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Elucidation of the Biosynthetic Pathway of Vitamin B Groups and Potential Secondary Metabolite Gene Clusters Via Genome Analysis of a Marine Bacterium Pseudoruegeria sp. M32A2M
1Department of Biological Sciences, Korea advanced institute of Science and Technology, Daejeon, Republic of Korea, 2KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, 3Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea
J. Microbiol. Biotechnol. 2020; 30(4): 505-514
Published April 28, 2020 https://doi.org/10.4014/jmb.1911.11006
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
According to the World Register of Marine Species (WoRMS) database, 535,681 biotas have been registered with marine taxonomic names among which 447,097 species are reported to inhabit the oceanic ecosystem [1]. Moreover, ~70% of all marine biomass has been estimated to be composed of microorganisms [2]. These abundant microorganisms compose complex marine networks, where the flux of dissolved organic carbon is described as the microbial loop [3]. However, there are more diverse interactions in the network between algae and bacteria beyond the predator-prey relationship [4, 5]. Algae form the phycosphere in their surroundings to exchange diverse metabolites with environmental bacteria [6]. Some bacteria such as
With the introduction of NGS technologies and bioinformatics tools, large amounts of nucleic acid sequence data can be obtained. These techniques and tools are also used in ecological studies [27]. A comparative genomics study of various algal species suggests that several algal groups need to obtain vitamins such as thiamin, cobalamin, and biotin from the environment [28]. Based on sequence data, the genetic background of the relationship between each bacteria-alga pair can be analyzed. In this study, we performed genome sequencing of
Materials and Methods
Isolation and Scanning Electron Microscopy
Genome Sequencing and Genome de novo Assembly
Genomic DNA of
Phylogenetic Analysis
Genome sequences of each strain used for phylogenetic analysis were downloaded from the NCBI genome portal. The phylogenetic tree was reconstructed by utilizing the Up-to-date Bacterial Core Gene (UBCG) analysis pipeline [30]. Randomized Axelerated Maximum Likelihood (RAxML) was used to generate the phylogenetic tree from calculated distance data [31].
Gene Annotation and Secondary Metabolite Biosynthesis Gene Prediction
Prediction of Genome Structure
To analyze the start and stop codon usage, the first three and last three nucleotides (TGA, TAG, or TAA) were extracted based on the predicted coding sequences (CDSs), and in pseudogenes, the start codons except the reinitiating position were used. To detect the Shine-Dalgarno sequence motif, the nucleotide sequences between 20 to 1 nt upstream from the start codon were subjected to MEME (Multiple Em for Motif Elicitation) [37]. The promoter motif was searched from nucleotides between the 100 to 1 nt upstream of RNA genes.
Results and Discussion
Morphology of Pseudoruegeria sp. M32A2M
We observed the morphology of
-
Fig. 1.
General features of (Pseudoruegeria sp. M32A2M.A ) Scanning electron microscopy ofPseudoruegeria sp. M32A2M. (B ) Circular representation of the draft genome ofPseudoruegeria sp. M32A2M . From the outside to the center: scaffolds in the order of length (black, ticks every 100 Kb), genes on the plus strand (blue), genes on the minus strand (light blue), tRNA (teal), rRNA (green), secondary metabolite biosynthetic gene clusters (red), and GC skew (orange and light purple). (C ) Start codon usage. (D ) Stop codon usage. (E-F ) Conserved motifs from nucleotides between 20 to 1 nt upstream of the CDSs (E ) and between 100 to 1 nt upstream of RNA genes (F ), were searched using MEME.
Genome Sequencing and Gene Annotation
We then performed genome sequencing of
-
Table 1 . Gene annotation statistics.
Features annotated Pseudoruegeria sp. M32A2MCoding sequences (CDSs) 5,049 Functional CDSs 4,927 Pseudogenes 122 RNA genes 52 rRNAs 1, 1, 1 (5S, 16S, 23S) tRNAs 46 ncRNAs 3 Total annotated features 5,101
From gene annotation results, we investigated the RNA polymerase subunits and sigma factors, which are core proteins for transcription. There were four kinds of RNA polymerase subunits: alpha (RpoA), beta (RpoB), beta’ (Rp°), and omega (RpoZ) along with several sigma factors, RpoD, and RpoH, and no assigned sigma factors were found together (Table 2). Sigma factors that are not searched with stringent e-value cut-offs from BLASTp seem to have degenerated sequences from known minor sigma factors such as
-
Table 2 . RNA polymerase subunit and sigma factors identified in Pseudoruegeria sp. M32A2M.
