Dataset Information

Processing of as-48ABC RNA in AS-48 enterocin production by Enterococcus faecalis.

ABSTRACT: 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.

SUBMITTER: Fernandez M

PROVIDER: S-EPMC2223719 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Processing of as-48ABC RNA in AS-48 enterocin production by Enterococcus faecalis.

Fernández Matilde M Sánchez-Hidalgo Marina M García-Quintáns Nieves N Martínez-Bueno Manuel M Valdivia Eva E López Paloma P Maqueda Mercedes M

Journal of bacteriology 20071102 1

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 o ...[more]

PMID: 17981958

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Project description:Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.

Dataset Information

Processing of as-48ABC RNA in AS-48 enterocin production by Enterococcus faecalis.

Publications

Processing of as-48ABC RNA in AS-48 enterocin production by Enterococcus faecalis.

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