A systematic study of the whole genome sequence of Amycolatopsis methanolica strain 239T provides an insight into its physiological and taxonomic properties which correlate with its position in the genus

Abstract

The complete genome of methanol-utilizing Amycolatopsis methanolica strain 239^T was generated, revealing a single 7,237,391 nucleotide circular chromosome with 7074 annotated protein-coding sequences (CDSs). Comparative analyses against the complete genome sequences of Amycolatopsis japonica strain MG417-CF17^T, Amycolatopsis mediterranei strain U32 and Amycolatopsis orientalis strain HCCB10007 revealed a broad spectrum of genomic structures, including various genome sizes, core/quasi-core/non-core configurations and different kinds of episomes. Although polyketide synthase gene clusters were absent from the A. methanolica genome, 12 gene clusters related to the biosynthesis of other specialized (secondary) metabolites were identified. Complete pathways attributable to the facultative methylotrophic physiology of A. methanolica strain 239^T, including both the mdo/mscR encoded methanol oxidation and the hps/hpi encoded formaldehyde assimilation via the ribulose monophosphate cycle, were identified together with evidence that the latter might be the result of horizontal gene transfer. Phylogenetic analyses based on 16S rDNA or orthologues of AMETH_3452, a novel actinobacterial class-specific conserved gene against 62 or 18 Amycolatopsis type strains, respectively, revealed three major phyletic lineages, namely the mesophilic or moderately thermophilic A. orientalis subclade (AOS), the mesophilic Amycolatopsis taiwanensis subclade (ATS) and the thermophilic A. methanolica subclade (AMS). The distinct growth temperatures of members of the subclades correlated with corresponding genetic variations in their encoded compatible solutes. This study shows the value of integrating conventional taxonomic with whole genome sequence data.

Keywords: AMS; AOS; ATS; Amycolatopsis methanolica; Complete genome sequence; One carbon metabolism; Sub-generic phyletic clades.

Fig. 1

Genome atlas of A. methanolica strain 239^Twith two characteristic episomes enlarged (A) and the gene clusters encoding biosynthetic functions for specialized metabolites (B). (A) The large circle represents the chromosome: the outer scale is numbered in megabases and indicates the core (blue) and non-core (yellow) regions. The circles are nominated to start from the outside in. The genes in circles 1 and 2 (forward and reverse strands, respectively) are color-coded according to COG functional categories. Circle 3 shows selected essential genes (cell division, replication, transcription, translation, and amino-acid metabolism; the paralogs of essential genes in the non-core regions are not included), circle 4 the phage (red), specialized metabolite clusters (orange) and integrated plasmid pMEA300 (pink), circle 5 the mobile genetic elements (transposases), circle 6 the RNAs, circle 7 the GC content with a calculated ratio of 71.53 mol % and circle 8 the GC bias (blue, values > 0; red, values < 0). The right panel of (A) demonstrates the two characteristic episomes of the A. methanolica genome of strain 239^T, i.e., the prophage with its 52 ORFs (upper) and the pMEA300-like integrated plasmid (lower). (B) Gene clusters in the genome of A. methanolica strain 239^Tencoding biosynthetic functions for specialized metabolites. The corresponding biosynthesis related genes are dark colored.

Fig. 2

Comparative analyses of 7 Pseudonocardiaceae genomes (A) and the chromosomal loci distribution of the highly conserved AMETH_3452 gene among actinobacterial genomes (B). In panel A, parallel straight lines represent the genomes of A. orientalis HCCB10007 (CP003410.1), A. mediterranei U32 (CP002000.1), A. methanolica 239^T (CP009110), S. viridis DSM 43017^T (CP001683.1), S. erythraea NRRL 2338^T (AM420293.1), A. mirum DSM 43827^T (CP001630.1) and P. dioxanivorans CB1190^T (CP002593.1), and are drawn to scale with oriC located at the very end of the lines. Vertical short bars representing different conserved genes are marked with distinct colors to demonstrate their genomic loci; the latter bars are connected by corresponding colored thin lines. The segregation of core, non-core and quasi-core regions are shown as clusters of conserved orthologous genes. Black arrows highlight the genomic loci of the highly conserved AMETH_3452 analogs located in the middle of the 7 genomes and are connected by thick black lines. In panel B, the distribution of chromosomal loci of the highly conserved genes orthologous to AMETH_3452 among species of the class Actinobacteria with circular genomes is plotted against their corresponding genome sizes. The horizontal coordinate represents the relative chromosomal positions of the genomes normalized to 0.0–1.0 with oriC located at both ends; the genomic loci of the conserved genes orthologous to AMETH_3452 are shown as scattered points. This conserved gene is located in the middle of the circular chromosomes (i.e., close to the 0.5 locus) in species that mainly belong to four taxa, namely the families Pseudonocardiaceae (12 genomes, dot, red), Corynebacteriaceae (50 genomes, dot, green), Micrococcaceae (9 genomes, asterisk, blue) and Mycobacteriaceae (59 genomes, dot, cyan). In contrast, the gene is located close to oriC in the species belonging to the families Acidimicrobiaceae (rhombus, gray). Catenulisporaceae (square, pink), Geodermatophilaceae (triangle, gray), Micromonosporaceae (triangle, purple), Nocardiopsaceae (triangle, pink) and Streptomycetaceae (only Streptomyces violaceusniger Tu 4113) (square, purple). This gene is located between these two positions in species belonging to the families Frankiaceae (square, gray), Microbacteriaceae (rhombus, gold), Nocardiaceae (rhombus, brown), Propionibacteriaceae (rhombus, plum), Actinomycetaceae, Beutenbergiaceae, Cellulomonadaceae, Dermabacteraceae, Dermacoccaceae, Glycomycetaceae, Intrasporangiaceae, Jonesiaceae, Nakamurellaceae, Nocardioidaceae, Promicromonosporaceae, Sanguibacteraceae, Segniliparaceae, Thermomonosporaceae, Tsukamurellaceae and in Thermobispora bispora, a member of the genus Thermobispora that was once classified in the family Pseudonocardiaceae but is now known to form a deep lineage in the 16S rRNA actinobacterial gene tree hence its reclassification as an order incertae sedis in the current edition of Bergey's Manual of Systematic Bacteriology (small dot, black). Detailed information on all of these genomes is given in Table S4.