Gene ID Gene Function Num. of Amino acid Size (Da) FPS10_11085 rpoA DNA-directed RNA polymerase subunit alpha 348 36,833 FPS10_15795 rpoB DNA-directed RNA polymerase subunit beta 1,390 153,105 FPS10_15790 rpoC DNA-directed RNA polymerase subunit beta' 1,427 157,458 FPS10_10510 rpoZ DNA-directed RNA polymerase subunit omega 126 13,252 FPS10_24745 rpoD RNA polymerase sigma factor RpoD 675 75,645 FPS10_02115 rpoH RNA polymerase sigma factor RpoH 312 33,691 FPS10_24655 - RNA polymerase factor sigma-32 300 33,358 FPS10_22820 - RNA polymerase sigma factor 203 21,954 FPS10_22950 - sigma-70 family RNA polymerase sigma factor 175 19,707 FPS10_23250 - RNA polymerase sigma factor 183 19,447
Interestingly, we found flagella-related gene clusters in
Genome Structure Analysis
With the assigned CDSs, the usage of the start and stop codon in
Like other bacterial species, the Shine-Dalgarno sequence of
From 52 RNA query sequences (3 rRNAs, 46 tRNAs, and 3 ncRNAs), DNA sequences were extracted from the 50 to 21 upstream and 20 to 1 upstream, respectively, to search promoter motifs. As a result, CTTG(a/c)(c/a) and TA motifs were found as -35 element and -10 element with E-values of E=7.2e-17 and E=2.8e-1, respectively (Fig. 1F). The distance between two elements was ~17 nt, which is in the normal spacing range of 16-19 nt. A high E-value at -10 element may be a result of variance due to a small number of query sequences. Further information such as transcription initiation sites is required to accurately determine the promoter sequence of an given organism [41]. The
Genome-Scale Phylogenetic Analysis of Pseudoruegeria sp. M32A2M
In the previous report, the phylogenetic analysis of another Pseudoruegeria species isolated from GeoJe island based on 16S rRNA sequences was performed [24]. The 16s rRNA sequence has been widely used for conventional phylogenetic analysis. However, advances in NGS technology have made it easier to obtain genome sequences, and comparison methods for multiple highly conserved genes have emerged [30]. The Up-to-date Bacterial Core Gene (UBCG) analysis pipeline was used to reconstruct the phylogenetic tree of
-
Fig. 2.
Genome level phylogenetic analysis. Phylogenetic analysis fromPseudoruegeria sp. M32A2M and 34 closely related taxa was performed based on their core genes.Stappia stellulata was selected as the outgroup. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were provided by UBCG and plotted by RAxML.
Functional Categorization
Further, functional annotation of CDS was performed using three pipelines, KEGG Orthology (KO), Clusters of Orthologous Groups of proteins (COG), and Gene Ontology (GO) (Table S2). In results, among 4,927 protein-coding genes, a total of 3,870 COG functions, 2,443 KO IDs, and 2,140 GO terms were annotated. A total of 4,025 genes were assigned to at least one functional category of three. In COG category assignment, except for poorly characterized categories (R and S), carbohydrate metabolism (G) and amino acid metabolism (E) showed high abundance (Fig. 3A). In KEGG analysis, carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and membrane transporter categories had a high abundance of genes assigned consistent with COG analysis (Fig. 3B). In general, carbohydrate and amino acid metabolism related genes are abundant in most cells. Notably, the high abundance of cofactors and vitamins metabolism-related genes in
-
Fig. 3.
KEGG pathway analysis and COG analysis. Among 4,927 coding genes, 3,870 genes were categorized by COG function (A ) and 2,443 genes were categorized by KEGG Orthology (B ).
Vitamin B Biosynthesis Pathways in Pseudoruegeria sp. M32A2M
Based on the inspection of KEGG functional annotation of
-
Fig. 4.
Vitamin B group biosynthesis metabolic pathways in In total, seven vitamin B (B1, B2, B3, B6, B7, B9, and B12) pathways were discovered based on the KEGG pathway database and the related enzymes were identified. Individual reactions are represented as arrows with the corresponding gene id (gene name).Pseudoruegeria sp. M32A2M.
Secondary Metabolites Biosynthesis Clusters in Pseudoruegeria sp. M32A2M
Secondary metabolites are not directly related to the cell but play a crucial role in defense against other microorganisms such as viruses or microbes, and against harmful stresses such as toxins or UV exposure. In addition, these metabolites are essential in a symbiotic relationship or in competition with other organisms. We thus examined the potential secondary metabolite-producing clusters in
-
Fig. 5.
