Ubiquity, diversity and physiological characteristics of Geodermatophilaceae in Shapotou National Desert Ecological Reserve

The goal of this study was to gain insight into the diversity of culturable actinobacteria in desert soil crusts and to determine the physiological characteristics of the predominant actinobacterial group in these crusts. Culture-dependent method was employed to obtain actinobacterial strains from desert soil samples collected from Shapotou National Desert Ecological Reserve (NDER) located in Tengger Desert, China. A total of 376 actinobacterial strains were isolated and 16S rRNA gene sequences analysis indicated that these isolates belonged to 29 genera within 18 families, among which the members of the family Geodermatophilaceae were predominant. The combination of 16S rRNA gene information and the phenotypic data allowed these newly-isolated Geodermatophilaceae members to be classified into 33 “species clusters,” 11 of which represented hitherto unrecognized species. Fermentation broths from 19.7% of the isolated strains showed activity in at least one of the six screens for antibiotic activity. These isolates exhibited bio-diversity in enzymatic characteristics and carbon utilization profiles. The physiological characteristics of the isolates from different types of crusts or bare sand samples were specific to their respective micro-ecological environments. Our study revealed that members of the family Geodermatophilaceae were ubiquitous, abundant, and diverse in Shapotou NDER, and these strains may represent a new major group of potential functional actinobacteria in desert soil.


Introduction
It has become increasingly clear that the overuse of antibiotics and the subsequent rise in antibioticresistant pathogens will force us to search for new antibiotics to meet urgent clinical needs (Talbot et al., 2006). Previous studies have indicated that environments considered to be extreme habitats are rich sources of novel actinobacteria (Subramani and Aalbersberg, 2013). It has been hypothesized that unusual climate conditions and ecological factors may endow the organisms in such habitats with the unique capacity to produce novel bioactive compounds (Bull et al., 2005;Okoro et al., 2008).
The Shapotou desert region (latitude 36 • 39 ′ -37 • 41 ′ N, elevation 104 • 25 ′ -105 • 40 ′ E) is recognized as the first "National Desert Ecological Reserve" (NDER) in China. This NDER is renowned worldwide as a teaching and scientific research base for studying controlled desertification. It is located on the southeast edge of the Tengger Desert, south of the Yellow River, in the northwest part of China. This region is at an altitude of 1300-1700 m, has an annual average precipitation of 186.2 mm, an annual mean temperature of 9.7 • C, and an annual average wind speed of 2.8 m/s with a typical temperate desert climate.
In desert regions, microbiotic crusts play a significant role in controlling desertification by providing surface stability. Microbiotic crusts are important in stabilization of the sandy surface, soil formation, and in carbon and nitrogen assimilation (Evans and Johansen, 1999). Microbiotic crusts in Shapotou NDER are generally categorized into the following three typical types: Cyanobacteria-dominated crusts (CC), Moss-dominated crusts (MC), and Lichen-dominated crusts (LC). Samples were therefore, collected from these three types of crusts and bare sands. Culture-dependent method was employed to evaluate the diversity of culturable actinobacteria in Shapotou NDER, and to explore the potential functional actinobacterial resources from this extreme environment.
Actinobacterial strains were discovered and identified from the three types of soil crusts and bare sands samples from the Shapotou NDER. We found that the members of the family Geodermatophilaceae were ubiquitous in the different types of crusts, as well as the bare sands samples. Based on the physiological characteristics of these diverse Geodermatophilaceae members, we characterized the influence of micro-ecological niche environments on the phenotypic characteristics of these isolates.

Sample Collection
A total of 50 samples for isolation of actinobacteria were collected from four different micro-ecological environments in Shapotou NDER (latitude 36 • 39 ′ -37 • 41 ′ N, elevation 104 • 25 ′ -105 • 40 ′ E). The detailed information regarding the sample number, type of sample, and specific collection location of the 50 samples is displayed in Table 1. All the samples were placed in sterilized envelopes following collection and taken to the laboratory within 1 week of collection. All samples were immediately processed for isolation after arriving at the laboratory.

Identification of Geodermatophilaceae
Purified isolates were transferred to PYG medium and International Streptomyces Project (ISP) medium 2 for observation of the morphological characteristics. Extraction of genomic DNA and PCR amplification of the 16S rRNA gene were performed as described in the methods section of Xu et al. (2003). The purified PCR products were sequenced with an ABI PRISM automatic sequencer. The sequences obtained were compared with available 16S rRNA gene sequences from GenBank using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net; Kim et al., 2012). The server was used to determine an approximate phylogenetic affiliation of each strain. Multiple alignments with sequences of the related strains and calculations of levels of sequence similarities were carried out using MEGA version 5 (Tamura et al., 2011). A phylogenetic tree was constructed using the neighbor-joining method described in Saitou and Nei (1987). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.

Bioactivity Screening
Antimicrobial activities of the isolated strains were investigated by using media containing Enterococcus faecalis HH22, Klebsiella pneumonia ATCC 700603, Mycobacterium smegmatis CPCC240556, Sporobolomyces salmonicolor SS04, and Xanthomonas campestris pv. oryzae PXO99A, respectively, all at a concentration of 10 8 colony forming units (CFU) per ml. The anti-viral activity against the human immunodeficiency virus (HIV) was investigated using the procedure described in Yang et al. (2013). Results were considered positive if the HIV inhibition ratio was above 90% and at least 80% of the cells survived. This assay was performed under conditions where the sample concentration was 1% (v/v).
Numerical comparative analysis of the physiological and biochemical characteristics tested was performed using the NTSYSpc package (version 2.2 for Windows; Exeter Software) (Rohlf, 2000). A binary 0/1 matrix was created based on the positive or negative respective values of 173 physiological characteristics, some of which are described above.

