RESUMO
A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF=tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of "locked" recombinant phages that carry foreign DNA but which cannot propagate themselves in nature.
Assuntos
Microbiologia de Alimentos , Genes Reporter , Luciferases/genética , Fagos de Salmonella/genética , Salmonella/isolamento & purificação , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Recombinante , DNA Viral/genética , Genoma Bacteriano , Mutagênese , Plasmídeos , RNA de Transferência/genética , Salmonella/enzimologia , Transdução GenéticaRESUMO
Felix 01 (F01) is a bacteriophage originally isolated by Felix and Callow which lyses almost all Salmonella strains and has been widely used as a diagnostic test for this genus. Molecular information about this phage is entirely lacking. In the present study, the DNA of the phage was found to be a double-stranded linear molecule of about 80 kb. 11.5 kb has been sequenced and in this region A + T content is 60%. There are relatively few restriction endonuclease cleavage sites in the native genome and clones show this is due to their absence rather than modification. A restriction map of the genome has been constructed. The ends of the molecule cannot be ligated although they contain 5' phosphates. At least 60% of the genome must encode proteins. In the sequenced portion, many open reading frames exist and these are tightly packed together. These have been examined for homology to published proteins but only 1 to 17 shows similarity to known proteins. F01 is therefore the prototype of a new phage family. On the basis of restriction sites, codon usage and the distribution of nonsense codons in the unused reading frames, a strong case can be made for natural selection that reacts to mRNA structure and function.
