Diversity and Physiological Characteristics of Antarctic Lichens-Associated Bacteria

The diversity of lichen-associated bacteria from lichen taxa Cetraria, Cladonia, Megaspora, Pseudephebe, Psoroma, and Sphaerophorus was investigated by sequencing of 16S rRNA gene amplicons. Physiological characteristics of the cultured bacterial isolates were investigated to understand possible roles in the lichen ecosystem. Proteobacteria (with a relative abundance of 69.7–96.7%) were mostly represented by the order Rhodospirillales. The 117 retrieved isolates were grouped into 35 phylotypes of the phyla Actinobacteria (27), Bacteroidetes (6), Deinococcus-Thermus (1), and Proteobacteria (Alphaproteobacteria (53), Betaproteobacteria (18), and Gammaproteobacteria (12)). Hydrolysis of macromolecules such as skim milk, polymer, and (hypo)xanthine, solubilization of inorganic phosphate, production of phytohormone indole-3-acetic acid, and fixation of atmospheric nitrogen were observed in different taxa. The potential phototrophy of the strains of the genus Polymorphobacter which were cultivated from a lichen for the first time was revealed by the presence of genes involved in photosynthesis. Altogether, the physiological characteristics of diverse bacterial taxa from Antarctic lichens are considered to imply significant roles of lichen-associated bacteria to allow lichens to be tolerant or competitive in the harsh Antarctic environment.


Introduction
Lichens form symbiotic relationships between fungi (mycobiont) and photosynthetic algae and/or cyanobacteria (photobiont) [1][2][3]. Establishment of symbiotic lifestyles through the proper combinations of fungal and algal species is considered to support the ecological success of lichens as one of the earliest colonizers of severe habitats and recently exposed land. Furthermore, lichens are usually highly tolerant to harsh environmental conditions such as extremes in temperature, low water content, low nutrient availability, and high ultraviolet light intensities. These properties enable more than 19,000 lichen species to thrive in practically any terrestrial environment from polar areas to tropical rainforests, covering up to 8% of the total land surface [3][4][5][6].
Despite the general definition of lichens as bipartite or tripartite according to the mycobiont and photobiont symbioses, the presence of other microorganisms such as lichenassociated fungi, microalgae, and bacteria in the lichen thalli as an additional and integral component of lichen has long been revealed [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. With the observation of high bacterial diversity and abundance in lichen, polyphasic approaches including metagenomic analysis and physiological determination of cultured isolates have been applied to understand the roles of bacterial communities in the lichen symbioses. As a consequence, several putative functional roles of bacteria such as nitrogen fixation, phosphate solubilization, amino acids and phytohormones production, nutrient supply via hydrolysis of major organic compounds, and defense mechanisms through antagonistic activities within the lichen symbiosis have been suggested, and these functions are considered to help lichens survive  (Table 1) and identified based on 28S rRNA gene sequences obtained by PacBio RSII platform (Pacific Bioscience, Menlo Park, CA, USA). The study area, the Barton Peninsula, is the southwestern part of King George Island, stretching 540 km off the northern end of the Antarctic Peninsula. The Barton Peninsula has a rugged topography with a wide and gentle slope in the central belt, having altitudes ranging from 90 to 180 m above sea level [48]. Fine fractions of soils on the Barton Peninsula are mostly composed of mineral and rock fragments with some volcanic ashes [48]. The Barton Peninsula is a cold moist maritime Antarctic region, which is characterized by annual average temperature ranges of −1.8~1.6 • C, an annual average relative humidity of 89%, and an annual precipitation rate of 437.6 mm [48]. Sixtyeight lichen species and 32 moss species with two vascular plants of Deschampsia antarctica and Colobanthus quitensis have been reported in this area [49]. A chisel was used to obtain the samples, and they were transported to the laboratory in Korea at −20 • C, where they were preserved at −20 • C until use.

