High-Resolution 3D Genome Map of Brucella Chromosomes in Exponential and Stationary Phases.

The three-dimensional (3D) genome structure of an organism or cell is highly relevant to its biological activities, but the availability of 3D genome information for bacteria, especially intracellular pathogens, is still limited. Here, we used Hi-C (high-throughput chromosome conformation capture) technology to determine the 3D chromosome structures of exponential- and stationary-phase Brucella melitensis at a 1-kb resolution. We observed that the contact heat maps of the two B. melitensis chromosomes contain a prominent diagonal and a secondary diagonal. Then, 79 chromatin interaction domains (CIDs) were detected at an optical density at 600 nm (OD600) of 0.4 (exponential phase), with the longest CID being 106 kb and the shortest being 12 kb. Moreover, we obtained 49,363 significant cis-interaction loci and 59,953 significant trans-interaction loci. Meanwhile, 82 CIDs of B. melitensis at an OD600 of 1.5 (stationary phase) were detected, with the longest CID being 94 kb and the shortest being 16 kb. In addition, 25,965 significant cis-interaction loci and 35,938 significant trans-interaction loci were obtained in this phase. Furthermore, we found that as the B. melitensis cells grew from the logarithmic to the plateau phase, the frequency of short-range interactions increased, while that of long-range interactions decreased. Finally, combined analysis of 3D genome and whole-genome transcriptome (RNA-seq) data revealed that the strength of short-range interactions in Chr1 is specifically and strongly correlated with gene expression. Overall, our study provides a global view of the chromatin interactions in the B. melitensis chromosomes, which will serve as a resource for further study of the spatial regulation of gene expression in Brucella. IMPORTANCE The spatial structure of chromatin plays important roles in normal cell functions and in the regulation of gene expression. Three-dimensional genome sequencing has been performed in many mammals and plants, but the availability of such data for bacteria, especially intracellular pathogens, is still limited. Approximately 10% of sequenced bacterial genomes contain more than one replicon. However, how multiple replicons are organized within bacterial cells, how they interact, and whether these interactions help to maintain or segregate these multipartite genomes are unresolved issues. Brucella is a Gram-negative, facultative intracellular, and zoonotic bacterium. Except for Brucella suis biovar 3, Brucella species have two chromosomes. Here, we applied Hi-C technology to determine the 3D genome structures of exponential- and stationary-phase Brucella melitensis chromosomes at a 1-kb resolution. Combined analysis of the 3D genome and RNA-seq data indicated that the strength of short-range interactions in B. melitensis Chr1 is specifically and strongly correlated with gene expression. Our study provides a resource to achieve a deeper understanding of the spatial regulation of gene expression in Brucella.

Genome-wide transcription start site mapping in the facultative intracellular pathogen Brucella melitensis by Capping-seq.

Brucella melitensis (B. melitensis) is an important facultative intracellular bacterium that causes global zoonotic diseases. Continuous intracellular survival and replication are the main obstruction responsible for the accessibility of prevention and treatment of brucellosis. Bacteria respond to complex environment by regulating gene expression. Many regulatory factors function at loci where RNA polymerase initiates messenger RNA synthesis. However, limited gene annotation is a current obstacle for the research on expression regulation in bacteria. To improve annotation and explore potential functional sites, we proposed a novel genome-wide method called Capping-seq for transcription start site (TSS) mapping in B. melitensis. This technique combines capture of capped primary transcripts with Single Molecule Real-Time (SMRT) sequencing technology. We identified 2,369 TSSs at single nucleotide resolution by Capping-seq. TSSs analysis of Brucella transcripts showed a preference of purine on the TSS positions. Our results revealed that -35 and -10 elements of promoter contained consensus sequences of TTGNNN and TATNNN, respectively. The 5' ends analysis showed that 57% genes are associated with more than one TSS and 47% genes contain long leader regions, suggested potential complex regulation at the 5' ends of genes in B. melitensis. Moreover, we identified 52 leaderless genes that are mainly involved in the metabolic processes. Overall, Capping-seq technology provides a unique solution for TSS determination in prokaryotes. Our findings develop a systematic insight into the primary transcriptome characterization of B. melitensis. This study represents a critical basis for investigating gene regulation and pathogenesis of Brucella.

Isolation of Flavonoids From Wild Aquilaria sinensis Leaves by an Improved Preparative High-Speed Counter-Current Chromatography Apparatus.

Four flavonoids including apigenin-7,4'-dimethylether, genkwanin, quercetin, and kaempferol were isolated in a preparative or semi-preparative scale from the leaves of wild Aquilaria sinensis using an improved preparative high-speed counter-current chromatography apparatus. The separations were performed with a two-phase solvent system composed of hexane-ethyl acetate, methanol-water at suitable volume ratios. The obtained fractions were analyzed by HPLC, and the identification of each target compound was carried out by ESI-MS and NMR. The yields of the above four target flavonoids were 4.7, 10.0, 11.0 and 4.4%, respectively. All these four flavonoids exhibited nitrite scavenging activities with the clearance rate of 12.40 ± 0.20%, 5.84 ± 0.03%, 28.10 ± 0.17% and 5.19 ± 0.11%, respectively. Quercetin was originally isolated from the Thymelaeaceae family, while kaempferol was isolated from the Aquilaria genus for the first time. In cytotoxicity test these two flavonoids exhibited moderate inhibitory activities against HepG2 cells with the IC50 values of 12.54 ± 1.37 and 38.63 ± 4.05 µM, respectively.