Predicted secondary metabolite biosynthetic gene clusters. Secondary metabolite biosynthetic pathways are predicted based on the genome sequence ofPseudoruegeria sp. M32A2M by AntiSMASH. In total, seven clusters were predicted; ectoine, homoserine lactone, beta-lactone, terpene, lasso peptide, bacteriocin, and NRPS/T1PKS. Each gene category is marked by a gray-scale contrast.
Taken together,
Supplemental Materials
Acknowledgments
This work was supported by the Basic Core Technology Development Program for the Oceans and the Polar Regions of the National Research Foundation (NRF), funded by the Ministry of Science, ICT, and Future Planning of Korea (2016M1A5A1027455 to S.C., 2016M1A5A1027453 to C.,-Y.A. and NRF-2016M1A5A1027458 to B.-K.C).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2020; 30(4): 505-514
Published online April 28, 2020 https://doi.org/10.4014/jmb.1911.11006
Copyright © The Korean Society for Microbiology and Biotechnology.
Elucidation of the Biosynthetic Pathway of Vitamin B Groups and Potential Secondary Metabolite Gene Clusters Via Genome Analysis of a Marine Bacterium Pseudoruegeria sp. M32A2M
Sang-Hyeok Cho 1, Eunju Lee 1, So-Ra Ko 3, Sangrak Jin 1, Yoseb Song 1, Chi-Yong Ahn 3, Hee-Mock Oh 3, Byung-Kwan Cho 1, 2* and Suhyung Cho 1, 2*
1Department of Biological Sciences, Korea advanced institute of Science and Technology, Daejeon, Republic of Korea, 2KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, 3Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea
Abstract
The symbiotic nature of the relationship between algae and marine bacteria is well-studied among the complex microbial interactions. The mutual profit between algae and bacteria occurs via nutrient and vitamin exchange. It is necessary to analyze the genome sequence of a bacterium to predict its symbiotic relationships. In this study, the genome of a marine bacterium, Pseudoruegeria sp. M32A2M, isolated from the south-eastern isles (GeoJe-Do) of South Korea, was sequenced and analyzed. A draft genome (91 scaffolds) of 5.5 Mb with a DNA G+C content of 62.4% was obtained. In total, 5,101 features were identified from gene annotation, and 4,927 genes were assigned to functional proteins. We also identified transcription core proteins, RNA polymerase subunits, and sigma factors. In addition, full flagella-related gene clusters involving the flagellar body, motor, regulator, and other accessory compartments were detected even though the genus Pseudoruegeria is known to comprise non-motile bacteria. Examination of annotated KEGG pathways revealed that Pseudoruegeria sp. M32A2M has the metabolic pathways for all seven vitamin Bs, including thiamin (vitamin B1), biotin (vitamin B7), and cobalamin (vitamin B12), which are necessary for symbiosis with vitamin B auxotroph algae. We also identified gene clusters for seven secondary metabolites including ectoine, homoserine lactone, beta-lactone, terpene, lasso peptide, bacteriocin, and nonribosomal proteins.
Keywords: Pseudoruegeria, whole-genome sequencing, Vitamin B, secondary metabolite
Introduction
According to the World Register of Marine Species (WoRMS) database, 535,681 biotas have been registered with marine taxonomic names among which 447,097 species are reported to inhabit the oceanic ecosystem [1]. Moreover, ~70% of all marine biomass has been estimated to be composed of microorganisms [2]. These abundant microorganisms compose complex marine networks, where the flux of dissolved organic carbon is described as the microbial loop [3]. However, there are more diverse interactions in the network between algae and bacteria beyond the predator-prey relationship [4, 5]. Algae form the phycosphere in their surroundings to exchange diverse metabolites with environmental bacteria [6]. Some bacteria such as
With the introduction of NGS technologies and bioinformatics tools, large amounts of nucleic acid sequence data can be obtained. These techniques and tools are also used in ecological studies [27]. A comparative genomics study of various algal species suggests that several algal groups need to obtain vitamins such as thiamin, cobalamin, and biotin from the environment [28]. Based on sequence data, the genetic background of the relationship between each bacteria-alga pair can be analyzed. In this study, we performed genome sequencing of
Materials and Methods
Isolation and Scanning Electron Microscopy
Genome Sequencing and Genome de novo Assembly
Genomic DNA of
Phylogenetic Analysis
Genome sequences of each strain used for phylogenetic analysis were downloaded from the NCBI genome portal. The phylogenetic tree was reconstructed by utilizing the Up-to-date Bacterial Core Gene (UBCG) analysis pipeline [30]. Randomized Axelerated Maximum Likelihood (RAxML) was used to generate the phylogenetic tree from calculated distance data [31].
