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Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae.

Moustafa DA, Jain N, Sriranganathan N, Vemulapalli R - PLoS ONE (2010)

Bottom Line: Brucella neotomae is not known to be associated with clinical disease in any host species.Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice.These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA.

ABSTRACT
Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.

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Related in: MedlinePlus

Specific activity of SOD enzyme in the periplasmic extracts of B. neotomae, B. neotomae/pBB4SOD, and B. abortus RB51 at different growth stages.The growth curves of the bacteria are shown in the top panel. The specific activities of SOD enzyme are shown in the bottom panel. For each Brucella, overnight culture was used at 1∶200 dilution to inoculate fresh 100 ml media and at different time intervals, the OD600 was measured and an aliquot of the culture was taken for preparing periplasmic extracts. With extracts of each time-point, the SOD assay was performed twice, each time in duplicates, and the results are shown as the mean ± standard deviation of specific activity (units/mg of protein). At each time point, the groups with one or two asterisks were significantly different from the B. neotomae group (P<0.001). At 4 and 48 hours time-points, there were significant differences between the groups with different number of asterisks (P<0.001).
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pone-0014112-g002: Specific activity of SOD enzyme in the periplasmic extracts of B. neotomae, B. neotomae/pBB4SOD, and B. abortus RB51 at different growth stages.The growth curves of the bacteria are shown in the top panel. The specific activities of SOD enzyme are shown in the bottom panel. For each Brucella, overnight culture was used at 1∶200 dilution to inoculate fresh 100 ml media and at different time intervals, the OD600 was measured and an aliquot of the culture was taken for preparing periplasmic extracts. With extracts of each time-point, the SOD assay was performed twice, each time in duplicates, and the results are shown as the mean ± standard deviation of specific activity (units/mg of protein). At each time point, the groups with one or two asterisks were significantly different from the B. neotomae group (P<0.001). At 4 and 48 hours time-points, there were significant differences between the groups with different number of asterisks (P<0.001).

Mentions: As shown in Fig. 2, SOD activity was detected in B. neotomae, B. neotomae/pBB4SOD and B. abortus RB51 at all time-points tested during the 48-hour culture period. The maximum enzyme activity was detected in B. neotomae during late log phase (24 hours) and stationary phase (48 hours). However, at all time-points tested, the SOD specific activity in B. neotomae was lower than that of B. abortus RB51 and B. neotomae/pBB4SOD.


Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae.

Moustafa DA, Jain N, Sriranganathan N, Vemulapalli R - PLoS ONE (2010)

Specific activity of SOD enzyme in the periplasmic extracts of B. neotomae, B. neotomae/pBB4SOD, and B. abortus RB51 at different growth stages.The growth curves of the bacteria are shown in the top panel. The specific activities of SOD enzyme are shown in the bottom panel. For each Brucella, overnight culture was used at 1∶200 dilution to inoculate fresh 100 ml media and at different time intervals, the OD600 was measured and an aliquot of the culture was taken for preparing periplasmic extracts. With extracts of each time-point, the SOD assay was performed twice, each time in duplicates, and the results are shown as the mean ± standard deviation of specific activity (units/mg of protein). At each time point, the groups with one or two asterisks were significantly different from the B. neotomae group (P<0.001). At 4 and 48 hours time-points, there were significant differences between the groups with different number of asterisks (P<0.001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991346&req=5

pone-0014112-g002: Specific activity of SOD enzyme in the periplasmic extracts of B. neotomae, B. neotomae/pBB4SOD, and B. abortus RB51 at different growth stages.The growth curves of the bacteria are shown in the top panel. The specific activities of SOD enzyme are shown in the bottom panel. For each Brucella, overnight culture was used at 1∶200 dilution to inoculate fresh 100 ml media and at different time intervals, the OD600 was measured and an aliquot of the culture was taken for preparing periplasmic extracts. With extracts of each time-point, the SOD assay was performed twice, each time in duplicates, and the results are shown as the mean ± standard deviation of specific activity (units/mg of protein). At each time point, the groups with one or two asterisks were significantly different from the B. neotomae group (P<0.001). At 4 and 48 hours time-points, there were significant differences between the groups with different number of asterisks (P<0.001).
Mentions: As shown in Fig. 2, SOD activity was detected in B. neotomae, B. neotomae/pBB4SOD and B. abortus RB51 at all time-points tested during the 48-hour culture period. The maximum enzyme activity was detected in B. neotomae during late log phase (24 hours) and stationary phase (48 hours). However, at all time-points tested, the SOD specific activity in B. neotomae was lower than that of B. abortus RB51 and B. neotomae/pBB4SOD.

Bottom Line: Brucella neotomae is not known to be associated with clinical disease in any host species.Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice.These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA.

ABSTRACT
Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.

Show MeSH
Related in: MedlinePlus