Samples were collected from the JPL-SAF, Pasadena, California from 10 locations of 1 m² area using sterile polyester wipes (23 cm × 23 cm; ITW Texwipe, Mahwah, NJ, United States) premoisented with phosphate buffer saline (PBS). Subsequently, each wipe was individually transferred to 200 mL PBS and vortexed at maximum speed for 5 s. The resulting suspension was then concentrated to appropriate volume (∼30-fold) with a concentration pipette CP-150 (Innova Prep, Drexel, MO, United States) using a 0.20 μm hollow fiber polysulfone tip (Kwan et al., 2011). The concentrated samples were then split in two 3 mL aliquots where one aliquot was treated with 100 μg/mL of chloramphenicol (Sigma, St. Louis, MO, United States) and the other aliquot was untreated. The sample amended with chloramphenicol was incubated at 25°C for 24 h and subsequently subjected to downstream processing utilizing both traditional microbiology and molecular biological techniques. At the time the experiments were conducted, the aim was the isolation of novel fungal species thriving in the JPL SAF, and hence a 100 μL suspension of the samples that were treated with and without chloramphenicol was spread onto antibiotic supplemented (chloramphenicol 100 μg/mL) media like potato dextrose agar (PDA) and Dichloran Rose Bengal Chloramphenicol Agar (DRBC) and incubated at 25°C for 5 days. Among several fungal colonies purified, some bacterial colonies also surfaced onto the PDA and DRBC media, and were selected for further characterization. Since the isolation was carried out on antibiotic treated samples, bacteria exhibiting resistance to chloramphenicol were interesting. The bacterial strains resistant to chloramphenicol that exhibited higher 16S rRNA gene similarities (>99%) with already described species were not further studied in this project. However, one of the several bacterial colonies (strain B12^T) showed 97.9% 16S rRNA gene sequence similarity with its closest neighbor (see below for details). Strain B12^T was further assayed for its polyphasic taxonomic and WGS characterization. Distinct colonies of strain B12^T were isolated and transferred to fresh PDA medium and subsequently archived in semi-solid R2A media and stored at room temperature as well as in glycerol stock for further characterization.

The cells of strain B12^T were fixed for scanning electron microscopy (SEM) by first suspending two separate colonies of the strain B12^T in 0.1 M phosphate buffered saline (PBS, pH 7.2; Sigma-Aldrich) in separate 1.5 mL microcentrifuge tubes. In order to obtain B12^T cells with decreased amounts of extracellular polysaccharides (EPS), the first tube was vigorously pipetted and then vortexed for 30 s, followed by filtration of the suspension through a 0.2 μm polycarbonate filter membrane on a vacuum manifold. The original tube was washed two more times with 0.1 M PBS, and then passed through the same filter. The filter membrane was then removed and placed into a fresh 1.5 mL microcentrifuge tube. The unbroken-up colony of B12^T (with preserved EPS) had excess PBS aliquoted off. The following steps were performed for both samples identically. Suitable aliquots (750 μL) of 2.5% glutaraldehyde in 0.1 M PBS was added to each tube and then incubated in the refrigerator at 4°C for 1 h. Without disturbing the colony or the filter membrane, the majority of the solution was aliquoted from the microcentrifuge tube and replaced with 1 mL of 0.1 M PBS, and returned to the refrigerator for a 10 min. incubation. This wash step was repeated for a total of three times. Next the samples underwent an ethanol (EtOH) dehydration series. The solution was aliquoted out of the microcentrifuge tubes and replaced with an increasing concentration of EtOH diluted in 0.1 M PBS. After each new EtOH solution was added, the tube was incubated in the refrigerator for 10 min. The EtOH concentrations were 50, 70, 80, 90, 95, and 100%. The 100% EtOH was aliquoted out and replaced with fresh 100% EtOH a total of three times, and stored in the refrigerator. The samples then underwent critical point drying in a Tousimis Automegasamdri 915B critical point dryer (Rockville, MD, United States). Samples were then adhered to carbon tape (Ted Pella Inc., Redding, CA, United States) and sputter coated with AuPd using an Anatech Hummer (Sparks, NV, United States) sputter coater. SEM was performed on a FEI Quanta 200F (Themo Fisher, Waltham, MA, United States).