Gene Annotation and Secondary Metabolite Biosynthesis Gene Prediction
Prediction of Genome Structure
To analyze the start and stop codon usage, the first three and last three nucleotides (TGA, TAG, or TAA) were extracted based on the predicted coding sequences (CDSs), and in pseudogenes, the start codons except the reinitiating position were used. To detect the Shine-Dalgarno sequence motif, the nucleotide sequences between 20 to 1 nt upstream from the start codon were subjected to MEME (Multiple Em for Motif Elicitation) [37]. The promoter motif was searched from nucleotides between the 100 to 1 nt upstream of RNA genes.
Results and Discussion
Morphology of Pseudoruegeria sp. M32A2M
We observed the morphology of
-
Figure 1.
General features of (Pseudoruegeria sp. M32A2M.A ) Scanning electron microscopy ofPseudoruegeria sp. M32A2M. (B ) Circular representation of the draft genome ofPseudoruegeria sp. M32A2M . From the outside to the center: scaffolds in the order of length (black, ticks every 100 Kb), genes on the plus strand (blue), genes on the minus strand (light blue), tRNA (teal), rRNA (green), secondary metabolite biosynthetic gene clusters (red), and GC skew (orange and light purple). (C ) Start codon usage. (D ) Stop codon usage. (E-F ) Conserved motifs from nucleotides between 20 to 1 nt upstream of the CDSs (E ) and between 100 to 1 nt upstream of RNA genes (F ), were searched using MEME.
Genome Sequencing and Gene Annotation
We then performed genome sequencing of
-
Table 1 . Gene annotation statistics..
Features annotated Pseudoruegeria sp. M32A2MCoding sequences (CDSs) 5,049 Functional CDSs 4,927 Pseudogenes 122 RNA genes 52 rRNAs 1, 1, 1 (5S, 16S, 23S) tRNAs 46 ncRNAs 3 Total annotated features 5,101
From gene annotation results, we investigated the RNA polymerase subunits and sigma factors, which are core proteins for transcription. There were four kinds of RNA polymerase subunits: alpha (RpoA), beta (RpoB), beta’ (Rp°), and omega (RpoZ) along with several sigma factors, RpoD, and RpoH, and no assigned sigma factors were found together (Table 2). Sigma factors that are not searched with stringent e-value cut-offs from BLASTp seem to have degenerated sequences from known minor sigma factors such as
-
Table 2 . RNA polymerase subunit and sigma factors identified in Pseudoruegeria sp. M32A2M..
Gene ID Gene Function Num. of Amino acid Size (Da) FPS10_11085 rpoA DNA-directed RNA polymerase subunit alpha 348 36,833 FPS10_15795 rpoB DNA-directed RNA polymerase subunit beta 1,390 153,105 FPS10_15790 rpoC DNA-directed RNA polymerase subunit beta' 1,427 157,458 FPS10_10510 rpoZ DNA-directed RNA polymerase subunit omega 126 13,252 FPS10_24745 rpoD RNA polymerase sigma factor RpoD 675 75,645 FPS10_02115 rpoH RNA polymerase sigma factor RpoH 312 33,691 FPS10_24655 - RNA polymerase factor sigma-32 300 33,358 FPS10_22820 - RNA polymerase sigma factor 203 21,954 FPS10_22950 - sigma-70 family RNA polymerase sigma factor 175 19,707 FPS10_23250 - RNA polymerase sigma factor 183 19,447
Interestingly, we found flagella-related gene clusters in
Genome Structure Analysis
With the assigned CDSs, the usage of the start and stop codon in
Like other bacterial species, the Shine-Dalgarno sequence of
From 52 RNA query sequences (3 rRNAs, 46 tRNAs, and 3 ncRNAs), DNA sequences were extracted from the 50 to 21 upstream and 20 to 1 upstream, respectively, to search promoter motifs. As a result, CTTG(a/c)(c/a) and TA motifs were found as -35 element and -10 element with E-values of E=7.2e-17 and E=2.8e-1, respectively (Fig. 1F). The distance between two elements was ~17 nt, which is in the normal spacing range of 16-19 nt. A high E-value at -10 element may be a result of variance due to a small number of query sequences. Further information such as transcription initiation sites is required to accurately determine the promoter sequence of an given organism [41]. The
Genome-Scale Phylogenetic Analysis of Pseudoruegeria sp. M32A2M
In the previous report, the phylogenetic analysis of another Pseudoruegeria species isolated from GeoJe island based on 16S rRNA sequences was performed [24]. The 16s rRNA sequence has been widely used for conventional phylogenetic analysis. However, advances in NGS technology have made it easier to obtain genome sequences, and comparison methods for multiple highly conserved genes have emerged [30]. The Up-to-date Bacterial Core Gene (UBCG) analysis pipeline was used to reconstruct the phylogenetic tree of
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Figure 2.
