Genomic and Physiological Characterization of the Verrucomicrobia Isolate Diplosphaera colitermitum gen. nov., sp. nov., Reveals Microaerophily and Nitrogen Fixation Genes

Abstract

Previously we reported the cultivation of novel verrucomicrobia, including strain TAV2 (93% 16S rRNA gene identity to its nearest cultivated representative, Opitutus terreae PB90-1) from the gut of the termite Reticulitermes flavipes. To gain better insight into the Verrucomicrobia as a whole and understand the role of verrucomicrobia within the termite gut ecosystem, we analyzed a draft genome and undertook a physiological characterization of TAV2. Strain TAV2 is an autochthonous member of the R. flavipes gut microbiota and groups phylogenetically among diverse Verrucomicrobia from R. flavipes and other termites that are represented by 16S rRNA gene sequences alone. TAV2 is a microaerophile, possessing a high-affinity cbb 3 -type terminal oxidaseencoding gene and exhibiting an optimum growth rate between 2 and 8% (vol/vol) oxygen. It has the genetic potential to degrade cellulose, an important function within termite guts, but its in vitro substrate utilization spectrum was limited to starch and a few mono-and disaccharides. Growth occurred on nitrogen-free medium, and genomic screening revealed genes for dinitrogenases, heretofore detected in only a few members of the Verrucomicrobia. This represents the first (i) characterization of a verrucomicrobial species from the termite gut, (ii) report of nif and anf genes in a nonacidophilic verrucomicrobial species, and (iii) description of a microaerophilic genotype and phenotype in this phylum of bacteria. The genetic and physiological distinctiveness of TAV2 supports its recognition as the type strain of a new genus and species, for which the name Diplosphaera colitermitum gen. nov., sp. nov., is proposed.

Development of an ecophysiological model for Diplosphaera colotermitum TAV2, a termite hindgut Verrucomicrobium

“…Our proteomic results indicated the presence of specific peptides corresponding to the dinitrogenase reductase (NifH) only when cells were grown with 2% O 2 , corroborating our previous findings for the presence of nitrogen fixation genes (nif) and growth on N-free medium (Wertz et al, 2012). Finally, the expression of a urea-binding signal peptide protein and the a-subunit of urease may indicate a role for TAV2 in the recycling of uric acid excreted by the host.…”

“…The results of this study confirm the microaerophilic nature of strain TAV2 (Wertz et al, 2012), with a higher growth rate observed for cells grown under 2% O 2 than those grown under 20% O 2 . The long lag-phase shown for cells grown in 20% O 2 suggests the need of an acclimation period and the possibility of cells being under oxidative stress.…”

“…In an effort to understand the ecological functions of this microbial group, we previously isolated and characterized Verrucomicrobia strains from the gut of R. flavipes, including the new species D. colotermitum strain TAV2. We confirmed their autochthonous nature, estimated the population number and unveiled the genomic properties (Isanapong et al, 2012, Wertz et al, 2012, suggesting new ecological roles for Verrucomicrobia within the termite gut.…”

“…First, our proteomic approach detected the presence of peptides derived from superoxide dismutase. This result provides an explanation on how TAV2 manages reactive oxygen species and helps to conciliate our previous experimental work, in which the activity of superoxide dismutase was not observed (Wertz et al, 2012). Second, TAV2 cells promote a shift in protein expression with a substantial increase of categories associated with amino-acid transport and metabolism, protein turnover and post-translational modifications, and signal transduction mechanisms.…”

“…Taken together, the above results imply that TAV2 can contribute to the nitrogen requirements of the termite. We remain cautious about establishing such a link based on the low Verrucomicrobia population size (1.2 Â 10 3 cells) residing in the hindgut (Köhler et al, 2012;Wertz et al, 2012), but even a small contribution could be important in an N-limited ecosystem.…”