- Extended genome report
- Open Access
Complete genome sequence of the thermophilic Acidobacteria, Pyrinomonas methylaliphatogenes type strain K22T
Standards in Genomic Sciencesvolume 10, Article number: 101 (2015)
Strain K22T is the type species of the recently- described genus Pyrinomonas, in subdivision 4 of the phylum Acidobacteria (Int J Syst Evol Micr. 2014; 64(1):220–7). It was isolated from geothermally-heated soil from Mt. Ngauruhoe, New Zealand, using low-nutrient medium. P. methylaliphatogenes K22T has a chemoheterotrophic metabolism; it can hydrolyze a limited range of simple carbohydrates and polypeptides. Its cell membrane is dominated by iso-branching fatty acids, and up to 40 % of its lipid content is membrane-spanning and ether lipids. It is obligately aerobic, thermophilic, moderately acidophilic, and non-spore-forming. The 3,788,560 bp genome of P. methylaliphatogenes K22T has a G + C content of 59.36 % and contains 3,189 protein-encoding and 55 non-coding RNA genes. Genomic analysis was consistent with nutritional requirements; in particular, the identified transporter classes reflect the oligotrophic nature of this strain.
Phylotypes from the phylum Acidobacteria Footnote 1 are commonly detected across a range of ecosystems, including marine and freshwater bodies, sediments, geothermal systems, and soils. Despite the apparent ubiquitous distribution acidobacterial phyotypes, particularly in soil environments, only 17 acidobacterial genera (represented by formal description and publication of respective type strains, in accordance with the International Code of Nomenclature of Prokaryotes ) have been validly published [2, 3]. Here we present a description of the complete genome sequence and annotation of Pyrinomonas methylaliphatogenes strain K22T (= DSM 25857 = ICMP 18710), the type species of the genus Pyrinomonas within subdivision 4 of Acidobacteria .
Pyrinomonas methylaliphatogenes K22T was isolated from a fumarole on the outer crater rim of the stratovolcano Mt. Ngauruhoe . It exhibits a Gram-negative cell wall, is non-spore-forming, and is catalase- and oxidase-positive (Table 1). It is a thermophilic and moderately acidophilic obligately aerobic chemoorganotroph. Of particular note is its unusual lipid composition that is dominated by odd-numbered saturated iso-branching fatty acids (iso-C15:0, iso-C17:0, iso-C19:0 and iso-C21:0 that total >88.5 % of the total fatty acid extract) . In addition, >40 % of the total membrane lipid content is made up by iso-branching glycerol ether analogues of the cellular fatty acids and membrane-spanning iso-diabolic acids . Membrane-spanning and ether lipids occur ubiquitously in Archaea, but in recent studies have also been commonly detected in cultivated representatives in subdivision groups 1, 3 and 4 of Acidobacteria [5, 6].
Subdivision 4 of the Acidobacteria has five validly-named species: P. methylaliphatogenes K22T, Chloracidobacterium thermophilum [7, 8], Blastocatella fastidiosa , Aridibacter famidurans , and Aridibacter kavangonensis . The latter three species are phylogenetically distant from P. methylaliphatogenes K22T, are mesophilic and have differing pH ranges and substrate utilization profiles from that of P. methylaliphatogenes K22T. Chloracidobacterium thermophilum is a moderately thermophilic facultatively anoxygenic photoheterotroph isolated from a hotspring microbial mat at Yellowstone National Park [7, 8]. An additional strain, Ellin6075 was isolated from an Australian pasture soil, and is a mesophilic heterotroph that derives its energy from complex carbohydrate sources, but has little information available regarding its phenotypic traits . Common features shared by subdivision 4 strains include an aerobic and heterotrophic phenotype [3, 4], and membrane lipid iso-diabolic acids .
