Introduction

The microbial communities have been deemed important organisms based on their metabolic capabilities and potential applications1. The co-cultured organisms can divide the labor reasonable and improve the robustness of the system2. For example, the ability to metabolize and degrade cellulose3, alkanes4, heavy metal toxins5 and natural products6. The multiple microbial species increased range of genes and metabolic capabilities in comparison to monocultures. Enumerating metabolic exchanges and the microbial diversity are still uncharacterized for complex metabolic needs and biosynthetic capabilities.

The microbial community of K. vulgare and Bacillus species have been widely used in the two-step vitamin C production7. In the community, K. vulgare is responsible for the bioconversion of sorbose to 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C). Mono-cultured K. vulgare grows poorly, even on rich natural media. Addition of amino acids8, vitamins9 and glutathione10 could enhance the growth of K. vulgare, implying the defects of oxidation metabolism, vitamin and amino acid synthesis11. The Bacillus species have been used to stimulate the growth of K. vulgare, including Bacillus megaterium, Bacilius cereus and B. endophyticus, and different 2-KLG productivities have been determined in the co-culture systems. Besides, mix cultured B. megaterium and B. cereus (1:3) could increase the 2-KLG yield compared to one-helper-strain co-culture system12. In our previous studies, the metabolomics approach has been demonstrated on the interactions between K. vulgare and Bacillus10,13. However, the different cooperative effects and mechanisms of companion bacteria are needed to interpret at molecular level.

Comparative genomics is a powerful approach for studying variation in physiological traits as well as the evolution and ecology of microorganisms14. Based on comparative genomics analysis, relationship of B. megaterium with other Bacillus species and numerous unique genetic traits were identified15. Through comparative genomics, the evolutionary relationships and major traits of Bacillus species were understood, including the diversity of sporulation and competence genes16. Whole-genome sequencing of B. thuringiensis 97-27 and B. cereus E33L was undertaken to identify shared and unique genes, the differences were revealed in terms of virulence, structural components and regulatory mechanisms17.

Identification the optimum companion bacterium has always been an important research topic in VC two-step fermentation. In present study, we observe the different phenotype of B. thuringiensis Bc601 and B. endophyticus Hbe603 and different 2- KLG productivities of K. vlugare in the co-culture system with different helper bacteria. In order to understand the genome evolution and the metabolic versatility, we have sequenced the complete genomes of B. thuringiensis Bc601 and eight plasmids of B. endophyticus Hbe603. Aided with comparative genomics analysis, characteristics of two species and cooperation mechanisms could be systematically understood, which are of great importance for consortium optimization.

Results

General genomic properties of two Bacillus species

The complete genome of B. thuringiensis Bc601 consists of one circular chromosome and six plasmids (Fig. S1 and Table S1). B. anthracis, B. cereus and B. thuringiensis are closely-related members, which have been named as the B. cereus-group. Comparison of their 16S rRNA sequences places them within the same species18. B. thuringiensis and B. cereus mix together in the phylogenetic tree by blasting the whole genome sequences. B. thuringiensis Bc601 is most similar with B. thuringiensis serovar kurstaki HD73, which contains 15 contigs (Fig. S2). The genes in plasmids are mainly encodes transposases, transcriptional regulators, recombinases, type VII secretion proteins and cell surface proteins. Besides, B. thuringiensis Bc601 also encodes thiamine biosynthesis protein, 3-oxoacyl-ACP synthase, ATPase and flagellar biosynthesis protein on plasmids for nutrients synthesis.

