Bacterial gall formation in Prionitis: Investigation of causation, development, physiology and molecular ecology of a marine red algal-microbial symbiosis

Prionitis is a common marine algal genus (Rhodophyta) from the Pacific coasts of North and South America. Several species (4) are found bearing bacterial galls characterized by hyperplastic algal growth and dense assemblages of intercellular bacteria. The bacterial symbiont of Prionitis lanceolata was identified by 16S rDNA sequencing and whole-cell in situ hybridization. Phylogenetic analysis based on 16S rDNA sequence comparisons places this microbial symbiont within the a sub-class of the Proteobacteria, most closely related to other Prionitis gall symbionts and the marine genus Roseobacter. In situ hybridization of the P. lanceolata symbiont was used to identify and monitor, in whole cells and in sections, this bacterium during consecutive rounds of laboratory gall induction. This allowed demonstration of the causative role or this microbe in gall and the elucidation of specific cellular level events resulting from the induction, establishment and development of galls. The presence of a single symbiotic bacterial phylotype in galls on P. lanceolata was confirmed by in situ hybridization of specimens collected from nature. Symbiont-host specificity of gall formation in the genus Prionitis was also investigated by sequence comparisons of symbiont small sub-unit ribosomal RNA's from galls on P. lanceolata, P. filiformis (Lufenholtz population, Arcata, California) and P. decipiens (Peru).Bacterial phylogeny inferred from sequence comparisons supports a pattern of gall symbiont-host coevolution when compared to algal host phylogeny inferred from nuclear ribosomal internal transcribed spacer region sequences (ITS). P. lanceolataand P. decipiens and their symbionts were closest relatives in this analysis, to the exclusion of P. filiformis and its symbiont. All three symbionts form a monophyletic clade (98.25-99.75% similarity) within the a sub-division of the Proteobacteria. Laboratory cross-inoculation trials confirmed the species-specificity of these associations, as did whole cell hybridization of bacterial symbionts from galls collected in nature. Analysis of P. lanceolata galls by gas chromatography-selective ion monitoring-mass spectrometry (GC-SIM-MS) demonstrated the presence of indole-3 acetic acid (IAA) with levels of this compound at least 2 times higher in galls than in apices and bladelets of the thalli. Molecular analysis of total gall and thallus DNA by standard and pulse field gel electrophoresis suggests the presence of bacterial plasmids in the bacterial gall symbiont but is not conclusive. This study addressed basic biological questions concerning the nature of the macroalgal-bacterial interactions leading to gall formation. Identification and characterization of the P. lanceolata symbiont is the first demonstration of the causative role of a specific microorganism in algal tumorization since Cantcauzene (1930). The quantification of IAA from P. lanceolata galls and thallus is similarly the first identification of this compound in the Florideophycean red algae. Species-specificity of gall formation is confirmed for 3 members of the genus Prionitis and their gall bacteria and the biological relevance of a low level of 16S rDNA sequence divergence elucidated for this group of closely related algae and their microbial symbionts. (Abstract shortened by UMI.).