Natronobiforma cellulositropha gen. nov., sp. nov., a novel haloalkaliphilic member of the family Natrialbaceae (class Halobacteria) from hypersaline alkaline lakes

Abstract

Six strains of extremely halophilic and alkaliphilic euryarchaea were enriched and isolated in pure culture from surface brines and sediments of hypersaline alkaline lakes in various geographical locations with various forms of insoluble cellulose as growth substrate. The cells are mostly flat motile rods with a thin monolayer cell wall while growing on cellobiose. In contrast, the cells growing with cellulose are mostly nonmotile cocci covered with a thick external EPS layer. The isolates, designated AArcel, are obligate aerobic heterotrophs with a narrow substrate spectrum. All strains can use insoluble celluloses, cellobiose, a few soluble glucans and xylan as their carbon and energy source. They are extreme halophiles, growing within the range from 2.5 to 4.8M total Na⁺ (optimum at 4M) and obligate alkaliphiles, with the pH range for growth from 7.5 to 9.9 (optimum at 8.5-9). The core archaeal lipids of strain AArcel5^T were dominated by C₂₀-C₂₀ dialkyl glycerol ether (DGE) (i.e. archaeol) and C₂₀-C₂₅ DGE in nearly equal proportion. The 16S rRNA gene analysis indicated that all six isolates belong to a single genomic species mostly related to the genera Saliphagus-Natribaculum-Halovarius. Taking together a substantial phenotypic difference of the new isolates from the closest relatives and the phylogenetic distance, it is concluded that the AArcel group represents a novel genus-level branch within the family Natrialbaceae for which the name Natronobiforma cellulositropha gen. nov., sp. nov. is proposed with AArcel5^T as the type strain (JCM 31939^T=UNIQEM U972^T).

Keywords: Cellulotrophic; Haloalkaliphilic; Hypersaline; Natronoarchaea; Soda lakes.

Fig. 1

Cell morphology of strain AArcel5 growing on amorphous cellulose at pH 9.5, 4 M total Na⁺ and 37 °C. (a) colonies; (b) phase contrast and (c) electron microscopy of thin sections of cells during absorption phase on cellulose; (d) electron microscopy of thin sections and (e) and whole free suspended cells from the second growth phase on cellulose.

Fig. 2

Influence of pH at 4 M total Na⁺ (a) and Na⁺ at pH 9 (b) on growth of strain AArcel5 with cellobiose at 37 °C. The results are mean values from two biological replicate experiments.

Fig. 3

Phylogeny of the AArcel strains. (a) Maximum Likelihood 16S rRNA gene sequence-based phylogenetic tree showing position of the AArcel strains (in bold) within the family Natrialbaceae. Branch lengths (see scale) correspond to the number of substitutions per site with corrections, associated with the model (GTR, G + I, 4 categories). All positions with less than 95% site coverage were eliminated. Totally 1359 positions were used in the alignment of 32 sequences (except for the partial AArcel4 and AArcel6 sequences, 100% identical to AArcel5^T). Numbers at nodes indicate bootstrap values of 1000 repetitions. Halomarina oriensis strain JCM 16495 (AB663390.1) was used as an outgroup. (b) Maximum Likelihood rpoB' gene sequence-based tree showing position of the AArcel2 and AArcel5 strains (in bold) within family Natrialbaceae. Totally 1827 positions were used in the alignment of 18 sequences. Halomarina oriensis JCM 16495 (KJ870934.1) was used as an outgroup. (c): Maximum Likelihood RpoB' protein sequence-based tree showing position AArcel2 and AArcel5 strains (in bold) within the family Natrialbaceae. Totally 608 positions were used in the alignment of 18 sequences. Halomarina oriensis JCM 16495 (KJ870934.1) was used as an outgroup. Sequences with accession numbers in italic were obtained from IMG, in roman – from the Genbank.