Genomic DNA Extraction, Amplification, and Bacterial Community Analysis
Samples were ground into fine powder using the TissueLyser II (Qiagen, Hilden, Germany) after freeze-drying, and genomic DNA was extracted from approximately 0.5 g of sample using an Exgene Soil DNA mini kit (GeneAll, Seoul, Korea) in accordance with the manufacturer's instructions. The V4-V5 regions of the bacterial 16S rRNA genes were amplified with polymerase chain reaction using the primer pair 515F/926R [43,50] at Integrated Microbiome Resource (Dalhousie University, Halifax, N.S., Canada, http: //cgeb-imr.ca accessed on 29 January 2021) in 2017. Sequencing of the amplicons was conducted by IMR using the paired-end (2 × 300 bp) Illumina MiSeq system (Illumina, San Diego, CA, USA). The adapter and primer sequences were removed using Cutadapt v2.10 [51], and the resultant sequences were processed by DADA2 v0.9.5 [52] to infer amplicon sequence variants (ASVs), which allowed a single nucleotide resolution. For quality trimming, a relaxed filtering option on the reverse reads as maxEE = c (2, 2) was applied, and the low-quality tails of each forward and reverse read were removed with truncLen = c (270, 210). Read pairs were then denoised based on a DADA2 error model, merged, and chimeras were removed by de novo approach. Representative ASV sequences were then taxonomically assigned against the EzBiocloud database [53]. All sequence data used in this study are deposited to the Sequence Read Archive (SRA) at NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) under the accession numbers SRR13490317-SRR13490322.

Isolation of Bacterial Strains
The specimens were washed for 10 min in 1 mL of 0.85% NaCl by vortexing in a Multi-EP tube vortexer (FinePCR, Gyeonggi, Korea) followed by centrifugation at 10,000 rpm (Eppendorf, Hamburg, Germany) for 5 min, discarding the supernatant. The process was repeated four times. After the final wash, the samples were crushed in a TissueLyzer II containing steel beads (Qiagen, Hilden, Germany) twice for 2 min. One hundred microliters of the final suspension was then spread on 0.01 × Reasoner's 2A (R2A), 0.1 × R2A, 0.01 × Inorganic-Salts Starch (ISP4), 0.1 × ISP4, Malt-Yeast extract (MY), and Nitrogenfixing bacteria (Nfb) [54] solid media. The plates were incubated at 10 • C for 25 days. After the incubation, bacterial colonies from the agar plates were picked based on their morphology and subcultured on fresh R2A agar medium three or more times until pure isolates were obtained. Pure cultures of the bacterial isolates were preserved in 20% (v/v) glycerol at −80 • C.

Identification of Bacterial Isolates
Genomic DNA of cultured isolates was extracted by using the Mini Tissue DNA kit (Cosmo Genetech Inc., Seoul, Korea) in accordance with the manufacturer's instructions. The 16S rRNA gene was amplified with two universal primers, 518F and 800R [55]. PCR products were purified using LaboPass PCR purification kit (Cosmo Genetech Inc., Seoul, Korea) and sequenced with the same primers used for amplification. The sequences of the 16S rRNA gene were compared with those of type strains available in the EzBioCloud database (ChunLab Inc., Seoul, Korea) [53] to find closely related species and to choose reference sequences for the phylogenetic analyses. Phylogenetic trees of the 16S rRNA gene sequences were reconstructed by the maximum likelihood method based on the GTR evolutionary model and the search options of best tree topology finding by branch swapping of NNIs and SPRs using MEGA X program [56]. The robustness of the tree topologies was assessed by bootstrap analyses based on 1000 replications of the sequences. The species affiliation of a bacterial isolate was determined when the isolate formed a monophyletic group with the reference species and the sequence similarity was 98.65% or higher. The sequences were deposited in GenBank (National Center for Biotechnology Information, Bethesda, MD, USA) under the accession numbers MW507603-MW507719.