Classification and features
Phylogenetic distances of closest-related phylotypes and cultivated subdivision 4 acidobacterial strains were determined by aligning the representative near full length 16S rRNA gene sequences (all sequences were > 1,400 nucleotides in length) and calculating sequence similarity via a pair-wise alignment within the ARB software environment . Analysis showed that the 16S rRNA gene sequence of P. methylaliphatogenes K22T (AM749787) is 85 % similar to B. fastidiosa strain A2-16T (JQ309130), and is 84 % similar to both A. famidurans strain A22_HD_4HT (KF245634), and A. kavangonensis Ac_23_E3T (KF245633) [3, 4, 9]. In addition, P. methylaliphatogenes K22T shares 85 % 16S rRNA gene sequence similarity with both Ellin6075 (AY234727)  and C. thermophilum BT (EF531339) . The most closely-related phylotypes to P. methylaliphatogenes K22T are two sequences from clonal libraries of environmental 16S rRNA genes (EU490264, EU490279) retrieved from geothermal soils on Mt. Erebus, Antarctica ; both of these shared 95 % 16S rRNA gene sequence similarity with P. methylaliphatogenes K22T. Phylogenetic comparison (Fig. 1) showed that P. methylaliphatogenes K22T is a taxonomically-distinct genus and species of subdivision 4 in the phylum Acidobacteria .
Pyrinomonas methylaliphatogenes K22T is non-motile and exhibits straight or bent rod cell morphology (0.3 – 0.6 μm in diameter and 1–4 μm in length) (Fig. 2). It has a temperature range (optimum) for growth of 50–69 °C (65 °C) and a pH range (optimum) of 4.1–7.8 (6.5). The bacterium has an obligately aerobic metabolism and can utilize a small selection of simple carbohydrates including glucose, lactate, alginate, mannose, xanthan, xylan, xylose, arabinose, and sucrose, as well as a limited variety of proteinaceous substrates including casamino acids, peptone, tryptone, yeast extract and nutrient broth (Table 1). It obtains nitrogen via the uptake of NO3 −, NH4 +, urea, yeast extract and casamino acids but cannot fix dinitrogen gas. The strain is not able to grow via photosynthesis, nor is it able grow autotrophically using CO2 as the sole source of carbon. However, optical density of culture is improved via the provision of additional CO2 in the headspace during heterotrophic growth, suggesting an assistive anapleurotic mechanism .
The primary cellular fatty acids are iso-C15:0 (40.8 %), iso-C17:0 (30.8 %), iso-C19:0 (12.1 %) and iso-C21:0 (4.8 %). P. methylaliphatogenes K22T also possesses membrane-spanning dicarboxylic acid 13,16-dimethyl octacosanedioic (iso-diabolic) acid and glyceryl ethers of alkyl analogues of iso-C15:0 and iso-C17:0 and iso-diabolic acid. Its primary cellular quinone is MK-8 and its primary cellular lipids are phosphatidylethanolamine and phosphatidylcholine .
Genome sequencing information
Genome project history
The genome of P. methylaliphatogenes K22T was selected for sequencing on the basis of its phylogenetic position and phenotypic dissimilarity to other cultured Acidobacteria strains. The quality draft (QD) assembly and annotation was completed in December 2013. The genome project is deposited in the Genomes OnLine Database Gp0050834. A summary of the project information is shown in Table 2. The EMBL-Bank project accession number is CBXV000000000 and consists of 16 scaffolds. Table 2 presents the project information and its association with MIGS version 2.0 compliance .
Growth conditions and genomic DNA preparation
Pyrinomonas methylaliphatogenes K22T was grown in 2 × 500 ml volumes of R2A liquid medium  at 60 °C in an air headspace (1 : 1 ratio of headspace to medium). The medium was sterilized at 121 °C (15 min, 15 psi) prior to inoculation. After three days of incubation, cells were collected via centrifugation. Culture purity was confirmed using an RFLP digestion (EcoR1) of the 16S rRNA gene PCR amplification product (amplification used the 9f/1492r primer set) . The restriction digest pattern was identical to known axenic cultures of P. methylaliphatogenes K22T. Genomic DNA was extracted from the wet biomass (200 mg) using the Nucleospin for Tissue extraction kit as per the manufacturer’s instructions (Macherey Nagel). The gDNA extract was purified via electrophoresis on a 0.8 % (w/v) agarose gel. The gel extracts were cleaned using a Gel Purification kit as per the manufacturer’s instructions (Macherey Nagel), giving a final concentration of 595 ng 100 μl−1. The purified gDNA was then frozen at −20 °C until sequenced.
