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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

Survival of B. melitensis 16 MΔotpR, 16 McΔotpR, and the wild-type strain under iron-limited conditions.The survival of the strains was measured in the TSB medium containing 2.5 mM, 5.0 mM and 10.0 mM Fe2+ chelator 2,2`-dipyridyl. The measurements were taken at 24 h, 48 h and 72 h after inoculation. Experiments were performed in triplicates and a significant difference was observed. Statistical analysis was performed by comparing the CFUs of 16 M versus 16 MΔotpR bacteria at different time points using the Student’s t-test. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represents different levels of significant differences. It is noted that in the presence of 10.0 mM DIP, the otpR mutant could be detected at 24 and 48 h after inoculation, but not at 72 h. When the CFU/mL was determined, 200 μL of culture sample was used in each condition, which equals to a detection limit of 5 CFU/ml (or ~0.7 LOG CFU). It is possible that there were still some viable otpR mutant cells survived in the iron-limited medium at 72 h; however, the level of survival was below the laboratory’s detection limit.
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f4: Survival of B. melitensis 16 MΔotpR, 16 McΔotpR, and the wild-type strain under iron-limited conditions.The survival of the strains was measured in the TSB medium containing 2.5 mM, 5.0 mM and 10.0 mM Fe2+ chelator 2,2`-dipyridyl. The measurements were taken at 24 h, 48 h and 72 h after inoculation. Experiments were performed in triplicates and a significant difference was observed. Statistical analysis was performed by comparing the CFUs of 16 M versus 16 MΔotpR bacteria at different time points using the Student’s t-test. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represents different levels of significant differences. It is noted that in the presence of 10.0 mM DIP, the otpR mutant could be detected at 24 and 48 h after inoculation, but not at 72 h. When the CFU/mL was determined, 200 μL of culture sample was used in each condition, which equals to a detection limit of 5 CFU/ml (or ~0.7 LOG CFU). It is possible that there were still some viable otpR mutant cells survived in the iron-limited medium at 72 h; however, the level of survival was below the laboratory’s detection limit.

Mentions: To confirm that OtpR regulates iron metabolism, the tolerance of 16 MΔotpR under an experimental condition of low iron was assessed after adding varying concentrations of the Fe2+ chelator DIP into the medium. In the presence of 2.5 mM, 5.0 mM, or 10 mM DIP, the survival capability of the mutant strain 16 MΔotpR was less than its parental strain 16 M (Fig. 4), suggesting that OtpR is critical to the utilization of iron in the low iron medium. The otpR gene complementation of 16 MΔotpR recovered the bacterial survival probably due to the recovered function of OtpR in the iron uptake. These results suggest that although the tolerance of 16 McΔotpR to low-iron medium was similar to that of 16 M, the otpR mutant appeared to affect longer-term survival in iron-limited medium.


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)

Survival of B. melitensis 16 MΔotpR, 16 McΔotpR, and the wild-type strain under iron-limited conditions.The survival of the strains was measured in the TSB medium containing 2.5 mM, 5.0 mM and 10.0 mM Fe2+ chelator 2,2`-dipyridyl. The measurements were taken at 24 h, 48 h and 72 h after inoculation. Experiments were performed in triplicates and a significant difference was observed. Statistical analysis was performed by comparing the CFUs of 16 M versus 16 MΔotpR bacteria at different time points using the Student’s t-test. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represents different levels of significant differences. It is noted that in the presence of 10.0 mM DIP, the otpR mutant could be detected at 24 and 48 h after inoculation, but not at 72 h. When the CFU/mL was determined, 200 μL of culture sample was used in each condition, which equals to a detection limit of 5 CFU/ml (or ~0.7 LOG CFU). It is possible that there were still some viable otpR mutant cells survived in the iron-limited medium at 72 h; however, the level of survival was below the laboratory’s detection limit.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Survival of B. melitensis 16 MΔotpR, 16 McΔotpR, and the wild-type strain under iron-limited conditions.The survival of the strains was measured in the TSB medium containing 2.5 mM, 5.0 mM and 10.0 mM Fe2+ chelator 2,2`-dipyridyl. The measurements were taken at 24 h, 48 h and 72 h after inoculation. Experiments were performed in triplicates and a significant difference was observed. Statistical analysis was performed by comparing the CFUs of 16 M versus 16 MΔotpR bacteria at different time points using the Student’s t-test. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represents different levels of significant differences. It is noted that in the presence of 10.0 mM DIP, the otpR mutant could be detected at 24 and 48 h after inoculation, but not at 72 h. When the CFU/mL was determined, 200 μL of culture sample was used in each condition, which equals to a detection limit of 5 CFU/ml (or ~0.7 LOG CFU). It is possible that there were still some viable otpR mutant cells survived in the iron-limited medium at 72 h; however, the level of survival was below the laboratory’s detection limit.
Mentions: To confirm that OtpR regulates iron metabolism, the tolerance of 16 MΔotpR under an experimental condition of low iron was assessed after adding varying concentrations of the Fe2+ chelator DIP into the medium. In the presence of 2.5 mM, 5.0 mM, or 10 mM DIP, the survival capability of the mutant strain 16 MΔotpR was less than its parental strain 16 M (Fig. 4), suggesting that OtpR is critical to the utilization of iron in the low iron medium. The otpR gene complementation of 16 MΔotpR recovered the bacterial survival probably due to the recovered function of OtpR in the iron uptake. These results suggest that although the tolerance of 16 McΔotpR to low-iron medium was similar to that of 16 M, the otpR mutant appeared to affect longer-term survival in iron-limited medium.

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