Examination of Bacterial Physiological Characteristics
To determine physiological characteristics, cell suspensions were replica plated on square dishes of diverse solid media with a 96-pin replicator (VP-408B, V&P Scientific, San Diego, CA, USA). Cell suspensions were prepared by adding a half-full loop (5 mm diameter) of cells from the agar plates to 500 µL of 0.85% NaCl with vigorous shaking using a vortex mixer. To inoculate bacterial isolates, 200 µL of the suspension was then transferred to 96-well plates. For the determination of optimal growth temperature, cell suspensions were replica plated on R2A with a 96-pin replicator and then incubated for 21 days at 0, 4, 10, 15, 20, 25, 30, or 37 • C. Growth was evaluated by scoring the size and turbidity of the colonies as described in Lee et al. [56]. Secretion of extracellular protease, lipase, amylase, and chitinase was examined by replica plating of the cell suspensions onto 0.1 × R2A plates supplemented with 1% skim milk (Fluka, Dorset, UK), 1% tributyrate (Sigma-Aldrich, St. Louis, MO, USA), 1% starch (Alfa Aesar, Ward Hill, MA, USA), or 1% chitin (Wako Chemicals, Richmond, VA, USA), respectively, and incubating at 10 • C for 21 days. Enzyme activity was expressed as scores based on the ratio of colony size to the width of the clear zone surrounding the colony as described in Lee et al. [57]. Hydrolysis of polymer and purine utilization were examined on 0.1 × R2A plates supplemented with Tween 20 (Duksan, Gyeonggi, Korea), 40, 60, or 80 (Sigma-Aldrich) and xanthine (Avocado Research Chemicals Ltd., London, UK) or hypoxanthine (Sigma-Aldrich), respectively.
Indole-3-acetic acid (IAA) excretion by bacterial strains was determined by means of a modified colorimetric analysis [60]. Cells from the agar plates were inoculated into 1 mL of R2B medium with L-tryptophan (0.2 g/L) in triplicate. After cultivation at 15 • C and 150 rpm shaking in the absence of light until OD600 of the cell suspension was approximately 0.5, and cultures were centrifuged at 10,000 rpm for 10 min. The cell-free supernatant was mixed with Salkowski reagent (50.0 mM FeCl 3 , 35.0% (v/v) perchloric acid) at the ratio of 3:2 and incubated for 30 min in the absence of light. IAA concentration was measured spectrophotometrically using an Envision microplate reader (PerkinElmer, Waltham, MA, USA) at 530 nm; the IAA concentration was calculated according to a standard curve.
Nitrogen fixation ability was tested by cultivation on Nfb solid medium. Isolates growing with scores of ≥3 on the Nfb solid medium were denoted as nitrogen-fixing bacteria.

Genome Sequencing and Analysis
Genomic sequences of PAMC 29362 and PAMC 29367 of the genus Polymorphobacter were obtained by sequencing with Illumina HiSeq (Macrogen, Seoul, Korea) and assembled with Unicycler assembler version 0.4.9b [61]. Genome annotation was performed using the Rapid Annotation using Subsystems Technology (RAST) server [62]. Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology was performed using KEGG Automatic Annotation Server (KAAS) (Kyoto University, Uji, Kyoto, Japan) [63]. The GenBank/EMBL/DDBJ accession numbers for the draft genome sequences of PAMC 29362 and PAMC 29367 are deposited under BioProject ID PRJNA693408.

Bacterial Community Structure Based on the Cultivation-Independent and -Dependent Approaches