Genome level phylogenetic analysis. Phylogenetic analysis fromPseudoruegeria sp. M32A2M and 34 closely related taxa was performed based on their core genes.Stappia stellulata was selected as the outgroup. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were provided by UBCG and plotted by RAxML.
Functional Categorization
Further, functional annotation of CDS was performed using three pipelines, KEGG Orthology (KO), Clusters of Orthologous Groups of proteins (COG), and Gene Ontology (GO) (Table S2). In results, among 4,927 protein-coding genes, a total of 3,870 COG functions, 2,443 KO IDs, and 2,140 GO terms were annotated. A total of 4,025 genes were assigned to at least one functional category of three. In COG category assignment, except for poorly characterized categories (R and S), carbohydrate metabolism (G) and amino acid metabolism (E) showed high abundance (Fig. 3A). In KEGG analysis, carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and membrane transporter categories had a high abundance of genes assigned consistent with COG analysis (Fig. 3B). In general, carbohydrate and amino acid metabolism related genes are abundant in most cells. Notably, the high abundance of cofactors and vitamins metabolism-related genes in
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Figure 3.
KEGG pathway analysis and COG analysis. Among 4,927 coding genes, 3,870 genes were categorized by COG function (A ) and 2,443 genes were categorized by KEGG Orthology (B ).
Vitamin B Biosynthesis Pathways in Pseudoruegeria sp. M32A2M
Based on the inspection of KEGG functional annotation of
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Figure 4.
Vitamin B group biosynthesis metabolic pathways in In total, seven vitamin B (B1, B2, B3, B6, B7, B9, and B12) pathways were discovered based on the KEGG pathway database and the related enzymes were identified. Individual reactions are represented as arrows with the corresponding gene id (gene name).Pseudoruegeria sp. M32A2M.
Secondary Metabolites Biosynthesis Clusters in Pseudoruegeria sp. M32A2M
Secondary metabolites are not directly related to the cell but play a crucial role in defense against other microorganisms such as viruses or microbes, and against harmful stresses such as toxins or UV exposure. In addition, these metabolites are essential in a symbiotic relationship or in competition with other organisms. We thus examined the potential secondary metabolite-producing clusters in
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Figure 5.
Predicted secondary metabolite biosynthetic gene clusters. Secondary metabolite biosynthetic pathways are predicted based on the genome sequence ofPseudoruegeria sp. M32A2M by AntiSMASH. In total, seven clusters were predicted; ectoine, homoserine lactone, beta-lactone, terpene, lasso peptide, bacteriocin, and NRPS/T1PKS. Each gene category is marked by a gray-scale contrast.
Taken together,
Supplemental Materials
Acknowledgments
This work was supported by the Basic Core Technology Development Program for the Oceans and the Polar Regions of the National Research Foundation (NRF), funded by the Ministry of Science, ICT, and Future Planning of Korea (2016M1A5A1027455 to S.C., 2016M1A5A1027453 to C.,-Y.A. and NRF-2016M1A5A1027458 to B.-K.C).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

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Table 1 . Gene annotation statistics..
Features annotated Pseudoruegeria sp. M32A2MCoding sequences (CDSs) 5,049 Functional CDSs 4,927 Pseudogenes 122 RNA genes 52 rRNAs 1, 1, 1 (5S, 16S, 23S) tRNAs 46 ncRNAs 3 Total annotated features 5,101
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Table 2 . RNA polymerase subunit and sigma factors identified in Pseudoruegeria sp. M32A2M..
Gene ID Gene Function Num. of Amino acid Size (Da) FPS10_11085 rpoA DNA-directed RNA polymerase subunit alpha 348 36,833 FPS10_15795 rpoB DNA-directed RNA polymerase subunit beta 1,390 153,105 FPS10_15790 rpoC DNA-directed RNA polymerase subunit beta' 1,427 157,458 FPS10_10510 rpoZ DNA-directed RNA polymerase subunit omega 126 13,252 FPS10_24745 rpoD RNA polymerase sigma factor RpoD 675 75,645 FPS10_02115 rpoH RNA polymerase sigma factor RpoH 312 33,691 FPS10_24655 - RNA polymerase factor sigma-32 300 33,358 FPS10_22820 - RNA polymerase sigma factor 203 21,954 FPS10_22950 - sigma-70 family RNA polymerase sigma factor 175 19,707 FPS10_23250 - RNA polymerase sigma factor 183 19,447
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