Isolation and Characterization of aRhizobiumBacterium Associated with the Toxic DinoflagellateGambierdiscus balechii

Abstract

25Algae-bacteria associations are increasingly being recognized to be important in 26shaping the growth of both algae and bacteria. Bacteria belonging to order 27Rhizobiales are important symbionts of legumes often developing as nodules on plant 28 roots, but have not been widely documented in association with algae. Here, we 29 detected, isolated, and characterized a Rhizobium species from the toxic benthic 30dinoflagellate Gambierdiscus culture. The sequence of 16S rDNA showed 99% 31 identity with that of Rhizobium rosettiformans. To further characterize the bacterium, 32we amplified and sequenced a cell wall hydrolase (CWH)-encoding gene; 33 phylogenetic analysis indicated that this sequence was similar to the homologs of 34Martellela sp. and Hoeflea sp, of order Rhizobiales. We performed PCR using nifH 35 primers to determine whether this bacterium can fix N2; however, the results of 36 sequencing analysis showed that it was closer to chlorophyllide a reductase-encoding 37 gene (bchX), which is similar to nifH. Results of 16S rDNA qPCR showed that 38 compared to that in the early exponential phase, the abundance of this bacterium 39 increased during the late exponential growth phase of Gambierdiscus. When the 40 dinoflagellate culture was subjected to N limitation, the abundance of the bacterium 41 represented by both 16S rDNA and CWH increased. Based on these results and 42 published literature, it is apparent that this Rhizobium bacterium benefits from the 43 association with Gambierdiscus by hydrolyzing and utilizing the extracellular organic 44 matter exudates released by the dinoflagellate. This is the first report of Rhizobium 45 4 species being associated with dinoflagellates, which will shed light on the algae-46 bacteria relationships. 47 7 RESULTS 105 Phylogenies of 16S rDNA and CWH gene sequences from the isolated bacterium 106G. balechii exhibited exponential growth when a sample was collected for 107 bacterium isolation. Random clones were selected when colonies appeared on the 108 marine agar plate. CWH primers were used to screen the bacterial clones that 109 contained the CWH gene. Out of these selected clones, 50 % yielded positive PCR 110 results. 111Sequencing of 16S rDNA amplicons from the CWH-positive colonies revealed 112 Bacterium spp., Bacillus spp., Rhizibium spp., and Flavobacterium spp. to be the 113 closest matches. A Rhizobium species was selected for further characterization. The 114 sequence of the isolated bacterium species (1,362 bp) was 99.93% identical to that of 115 Rhizobium rosettiformans strain W3, which was isolated from groundwater from 116 Lucknow, India [31]. Based on the commonly used species-delineating criterion of 117 16S rDNA identity (97% for prokaryotes), this bacterial strain was classified as 118Rhizobium rosettiformans strain GAMBA-01. The result of phylogenetic analysis 119 verified that this bacterial strain belonged to the same sub-cluster as other Rhizobium 120 strains (Fig. 1). 121 122Conserved domain searches in BLASTp revealed that the CWH sequence c...

From the microbial to the mechanical : environmental impacts on coral nitrogen cycling, microbial communities, and skeletal properties

“…diazotrophs has yet to be directly documented, such a relationship may explain why some of the highest rates of DDN assimilation reported in the literature were measured in short, day-time experiments (Benavides et al, 2016;Sangsawang et al, As a number of potentially diazotrophic Alphaproteobacteria were abundant in the 16S rRNA community, notably Rhizobiales, it is surprising that these taxa were not equally abundant in the nifH Cluster I-III sequences. Rhizobiales contains many known diazotrophs that may associate with microalgae (Kim et al, 2014;Wu et al, 2019) and have been found in varying abundance in coral microbial communities (Lesser et al, 2018;Davey et al, 2007;Lema et al, 2012). While Rhizobiales have been proposed as possible diazotrophic coral symbionts (Lema et al, 2012), their role in coral N 2 fixation is still debated, particularly as some species contain only Cluster V nifH homologs and do not appear to fix nitrogen (Lesser et al, 2018;Wu et al, 2019).…”

“…Rhizobiales contains many known diazotrophs that may associate with microalgae (Kim et al, 2014;Wu et al, 2019) and have been found in varying abundance in coral microbial communities (Lesser et al, 2018;Davey et al, 2007;Lema et al, 2012). While Rhizobiales have been proposed as possible diazotrophic coral symbionts (Lema et al, 2012), their role in coral N 2 fixation is still debated, particularly as some species contain only Cluster V nifH homologs and do not appear to fix nitrogen (Lesser et al, 2018;Wu et al, 2019). We found that Rhizobiales had low relative activity in both the tissue and skeleton (16S RNA:DNA < 1), and that the majority of Rhizobiales ).…”