The complete genome of B. endophyticus Hbe603 consists of one circular chromosome and eight plasmids (Table S2). Our previous research published the chromosome of B. endophyticus Hbe60319. Besides, it owns eight plasmids, which have the special sequence information and low similarity compared to diverse Bacillus spp. With the RAST analysis platform, we identify 468 genes in eight plasmids and almost half of them encode the hypothetical proteins or unknown enzymes. Six plasmids contain phage proteins which may derive from horizontal transfer. The genes in plasmids are mainly about sporulation, germination, regulation and transportation. The sporulation cycle is complete in B. endophyticus and plasmids also harbor some sporulation-related proteins, including AbrB family transcription regulatory proteins, spores coat protein and germination proteins GerKA, GerKC and GerKB. B. endophyticus has abundant transcriptional regulatory proteins in response to changes of external environment, and the plasmids harbor sigma-W, sigma-B and two-component signal transduction system for the regulation of cell density. In addition, it contains a large number of other transcription factors, covering MerR, ArsR, MarR, AbrB, GntR, AraC and LacI family. Besides, B. endophyticus also encodes five cell division proteins and 36 mobile element proteins on plasmids for cell division and proliferation.

The different morphologies and companion effects of two Bacillus species

By microscopic observation, we found the obvious differences in the morphology of two Bacillus species. B. thuringiensis has a long rod-shaped with no obvious changes before and after sporulation (Fig. 1A,B). B. endophyticus keeps the long chain distribution characteristics in the vegetative state, and becomes elliptic and reduces half of its volume after sporulation (Fig. 1C,D). We speculate that its cell structure is changeable. The different morphologies after sporulation may be connected with the characteristics of two species, particularly the cell structure related proteins. The highest cell density of single-cultured B. thuringiensis was 1.59 times of single-cultured B. endophyticus, and the cell density decreased significantly after 20 hours due to the sporulation (Fig. 2A). B. thuringiensis may absorb more nutrients for its strong growth ability compared to B. endophyticus in the mixed bacteria system. In the co-culture system, the 2-KLG productivity of K. vulgare with B. endophyticus was higher than that with B. thuringiensis after 36 hours and tended to be stable in 72 hours (Fig. 2B). Swarming motility is a multi-cellular behavior which can help us to better understand the metabolic interaction and the cooperative mechanism between the two species. We orthogonal cultured B. thuringiensis, B. endophyticus and K. vulgare on an agar plate. The horizontal cultivation was K. vulgare, and vertical cultivation was B. thuringiensis Bc601 and B. endophyticus Hbe603. Swarming motility of B. thuringiensis could be induced when it was co-cultured with K. vulgare, while B. endophyticus didn’t swarm to K. vulgare (Fig. 1E).

Figure 1: The growth state of B. endophyticus Hbe603 and B. thuringiensis Bc601 by microscopic observation.
figure 1

The vegetative state (A) and sporulation (B) of B. thuringiensis Bc601 and the vegetative state (C) and sporulation (D) of B. endophyticus Hbe603. Swarming pattern of the ecosystem via chemotaxis of species (E). The photographs show monocultures of B. thuringiensis, B. endophyticus and K. vulgare and coculture after 96 hours of growth at 30 °C on soft agar.

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Figure 2: The two Bacillus species cooperated with K. vulgare in fermentation, respectively.
figure 2

(A) Cell density of Bacillus species, the Y axis represents the average OD600nm at each time point; (B) Concentration of 2-KLG.

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Comparative analysis of the versatile metabolism in two Bacillus species

To gain further insight into the relationship between B. thuringiensis Bc601 and B. endophyticus Hbe603, the common and unique genes were calculated using the CD-HIT rapid clustering20. B. thuringiensis Bc601 and B. endophyticus Hbe603 have 1524 common genes, and the number of specific genes is 4189 and 3698, respectively. The distribution of COG classification was compared to facilitate the difference of gene function in two species (Table S3). In B. thuringiensis Bc601, the number of genes related to carbohydrate transport and metabolism (G), Energy production and conversion (C) and lipid metabolism (I) is less than that in B. endophyticus Hbe603, respectively. We speculate that B. endophyticus Hbe603 may have greater promotion for K. vulgare due to greater capacity of nutritional supplements. From our observation, the cell wall and membrane structure of B. thuringiensis Bc601 are significantly thicker than B. endophyticus Hbe603, and the ratio of peptidoglycan biosynthesis related gene number is 29:21. Only B. thuringiensis Bc601 harbors femX, femA, femB, murN, mtgA, pbpC and pbpB. Besides, D-alanyl-D-alanine carboxypeptidases, collagenases, ATPases, spore coat and two-component system related proteins in B. thuringiensis Bc601 are more than those in B. endophyticus Hbe603. We speculate these factors may affect B. endophyticus Hbe603’s ability to communicate with the external environment. Furthermore, B. endophyticus Hbe603 has more PTS related transporters and ferredoxins than B. thuringiensis Bc601 (Table 1).

