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Applied and Environmental Microbiology, February 2003, p. 1229-1236, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1229-1236.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of a New Operon, as-48EFGH, from the as-48 Gene Cluster Involved in Immunity to Enterocin AS-48

Marta Diaz,1 Eva Valdivia,1 Manuel Martínez-Bueno,1 Matilde Fernández,1 Andrés Santos Soler-González,2 Hilario Ramírez-Rodrigo,2 and Mercedes Maqueda1*

Departamento de Microbiología,1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Granada, Granada, Spain2

Received 1 April 2002/ Accepted 1 November 2002

Enterocin AS-48 is a cyclic peptide produced by Enterococcus faecalis S-48 whose genetic determinants have been identified in the conjugative plasmid pMB2. A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52). In this region, the as-48A structural gene and as-48B, as-48C, as-48C1, as-48D, and as-48D1 genes and open reading frame 6 (ORF6) and ORF7 had been identified. The sequence analysis carried out in this work in the BglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs, as-48G and as-48H, necessary for full AS-48 expression. Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level. Tn5 disruption experiments in the last genes, as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid. The as-48EFGH operon encodes a new ABC transporter that could be involved in producer self-protection. On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed. This cluster of genes is expressed by two polycistronic mRNAs, T2 and T3, in JH2-2(pAM401-81) in coordinate expression. Our results also suggest that expression of T3 could be regulated, because in JH2-2(pAM401EH) transformants, T3 was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for the as-48D1 gene for immunity against AS-48.

* Corresponding author. Mailing address: Dpto. Microbiología (Facultad de Ciencias), Univ. Granada, Fuentenueva s/n, E-18071-Granada, Spain. Phone: (349) 58 242857. Fax: (349) 58 249486. E-mail: mmaqueda{at}ugr.es.

Applied and Environmental Microbiology, February 2003, p. 1229-1236, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1229-1236.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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