Comparative genomics analysis of the companion mechanisms of Bacillus thuringiensis Bc601 and Bacillus endophyticus Hbe603 in bacterial consortium

Bacillus thuringiensis and Bacillus endophyticus both act as the companion bacteria, which cooperate with Ketogulonigenium vulgare in vitamin C two-step fermentation. Two Bacillus species have different morphologies, swarming motility and 2-keto-L-gulonic acid productivities when they co-culture with K. vulgare. Here, we report the complete genome sequencing of B. thuringiensis Bc601 and eight plasmids of B. endophyticus Hbe603, and carry out the comparative genomics analysis. Consequently, B. thuringiensis Bc601, with greater ability of response to the external environment, has been found more two-component system, sporulation coat and peptidoglycan biosynthesis related proteins than B. endophyticus Hbe603, and B. endophyticus Hbe603, with greater ability of nutrients biosynthesis, has been found more alpha-galactosidase, propanoate, glutathione and inositol phosphate metabolism, and amino acid degradation related proteins than B. thuringiensis Bc601. Different ability of swarming motility, response to the external environment and nutrients biosynthesis may reflect different companion mechanisms of two Bacillus species. Comparative genomic analysis of B. endophyticus and B. thuringiensis enables us to further understand the cooperative mechanism with K. vulgare, and facilitate the optimization of bacterial consortium.

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).
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 clustering 20 . 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 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).
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 repair 21 . In the process of sporulation, the companion Bacillus bacterium further releases abundant nutrients for the growth and 2-KLG production of K. vulgare 22 . 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).
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 mechanism 23,24 . 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).
By the means of metabolomics, the metabolites changes were identified by B. thuringiensis, K. vlugare and consortium 10 . 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 acid 25 . 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-cultivation 26 . 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.

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 H 2 SO 4 at 65 °C with a flow rate of 0.6 mL/min. The cell density was measured as optical density at 600 nm (OD 600 ) with a spectrophotometer after dissolving CaCO 3 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 GeneMarkS 27 . Transfer RNAs (tRNAs) were predicted with tRNAscan-SE 28 , ribosome 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).