Table 1 The major different genes among two Bacillus species.
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Based on the transcriptome analysis of B. thuringiensis sporulation process, 1646 genes were differentially expressed and most of them were connected with transport, transcriptional regulation, cell motility and DNA repair21. In the process of sporulation, the companion Bacillus bacterium further releases abundant nutrients for the growth and 2-KLG production of K. vulgare22. In order to analyze the different metabolic capacities in B. thuringiensis Bc601 and B. endophyticus Hbe603, metabolic network of two species was obtained, including the central carbon, amino acid and cofactor metabolism (Fig. 3).

Figure 3
figure 3

Comparative analysis of the mainly different pathway related to TCA cycle between B. endophyticus Hbe603 and B. thuringiensis Bc601.

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In the central carbon metabolism, we identified the complete glycolysis, citrate cycle (TCA cycle) and pentose phosphate pathway in the two species. Besides, B. thuringiensis Bc601 owns glyceraldehyde-3-phosphate dehydrogenases and 2- oxoglutarate ferredoxin oxidoreductase, which converses glyceraldehyde-3-phosphate to glycerate-3-phosphate, and converts 2-oxoglutarate into succinyl-CoA, respectively. In B. thuringiensis Bc601, the alpha-galactosidases, pentose and glucuronate conversion related proteins, which can utilize D-galacturonate, D-altronate, D-mannonate, xylitol, xylose and galactose for carton implying, are less than that in B. endophyticus Hbe603. In the propanoate metabolism, only B. endophyticus Hbe603 harbors a complete pathway of six steps to converse 2-oxobutanoate to succinyl-CoA, thus implies for the citrate cycle. The absence of propionyl-CoA carboxylase and methylmalonyl-CoA mutase in B. thuringiensis Bc601 significantly impedes the metabolic flux to citrate cycle. Particularly, three methylmalonyl-CoA mutases are identified in B. endophyticus Hbe603, which have only been found in B. megaterium, Geobacillus kaustophilus and Bacillus halodurans.

In the amino acid metabolism, B. endophyticus Hbe603 has more lysine degradation related genes than B. thuringiensis Bc601, the ratio of gene number is 26:18. In the tyrosine metabolism, the two species are both lack of adequate genes. B. thuringiensis Bc601 uses 4- hydroxyphenylpyruvate dioxygenase and homogentisate 1, 2-dioxygenase to converse tyrosine to 4-maleyl-acetoacetate, and phenylalanine-4-hydroxylase to converse phenylalanine to tyrosine. B. endophyticus Hbe603 uses tyrosinase (EC 1.14.18.1) to converse tyrosine to pheomelanin and eunelanin. In the tryptophan metabolism, the two species are both lack of the degradation pathway. They use tryptophan 2, 3- dioxygenase and kynureninase to converse tryptophan to formyl-anthranilate. The two species both have the complete phenylalanine, tyrosine and tryptophan biosynthesis pathway while only B. thuringiensis Bc601 has the phenylalanine-4-hydroxylase to converse the phenylalanine to tyrosine. The glutathione metabolism in B. thuringiensis Bc601 is not complete and B. endophyticus Hbe603 contains five gamma-glutamyl-transpeptidases to converse glutathione to glycine, cysteine and glutamate.

