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RNA-seq reveals the critical role of OtpR in regulating Brucella melitensis metabolism and virulence under acidic stress.

Liu W, Dong H, Li J, Ou Q, Lv Y, Wang X, Xiang Z, He Y, Wu Q - Sci Rep (2015)

Bottom Line: For instance, the virB operon encoding type IV secretion system was significantly down-regulated, and 36 known transcriptional regulators (e.g., vjbR and blxR) were differentially expressed in 16 MΔotpR.Selected RNA-seq results were experimentally confirmed by RT-PCR and RT-qPCR.Overall, these results deciphered differential phenomena associated with virulence, environmental stresses and cell morphology in 16 MΔotpR and 16 M, which provided important information for understanding the detailed OtpR-regulated interaction networks and Brucella pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China [2] Unit for Laboratory Animal Medicine and Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, MI 48109 [3] Center for Computational Medicine and Bioinformatics, The University of Michigan Medical School, Ann Arbor, MI 48109 [4] Beijing Senkang Biotech Development Co., LTD, Beijing 101400, People's Republic of China.

ABSTRACT
The response regulator OtpR is critical for the growth, morphology and virulence of Brucella melitensis. Compared to its wild type strain 16 M, B. melitensis 16 MΔotpR mutant has decreased tolerance to acid stress. To analyze the genes regulated by OtpR under acid stress, we performed RNA-seq whole transcriptome analysis of 16 MΔotpR and 16 M. In total, 501 differentially expressed genes were identified, including 390 down-regulated and 111 up-regulated genes. Among these genes, 209 were associated with bacterial metabolism, including 54 genes involving carbohydrate metabolism, 13 genes associated with nitrogen metabolism, and seven genes associated with iron metabolism. The 16 MΔotpR also decreased capacity to utilize different carbon sources and to tolerate iron limitation in culture experiments. Notably, OtpR regulated many Brucella virulence factors essential for B. melitensis intracellular survival. For instance, the virB operon encoding type IV secretion system was significantly down-regulated, and 36 known transcriptional regulators (e.g., vjbR and blxR) were differentially expressed in 16 MΔotpR. Selected RNA-seq results were experimentally confirmed by RT-PCR and RT-qPCR. Overall, these results deciphered differential phenomena associated with virulence, environmental stresses and cell morphology in 16 MΔotpR and 16 M, which provided important information for understanding the detailed OtpR-regulated interaction networks and Brucella pathogenesis.

No MeSH data available.


Related in: MedlinePlus

Growth curves of the parental B. melitensis strain 16 M, its otpR mutant 16 MΔotpR, and the mutant complementation strain 16 McΔotpR in a synthetic minimal medium.The minimal medium contains only carbon and nitrogen as the nutrient resources. Compared to the wild type strain, the otpR mutant had a decreased OD600 value. The gene complementation of the otpR mutant resumed the OD600 level. A curve of the optical density values at OD600 determined at several time points reflects the growth dynamics of a bacterial strain in the culture medium over the different time points.
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f3: Growth curves of the parental B. melitensis strain 16 M, its otpR mutant 16 MΔotpR, and the mutant complementation strain 16 McΔotpR in a synthetic minimal medium.The minimal medium contains only carbon and nitrogen as the nutrient resources. Compared to the wild type strain, the otpR mutant had a decreased OD600 value. The gene complementation of the otpR mutant resumed the OD600 level. A curve of the optical density values at OD600 determined at several time points reflects the growth dynamics of a bacterial strain in the culture medium over the different time points.

Mentions: To further investigate the importance of OtpR in regulating carbon and nitrogen metabolisms, we used a defined minimal medium that contains only carbon and nitrogen nutrients (without amino acids and growth factors). The minimal medium was used to separately culture parental strain 16 M, 16 MΔotpR, and the mutant complementing strain 16 McΔotpR, followed by the measuring of their dynamic growth profiles. All these three strains were able to grow in the minimal medium, indicating that the inorganic carbon and nitrogen resources provide sufficient nutrients for Brucella growth and replication. Compared to 16 M, the mutant 16MΔotpR showed reduced growth at the late log phase (Fig. 3). The phenomenon suggested that OtpR was important to sustain regular cell growth through the regulation of the carbon and nitrogen metabolism. The observation was further confirmed by the complementation of the gene in the mutant as shown by the full recovery of the cell growth in 16 McΔotpR (Fig. 3).