Phenotypic characterization of the strain B12^T was performed by following standardized protocols (Jones, 1981). For phenotypic tests, strain B12^T was grown in sterile peptone-tryptone-yeast extract-glucose (PTYG; 5 g each per liter) medium incubated at 32°C with pH 7 and 0.5% NaCl, unless otherwise stated. Morphology, size, and pigmentation were observed on PTYG medium after 72 h of incubation. A commercially available kit (BD Difco) was used to determine the Gram-staining status of the strain. Motility was determined by inoculating a loopful of culture into a PTYG broth and incubating it for a period of 72 h. Subsequently, a loopful of broth was tested for motility using a previously established technique (Skerman, 1960). Growth in various temperature conditions (5–45°C) was tested by increasing incubation temperature in increments of 5°C and grown in PTYG broth. Similarly, the pH tolerance (4–10) was tested by adjusting the pH of the PTYG broth with biological buffers (Xu et al., 2005). The NaCl tolerance (0–5%) tests were carried out in 1% sterile peptone broth containing appropriate amounts of NaCl. The carbon substrate utilization profile was carried out as per the BioLog protocol for actinobacteria using GEN III MicroPlate test assay with a Biolog system. The test panel comprises 71 carbon sources with 23 chemical sensitivity assays and thus provides a “Phenotypic Fingerprint” of the tested microorganism. Since B12^T cells grown either in tryptic soy broth or Luria broth and washed in buffer before placing in BioLog plates did not exhibit any carbon substrate utilization profile, cells were grown in PTYG medium but such modified growth medium also did not show carbon utilization in BioLog plates. This is unusual for the bacterium not to utilize any of the carbon substrate provided in BioLog. The BioLog system determines respiratory activity and the tetrazolium dye used to assay the respiratory activity might be potentially toxic to certain organisms. More research is needed for identifying appropriate growth promoting substances before defining the carbon substrate utilization profile of strain B12^T.

Cellular fatty acids were analyzed by collecting biomass of a freshly grown culture at optimum growth conditions stated above. The cellular fatty acids were extracted, methylated, and analyzed by gas chromatography, as per the Sherlock Microbial Identification System (MIDI version 4.0) described previously (Muller et al., 1998; Pandey et al., 2002). A combined analysis by gas chromatography coupled to a mass spectrometer was used to confirm the identity of the fatty acids based on retention time and mass spectral data. The position of the double bond was confirmed by a derivatization to the corresponding dimethyl disulfide adduct (Moss and Lambert-Fair, 1989). Whole cell sugars and the peptidoglycan structure were determined according to Schumann (2011). The analyses of respiratory quinones and polar lipids were performed with 200 mg freeze-dried cells, which were previously incubated in glucose yeast extract malt extract medium (GYM) medium at 28°C and harvested in the stationary phase. The extraction of quinones was carried out using the two-stage method (Tindall, 1990a).

Polar lipids were separated by two-dimensional silica gel thin layer chromatography (Macherey-Nagel, 2007). The first direction was developed in chloroform:methanol:water (65:25:4, v/v/v), and the second in chloroform:methanol:acetic acid:water (80:12:15:4, v/v/v/v). Total lipid material was detected using molybdatophosphoric acid and specific functional groups by using spray reagents specific for the groups (Tindall, 1990b).

A loopful of purified B12^T culture was subjected to DNA extraction with the Quick DNA Fungal/Bacterial Miniprep kit (Zymo Research, Irvine, CA, United States), as per the manufacturer’s protocol. The extracted DNA was eluted in 50 μL of molecular grade water and stored at −20°C until further analysis. The 16S rRNA gene (Suzuki et al., 2001; Checinska et al., 2015) was amplified using a universal primer set (Yamamoto and Harayama, 1995) as per previously established protocols. The amplified products were treated with Antarctic phosphatase and exonuclease (New England Biolabs, Ipswich, MA, United States) to remove 5′- and 3′-phosphates from unused dNTPs before sequencing. The resulting sequences were assembled using SeqMan Pro from the DNAStar Lasergene package (DNASTAR Inc., Madison, WI, United States). Bacterial sequences were compared with the EzTaxon-e and EzBioCloud databases (Kim et al., 2012; Yoon et al., 2017) and identified based on the closest percentage similarity to previously identified microbial type strains. An alignment of the B12^T 16S rRNA Sanger sequence and those collected from 50 members of class Actinobacteria found in public database was created using Clustal Omega (v. 1.2.1). A maximum-likelihood phylogeny based on this 16S rRNA gene alignment was generated using FastTree (v. 2.1.10), and bootstrap values were calculated using PHYLIP Seqboot (v. 3.696) and a script provided by the authors of FastTree, CompareToBootstrap.pl. Neighbor-joining and maximum-parsimony phylogenies based on the same 16S rRNA gene alignment were generated using PHYLIP (v. 3.696) and nodes with the same branching pattern in all three algorithms are highlighted in the phylogenetic tree (Figure 2).