Sequencing of the 16S rRNA genes in genomic DNA extracted from six lichens of the genera Cetraria, Cladonia, Megaspora, Pseudephebe, Psoroma, and Sphaerophorus produced 6501 to 14,292 reads after the quality check (Table S1). Plastid and non-bacterial sequences were excluded from further analyses. A total of 297 ASVs were observed, and each sample included from 23 to 112 ASVs (Table S1). Proteobacteria (69.7-96.7%) and Acidobacteria (0.3-30.2%) dominated across the samples ( Figure S1). At the class level of Proteobacteria, Alphaproteobacteria accounted for most of the Proteobacteria, ranging from 69.5 to 95.9% ( Figure S1). At the order level, Alphaproteobacteria was mostly represented by Rhodospirillales (64.2-87.7%), followed by Rhizobiales (0.2-13.0%), Caulobacterales (0.0-2.1%), and Sphingomonadales (0.0-0.5%) ( Figure S1). Rhodospirillales accounted for a higher proportion than Rhizobiales across the six samples; the proportion of Rhizobiales was more than 10% of the bacterial communities only in lichens of Megaspora (12.1%) and Psoroma (13.0%) ( Figure S1). Approximately 400 lichen species are known in Antarctica, and lichens are a major terrestrial vegetation type in Antarctica [64,65]. Lichen-associated bacteria are considered to contribute to the tolerance of lichens to the extreme environments of Antarctica. However, only a few studies on the bacterial communities of lichens from Antarctica based on culture-independent approaches have been performed to date [21,25,38]. This study provides the first report of the bacterial communities of Megaspora, Pseudephebe, Psoroma, and Sphaerophorus based on the cultivation-independent approach. Bacterial communities of lichens are structured by various factors such as host, photobiont, geography, and substrates [15,21,25,26,37,38]. Consistent with previous studies on the Antarctic lichen microbiome of Amandinea, Buellia, Cetraria, Cladonia, Ochrolechia, Umbilicaria, and Usnea, Alphaproteobacteria was the major bacterial class with higher proportions of Rhodospirillales than that of Rhizobiales at the order level [21,25,38].
The overall similarity of 16S rRNA gene sequences of the bacterial isolates to the known type strains ranged from 92.6% to 100%, and 46 isolates had ≤98.65% similarity, the threshold for differentiating two species [66], indicating that they are potential novel species. When comparing the cultivation-independent results with cultured isolates, 28 ASVs out of 50 major ASVs with more than 1% in relative abundance of bacterial communities in at least one sample showed low similarity (91.5-97.9%) with cultured isolates obtained in this study (Table S3). In particular, ASV00007, ASV00020, and ASV00027 of the order Rhodospirillales with proportions of >24% of bacterial communities in each sample showed 95.4-96.3% 16S rRNA gene similarity with PAMC 29349 (Table S3), indicating that traditional cultivation methods have failed to obtain ecologically more relevant microorganisms in this study. Nonetheless, lichen-associated isolates of the genus Polymorphobacter from crustose Megaspora cultured only in this study are very interesting. The strains of the genus Polymorphobacter growing at low temperatures (−4 • C to 4 • C) were inferred to be an aerobic anoxygenic photoheterotrophic bacterium, based on the presence of the photosynthetic gene pufML or bacteriochlorophyll a [67,68]. Polymorphobacter sp. strains in this study had 94.8-97.0% 16S rRNA gene sequence similarity with Polymorphobacter arshaanensis and genome sequence of PAMC 29362 and PAMC 29367 revealed that these strains include most of genes involved in photosynthesis and synthesis of bacteriochlorophyll a (Table S4). Phototrophy by lichen-associated bacteria that harvest sunlight abundantly present during the austral summer in Antarctica and convert it into chemical energy might enhance the survival of lichens in the oligotrophic environment [69]. Thus, despite the cultivation anomaly not fully reflecting the environmental diversity, cultured isolates provide the ability to test putative functions to fill the gap regarding the functional capacities inferred from molecular approaches.