Isolation of Actinobacteria
A total of 470 purified isolates were obtained in the present study. The 16S rRNA gene sequences revealed that 376 actinobacterial strains were isolated from the 50 samples. These isolates belonged to 18 families and 29 genera, among which the members of Geodermatophilaceae were predominant, including 70 strains of three genera. (Supplementary Figure S1). Among the four types of isolation media, M2 resulted in the most successful isolation of actinobacterial strains. Specifically, 35% of the actinobacterial strains were obtained from M2. While 29, 26, and 10% of the actinobacterial isolates were purified from M1, M4, and M3, respectively (Supplementary Figure S2).

Diversity of Geodermatophilaceae
In total, 70 Geodermatophilaceae strains, including 34 Blastococcus spp., 11 Geodermatophilus spp., and 25 Modestobacter spp. were collected from the 50 samples ( Table 2). In the phylogenetic dendrogram based on 16S rRNA gene sequences analysis of the isolates and the type stains of 25 validly described species in the family Geodermatophilaceae, these 70 newly-isolated members of the family Geodermatophilaceae fell into 23 "species clusters, " with the 16S rRNA gene sequence similarity below 98.65% to the closest homolog as the threshold for differentiating two species (Kim et al., 2014) (Figure 1). As indicated in the phylogenetic dendrogram, six Modestobacter "species clusters, " two Blastococcus "species clusters" and three Geodermatophilus "species clusters" may represent hitherto unrecognized species.

Physiological Characteristics of Newly-isolated Strains
The strains assayed for physiological characteristics were similar in their physiological characteristic profiles in the following capacity: more than 60% of the strains tested could utilize dextrin, D-fructose, D-fructose-6-PO 4 , D-galactose, α-D-glucose, glucuronamide, α-keto-glutaric acid, D-malic acid, D-maltose, D-mannose, D-trehalose, D-turanose and sucrose as their sole carbon source, and 91% of the strains tested assimilated esculin ferric citrate and potassium 5-ketogluconate and produced acid. In the API ZYM assay, none of the strains tested was positive for β-fucosidase, N-acetyl-β-glucosaminidase, or α-mannosidase. Twenty-nine strains showed the enzymatic activities of acid phosphatase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, lipase (C14), and valine arylamidase. Most of the tested strains were resistant to aztreonam (1500 µg/ml), sulfanilamide (15,000 µg/ml), and sulfamethoxazole/trimethoprim (1187.5 µg/ml and 62.5 µg/ml). The phylogenetic dendrogram based on 173 physiological characteristics of the tested strains showed that the microecological environment from which the strains were isolated was an important factor correlating with the physiological characteristic profiles of the isolates. The strains exhibited characteristics specific to the micro-ecological environment where they were found (Figure 2).

Discussion
The family Geodermatophilaceae is a newly-established actinobacterial taxon. Normand et al. (1996) proposed the family Geodermatophilaceae in 1996, which was regarded as an invalid taxon at that time. In 2006, based on the common characteristics of the genera Geodermatophilus, Blastococcus, and Modestobacter, Normand (2006) summarized the typical characteristics of Geodermatophilaceae. Subsequently, the family Geodermatophilaceae was finally accommodated as a validly described taxon in the phylum Actinobacteria. To date, the family Geodermatophilaceae consists of three genera: Geodermatophilus, Blastococcus, and Modestobacter, that includes 25 validly described species. The members of Geodermatophilaceae were found from various environments, including soil samples (Zhang et al., 2011;Jin et al., 2013), soil crusts (Reddy et al., 2007), deep subseafloor sediment (Ahrens and Moll, 1970), even stone habitats (Salazar et al., 2006;Chouaia et al., 2012;Gtari et al., 2012;Normand et al., 2012), dry-hot valley (Nie et al., 2012), and arid sand from desert (Montero-Calasanz et al., 2012, 2013a. In this study, we found Geodermatophilaceae members ubiquitously in desert soil samples, and we obtained Geodermatophilaceae cultures from three different types of desert soil crusts, as well as from the bare sands. These four environments represent typical micro-ecological environments in the Shapotou region. As we have observed, most Geodermatophilaceae members could form tiny motile spores or dormant spores, allowing them to spread around and survive long periods of desiccation. Moreover, most of the Geodermatophilaceae members we tested formed pink to black colonies on different types of agar plates. The pigmentation, cell wall composition and a high G+C content may increase protection of these strains from UV damage in the desert environments, where the UV transparency is often high.
The abundance and ubiquitous distribution of the Geodermatophilaceae in desert environments exhibited in relation to their resident microbiota, and in turn, the microecological environments endowed the microorganisms with some specific metabolic characteristics. We found that the abundance and diversity of the Geodermatophilaceae in lichenand cyanobacteria-dominated soil crusts were much higher than those of the bacteria found in moss-dominated soil crusts or bare sands. In the desert, the moisture, organic, and nitrogen content of the soil were the vital factors in determining physiological characteristics of the organisms. The lichen-and cyanobacteriadominated soil crusts may contain a much higher proportion of clay and humic colloidal material, which can markedly affect the physiological activities of the strains from different micro-ecological environments. Blastococcus spp.