RESUMO
We have isolated and characterized a lytic double-stranded DNA Xanthomonas campestris pv. campestris bacteriophage (XTP1) capable of mediating generalized transduction. The phage transduces chromosomal markers at frequencies of 10(-5) to 10(-6) transductants per PFU. We demonstrated its genetic utility by the isolation and cotransduction of linked transposon insertions to a nonselectable locus, xgl, required for the cleavage of 5-bromo-3-chloro-indoyl-beta-D-galactoside and showed that rif and str alleles in X. campestris are 75% linked. One-step growth experiments showed that the latent and rise periods were each 2 h and the average burst size was 35. The DNA genome is approximately 180 kb, presumably modified in a sequence-specific manner, and may be covalently attached to protein(s). Electron micrographs show the phage particle to have an icosahedral head and contractile tail with tail fibers uniquely attached to a location 40 nm proximal from the end of the tail.
Assuntos
Bacteriófagos/genética , Vírus de DNA/genética , Transdução Genética , Xanthomonas campestris/genética , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Vírus de DNA/crescimento & desenvolvimento , Vírus de DNA/isolamento & purificação , Vírus de DNA/ultraestrutura , DNA Viral/isolamento & purificação , Galactosídeos/metabolismo , Genoma Viral , Mutagênese Insercional , Microbiologia do Solo , Replicação ViralRESUMO
We have constructed (in vitro) a set of P1 miniplasmids. The smallest of these that could function as an independent replicon contained the right side of EcoRI-5 plus all of EcoRI-8. Those miniplasmids that lack EcoRI-6 induce the SOS pathway of the cell as shown by (i) increased expression of the recA operon, (ii) excision of the cryptic genetic element e14, (iii) spontaneous induction of lambda, and (iv) dependence of e14 excision on recA+ function. This induction was contingent upon the replication of the P1 Km miniplasmids from their P1 origin and, thus, was apparently caused by an aberrant initiation of DNA replication. When P1 EcoRI-6 was present in cis or trans with a P1 Km miniplasmid, neither e14 nor lambda was excised, but the expression of the recA operon was still induced. These results suggest that P1 EcoRI fragments 5 and 8 are insufficient for normal replication, and thus our P1 Km miniplasmids induced SOS functions. A product of EcoRI-6 may partially restore normal replication.
Assuntos
Colífagos/genética , Reparo do DNA , Plasmídeos , Proteínas de Bactérias/genética , Bacteriófago lambda/genética , Bacteriófago lambda/efeitos da radiação , Replicação do DNA , DNA Bacteriano/análise , DNA Viral/análise , Escherichia coli/genética , Regulação da Expressão Gênica , Recombinases Rec A , Raios Ultravioleta , Ativação ViralRESUMO
In Escherichia coli K-12 strains that carry the mutation ssb-1 in the gene for single-stranded DNA binding protein, the presence of the F sex factor partially reverses the temperature-sensitive growth phenotype caused by the mutation. The region of F (EcoRI fragment 3) responsible for this compensation has been identified and subcloned onto pBR322. A BamHI cleavage site has been found to intersect the essential coding region for this F function. By using this site, mutational blocks in the function have been constructed and used to identify a protein product (Mr approximately 22,000, slightly larger than the E. coli K-12 single-stranded DNA binding protein) which is correlated with the ssb-1-complementing activity. Labeled extracts from maxicells were used to show that this protein binds tightly to single-stranded DNA. The gene on F that codes for this protein is denoted ssf and is located at approximately 55.2 kilobases on the standard map of F, in the region transferred very early during bacterial conjugation.
