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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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Evaluation of strength of sodC promoters from B. abortus, B. neotomae, B. suis biovars 2 (strain Thomsen) and 4 (strain 40).A) Nucleotide sequence features of the promoter-containing 5′ flanking region of B. abortus sodC gene as cloned in pBaSODpro/lacZ. The sodC start codon, ribosomal binding site (RBS), and 5′ ends of cDNA are indicated in bold. The asterisk indicates the site of nucleotide insertion polymorphism with B. neotoame and B. suis biovars. The two potential −10 sequences are boxed (see Discussion). B) Expression levels of β-galactosidase in late log phase cultures of B. neotomae cultures harboring plasmids with lacZ gene cloned under the control of sodC promoters obtained from the indicated Brucella spp. For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. Means with the same number of asterisks were not significantly different from each other (P>0.05). There were significant differences between means with different number of asterisks (P<0.001).
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pone-0014112-g005: Evaluation of strength of sodC promoters from B. abortus, B. neotomae, B. suis biovars 2 (strain Thomsen) and 4 (strain 40).A) Nucleotide sequence features of the promoter-containing 5′ flanking region of B. abortus sodC gene as cloned in pBaSODpro/lacZ. The sodC start codon, ribosomal binding site (RBS), and 5′ ends of cDNA are indicated in bold. The asterisk indicates the site of nucleotide insertion polymorphism with B. neotoame and B. suis biovars. The two potential −10 sequences are boxed (see Discussion). B) Expression levels of β-galactosidase in late log phase cultures of B. neotomae cultures harboring plasmids with lacZ gene cloned under the control of sodC promoters obtained from the indicated Brucella spp. For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. Means with the same number of asterisks were not significantly different from each other (P>0.05). There were significant differences between means with different number of asterisks (P<0.001).

Mentions: In order to verify if the identified nucleotide insertion in the sodC upstream region of B. neotomae and B. suis biovar 2 affected the promoter activity, we assessed the strength of the sodC promoters by constructing translational fusions with E. coli β-galactosidase enzyme by generating plasmids pBnSODpro/laZ, pBaSODpro/lacZ, pBs2SODpro/lacZ, and pBs4SODpro/lacZ (Fig. 5A). The level of β-galactosidase expression in B. neotomae harboring these plasmids was determined by measuring the enzyme activity during late log phase (24 hour cultures). The expression of β-galactosidase under the B. neotomae and B. suis biovar 2 promoters was considerably lower than the expression under B. abortus and B. suis biovar 4 promoters (Fig. 5B).


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)

Evaluation of strength of sodC promoters from B. abortus, B. neotomae, B. suis biovars 2 (strain Thomsen) and 4 (strain 40).A) Nucleotide sequence features of the promoter-containing 5′ flanking region of B. abortus sodC gene as cloned in pBaSODpro/lacZ. The sodC start codon, ribosomal binding site (RBS), and 5′ ends of cDNA are indicated in bold. The asterisk indicates the site of nucleotide insertion polymorphism with B. neotoame and B. suis biovars. The two potential −10 sequences are boxed (see Discussion). B) Expression levels of β-galactosidase in late log phase cultures of B. neotomae cultures harboring plasmids with lacZ gene cloned under the control of sodC promoters obtained from the indicated Brucella spp. For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. Means with the same number of asterisks were not significantly different from each other (P>0.05). There were significant differences between means with different number of asterisks (P<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991346&req=5

pone-0014112-g005: Evaluation of strength of sodC promoters from B. abortus, B. neotomae, B. suis biovars 2 (strain Thomsen) and 4 (strain 40).A) Nucleotide sequence features of the promoter-containing 5′ flanking region of B. abortus sodC gene as cloned in pBaSODpro/lacZ. The sodC start codon, ribosomal binding site (RBS), and 5′ ends of cDNA are indicated in bold. The asterisk indicates the site of nucleotide insertion polymorphism with B. neotoame and B. suis biovars. The two potential −10 sequences are boxed (see Discussion). B) Expression levels of β-galactosidase in late log phase cultures of B. neotomae cultures harboring plasmids with lacZ gene cloned under the control of sodC promoters obtained from the indicated Brucella spp. For each promoter construct, the assays were performed using 3 separate colony cultures. Results are shown as the mean ± standard deviation of Miller units. Means with the same number of asterisks were not significantly different from each other (P>0.05). There were significant differences between means with different number of asterisks (P<0.001).
Mentions: In order to verify if the identified nucleotide insertion in the sodC upstream region of B. neotomae and B. suis biovar 2 affected the promoter activity, we assessed the strength of the sodC promoters by constructing translational fusions with E. coli β-galactosidase enzyme by generating plasmids pBnSODpro/laZ, pBaSODpro/lacZ, pBs2SODpro/lacZ, and pBs4SODpro/lacZ (Fig. 5A). The level of β-galactosidase expression in B. neotomae harboring these plasmids was determined by measuring the enzyme activity during late log phase (24 hour cultures). The expression of β-galactosidase under the B. neotomae and B. suis biovar 2 promoters was considerably lower than the expression under B. abortus and B. suis biovar 4 promoters (Fig. 5B).

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