Belnapia mucosa sp. nov. and Belnapia arida sp. nov., isolated from desert biocrust

Two novel Gram-staining-negative, aerobic, cocci-shaped, non-motile, non-spore forming, pink-pigmented bacteria designated strains T6T and T18T, were isolated from a biocrust (biological soil crust) sample from the vicinity of the Tabernas Desert (Spain). Both strains were catalase-positive and oxidase-negative, and grew under mesophilic, neutrophilic and non-halophilic conditions. According to the 16S rRNA gene sequences, strains T6T and T18T showed similarities with Belnapia rosea CGMCC 1.10758T and Belnapia moabensis CP2CT (98.11 and 98.55% gene sequence similarity, respectively). The DNA G+C content was 69.80 and 68.96% for strains T6T and T18T, respectively; the average nucleotide identity by blast (ANIb) and digital DNA–DNA hybridization (dDDH) values confirmed their adscription to two novel species within the genus Belnapia. The predominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C16 : 0, C18 : 1 2-OH and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c). According to he results of the polyphasic study, strains T6T and T18T represent two novel species in the genus Belnapia (which currently includes only three species), for which names Belnapia mucosa sp. nov. (type strain T6T = CECT 30228T=DSM 112073T) and Belnapia arida sp. nov. (type strain T18T=CECT 30229T=DSM 112074T) are proposed, respectively.

The genus Belnapia was first described by Reddy et al. [1] and it is, at the time of writing, comprised of three species, which were all isolated from soil samples: Belnapia moabensis [1], Belnapia rosea [2] and Belnapia soli [3]. In this study we describe the polyphasic characterization of two strains, namely T6 T and T18 T , which were isolated from biocrust (biological soil crust) samples from south-eastern Spain during a study on the microbial diversity of European arid regions.
Strains T6 T and T18 T were isolated in the vicinity of the Tabernas Desert (Almería, Spain) during a study on the culturable microbial diversity in European drylands [4]. The Tabernas Desert is considered the only arid desert in Europe. Biocrust samples were obtained from near the Tabernas Desert Parc (37.002404° N, 2.450655° W) and homogenized in phosphate buffered saline (PBS; NaCl 8.0 g l −1 , KCl 0.2 g l −1 , Na 2 HPO 4 1.44 g l −1 , KH 2 PO 4 0.24 g l −1 ) pH 7.4 (1 g in 1 ml). The suspensions were then spread on 1, 0.1 and 0.01× trypticase soy agar (TSA; 15 g l −1 tryptone, 5 g l −1 NaCl, 5 g l −1 soya peptone), and Reasoner's 2A Agar (R2A; 1 g l −1 peptone, 0.5 g l −1 yeast extract, 0.5 g l −1 dextrose, 0.5 g l −1 soluble starch, 0.3 g l −1 dipotassium phosphate, 0.05 g l −1 magnesium sulphate heptahydrate, 0.3 g l −1 sodium pyruvate). Agar was autoclaved separately and added before plating at a final concentration of 15 g l −1 . The plates were incubated at 23 °C for 1 week. Strain T6 T was isolated from 0.1× TSA plates, whereas T18 T was isolated from a 0.01× TSA plate. The isolation of the strains was carried out by re-streaking on fresh media until a pure culture was obtained. Cell suspensions in TSA and R2A were cryopreserved at −80 °C with 15% glycerol (v/v). Their taxonomic status was determined by a polyphasic approach. On the basis of the results from phylogenetic, phenotypic and chemotaxonomic analysis, it is concluded that strains T6 T and T18 T are related to members of the genus Belnapia and representatives of two novel species. In the present work, OPEN ACCESS the reference strains B. rosea DSM 23312 T , B. moabensis DSM 16746 T and B. soli DSM 28067 T , from the DSMZ (German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany), and strains T6 T and T18 T were all grown in parallel on R2A media at 30 °C, unless otherwise specified.
The phenotypic characteristics of T6 T and T18 T were analysed after 1 week of growth at 30 °C. A Gram staining test was carried out with KOH 3 % (w/v), recording viscosity as a negative result. Oxidase activity was tested by using the commercial Oxidase Test Stick for microbiology (PanReac AppliChem). Catalase activity was tested with hydrogen peroxide 30% (v/v), recording bubble formation as a positive result. Cell morphology was observed under an optical microscope with crystal violet glass stain. Growth at different temperatures (4,10,15,20,23,30,37,40, 42 and 45 °C) and NaCl concentrations (0.0-4.0% at intervals of 0.5%) was checked on R2A. Growth at different pH values (4.0-10.0 at intervals of 1.0 pH unit) was examined by growing the strains in liquid R2A using the buffers MES (pH 4-6), HEPES (pH 7-8) and CHES (pH 9-10) at a final concentration of 10 mM. Growth under microaerophilic and anaerobic conditions was tested by incubating the plates in a candle jar and with the BD GasPak EZ pouch system (Becton, Dickinson), respectively. Carbon source assimilation and enzymatic activities were checked using the API 20NE and API ZYM system strips (bioMérieux) according to manufacturer's instructions. BIOLOG GEN III MicroPlates (BIOLOG) were also used to determine carbon source assimilation.
Strains T6 T , T18 T and the reference strains B. rosea DSM 23312 T , B. moabensis DSM 16746 T and B. soli DSM 28067 T were grown on R2A medium at 30 °C for 72 h for analysis of cellular fatty acids. The analysis was carried out following the protocol recommended by MIDI Microbial Identification System (version 6.1, MIDI, Inc, Newark, DE, USA) [5]. The fatty acids content was analysed on a 6850 gas chromatography system (Agilent) using the TSBA6 method [6].
Genomic DNA extraction was carried out using the DNeasy Power Soil kit (Qiagen), according to the manufacturer's instructions, but incubating at 65 °C after the addition of C1. Whole 16S rRNA gene PCR was carried out with universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) [7] and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [8] following procedures described previously [4]. Phylogenetic trees based on the 16S rRNA gene sequences were reconstructed using the maximum-likelihood (ML) [9] and neighbour-joining (NJ) [10] methods with the software mega X v.10.1.7. The TamuraNei G+I evolutionary model and the Kimura twoparameter model were used for the ML and NJ trees, respectively. The reliability of the branch patterns was assessed using bootstrap analysis based on 500 and 1000 replicates, respectively, for the ML and the NJ trees [11].
The draft genome of strains T6 T and T18 T were sequenced with the NovaSeq 6000 system (Illumina; 2×150 bp paired-end sequencing). The genomic DNA was randomly fragmented by sonication, then DNA fragments were end polished, A-tailed and ligated with the full-length adapters for Illumina sequencing. Further PCR amplification was carried out with P5 and indexed P7 oligonucleotides, and PCR products for the final construction of the libraries were purified with an AMPure XP system. Libraries were then checked for size distribution by Agilent 2100 Bioanalyzer (Agilent Technologies), and quantified by real-time PCR. The FastQC tool (v0.11.5) [12] was utilized to assess the quality of the sequence reads. There were 14 375 848 and 12 879 132 paired-end reads for strains T6 T and T18 T , respectively before filtering. After quality filtering, there were considered to be 13 106 155 and 11 792 897 paired-end sequences the genomes of strains T6 T and T18 T , respectively. Genome assembly of paired reads was performed using the '--isolate' mode in SPAdes (3.14.1) [13]. Assembly statistics were calculated with QUAST (v.5.0.2) [14] and the completeness and contamination levels were evaluated with CheckM (v.1.1.3) [15]. The draft genomes were annotated using the RAST tool kit (RAStk) [16] integrated in PATRIC v.3.6.8. The draft genomes were analysed with the TYGS tool [17] in order to identify the most closely related type strains to T6 T and T18 T with publicly available genomes and to calculate digital DNA-DNA hybridization (dDDH) indexes. JSpecies [18] was used for calculating the average nucleotide identities according to blast (ANIb) between genome pairs. UBCG (v.3.0) [19] was used for reconstructing the phylogenomic tree among the selected strains based on a multiple alignment of a set of 92 housekeeping genes. We selected the alignment method codon and inferred the phylogenetic relationships with FastTree. The reliability of the branch patterns was assessed using bootstrap analysis based on 100 replicates.
Strains T6 T and T18 T were aerobic, Gram-staining-negative, non-motile and coccus-shaped (0.8-1.0 µm in diameter). The cells of both strains occurred singly, as in other members of the genus Belnapia. Colonies were pink, irregular and mucous. T6 T colonies were paler than those of the rest of the members of the genus Belnapia. After 3-5 days of growth at 30 °C, the colonies of both strains displayed a diameter of around 3-4 mm.
Both strains were able to grow at between 4 and 40 °C (optimum at 30 °C). Moreover, T6 T was able to grow at up to 42 °C. Both strains showed tolerance to up to 1.5% (w/v) of NaCl (optimum 0-1%). B. moabensis DSM 16746 T and B. rosea DSM 23312 T showed similar NaCl tolerances, in contrast with B. soli DSM 28067 T , which was able to grow at concentrations of up to 3%. All five strains were able to grow at between pH 5 and 9, with an optimum at 6-7 (Table 1).

