Processing of as-48ABC RNA in AS-48 enterocin production by Enterococcus faecalis.
Enterocin AS-48 production and immunity characters are encoded by 10 genes (as-48ABCC(1)DD(1)EFGH) of the pMB2 plasmid from the Enterococcus faecalis S-48 strain. Among these, as-48A, encoding the AS-48 peptide, and the as-48BC genes constitute a cluster required for AS-48 biogenesis and full immunity. In this study, the levels of expression of this cluster have been altered by insertion and site-directed mutagenesis as well as by expression coupled to trans complementation. Phenotypic studies of the mutants have indicated cotranscription of the three genes and revealed that the inactivation of as-48B prevents the production of AS-48, thus confirming its essentiality in AS-48 biogenesis. These studies have also supported the involvement of as-48C in enterocin immunity. In addition, they established that the intergenic region between the as-48A and as-48B genes is decisive for AS-48 expression, since a 3-bp substitution, which should disrupt a potential 47-nucleotide complex secondary structure, resulted in a hypoproducing phenotype. Transcriptional analyses of the E. faecalis wild-type and mutant strains supports the possibility that the as-48ABC genes are transcribed from the P(A) promoter located upstream of as-48A. Moreover, analysis and bioinformatic predictions of RNA folding indicate that as-48ABC mRNA is processed at the secondary structure located between as-48A and as-48B. Thus, synthesis of the AS-48 peptide appears to be controlled at the posttranscriptional level and is uncoupled from as-48BC translation. This mechanism of genetic regulation has not been previously described for the regulation of bacteriocin expression in enterococci.
Fernández
Matilde
M
Departamento Microbiología, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, E-18071 Granada, Spain.
Sánchez-Hidalgo
Marina
M
García-Quintáns
Nieves
N
Martínez-Bueno
Manuel
M
Valdivia
Eva
E
López
Paloma
P
Maqueda
Mercedes
M
D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D013293:Enterococcus faecalis / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000235:genetics* / Q000276:immunology; D010957:Plasmids; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010455:Peptides; D012329:RNA, Bacterial; D011994:Recombinant Proteins; C051034:BacA protein, Enterococcus faecalis