Physiological Characteristics
All 117 bacterial isolates grew at 10 and 15 • C and most of the strains (105) were able to grow at 4 • C. However, the number of strains that can grow at higher temperature decreased to 96, 77, and 55 at 20, 25, and 30 • C, respectively. No strains could grow at 37 • C ( Figure 2). Extracellular protease activities were detected in 9 isolates affiliated with Streptacidiphilus carbonis (1), Sphingomonas gracilis  (Table 3 and Table S2).  Table 3). Hydrolysis of xanthine was observed in 14 isolates of the genera Streptacidiphilus (1) and Caballeronia (13), and hydrolysis of hypoxanthine was observed in 12 isolates of the genera Subtercola (1), Streptacidiphilus (1), Sphingomonas (3), and Caballeronia (7), supporting the putative involvement of lichen-associated bacteria in nutrient cycling in the lichens.

Physiological Characteristics
All 117 bacterial isolates grew at 10 and 15 °C and most of the strains (105) were able to grow at 4 °C. However, the number of strains that can grow at higher temperature decreased to 96, 77, and 55 at 20, 25, and 30 °C, respectively. No strains could grow at 37 °C (Figure 2). (1), and Caballeronia mineralivorans (6) ( Table 3). Hydrolysis of xanthine was observed in 14 isolates of the genera Streptacidiphilus (1) and Caballeronia (13), and hydrolysis of hypo-  (Table 3 and Table S2). High phosphate solubilization activity (≥3) was observed in the strains belonging to Lichenicola, Caballeronia, Massilia, and Pseudomonas (Table S2). Phosphorus is a primary element involved in all major metabolic pathways [70,71]. In nature, large portions of phosphorus are present in insoluble form that cannot be directly used by organisms [72]. Interestingly, isolates of Lichenicola (order Rhodospirillales) and Lichenihabitans psoromatis (order Rhizobiales) were capable of solubilizing inorganic phosphate. Although bacterial taxa belonging to orders Rhodospirillales and the LAR1 lineage of Rhizobiales dominated across diverse lichens, only a few bacterial taxa belonging to orders Rhodospirillales and the LAR1 lineage of Rhizobiales have been successfully cultivated from lichen [18,33,[44][45][46][47]. The phosphate-solubilizing ability of those strains has not yet been determined [27]. Most strains (10/13) of the genus Caballeronia solubilized inorganic phosphate in this study. Considering that members of Caballeronia were commonly obtained from Arctic and Antarctic lichens [18] and identification of the capability of Caballeronia strains in this study to solubilize inorganic phosphate, phosphate solubilizing lichen-associated bacteria could be of great importance for lichen colonization in the oligotrophic environments of Antarctica.
IAA was detected in the supernatant of 67 bacterial culture (57%) ( Table 3). Secretion of IAA (more than 5 ng/µL) was detected in isolates belonging to the genera Frondihabitans, Galbialea, Lacisediminihabitans, Subtercola, Rhodococcus, Lichenibacterium, Lichenihabitans, Methylobacterium, Lichenicola, Polymorphobacter, Sphingomonas, Caballeronia, Massilia, Rhodanobacter, Pseudomonas, and Pedobacter. Production of IAA has been reported in lichen, lichen-forming fungi and lichen-associated bacteria [8,29,[73][74][75]. IAA from lichen-forming fungi enhances the water uptake in higher plants and is also related to promotion of cell growth, biomass production, and intracellular concentrations of chlorophyll a, carotenoids, and lipids biosynthesis of the green algae Scenedesmus quadricauda [76,77]. Pichler et al. showed that three lichen forming fungi-Cladonia grayi Sandst., Xanthoria parietina (L.) Th. Fr., and Tephromela atra (Huds.) Hafellner-produced IAA that was secreted exogenously and increased the water contents of their microalgae by up to 4.4% [74]. Thus, it was suspected that bacterial IAA in lichen supports the amicable interaction between lichen-associated bacteria and the microalgae as well as the lichen forming fungi in lichen symbiosis [12,29]. This could dramatically help the symbiotic relationship between algae with lichen forming fungi when it comes to a certain environmental status that can resume photosynthesis and thus support lichen survival.
Thirty-five strains of the phylotypes Frondihabitans peucedani   (Table 3). All cultured strains of Caballeronia in this study and Methylorosula sp., Sphingomonas aerolata, and S. glacialis grew on Nfb medium. The ability of members of Caballeronia (previously known as Burkholderia) to fix atmospheric nitrogen is known [78] and the nifH gene, responsible for production of the nitrogenase Fe protein subunit, was successfully amplified in some cultured isolates of this genus from lichen Lobaria retigera [33]. Because acetylene reduction assay-positive but nifH-negative isolates were reported [8,79], we cannot exclude the possibility that nifH-negative isolates can fix nitrogen via an alternative system or have divergent genes. Furthermore, 35% of the total isolates grown on Nfb medium in this study, may grow using the nitrogenous elements in agar instead of atmospheric nitrogen fixation. Order Rhodospirillales and the LAR1 lineage of Rhizobiales (Alphaproteobacteria) include many nitrogen-fixing taxa that can form plant-associated root nodules [40][41][42][43]80,81]. Previous studies based on molecular approaches suggested the nitrogen fixing function of these bacteria in lichen symbioses [12,27,39]. Interestingly, most of the bacterial isolates of orders Rhodospirillales and the LAR1 lineage obtained in this study did not grow on Nfb medium. Instead, many strains of the genus Sphingomonas of Sphingomonadales could fix nitrogen in this study. Nitrogen-fixing gammaproteobacterial isolates have been cultivated from cyanobacteria-deprived lichens [8]. However, gammaproteobacterial isolates in this study did not grow on Nfb medium. Both cyanolichens, which have symbiotic relationships with cyanobacteria to fix nitrogen in the cephalodium, and many chlorolichens can grow in nutrient poor environments [3,82]. Lichens harboring nitrogen fixers of diverse taxa of Actinobacteria, Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes, which were detected across the lichens, could be competitive to survive in nitrogen-limiting environments.
In this study, the comparison between culture-dependent and culture-independent bacterial communities revealed a limited overlap. However, psychrotolerant bacterial isolates of diverse phylotypes showed lytic, nitrogen-fixing, and phosphate-solubilizing ability that could significantly enhance the mobilization of nutrients in lichen symbioses. In particular, strains of the orders Rhodospirillales and the LAR1 lineage of Rhizobiales, which were predominant in lichen-associated bacterial communities, did not have nitrogenfixing ability. Instead, they solubilized inorganic phosphates, showing the importance of cultivation for testing the metabolic potential and expanding knowledge about the functions of dominant bacterial taxa in lichens. Furthermore, the phototrophic potential of lichen-associated bacteria suggested for the first time in this study will contribute to the significant roles of lichen-associated bacteria for lichens to adapt to the oligotrophic environment. Although the knowledge gap about bacterial functions in the context of lichen symbioses still exists, the bacterial isolates obtained in this study can be used to verify and better understand the functions of lichen-associated bacteria.