Enzymatic activity (API ZYM)
The draft genomes of strains T6 T and T18 T consisted of 220 and 355 contigs, respectively, which constituted a total length of 6449681 and 6937094 bp, respectively. The N50 values were 328 210 and 194 160 for T6 T and T18 T respectively. The genomic DNA G+C contents were 69.80 and 68.96% for T6 T and T18 T respectively, which is in accordance with the values previously described for the rest of the species within the genus Belnapia and further confirms their adscription to this genus [1][2][3]. A total of 6369 and 7450 coding sequences (CDSs) were predicted for strains T6 T and T18 T , of which 3380 and 3480, respectively, corresponded to proteins with functional assignment. Regarding the prediction of tRNA and rRNAs, a total of 49 and 47 tRNAs, and 3 and 2 rRNAs were predicted for strains T6 T and T18 T , respectively. The 16S rRNA gene sequences of strains T6 T and T18 T were also extracted from the genome, which were 1496 and 1494 bp long, respectively (accession numbers MW960268 and MW960269, respectively In order to obtain a more accurate phylogenetic inference of novel strains, a phylogenomic tree based on nucleotide sequences was reconstructed (Fig. 2). The phylogenomic tree corroborated that the two strains represent members of the genus Belnapia. Strain T18 T was most closely related to B. moabensis DSM 16746T, while T6 T showed an external position to the rest of the members of the genus Belnapia. The type strain of B. soli was not included in this analysis because its genome was not publicly available at the time of writing.
The ANIb and digital DDH values between strains T6 T and T18 T and other related species were calculated (  [20] and 70 % for dDDH [21], both genome indexes confirmed the classification of strains T6 T and T18 T as representing novel species [22].   Table 2). This is in accordance with the profiles obtained for the members of the genus Belnapia, which also showed high amounts of summed feature 8, summed feature 3, C 18 : 1 2-OH and C 16 : 0 , thus confirming the inclusion of both strains within the genus Belnapia.
The results of the phenotypic, chemotaxonomic, genomic and phylogenetic analyses confirm that strains T6 T and T18 T should be considered as each representing a novel species within the genus Belnapia, for which the names Belnapia mucosa sp. nov. and Belnapia arida sp. nov., respectively, are proposed.