The sequencing and analysis of the WGS were carried out as previously described (Singh et al., 2017) with minor modifications. In brief, sequencing libraries from isolated B12^T strain DNA was prepared using the Nextera DNA Library Preparation Kit from Illumina as per the manufacturer’s instructions. Paired-end sequencing (100 bp) was performed on an Illumina HiSeq 2500 instrument. The data was filtered for high-quality vector, and adapter free reads using cutoff read length of 80 bp and quality score of 20 for genome assembly by using the NGS QC Toolkit v2.3 (Patel and Jain, 2012). The high-quality vector filtered reads were then assembled using the SPAdes genome assembler (Nurk et al., 2013) with default parameters. Subsequently, the assembled genome was annotated using Rapid Annotations using Subsystems Technology (RAST) (Aziz et al., 2008), and their quality was assessed using the Quast package (Gurevich et al., 2013). Furthermore, pairwise ANI was calculated using the established algorithm (Nurk et al., 2013) with EzTaxon-e (Kim et al., 2012). Pairwise AAI comparisons were calculated using an established method (Konstantinidis and Tiedje, 2005). Additionally, dDDH analysis was performed using the Genome-to-Genome Distance Calculator 2.0 (GGDC 2.0) (Meier-Kolthoff et al., 2013). A whole-genome alignment was generated using the CLC (v. 20.0.2) whole genome alignment plugin, and a phylogenetic tree was generated from the alignment using FastTree (v. 2.1.10).

Multi-locus sequence analysis (MLSA) based phylogenetic affiliation was performed as reported elsewhere to interpret the phylogenetic affiliation of the Kineosporiaceae members considered in this study (Gao and Gupta, 2005). Representative genomes of Angustibacter, Kineococcus, Kineosporia, Pseudo kineococcus, Quadrisphaera, and Thalassiella were used to determine the correct phylogenetic position of strain B12^T. Nakamurella multipartita DSM 44233^T was included in the phylogeny to serve as the out-group. Members of genus Kineosporia and Thalassiella were not selected for MLSA due to the absence of full-length genes in the publicly available database. Full-length DNA sequences for housekeeping genes frr, gyrB, nusA, rplS, rpsB, and tsf were retrieved from eight genomes (including strain B12^T), and an MLSA phylogenetic tree was generated. Gene sequences were individually aligned using Clustal Omega (v. 1.2.1), and concatenated together using a custom Perl script in the order listed. An approximate maximum-likelihood phylogenetic tree was generated from concatenated sequences using FastTree (v. 2.1.10), and bootstrap values were added to the tree using PHYLIP Seqboot (v. 3.696) and the CompareToBootstrap.pl script provided by the authors of FastTree.

Cells of strain B12^T were non-spore forming, Gram-positive, motile, and strictly aerobic. Orange pigmented, round with rough edges and cluster forming colonies (0.6–1.0 mm diameter) were isolated after 72 h of incubation. The optimum growth conditions for the cells of strain B12^T were 32°C, 7 to 8 pH, and 0.5% NaCl concentration. Since strain B12^T is a slow grower, at least 3–5 days of incubation time was required to see the first visible growth when incubated at the optimum cultural conditions in PTYG medium. Since strain B12^T was isolated from chloramphenicol supplemented medium, the purified strain was re-streaked again in PTYG agar medium supplemented with 100 μg chloramphenicol, confirming that strain B12^T was resistant to chloramphenicol. The SEM images revealed that the cells of strain B12^T are coccoid in shape with 1.0 μm diameter (Figure 1). In general, cells of strain B12^T form in clusters due to extracellular polymeric substance (Figure 1), but individual motile cells were also observed.