In the cofactor and vitamin metabolism, the two species both have the complete biosynthesis pathway of folate, protoheme, pantothenate and CoA, while the lipoic acid and biotin biosythensis pathway are defect. In the inositol phosphate metabolism, only B. endophyticus Hbe603 contains a complete pathway of thirteen key enzymes, including myo-inositol 2-dehydrogenase, inosose dehydratase, glucuronate isomerases, 5-dehydro-2-deoxygluconokinase, 6-phospho-5-dehydro-2-deoxy-D-gluconate aldolase, methylmalonate-semialdehyde dehydrogenase and triosephosphate isomerase. That pathway converses inositol to acetyl-CoA and glyceraldehyde-3-phosphate, which participates in TCA cycle and glycolysis, respectively. In the nicotinate and nicotinamide metabolism, the two species both can transfer L-aspartate to nicotinate, but only B. thuringiensis Bc601 contains three nucleotidases that can transfer L-aspartate to nicotinamide.

Discussion

Microorganisms can often utilize and secrete a large number of metabolites. This plastic network is readily adapted and regulated in response to nutrients. Swarming motility is a multi-cellular behavior which can help us to better understand the metabolic interaction and cooperative mechanism23,24. Swarming motility of B. thuringiensis could be induced when it was co-cultured with K. vulgare, while B. endophyticus didn’t swarm to K. vulgare. The metabolic interaction and companion mechanism of the two Bacillus species are completely different in bacterial consortium (Fig. 4).

Figure 4: Schematic representation of the companion mechanisms of Bacillus thuringiensis Bc601 and Bacillus endophyticus Hbe603 in bacterial consortium at a genome-wide scale.
figure 4

For the ability of response to the external environment, B. thuringiensis Bc601 has more collagenases, ATPases, two-component system and peptidoglycan biosynthesis related proteins than B. endophyticus Hbe603 (marked in blue words). For the ability of nutrients biosynthesis, B. endophyticus Hbe603 has more PTSsystem, propanoate, glutathione and inositol phosphate metabolism, and amino acid degradation related proteins than B. thuringiensis Bc601 (marked in red words).

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By the means of metabolomics, the metabolites changes were identified by B. thuringiensis, K. vlugare and consortium10. The contents of nutritional compounds in the medium surrounding K. vulgare were fairly higher than in fresh medium. Erythrose, erythritol, guanine and inositol accumulated around B. thuringiensis were consumed by K. vulgare, and the oxidization products of K. vulgare were sharply increased. For the ability of response to the external environment, B. thuringiensis Bc601 has more two-component system, sporulation coat and peptidoglycan biosynthesis related proteins than B. endophyticus Hbe603. B. thuringiensis is capable of crawling along K. vulgare, indicating that it has a stronger ability to communicate to the external environment and respond to the nutrients surrounding K. vulgare.

Our previous research showed that the sub-cultivated B. thuringiensis and K. vulgare significantly increased the productivity of 2-keto-L-gulonic acid25. By culturing the B. thuringiensis and K. vulgare orthogonally on agar plates, the swarming distance of B. thuringiensis along the trace of K. vulgare decreased after 150 days’ sub-cultivation26. Metabolomic analysis showed that the ability of nutrients searching and intaking was increasing in the evolved B. thuringiensis and it provided more nutrients to K. vulgare. For the ability of nutrients biosynthesis, B. endophyticus Hbe603 has more alpha-galactosidases, propanoate, glutathione and inositol phosphate metabolism, and amino acid degradation related proteins than B. thuringiensis Bc601. B. endophyticus didn’t swarm to K. vulgare, probably due to its adequate metabolic capacity in consortium. The high production of 2-KLG is also connected with the abundant nutrition that B. endophyticus provided.

Although thousands of B. thuringiensis strains were isolated, less than 30 complete genomes were obtained. The complete genome sequencing of B. thuringiensis Bc601 adds a new member in genome library of B. thuringiensis family, and the genomic analysis will give us the opportunity to investigate the diversity and evolution among B. thuringiensis-B. cereus family. B. endophyticus Hbe603 is the first in that species with complete genome, which will provide the important genetic background and molecular information for gene modification. All in all, comparative genomic analysis of B. endophyticus and B. thuringiensis enables us to identify the unique genes for each species and understand the companion mechanism for system optimization.

