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BIOL 398: Bioinformatics Laboratory

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1 BIOL 398: Bioinformatics Laboratory
σB is required for M. tuberculosis stress response in vitro, but has little effect on survival in vivo Fontán P. A., Voskuil M. I., Gomez M., Tan D., Pardini M., Manganelli R., Fattorini L., Schoolnik G. K., Smith I. (2009 July 10). J. Bacteriol, 191, doi: /JB Journal Club 3 Nicole Anguiano November 19, 2014 BIOL 398: Bioinformatics Laboratory

2 OUTLINE σB plays a role in the stress response of M. tuberculosis
In vitro, sigB mutants display higher sensitivity to all forms of stress outside of heat stress DNA microarray results for σB show little overlap in regulated genes with σE and σH, but did confirm three new genes regulated by σB In vitro growth does not follow the trends seen in the in vivo results

3 OUTLINE σB plays a role in the stress response of M. tuberculosis
In vitro, sigB mutants display higher sensitivity to all forms of stress outside of heat stress DNA microarray results for σB show little overlap in regulated genes with σE and σH, but did confirm three new genes regulated by σB In vitro growth does not follow the trends seen in the in vivo results

4 Sigma factors bind to RNA polymerases and increase promoter specificity
A sigma factor (σB) allows the RNA polymerase to bind to the promoter and begin transcription Different sigma factors are used when the cell is under different environmental stresses More sigma factors → more adaptability to stress M. tuberculosis has 13 sigma factors σB is closely related to σA, the primary sigma factor

5 Transcription of sigB is induced during the stress response
σB is closely related to σA, the primary sigma factor The structural gene for σB, sigB, is positively regulated by σE, σH, and σL σE regulates the response to cell envelope stress, and σH regulates the response to heat and oxidative stress σB is a member of both regulons Due to sigB expression being controlled by many regulatory pathways, it likely plays a central role in the stress response

6 sigB importance in the stress response was tested both in vivo and in vitro
Three strains were used in the tests: A wild type strain A sigB mutant with a disrupted sigB gene A complemented strain that re-introduces the sigB gene elsewhere in the DNA In vitro, cell envelope, heat, and oxidative stress was tested: Cell envelope stress: application of 0.05% SDS and 5mM diamide Heat stress: heated to 45°C for 24 hours Oxidative: sealed for 4 days In vivo, the three strains were raised in mice, guinea pigs, and macrophage-like cells

7 OUTLINE σB plays a role in the stress response of M. tuberculosis
In vitro, sigB mutants display higher sensitivity to all forms of stress outside of heat stress DNA microarray results for σB show little overlap in regulated genes with σE and σH, but did confirm three new genes regulated by σB In vitro growth does not follow the trends seen in the in vivo results

8 The sigB mutant was more sensitive to SDS than the complemented or wild type strains
Gray: wild type Black: sigB mutant White: complemented strain Lower colony diameter seen in sigB mutant

9 Heat stress showed no difference in rolling cultures
Standing cultures showed decreased survival in the mutant strain Due to discrepancy, low levels of oxygen in standing culture were suspected Gradual loss of oxygen was tested to prove this hypothesis

10 Effect of hypoxia on mutant survival is not apparent when represented as ODs
OD = optical density Results not easily apparent initially

11 sigB mutants had significantly decreased survival in low oxygen
Proved hypothesis that low oxygen was the reason for the discrepancy in the heat stress test sigB mutant has a 3-log-order lower survival

12 OUTLINE σB plays a role in the stress response of M. tuberculosis
In vitro, sigB mutants display higher sensitivity to all forms of stress outside of heat stress DNA microarray results for σB show little overlap in regulated genes with σE and σH, but did confirm three new genes regulated by σB In vitro growth does not follow the trends seen in the in vivo results

13 σB regulates genes relating to both cell envelope and oxidative stress
Used in comparing results with the wild type and the sigB mutant

14 There was little overlap between the genes regulated by σE, σH, and σB in the mutant
Only a single gene overlapped between the σB and σE regulons during the SDS and oxidative stress tests Rv0465c, a putative transcriptional regulator Under oxidative stress, 40 genes were found to be controlled by σB Only 2 overlapped with the σH regulon Rv0251c and Rv0384c, both heat shock proteins

15 Decrease in expression of genes in the mutant indicates regulation by σB
All genes saw differences in expression during various tests with the exception of Rv1361c: Rv1361c: non-stressed bacteria ideR: SDS treatment Rv0251c: diamide treatment Confirmed by RT-PCR

16 The proposed σB consensus sequence is seen in genes regulated by σB
The putative promoter consensus sequence was seen in genes expressed by σB under stress conditions

17 OUTLINE σB plays a role in the stress response of M. tuberculosis
In vitro, sigB mutants display higher sensitivity to all forms of stress outside of heat stress DNA microarray results for σB show little overlap in regulated genes with σE and σH, but did confirm three new genes regulated by σB In vitro growth does not follow the trends seen in the in vivo results

18 No differences are seen in vivo between strains in macrophages

19 No differences in growth between strains are seen in mice

20 No differences in growth are seen between strains in guinea pigs

21 The difference in in vivo and in vitro growth of mutants had been previously observed
In a study analyzing dosR, the same difference in in vivo and in vitro survivability was seen (Rustad) Likely due to poor representation in available animal models Indicates complexity in regulatory networks

22 Summary σB plays a role in the stress response of M. tuberculosis
In vitro, sigB mutants display higher sensitivity to all forms of stress outside of heat stress, and results from heat stress were due to low oxygen DNA microarray results for σB show little overlap in regulated genes with σE and σH, but did confirm three new genes regulated by σB In vitro growth does not follow the trends seen in the in vivo results, which was also seen in previous studies

23 Citation Fontán P. A., Voskuil M. I., Gomez M., Tan D., Pardini M., Manganelli R., Fattorini L., Schoolnik G. K., Smith I. (2009 July 10). The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth. J. Bacteriol, 191, doi: /JB Rustad, T. R., M. I. Harrell, R. Liao, and D. R. Sherman. (2008). The enduring hypoxic response of Mycobacterium tuberculosis. PLoS One 3:e1502.

24 ACKNOWLEDGMENTS Dr. Kam D. Dahlquist Stephen Louie Loyola Marymount University

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