Comparison of phenotypic characteristics of strain B12^T with other members of genus Kineococcus are provided in Table 1. Briefly, B12^T most closely matches characteristics of its closest neighbor, K. radiotolerans, except B12^T strain was oxidase positive. Since B12^T strain was not able to grow in a minimal media with less than 0.1% glucose or 0.1% yeast extract, phenotypic screening for carbon sources was not successful; instead, genome based phenotypic characterization was performed (Supplementary Table S1). All Kineococcus species, including the B12^T strain, could be differentiated from K. radiotolerans by the utilization of d-fructose and l-alanine as sole carbon and nitrogen sources, respectively. The absence of sucrose utilization by K. endophytica and K. rhizosphaerae might differentiate them from other members of the Kineococcus genera, including the B12^T strain. Likewise, d-xylitol assimilation and milk peptonization were observed in K. glutinatus, but not in other Kineococcus species. L-tryptophan was utilized by most Kineococcus species, including strain B12^T, except by K. glutinatus and K. gypseus. Gelatin was hydrolyzed by only K. aureolus and K. rhizosphaerae. K. aurantiacus and K. radiotolerans were the only members of the genus, including B12^T, to lack the capability for nitrate reduction. In addition, strain B12^T had different characteristic cell wall sugars (Gal, Glu, and Man instead of Ara, and Gal) when compared to other members of the Kineococcus genus.

FAME profiles of strain B12^T and related Kineococcus genera are shown in Table 2. The majority of the fatty acids produced by strain B12^T consisted of anteiso 15:0 (66.3%), similar to other members of Kineococcus genera (58–77%). Minor amounts of other fatty acids were detected including anteiso-C_15:1 ω10c (4.6%), iso-C_14:0 (1.0%), iso-C_15:0 (1.1%), and C_14:0 (1.6%). The peak assigned to C_14:0 2OH by the MIDI system was identified as anteiso-C_15:0 dimethyl acetal (DMA) (2.9%) by GC-MS. Further two peaks that were assigned as C_20:4 (16.4%) and C_17:0 2OH (2.2%) by the MIDI system, could not be confirmed to represent fatty acid methyl esters by GC-MS and have to be assigned as non-identified hydrophobic compounds. The presence of DMA derivatives in the sample appeared at first sight to be surprising as they are usually only detected in anaerobic bacteria but they show a clear diagnostic fragment at m/z 75 [(M-OCH₃)⁺] and a typical [M–31]⁺ fragment in the GC-MS analysis (Maulik et al., 1993; Alves et al., 2013). The presence of significant amounts of branched chain dimethylacetals has been detected in the aerobic taxa Subtercola boreus and Subtercola frigoramans (Männistö et al., 2000) that was also confirmed by GC-MS. The misidentification of dimethylacetals may be attributed to the fact that the identification of DMA derivatives by the MIDI system is only possible with the use of the ANAEROBE six reference database. This is in contrast to the misidentification of hydroxylated fatty acids as dimethylacetals in members of the Selenomonadales (Moore et al., 1994; Helander and Haikara, 1995; Strompl et al., 2000). It is possible that DMA derivatives may be present in other members of genera Kineococcus but have been misidentified as hydroxy fatty acids by the MIDI system. The presence of dimethylacetals in the hydrolyzed cellular fatty acid fraction is also indicative of plasmalogens in the polar lipids.

The differential characteristics of various genera of the family Kineosporiaceae based on chemotaxonomic profiles are shown in Table 3. Most of these chemotaxonomic characteristics are similar to the other members of genus Kineococcus. The major menaquinone of strain B12^T is MK-9(H2), with minor amounts MK-9 and MK-8(H2) detected (Supplementary Figure S1). Mycolic acids were not detected. The polar lipid analysis of strain B12^T showed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, a monoacylated dimannosylphosphatdylinositol, several unidentified phosph- olipids, an unidentified glycolipid, and an unidentified lipid (Supplementary Figure S2). The polar lipids may contain plasmalogens. However, the novel strain B12^T showed major differences in its cell-wall diamino acid composition with the members of genus Kineococcus. The cell wall of strain B12^T contain meso-diaminopimelic acid, A1 gamma, and A31, whereas Kineococcus species were reported to contain only meso-diaminopimelic acid (Lee, 2006; Tamura et al., 2010). Furthermore, whole cell wall sugars detected for strain B12^T are galactose, glucose, and mannose, while other Kineococcus species possess galactose, and arabinose. Except a few, there were no major cell-wall and polar-lipid characteristic differences between the Kineococcus and related genera as shown in Table 3.

