as AAC(6)-I. The chloramphenicol acetyltransferase encoded by the catB3 gene is closely related to members of a recently identified family of chloramphenicol acetyltransferases. The catB3 gene displays a relatively high degree of sequence identity to a chromosomally located open reading frame in Pseudomonas aeruginosa, and this may represent evidence for the acquisition by a cassette of a chromosomal gene.pBWH301 is a 69-kb multidrug-resistant conjugative plasmid isolated from two bacterial species, Enterobacter aerogenes and Enterobacter cloacae, during an outbreak of amikacin resistance in Hospital Vargas in Venezuela (19). Most of the drug resistance genes in pBWH301 were found clustered within a 6.0-kb BamHI fragment that confers resistance to the aminoglycosides amikacin (Ak . Two aminoglycoside resistance genes were identified on the basis of aminoglycoside resistance profiles: an aacA gene encoding a type I aminoglycoside acetyltransferase [AAC(6Ј)-I] that confers resistance to amikacin, netilmicin, and tobramycin and an aadB gene encoding an aminoglycoside adenylyltransferase [AAD(2Љ)] that confers resistance to gentamicin, kanamycin, and tobramycin (19). A chloramphenicol resistance gene and a sulfonamide resistance gene were also identified. The presence of the restriction sites characteristic of the sulI gene, which is normally found in the 3Ј-conserved segment of sulI-associated integrons (37), suggested that the resistance genes may be integron associated (19).The ability of bacteria to develop multiple-drug resistance is due in part to their ability to acquire new antibiotic resistance genes. Mobile elements called integrons determine a site-specific recombination system that is responsible for the acquisition of many antibiotic resistance determinants (14,15,37). A large number of antibiotic resistance genes (conferring resistance to aminoglycosides, ␤-lactams, chloramphenicol, and trimethoprim), as well as several unidentified open reading frames, have been found as inserts in integrons (13). These genes are contained in individual mobile units called gene cassettes that can be inserted into and excised from an integron by site-specific recombination (3-5, 13). Gene cassettes consist of a gene coding region (or open reading frame) and a recombination site known as a 59-base element which is located 3Ј to the gene in the linear integrated form (4,13,14). The 59-base elements vary in sequence and length but are all imperfect inverted repeats and are related to a consensus sequence at their outer ends (1, 5, 13). The 59-base elements play an essential role in the process of gene acqui...

The LexA protein of Escherichia coli represses the damage-inducible SOS regulon, which includes genes for repair of DNA. Surprisingly, lexA null mutations in Salmonella enterica are lethal even with a sulA mutation, which corrects lexA lethality in E. coli. Nine suppressors of lethality isolated in a sulA mutant of S. enterica had lost the Fels-2 prophage, and seven of these (which grew better) had also lost the Gifsy-1 and Gifsy-2 prophages. All three phage genomes included a homologue of the tum gene of coliphage 186, which encodes a LexA-repressed cI antirepressor. The tum homologue of Fels-2 was responsible for lexA lethality and had a LexA-repressed promoter. This basis of lexA lethality was unexpected because the four prophages of S. enterica LT2 are not strongly UV inducible and do not sensitize strains to UV killing. In S. enterica, lexA(Ind ؊ ) mutants have the same phenotypes as their E. coli counterparts. Although lexA null mutants express their error-prone DinB polymerase constitutively, they are not mutators in either S. enterica or E. coli.