Genome sequencing and assembly
Genomic sequencing was conducted using a combination of the Illumina MiSeq and 454 GS Junior platforms. A single-end 454 library was constructed according to the protocols of 454 GS FLX Titanium Rapid Library kits and GS Junior Titanium emPCR kits (Additional file 1). The sequencing of the 454 library yielded 75,215 reads with an average length of 492 bps. The paired-end Illumina library was constructed using the Nextera XT DNA Sample Preparation kit (Illumina), according to the manufacturer's protocol (Additional file 1), and sequenced on a MiSeq (2 × 150 bp paired-end reads), yielding 1,196,578 reads. The combined 454 (28.9 Mbp) and Illumina (301 Mbp) sequencing data were assembled together using the hybrid assembly capability of MIRA 4.0 rc4  (parameter and methodologies provided in Additional file 1). The resulting contigs were manually curated via the Staden package , generating scaffolds with an average 75 × coverage. Scaffolds with average coverage two standard deviations below the aforementioned overall genome average were discarded (i.e. 32.5 × coverage threshold). The resulting 16 scaffolds contained 2,302,690 assembled reads and 3188 protein coding genes. The abundance of clustered regularly interspaced short palindromic repeats (CRISPRs) and other repeating elements (e.g. transposons and RHS repeat-encoded genes) may have contributed to the scaffolds junctions, such as those observed in scaffold CBXV010000001, CBXV010000004, CBXV010000005, and CBXV010000006.
Genome annotation was processed via the DOE-JGI Integrated Microbial Genome – Expert Review (IMG-ER) annotation pipeline  using the following steps/components: Coding sequences (CDSs) were predicted using Prodigal . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to ascribe descriptions of the protein tRNAScan-SE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by searching against models of the ribosomal RNA genes built from SILVA. Other non-coding RNA such as the RNA components of the protein secretion complex and the RNaseP were identified by searching the genome for the corresponding Rfam profiles using INFERNAL . Transmembrane helices and signal peptide cleavage sites within the putative proteins were predicted via TMHMM , and SignalP  respectively. Additional annotation and gene function prediction as well as data visualization was conducted within the IMG-ER system .
The QD assembly of the genome consists of 16 scaffolds totaling 3,788,560 bp in length (59.36 % GC content). Of the 3,244 genes predicted, 3,189 were protein-coding genes, and 55 were non-coding RNA genes. A majority (79.0 %) of genes were assigned putative functions, and the remainder were annotated as hypothetical proteins. The properties and the statistics of the P. methylaliphatogenes K22T genome and the distribution of genes into COG functional categories are presented in Table 3, Table 4, and Fig. 3.
Insights from the genome sequence
The P. methylaliphatogenes K22T genome assembly has a size of 3.79 Mb with a %G + C content of 59.3, both of which are comparable with the genomes of other sequenced Acidobacteria . It possesses complete citric acid and pentose phosphate cycles. A complete electron transport pathway with an F-type ATPase, NADH dehydrogenase and cytochrome C complex, and the presence of genes encoding superoxide dismutase (PYK22_00483-00484) and catalase (PYK22_02691) are consistent with the observed aerobic phenotype. Genes encoding outer membrane secretion (for example, a type II secretion system, PYK22_02507-02511) and protein assembly (Bam complex, PYK22_02371 & 01777) are present, confirming the observed Gram-negative cell wall structure . Interestingly, P. methylaliphatogenes K22T possesses a near-complete complement of flagella encoding-genes (possibly missing the proximal rod flgF gene) despite having no observed motility. Key genes for all autotrophic carbon fixation pathways were absent. However, it was previously noted that while P. methylaliphatogenes K22T was unable to fix carbon, additional CO2 to the headspace while growing heterotrophically improved growth . The presence of phosphoenolpyruvate carboxylase and isocitrate dehydrogenase confirmed the ability of P. methylaliphatogenes K22T to supplement carbon anapleurotically. No genes encoding the ability to fix dinitrogen gas were found, again confirming previous phenotypic observations. Interestingly, the genome contains a gene cluster encoding a group 5-type [NiFe] hydrogenase (PYK22_03058-03084) similar to that found in Mycobacterium smegmatis ; this may confer an ability to oxidize tropospheric concentrations of hydrogen for cell maintenance.
Previous phenotypic characterization of P. methylaliphatogenes K22T indicated that it possessed a heterotrophic phenotype with the ability to grow on a range of simple carbohydrates. The P. methylaliphatogenes K22T genome encodes for a large number of beta-glucosidase and exoglucanase-acting glycosyl hydrolases, reflecting its ability to grow on primarily simple oligosaccharides such as cellobiose, sucrose, and maltose. A single C6 endoglucanase-acting glycosyl hydrolase (PYK22_03181) was identified in the genome despite having no reported growth on complex or crystalline cellulose as energy sources . Two endo-1,4-beta-xylanases genes confer an ability to grow on xylan and xanthan gum.
