Investigation of the role of a {beta}(1-4) agarase produced by Pseudoalteromonas gracilis B9 in eliciting disease symptoms in the red alga Gracilaria gracilis

• Published in: Microbiology (Society for General Microbiology) 2003-10, Vol.149 (10), p.2919
• Main Authors: Schroeder, Declan C, Jaffer, Mohamed A, Coyne, Vernon E
• Format: Article
• Language: English
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.26513-0

1 Department of Molecular and Cell Biology, University of Cape Town, South Africa 2 Electron Microscope Unit, University of Cape Town, South Africa Correspondence Vernon E. Coyne vernon{at}science.uct.ac.za Gracilaria species are an important source of agar. The South African Gracilaria industry 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 in Gracilaria gracilis . The recombinant plasmid pDA1, isolated from a Pseudoalteromonas gracilis B9 genomic library, was responsible for the agarolytic activity exhibited by Escherichia coli transformants when grown on solid medium. A BLAST search of the GenBank database showed that an 873 bp ORF ( aagA ) located on pDA1 had 85 % identity to the -agarase ( dagA ) from Pseudoalteromonas atlantica ATCC 19262 T (or IAM 12927 T ) at the amino acid level. AagA was purified from the extracellular medium of an E. coli transformant harbouring pDA1 by using a combination of gel filtration and ion-exchange chromatography. AagA has an M r of 30 000 on SDS-PAGE. TLC of the digestion products of AagA showed that the enzyme cleaves the -(1,4) linkages of agarose to yield predominately neoagarotetraose. Western hybridization confirmed that the cloned agarase was in fact the extracellular -agarase of P. gracilis B9. The observed relationship between disease symptoms of G. gracilis and the agarolytic phenotype of P. gracilis B9 was confirmed. Transmission electron microscope examination of cross sections of both healthy G. gracilis and G. gracilis infected with P. gracilis , revealed a weakening of the cell structure in the latter plants. Immunogold-labelled antibodies localized the agarase in situ to the cell walls of bleached G. gracilis . Thus, the weakening observed in the cell structure of G. gracilis infected with P. gracilis can be attributed to degradation of the mucilaginous component of the cell wall of the bleached thalli. Abbreviations: Ap, ampicillin; LA, LB agar; SSW, sterile seawater The GenBank accession number for the sequence reported in this paper is U61972 . Present address: The Marine Biological Association of the UK, Citadel Hill, Plymouth, UK.