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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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β-Galactosidase activity in B. abortus virulent strain 2308 (A) and B. abortus vaccine strain RB51 (B) transformed with plasmids containing lacZ gene under the control of wildtype and the indicated mutated B. neotomae sodC promoters.For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. In each panel, means with the same number of asterisks were not significantly different from each other (P>0.05), but there were significant differences between means with different number of asterisks (P<0.001).
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pone-0014112-g008: β-Galactosidase activity in B. abortus virulent strain 2308 (A) and B. abortus vaccine strain RB51 (B) transformed with plasmids containing lacZ gene under the control of wildtype and the indicated mutated B. neotomae sodC promoters.For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. In each panel, means with the same number of asterisks were not significantly different from each other (P>0.05), but there were significant differences between means with different number of asterisks (P<0.001).

Mentions: Activities of the B. neotomae wild-type and the mutated sodC promoters were also assessed in B. abortus virulent strain 2308 and vaccine strain RB51. The β-galactosidase expression analysis in B. abortus strains harboring the plasmid constructs showed that the original B. neotomae sodC promoter and its two single-nucleotide mutation variants were significantly less active than the variant with double-nucleotide mutation (Fig. 8).


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)

β-Galactosidase activity in B. abortus virulent strain 2308 (A) and B. abortus vaccine strain RB51 (B) transformed with plasmids containing lacZ gene under the control of wildtype and the indicated mutated B. neotomae sodC promoters.For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. In each panel, means with the same number of asterisks were not significantly different from each other (P>0.05), but there were significant differences between means 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-g008: β-Galactosidase activity in B. abortus virulent strain 2308 (A) and B. abortus vaccine strain RB51 (B) transformed with plasmids containing lacZ gene under the control of wildtype and the indicated mutated B. neotomae sodC promoters.For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. In each panel, means with the same number of asterisks were not significantly different from each other (P>0.05), but there were significant differences between means with different number of asterisks (P<0.001).
Mentions: Activities of the B. neotomae wild-type and the mutated sodC promoters were also assessed in B. abortus virulent strain 2308 and vaccine strain RB51. The β-galactosidase expression analysis in B. abortus strains harboring the plasmid constructs showed that the original B. neotomae sodC promoter and its two single-nucleotide mutation variants were significantly less active than the variant with double-nucleotide mutation (Fig. 8).

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