The16S rRNA gene sequences of ∼1,000 strains archived from the SAF were queried against the 16S rRNA gene retrieved from the WGS of strain B12^T. Phylogenetic analysis of the 16S rRNA gene (1,508 bp) indicated that it formed a distinct cluster with members of the genus Kineococcus within the radiation tolerant members of the family Kineosporiaceae. The 16S rRNA sequence of strain B12^T exhibited high sequence similarity with K. radiotolerans ATCC BAA 149 (97.9%). Low 16S rRNA gene sequence similarities (93.2–97.9%) with 28 members of the family Kineosporiaceae (Figure 2), including Angustibacter (93%), Kineococcus (96% to 97%), Kineosporia (94% to 95%), Pseudokineococcus (93%), Quadrisphaera (93%), and Thalassiella (94%), showed that strain B12^T belong to the members of this family. Similarly, when 1-kb gyrB sequences were retrieved from WGS of the strain B12^T and compared with available gyrB sequences (Figure 3) of other members of the family Kineosporiaceae, K. radiotolerans ATCC BAA 149 was the closest relative, but the low similarity percentage (85.6%) confirmed that strain B12^T is phylogenetically distinct from K. radiotolerans.

The MLSA phylogenetic tree with full-length DNA sequences for housekeeping genes frr, gyrB, nusA, rplS, rpsB, and tsf was generated and shown in Figure 4. Genomes belonging to Angustibacter, Kineococcus, Pseudokineococcus, and Quadrisphaera formed a family level clade but showed inter-genus distinction among themselves. Similar to the gyrB phylogeny, the MLSA tree clearly showed strain B12^T far separated from the type strain of K. radiotolerans, but within the Kineococcus clade.

The Illumina HiSeq 2500 platform yielded 5,435,202 paired-end reads from the sequencing of strain B12^T. Subsequent trimming and quality filtering of the paired-end sequences resulted in a total of 5,352,711 reads. The final assembled draft genome consists of 119 contigs comprising 4,880,137 bp with an N50 contig length of 66,629 bp. The largest contig assembled accounted for 392,027 bp. The final draft genome has a mean coverage of 100×, and G + C mol% of 74.16%, similar to other members of the Kineococcus genera. Table 4 provides a complete summary of genome assembly statistics.

The ANI, AAI, and dDDH values of the strain B12^T were compared with the members of the Kineococcus genus (Table 5). The closest genomic neighbor to strain B12^T was Kineococcus radiotolerans GCF_000017305.1, which was evident from the ANI, AAI, and dDNA-DNA hybridization values (Table 5). The ANI values showed 72–78% similarity, and AAI values showed 66–72% with other members of the Kineococcus genera, demonstrating that B12^T is a novel species within this genus (<95% for ANI and AAI). The dDDH comparison of strain B12^T genome with Kineococcus genera showed only 20–22% similarity, demonstrating that B12^T is a novel species (<70%) that is distantly related to other members of genera Kineococcus.

A phylogenetic tree (Figure 5) was generated from a whole-genome alignment of 17 genomes, employing FastTree (v. 2.1.10) and PHYLIP Seqboot (v. 3.696). Even though MLSA clearly placed strain B12^T within the Kineococcus genus, whole genome phylogenetic analysis was carried out to validate these results using all available WGS of Kineosporiaceae reference genomes (n = 17) from GenBank. The whole-genome analyses confirmed and gave a strong validation to the MLST/gyrB data, confirming that strain B12^T is a novel member of a genus Kineococcus.