RNA-seq reveals the critical role of OtpR in regulating Brucella melitensis metabolism and virulence under acidic stress.

Liu W, Dong H, Li J, Ou Q, Lv Y, Wang X, Xiang Z, He Y, Wu Q - Sci Rep (2015)

Growth curves of the parental B. melitensis strain 16 M, its otpR mutant 16 MΔotpR, and the mutant complementation strain 16 McΔotpR in a synthetic minimal medium.The minimal medium contains only carbon and nitrogen as the nutrient resources. Compared to the wild type strain, the otpR mutant had a decreased OD600 value. The gene complementation of the otpR mutant resumed the OD600 level. A curve of the optical density values at OD600 determined at several time points reflects the growth dynamics of a bacterial strain in the culture medium over the different time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542472&req=5

f3: Growth curves of the parental B. melitensis strain 16 M, its otpR mutant 16 MΔotpR, and the mutant complementation strain 16 McΔotpR in a synthetic minimal medium.The minimal medium contains only carbon and nitrogen as the nutrient resources. Compared to the wild type strain, the otpR mutant had a decreased OD600 value. The gene complementation of the otpR mutant resumed the OD600 level. A curve of the optical density values at OD600 determined at several time points reflects the growth dynamics of a bacterial strain in the culture medium over the different time points.
Mentions: To further investigate the importance of OtpR in regulating carbon and nitrogen metabolisms, we used a defined minimal medium that contains only carbon and nitrogen nutrients (without amino acids and growth factors). The minimal medium was used to separately culture parental strain 16 M, 16 MΔotpR, and the mutant complementing strain 16 McΔotpR, followed by the measuring of their dynamic growth profiles. All these three strains were able to grow in the minimal medium, indicating that the inorganic carbon and nitrogen resources provide sufficient nutrients for Brucella growth and replication. Compared to 16 M, the mutant 16MΔotpR showed reduced growth at the late log phase (Fig. 3). The phenomenon suggested that OtpR was important to sustain regular cell growth through the regulation of the carbon and nitrogen metabolism. The observation was further confirmed by the complementation of the gene in the mutant as shown by the full recovery of the cell growth in 16 McΔotpR (Fig. 3).

Bottom Line: For instance, the virB operon encoding type IV secretion system was significantly down-regulated, and 36 known transcriptional regulators (e.g., vjbR and blxR) were differentially expressed in 16 MΔotpR.Selected RNA-seq results were experimentally confirmed by RT-PCR and RT-qPCR.Overall, these results deciphered differential phenomena associated with virulence, environmental stresses and cell morphology in 16 MΔotpR and 16 M, which provided important information for understanding the detailed OtpR-regulated interaction networks and Brucella pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China [2] Unit for Laboratory Animal Medicine and Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, MI 48109 [3] Center for Computational Medicine and Bioinformatics, The University of Michigan Medical School, Ann Arbor, MI 48109 [4] Beijing Senkang Biotech Development Co., LTD, Beijing 101400, People's Republic of China.

ABSTRACT
The response regulator OtpR is critical for the growth, morphology and virulence of Brucella melitensis. Compared to its wild type strain 16 M, B. melitensis 16 MΔotpR mutant has decreased tolerance to acid stress. To analyze the genes regulated by OtpR under acid stress, we performed RNA-seq whole transcriptome analysis of 16 MΔotpR and 16 M. In total, 501 differentially expressed genes were identified, including 390 down-regulated and 111 up-regulated genes. Among these genes, 209 were associated with bacterial metabolism, including 54 genes involving carbohydrate metabolism, 13 genes associated with nitrogen metabolism, and seven genes associated with iron metabolism. The 16 MΔotpR also decreased capacity to utilize different carbon sources and to tolerate iron limitation in culture experiments. Notably, OtpR regulated many Brucella virulence factors essential for B. melitensis intracellular survival. For instance, the virB operon encoding type IV secretion system was significantly down-regulated, and 36 known transcriptional regulators (e.g., vjbR and blxR) were differentially expressed in 16 MΔotpR. Selected RNA-seq results were experimentally confirmed by RT-PCR and RT-qPCR. Overall, these results deciphered differential phenomena associated with virulence, environmental stresses and cell morphology in 16 MΔotpR and 16 M, which provided important information for understanding the detailed OtpR-regulated interaction networks and Brucella pathogenesis.

No MeSH data available.


Related in: MedlinePlus