Biostimulation of Estuarine Microbiota on Substrate Coated Agar Slides: A Novel Approach to Study Diversity of Autochthonous Bdellovibrio- and Like Organisms |
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Authors: | Ashvini Chauhan Henry N Williams |
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Institution: | (1) Marine Molecular Microbial Ecology Laboratory, Environmental Sciences Institute, Florida A&M University, 1515, S. MLK Blvd., 305 FSHSRC, Tallahassee, FL 32307, USA |
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Abstract: | Characterization of Bdellovibrio- and like organisms (BALOs) from environmental samples involves growing them in the presence of Gram-negative prey bacteria
and isolation of BALO plaques. This labor-intensive enrichment and isolation procedure may impede the detection and phylogenetic
characterization of uncultivable BALOs. In this article, we describe a simple slide biofilm assay to improve detection and
characterization of BALO microbiota. Agar spiked with biostimulants such as yeast extract (YE), casamino acids (CA), or concentrated
cells of Vibrio parahaemolyticus P5 (most widely used prey bacteria for isolation of halophilic BALOs) was plated onto buffed glass slides and exposed to
water samples collected from Apalachicola Bay, Florida. After incubating for a week, diversity of the biofilm bacterial community
was studied by culture-dependent and culture-independent molecular methods. The results revealed that most probable numbers
(MPNs) of BALOs and total culturable bacteria recovered from YE agar slide were significantly higher than the numbers on CA-
or P5-spiked agar slides. Polymerase chain reaction–restriction fragment length polymorphism followed by 16S rDNA sequencing
of clones from different biostimulants resulted in identification of a plethora of Gram-negative bacteria predominantly from
the alpha, gamma, delta-proteobacteria, and the Cytophaga–Flavobacterium–Bacteroides group. Corresponding to the higher biomass on the YE agar slide, the BALO clone library from YE was most diverse, consisting
of Bacteriovorax spp. and a novel clade representing Peredibacter spp. Microbiota from all three biostimulated biofilms were exclusively Gram-negative, and each bacterial guild represented
potential prey for BALOs. We propose the use of this simple yet novel slide biofilm assay to study oligotrophic aquatic bacterial
diversity which could also potentially be utilized to isolate marine bacteria with novel traits. |
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