Calcium Inhibits Bap-Dependent Multicellular Behavior in Staphylococcus aureus

Abstract

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 Ca 2؉ with low affinity and that Ca 2؉ 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.

SarA Positively Controls Bap-Dependent Biofilm Formation in Staphylococcus aureus

“…Our results also demonstrated that the Ca 2ϩ inhibition of the Bap-mediated bacterial multicellular behavior was not due to repression of Bap expression. Instead, our results were consistent with the hypothesis that calcium causes a conformational change in Bap that affects its ability to form biofilms (1). The fact that calcium inhibition of Bapdependent biofilm formation takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition could be important to the pathogenesis of the bacteria.…”

“…Interestingly, in addition to the genetic control here reported, we found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion and biofilm formation by Bappositive strain V329 (1). Our results also demonstrated that the Ca 2ϩ inhibition of the Bap-mediated bacterial multicellular behavior was not due to repression of Bap expression.…”

Calcium Causes Multimerization of the Large Adhesin LapF and Modulates Biofilm Formation by Pseudomonas putida

“…Calcium has been related to a variety of biological processes in bacteria (4,7,10), but its role in biofilm development is somewhat controversial. Previous research has shown that increasing amounts of calcium disable Bap-mediated biofilm formation and intercellular adhesion of Staphylococcus aureus (1). However, recent reports indicate that calcium promotes biofilm formation in other microorganisms, such as Xyllela fastidiosa or Vibrio vulnificus (4,7).…”

“…A significant number of large proteins involved in cell surface and cell-cell interactions contain putative calcium binding domains (1,11,28). Calcium has been related to a variety of biological processes in bacteria (4,7,10), but its role in biofilm development is somewhat controversial.…”

σ B Regulates IS 256 -Mediated Staphylococcus aureus Biofilm Phenotypic Variation

“…The fragment was then cloned into the BamHI site of the shuttle plasmid pMAD (1), and the resulting plasmid was transformed into the ⌬ B strain by electroporation, using a previously described protocol (13). Allelic exchange in the absence of a selection marker was performed as previously described (2). Briefly, the pMAD plasmid harboring a wild-type copy of the B gene was integrated into the chromosome through homologous recombination at a nonpermissive temperature (43.5°C).…”