The strain B12^T genome was analyzed to understand its genetic makeup and its metabolic potential. RAST annotation detected 4,872 coding sequences in the B12^T WGS. A major fraction of the annotated genes was comprised of amino acids and derivatives (299), carbohydrate metabolism (246), protein metabolism (207), genes associated with cofactors, vitamins, prosthetic groups, pigments metabolism (141), and DNA (100) and RNA (33) metabolism (Table 6). Genes responsible for motility and chemotaxis (36), metabolism of aromatic compounds (26), and stress response (48) were also observed.

A close look at the annotated draft genome (Supplementary Dataset 1) predicts that strain B12^T may be highly resistant to osmotic, oxidative, and periplasmic stress, a prime requirement for survival in a SAF-like ultra-low biomass environment regularly cleaned with industrial reagents (La Duc et al., 2012). Strain B12^T also harbors the degradation pathway for geraniol, dichlorodiphenyltrichloroethane, chlorocyclohexane, chlorobenzene, benzoate, bisphenol, fluorobenzoate, and furfural, which may provide a pathway for survival from industrial-strength cleaning reagents. Actinobacteria have been known to produce antibiotics, and the WGS of the strain B12^T also revealed genetic pathways related to the production of these antibiotics. Pathway analysis predict the production of novobiocin, puromycin, tetracycline, penicillin, and cephalosporin from the draft genome. The production of antibiotics would be helpful in reducing competition for B12^T, and may provide an advantage in the ultralow biomass SAF environment. This makes the organism B12^T a potential candidate for industrial use.

Strain B12^T shares a maximum 97.9% 16S rRNA similarity with K. radiotolerans, its closest phylogenetic neighbor within the Kineosporiaceae family. These values fall below the 98.7% threshold as demonstrated to delineate bacterial species (Stackebrandt and Ebers, 2006). The phenotypic, phylogenetic, morphological, and genomic characteristics provide evidence to differentiate strain B12^T from the members of family Kineosporiaceae. Similarly, the ANI, AAI, and dDDH values were lower than the threshold, further corroborating the findings to classify strain B12^T as a new species within the family Kineosporiaceae. Based on polytaxonomic and WGS analyses, a novel species, K. rubinsiae sp. nov., is proposed, with strain B12^T (=FJII-L1-CM-PAB2^T = DSM 110506^T) being the type strain of the species K. rubinsiae.

Kineococcus rubinsiae sp. nov. (ru.bin.si.ae. N.L. fem. n. rubinsiae named in honor of a NASA astronaut (Kate Rubins) who is a molecular microbiologist and the first person to perform DNA sequencing in space).

Cells are Gram-positive cocci that are 0.6–1.0 mm in diameter and occur in tetrads or clusters due to extracellular polysaccharide secretion. Colonies are circular, convex with a diameter of approximately 0.6–1.0 mm, and orange in color after 72 h of incubation on PDA or PTYG medium at 28 to 32°C. A slow growing aerobic bacterium with an optimum temperature of 32°C and incubation period of 3–5 days. Bacteria can exhibit motility after 72 h of growth in a liquid medium. Cells exhibit growth in a cluster formation. The pH tolerance is between 6.0 and 9.0, and shows positive growth at 0–5% NaCl. Cells will not utilize any of the carbon substrates in the BioLog system. Cells are resistant to chloramphenicol and multiply in the PDA supplemented with 100 μg/mL. The predominant fatty acid is anteiso-C_15:0. Whole cell sugars were galactose, glucose and mannose with minor amounts of arabinose and ribose. The major menaquinone is MK-9(H2) (Supplementary Figure S1). Polar lipids comprise diphosphatidylglycerol, phosphatidylglycerol, phosphatidyl inositol, an unidentified phospholipid, an unidentified glycolipid and an unidentified phosphoglycolipid (Supplementary Figure S2). The DNA G + C content of the type strain is 74.16 mol%.

The type strain, B12^T (=FJII-L1-CM-PAB2^T; NRRL B-65556^T = DSM 110506^T), was isolated from the East side of the JPL-SAF cleanroom where crew enter the room. WGS (VWPY00000000) and 16S rRNA Sanger sequence (MN493040) are available in NCBI GenBank.

The draft genome sequence of type strain B12^T was deposited in NCBI GenBank. The version described in this paper is the first version, and the accession number for the K. rubinsiae strain B12^T is VWPY00000000. The Sanger sequence of the 16S rRNA gene is deposited in GenBank under accession number MN493040.