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Journal of Bacteriology, November 2004, p. 7490-7498, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7490-7498.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Calcium Inhibits Bap-Dependent Multicellular Behavior in Staphylococcus aureus

María Jesús Arrizubieta,1* Alejandro Toledo-Arana,1 Beatriz Amorena,1 José R. Penadés,2 and Iñigo Lasa1

Instituto de Agrobiotecnología y Recursos Naturales, Universidad Pública de Navarra-CSIC, Pamplona,1 Instituto Valenciano de Investigaciones Agrarias (IVIA)-Universidad Cardenal Herrera-CEU, Moncada, Spain2

Received 14 May 2004/ Accepted 29 July 2004

Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.

* Corresponding author. Mailing address: Instituto de Agrobiotecnología y Recursos Naturales, Universidad Pública de Navarra, Mutilva Baja, Pamplona 31006, Spain. Phone: 34 948 168470. Fax: 34 948 232191. E-mail: mjesus.arrizubieta{at}unavarra.es.

Journal of Bacteriology, November 2004, p. 7490-7498, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7490-7498.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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