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

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 β-agarase (dagA) from Pseudoalteromonas atlantica ATCC 19262T (or IAM 12927T) 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 β-(1,4) linkages of agarose to yield predominately neoagarotetraose. Western hybridization confirmed that the cloned agarase was in fact the extracellular β-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.

Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

“…Therefore, AgaB was functionally different from currently known ␤-agarases grouped into families GH 16, 50, and 86. Some agarases of family GH 16 degrade agarose and agarose oligosaccharides comprising at least six sugars to yield DP4 as a main product (10,11). AgaA of family GH 50 from Vibrio sp.…”

“…They are grouped into ␣ and ␤ types that hydrolyze ␣-1,3 linkages and ␤-1,4 linkages, respectively (4). Agarases have been isolated from several bacteria genera, including Cytophaga (5), Pseudomonas (6 -9), Pseudoalteromonas (10), Zobellia (11), Streptomyces (12), Alteromonas (13), Vibrio (14,15), Microscilla (16), Microbulbifer (17), and Aeromonas. Most known agarases are ␤-agarases, with the exception of two ␣-agarases found from Alteromonas agarlyticus GJ1B (18,19) and Thalassomonas sp.…”

Optimization of Anion-exchange Chromatography for the Separation of Agarase from Culture Broth of Pseudoalteromonas sp.

“…아가레이즈는 아가로오스를 가수분해하는 효소 로 그 분해 특성에 따라 두 그룹으로 나뉜다 [1][2][3]. β-아가레이즈는 α-1,3 결합을 끊어 agarooligosaccharide를 생성하는 반면 [4][5][6], β-아 가레이즈는 β-1,4 결합을 끊어 neooligosaccharide를 생성한다 [7][8][9][10].…”

An agar degrading diazotrophic actinobacteria from hyperalkaline meteoric lonar crater lake - a primary study

“…have quite been isolated from marine environment. [2][3][4][5][6][7][8][9][10][11][12][13][14] Since agar is a polysaccharide produced by marine red algae, it is natural that most agar degrading bacteria are inhabitants of marine habitats. However, there are very few reports of nitrogen fixing bacteria degrading agar except for an agar degrading marine nitrogen fixing bacterium placed in the family Vibrionaceae 15 There are also no reports of any actinobacteria other than Streptomyces coelicolor reported by Stanier.…”