Transporters encoded in the P. methylaliphatogenes K22T genome mainly belong to the ABC-type transporter superfamily and the major facilitator superfamily. This is consistent with previous study of acidobacterial genomes, which suggest these transporters types were adapted for low-nutrient conditions . ABC transporters in P. methylaliphatogenes K22T appear to be involved in the transport of carbohydrates (and derivatives) such as ribose, D-xylose, lipopolysaccharide (rfbAB, e.g. PYK22_01076-77, PYK22_01839-40, PYK22_02287-88), and lipo-oligosaccharide (nodJI, PYK22_00778 and PYK22_00785). These reflect the carbohydrate and polypeptide utilizing phenotype of the bacterium. Pyrinomonas methylaliphatogenes K22T also possesses putative ABC transporters targeting amino acid cysteine, oligopeptides (oppABCDF, e.g. the PYK22_01277-281 cluster), and lipoproteins (lolCDE, PYK22_02373-4). Nitrogen assimilation is facilitated via an ammonia permease (PYK22_02853), the importation of oligopeptides by an oppABCDF ABC transporter system (similar to the system in Salmonella typhimurium ), and major facilitator superfamily nitrate/nitrite permeases (PYK22_00018 & PYK22_00946). Additionally, the P. methylaliphatogenes K22T genome contained a cluster of genes tonB-exbB-exbD-exbD (PYK22_00991-94) associated with siderophore transport in some other acidobacterial species . However, genes involved in siderophore synthesis, polyketide synthase, and nonribosomal peptide synthetase were not found, suggesting that it scavenges siderophores produced by other bacteria.
Based upon 16S rRNA gene sequence similarity, the most closely related and cultivated strain to P. methylaliphatogenes K22T is C. thermophilum BT  (Fig. 1). The sequence similarity (~86 %) indicates that the two strains may belong to the same subdivision based on taxonomic sequence identity thresholds calculated for other prokaryotic taxa . This phylogenetic dissimilarity between the two strains is also reflected in a comparison of the genomic content and the different metabolic modes of existence (chemoheterotrophic P. methylaliphatogenes K22T vs. photoheterotrophic C. thermophilum BT) of the two strains. For example, the C. thermophilum BT genome encodes for genes for chlorosomes, bacteriochlorophyll pigments a and c and a pigment protein complex for phototrophic growth, whereas no genes encoding for phototrophy were found in K22T. The C. thermophilum BT genome also contained significantly more COGs (15 vs 50) related to signal transduction kinases (COG0515 and COG0642) than were encoded in P. methylaliphatogenes K22T. Conversely, P. methylaliphatogenes K22T contained more genes related to amino acid utilization, such as amino acid transporters (COG0531) and amidohydrolases (COG1228), reflecting its ability to grow using proteinaceous media as the carbon and energy source. While both species possess carbohydrate-related metabolisms, the P. methylaliphatogenes K22T genome encodes a much larger number of glycosyltransferases (COG0438 and COG0463) and beta-glucosidase-related glycosidases (COG1472) than that of C. thermophilum BT.
Acidobacteria is one of the most widely-distributed bacterial phyla, particularly in soils [30–32]. Despite the wide distribution, the number of cultivated and sequenced representatives within most subdivisions within Acidobacteria remains low . The sequencing and annotation of the P. methylaliphatogenes K22T genome presented here links the phenotypic traits of P. methylaliphatogenes K22T  with its genetic characteristics, and represents a step that will assist future studies describing the ecological and metabolic capabilities of this widespread phylum.
Editor’s note: Although the name Acidobacteria is in common use at the phylum and class level, readers are advised that it appears on the list of rejected names. By definition, a rejected name must not be used to designate any taxon (Rule 23 a Note Note 4 (i)) at any rank.
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Support for this work was provided by Geothermal Resources of New Zealand (GRN) Programme at GNS Science.
The authors declare that they have no competing interests.
KCYL, XCM, PFD, CH and MBS drafted the manuscript. MBS conducted the phylogenetic studies. JFP, PFD and MBS performed the laboratory experiments. KCYL, XCM, PFD, CH and MBS sequenced, assembled and annotated the genome. All authors read and approved the final manuscript.
Associated MIGS Record and Sequencing and Assembly Methodologies. (DOCX 39 kb)