Investigation of the role of a β(1–4) agarase produced by Pseudoalteromonas gracilis B9 in eliciting disease symptoms in the red alga Gracilaria gracilis

Abstract

Gracilariaspecies are an important source of agar. The South AfricanGracilariaindustry has experienced a number of setbacks over the last decade in the form of complete or partial die-offs of the agarophyte growing in Saldanha Bay, which may be attributed to bacterial infection. Since a positive correlation was observed between the presence of agarolytic epiphytes and bacterial pathogenicity, we investigated the role of an agarase in the virulence mechanism employed by a bacterium that elicits disease inGracilaria gracilis. The recombinant plasmid pDA1, isolated from aPseudoalteromonas gracilisB9 genomic library, was responsible for the agarolytic activity exhibited byEscherichia colitransformants when grown on solid medium. Ablastsearch of the GenBank database showed that an 873 bp ORF (aagA) located on pDA1 had 85 % identity to theβ-agarase (dagA) fromPseudoalteromonas atlanticaATCC 19262T(or IAM 12927T) at the amino acid level. AagA was purified from the extracellular medium of anE. colitransformant harbouring pDA1 by using a combination of gel filtration and ion-exchange chromatography. AagA has anMrof 30 000 on SDS-PAGE. TLC of the digestion products of AagA showed that the enzyme cleaves theβ-(1,4) linkages of agarose to yield predominately neoagarotetraose. Western hybridization confirmed that the cloned agarase was in fact the extracellularβ-agarase ofP. gracilisB9. The observed relationship between disease symptoms ofG. gracilisand the agarolytic phenotype ofP. gracilisB9 was confirmed. Transmission electron microscope examination of cross sections of both healthyG. gracilisandG. gracilisinfected withP. gracilis, revealed a weakening of the cell structure in the latter plants. Immunogold-labelled antibodies localized the agarasein situto the cell walls of bleachedG. gracilis. Thus, the weakening observed in the cell structure ofG. gracilisinfected withP. graciliscan be attributed to degradation of the mucilaginous component of the cell wall of the bleached thalli.