Methods

Bacteria and cultivation conditions

The growth medium was composed of 2% L-sorbose, 0.3% corn-steep liquor (CSL), 1% peptone, 0.3% yeast extract, 0.3% beef extract, 0.1% urea, 0.1% KH2PO4, 0.02% MgSO4·7H2O and 0.1% CaCO3. The fermentation medium contained 8% L-sorbose, 2% corn-steep liquor (CSL), 1.2% urea, 0.1% KH2PO4, 0.05% MgSO4.7H2O and 0.1% CaCO3. The seed cultures of K. vulgare and Bacillus species were cultivated in 250 mL flasks with 50 mL growth medium (30 °C, 250 rpm) for 24 hours. Subsequently, the two species were co-inoculated into 250 mL flasks with 50 mL fermentation medium at 250 rpm, 30 °C for 96 hours. Swarming motility was observed by culturing the two Bacillus species with K. vulgare orthogonally on agar plates. Bacillus species and K. vulgare were cultivated for 12 hours and 36 hours, respectively, and then inoculated separately in solid sorbose-CSL medium containing 1.5% agar and cultivated at 30 °C for 96 hours.

Analyses of 2-KLG and biomass

The concentration of extracellular 2-KLG were determined by the High Performance Liquid Chromatography (HPLC) (Waters Corp., Massachusetts, USA), equipped with an Aminex HPX-87H column (Bio-Rad, CA) and a refractive index detector. The mobile phase used in the HPLC system was 5 mM H2SO4 at 65 °C with a flow rate of 0.6 mL/min. The cell density was measured as optical density at 600 nm (OD600) with a spectrophotometer after dissolving CaCO3 in 100 mM HCl.

DNA extraction, genome sequencing and assembly

Isolation of genomic DNA was carried out using SDS method. Total DNA obtained was subjected to quality control by agarose gel electrophoresis and quantified by Qubit. DNA was used to construct a 10 kb SMRTbell library, and the genome was sequenced by Single Molecule, Real-Time (SMRT) technology. Sequencing was performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. SMRT Analysis 2.3.0 were used to filter low quality reads and the filtered reads were assembled by SOAPdenovo (http://soap.genomics.org.cn/soapdenovo.html) to generate the complete genome, which has been confirmed by PCR amplification.

Gene prediction, annotation and protein classification

Gene prediction was performed on the genome assembly by GeneMarkS27. Transfer RNAs (tRNAs) were predicted with tRNAscan-SE28, ribosome RNAs (rRNAs) were predicted with rRNAmmer29 and sRNAs were predicted by BLAST against Rfam database30. PHAST31 was used for prophage prediction and CRISPRFinder32 was used for CRISPR identification. A whole genome Blast33 search was performed against KEGG database (Kyoto Encyclopedia of Genes and Genomes)34, COG database (Clusters of Orthologous Groups)35, NR database (Non-Redundant Protein Database) and Swiss-Prot database36. The origin of replication (oriC) and putative DnaA boxes were identified by Ori-Finder37. CVTree was performed for the phylogenetic analysis38 and the phylogenetic tree was generated using the MEGA program39. The GC-Profile was used to compute the GC content variation in DNA sequences and to predict the horizontal gene transfer40. CGView Server was used for the visualization of circular genomes41 and the metabolic network was constructed by KEGG automatic annotation server KAAS42.

Nucleotide sequence accession numbers

The genome sequence of the B. thuringiensis Bc601 has been deposited at DDBJ/EMBL/GenBank under the accession numbers CP015150 (chromosome), CP015151 to CP015156 (six plasmids, respectively). The genome sequence of the B. endophyticus HBe603 has been deposited at DDBJ/EMBL/GenBank under the accession numbers CP011974 (chromosome), CP015323 to CP015330 (eight plasmids, respectively).

Additional Information

How to cite this article: Jia, N. et al. Comparative genomics analysis of the companion mechanisms of Bacillus thuringiensis Bc601 and Bacillus endophyticus Hbe603 in bacterial consortium. Sci. Rep. 6, 28794; doi: 10.1038/srep28794 (2016).