Fig. 3

Collinearity analyses of the genome of A. methanolica strain 239^Tagainst the genomes of A. mediterranei strain U32 (A), A. orientalis strain HCCB10007 (B), S. viridis DSM 43017^T(C), and S. erythraea NRRL 2338^T(D), respectively. The ortholog ordering in the defined core region of the A. methanolica genome is highlighted in light red while the corresponding consecutive genomic segments in the non-core region, which are matched with the quasi-core regions of the corresponding genomes, are shown in light green.

Fig. 4

Neighbour-joining phylogenetic tree based on 16S rDNA of 62 Amycolatopsis type strains employing S. viridis DSM 43017^Tas the outgroup (A) together with corresponding growth and temperature tolerance properties (B) In panel A, numbers at the nodes are percentage bootstrap values based on 1000 resampled datasets. The scale bar indicates 5 nucleotide substitutions per 1000 nucleotides. The subclades are designated AOS, ATS and AMS, respectively. Subclade groups (A–G) defined by Everest and Meyers and Everest et al. are shown, the F group is divided into F1 and F2. The subclades H-J were defined in this study; the J group lies at the periphery of AOS subclade hence the corresponding vertical bar is dashed. In panel B, the phenotypic characteristics of the 62 Amycolatopsis type strains are listed according to their phylogenetic locations. All of the growth condition data were taken from the initial published descriptions of the species. Abbreviations: +, positive growth, w, weak growth, -, no growth and ND, no data available.

Fig. 5

Pairwise comparison of 16S rDNA sequences from A. mediterranei strain U32, A. orientalis strain HCCB10007, A. japonica strain MG417-CF17^Tand A. methanolica strain 239^T(A) and their overall phylogenetic relationships (B). In panel A, the percentage of sequence identity is shown for each pair of the different copies of 16S rDNAs. The brackets on the diagonal line of the table represent comparisons between the same 16S rDNAs. The pairs circled by triangles represent intra-species comparisons while those circled by squares represent inter-species comparisons comparable to those of the corresponding intra-species comparisons shown as square circled data inside the triangle. In panel B, the small differences between the intra- and inter-species 16S rDNA comparisons accounted for the abnormal clustering of the corresponding species in the phylogenetic tree are shown.

Fig. 6

Neighbour-joining trees based on AMETH_3452 orthologues with (A) or without (B) the corresponding 16S rDNA sequences, and the 16S rDNA sequences alone (C) from the complete or draft genomes of the 18 Amycolatopsis type strains. The AOS/ATS/AMS classification and the A-F and J groupings are shown. Half square brackets in red denote the AMS branches, the green color the ATS branch and the blue color the AOS branches. Numbers at the nodes are bootstrap values based on 1000 replicates. The scale bar indicates 0.02, 0.05 or 0.005 nucleotide substitutions per site.

Fig. 7

Schematic representation of the pathways for methanol, glucose and gluconate metabolism and for the biosynthesis of aromatic amino acids in A. methanolica strain 239^T(A) and comparison of the corresponding genes against those of other Amycolatopsis strains (B). AMETH_5577 represents methanol:N,N-dimethyl-4-nitrosoaniline oxidoreductase, K17067, EC:1.2.99.4/EC:1.1.99.37; AMETH_3767 NAD/mycothiol-dependent formaldehyde dehydrogenase, K00153, EC:1.1.1.306; AMETH_3397 formate dehydrogenase, K00123, EC:1.2.1.2, and AMETH_3398 formate dehydrogenase iron-sulfur subunit, K00124, EC:1.2.1.2. In panel B, Y denotes the identified genes, N denotes the unidentified genes in complete genomes, and ND denotes the genes that are not detected in the incomplete genomes.

Fig. 8

Phylogenetic tree of HPI (A) and HPS (B) protein sequences and comparison of their encoding cluster in A. methanolica strain 239^Tagainst those in the two Pseudonocardiaceae strains with the same two proteins (C). Maximum-likelihood tree (A and B) showing 6-phospho-3-hexuloisomerase (HPI) and 3-hexulose-6-phosphate synthase (HPS) in relation to the orthologous proteins selected from organisms which have available whole genome sequences and the CDSs for both HPS and HPI. The protein sequences of HPS and HPI from A. methanolica strain 239^T are marked by solid triangles. Numbers at the nodes are percentage bootstrap values based on a maximum-likelihood analysis of 1000 resampled datasets. Bar indicates 0.1 substitutions per site. (C) Comparison of chromosomal loci of genes involved in RuMP and related carbon metabolism from A. methanolica strain 239^T, A. benzoatilytica AK 16/56^T, S. marina XMU15, R. jostii RHA1, Arthrobacter aurescens TC1 and B. subtilis BSn5. The clusters from A. methanolica, A. benzoatilytica and S. marina strains are conserved. Genes marked in red are specific for the corresponding strains, those in green have corresponding orthologs in most of the actinobacteria, the hps and hpi genes are marked in black.