Wang et al Anticancer Exopolysaccharide From B breve Iwo1 TABLE 1 I Features of B breve lol genome TABLE 2 COG categories of coding proteins in B. breve lwo1 genome Features CoG Name Count Percent (p) GC content (%6 INA processing and modification RNAS Energy production and conversion TRNAS 2,2,25s,168,23s)D Cell cycle control, cell avision tRNAs Amino acid transport and 10.69 metabolism CDSs ( with protein 4.09 metabolism Statistical Analysis G carbohydrate transport an 253 1294 The absorbance value in CCK-8 assay and western blot results were expressed as means standard deviation(SD). One-way enzyme transport and ANOVa was used for comparison among different concentration group and Fishers least significant difference(LSD)method was d transport and metabolism Translation ribosomal structure and used for multiple comparisons in CCK-8 assay. Independent biogenesis sample t-test was used to compare the treatment group andK Transcription control group in western blot assay. All calculations and L replication, recombination and analyses were performed using SPSS 20.0 software(SPSS Inc Chicago, IL, United States). A P-value 0.05 was considered M statistically significant. Cell motility 061 Posttranslational modification RESULTS protein tumover, chaperones Inorganic ion transport and 4.81 metabolism Isolation of bifidobacterium Secondary metabolites a total of 653 strains bacteria were isolated from the infant fecal samples. Among those, 23 viscous strains were isolated catabolism d transferred in a modified mrs-C medium. one strain R General function prediction only was typically rod-shaped or bifurcated, non-spore-forming. S Function unknown Consequently, this clone was analyzed using 16S rRNA PCR (16S T Signal transduction mechanisms rRNA-F: 5-AGAGTTTGATCMTGGCTCAG-3' and 16S rRNA- U Intracellular traficking, secretion, R: 5'-TACGGYTACCTTGTTACGACTT-3). The 16S RNA and vesicular transport sequence analyses showed 99% identity with B breve DSM20213 V 67 3.43 (Supplementary Figure S1) Extrace ular structures Mobilome: prophages, transposons Genome Information of b, breve lwo1 The complete genome sequence of our new isolated B breve strain was submitted to GenBank under the accession Number genes. Priming-GTF (P-atf) is the enzyme that catalyzes CP034192. The complete genome of this strain is composed of the initial step of EPS-unit synthesis; consequently,we one circular chromosome of 2, 313, 172 bp with a GC content of first found this enzyme: UDP-galactosephosphotransferase 58.7%, which is similar to other reported B breve strains(GC (EH245_02110)coding for proteins of 576 amino acids ntent of 58.5-62.8%). In addition, the new strain contains Five genes were coding for the GTFs(EH245_02130, total of 63 RNAs including 54 tRNA genes, 2 rRNA, and 3 EH245_02135, EH24502140, EH245_02145, EH245 02155 ncRNAs. A total of 1862 coding genes out of 2077 total genes Two types of genes could be responsible for transporting were classified into 23 COG categories(Tables 1, 2 and Figure 1). of the repeat EPS-units across the cytoplasmic membrane The genome comparative analyses among B breve lwol and other flippase(EH245_02150) and membrane spanning protein seven Bifidobacterium strains are presented in Supplementary (EH245-_02120, EH245-_02180). The chain length regulation Table S1. Consequently, this strain, named B. breve Iwol was and polymerization system were also found in this EPS deposited in China Center of Industrial Culture Collection unit, which included the polymerase(EH245-_02190), chain (CICC)under the number CICC 24633 length regulator(EH245-_02200)and the protein tyrosine phosphatase(EH245-_02115). Another discovered feature was In silico Analysis of Bifido-EPS Cluster the presence of transposase (EH245-_02125, EH245-02165 Bifidobacterium breve lwol harbors an EPS-encoding EH245-_02170, EH245-_02175, EH245-_02185), an enzyme cluster with 14 predicted genes and 5 transposase coding participated putative horizontal transfer of EPS among FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044fmicb-10-01044 May 8, 2019 Time: 14:36 # 4 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 TABLE 1 | Features of B. breve lw01 genome. Features Chromosome Genome size (bp) 2,313,172 GC content (%) 58.7 RNAs 63 rRNAs 2, 2, 2 (5S, 16S, 23S) tRNAs 54 ncRNAs 3 CDSs (with protein) 1862 Statistical Analysis The absorbance value in CCK-8 assay and western blot results were expressed as means ± standard deviation (SD). One-way ANOVA was used for comparison among different concentration group and Fisher’s least significant difference (LSD) method was used for multiple comparisons in CCK-8 assay. Independent sample t-test was used to compare the treatment group and control group in western blot assay. All calculations and analyses were performed using SPSS 20.0 software (SPSS Inc., Chicago, IL, United States). A P-value < 0.05 was considered statistically significant. RESULTS Isolation of Bifidobacterium A total of 653 strains bacteria were isolated from the infant fecal samples. Among those, 23 viscous strains were isolated and transferred in a modified MRS-C medium. One strain was typically rod-shaped or bifurcated, non-spore-forming. Consequently, this clone was analyzed using 16S rRNA PCR (16S rRNA-F: 50 -AGAGTTTGATCMTGGCTCAG-30 and 16S rRNA￾R: 50 -TACGGYTACCTTGTTACGACTT-30 ). The 16S rRNA sequence analyses showed 99% identity with B. breve DSM20213 (Supplementary Figure S1). Genome Information of B. breve lw01 The complete genome sequence of our new isolated B. breve strain was submitted to GenBank under the accession Number CP034192. The complete genome of this strain is composed of one circular chromosome of 2,313,172 bp with a GC content of 58.7%, which is similar to other reported B. breve strains (GC content of 58.5–62.8%). In addition, the new strain contains a total of 63 RNAs including 54 tRNA genes, 2 rRNA, and 3 ncRNAs. A total of 1862 coding genes out of 2077 total genes were classified into 23 COG categories (Tables 1, 2 and Figure 1). The genome comparative analyses among B. breve lw01 and other seven Bifidobacterium strains are presented in Supplementary Table S1. Consequently, this strain, named B. breve lw01 was deposited in China Center of Industrial Culture Collection (CICC) under the number CICC 24633. In silico Analysis of Bifido-EPS Cluster Bifidobacterium breve lw01 harbors an EPS-encoding cluster with 14 predicted genes and 5 transposase coding TABLE 2 | COG categories of coding proteins in B. breve lw01 genome. COG Name Count Percent (%) A RNA processing and modification 2 0.10 C Energy production and conversion 53 2.71 D Cell cycle control, cell division, chromosome partitioning 31 1.59 E Amino acid transport and metabolism 209 10.69 F Nucleotide transport and metabolism 80 4.09 G carbohydrate transport and metabolism 253 12.94 H Coenzyme transport and metabolism 77 3.94 I Lipid transport and metabolism 54 2.76 J Translation, ribosomal structure and biogenesis 167 8.54 K Transcription 153 7.83 L Replication, recombination and repair 90 4.60 M Cell wall/membrane/envelope biogenesis 106 5.42 N Cell motility 12 0.61 O Posttranslational modification, protein turnover, chaperones 74 3.79 P Inorganic ion transport and metabolism 94 4.81 Q Secondary metabolites biosynthesis, transport and catabolism 21 1.07 R General function prediction only 153 7.83 S Function unknown 93 4.76 T Signal transduction mechanisms 92 4.71 U Intracellular trafficking, secretion, and vesicular transport 15 0.77 V Defense mechanisms 67 3.43 W Extracellular structures 4 0.20 X Mobilome: prophages, transposons 55 2.81 genes. Priming-GTF (p-gtf) is the enzyme that catalyzes the initial step of EPS-unit synthesis; consequently, we first found this enzyme: UDP-galactosephosphotransferase (EH245_02110) coding for proteins of 576 amino acids. Five genes were coding for the GTFs (EH245_02130, EH245_02135, EH245_02140, EH245_02145, EH245_02155). Two types of genes could be responsible for transporting of the repeat EPS-units across the cytoplasmic membrane: flippase (EH245_02150) and membrane spanning protein (EH245_02120, EH245_02180). The chain length regulation and polymerization system were also found in this EPS￾unit, which included the polymerase (EH245_02190), chain length regulator (EH245_02200) and the protein tyrosine phosphatase (EH245_02115). Another discovered feature was the presence of transposase (EH245_02125, EH245_02165, EH245_02170, EH245_02175, EH245_02185), an enzyme participated putative horizontal transfer of EPS among Frontiers in Microbiology | www.frontiersin.org 4 May 2019 | Volume 10 | Article 1044