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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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Multiple alignment of nucleotide sequences of the 5′ flanking region of sodC start codon from B. abortus, B. suis biovars 4 (strain 40) or 1 (strain 1330) and 2 (strain Thomsen), and B. neotomae.Alignment was performed with the Clustal method of the MegAlign program of LaserGene software. Gaps are indicated by dashes. The nucleotides differing between B. suis and B. abortus or B. neotomae are in bold and underlined. Note: B. melitensis nucleotide sequences are identical to those of B. abortus shown above.
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pone-0014112-g003: Multiple alignment of nucleotide sequences of the 5′ flanking region of sodC start codon from B. abortus, B. suis biovars 4 (strain 40) or 1 (strain 1330) and 2 (strain Thomsen), and B. neotomae.Alignment was performed with the Clustal method of the MegAlign program of LaserGene software. Gaps are indicated by dashes. The nucleotides differing between B. suis and B. abortus or B. neotomae are in bold and underlined. Note: B. melitensis nucleotide sequences are identical to those of B. abortus shown above.

Mentions: In order to verify if the low level of SOD expression in B. neotomae is because of mutations within the coding or the upstream regions of sodC gene, we first performed PCR amplification using B. neotomae genomic DNA as template and a primer-pair designed based on the known sodC gene sequence from B. abortus. The PCR amplification resulted in a 575 bp product, the expected size in the presence of a complete sodC gene. Nucleotide sequence of the amplified product showed 99.7% identity with the corresponding region from B. suis and 99.5% identity with that from B. abortus or B. melitensis. The B. neotomae sequence differed in 2 and 3 nucleotides from that of B. suis and B. abortus or B. melitensis, respectively. All the nucleotide differences were located within the open reading frame of the sodC gene and only one of the differed nucleotides caused a change in the deduced amino acid sequence, a F→V change at amino acid position 52 of the precursor polypeptide (data not shown). We then amplified a 350 bp region flanking the 5′ end of the start codon from the B. neotomae DNA. Nucleotide sequence analysis of this region revealed that B. neotomae differed in 2 nucleotides from that of the B. suis strain 1330 (biovar 1) sequence (GenBank accession no. NC_009504), one was a substitution of A with G and the other was an insertion of A (Fig. 3). Both the changes were within the 138 bp region upstream to the start codon that was previously shown to contain the sodC promoter element in B. abortus [16]. We also PCR amplified and sequenced the upstream region from strains belonging to B. suis biovars 2 (strain Thomsen) and 4 (strain 40). While the B. suis biovar 4 sequence was 100% identical with that of B. suis biovar 1, the sequence from B. suis biovar 2 differed in just 1 nucleotide, an insertion of A at the same location as that found in the B. neotomae sequence. Computer analysis of nucleotide sequences reported in the databases indicated 100% identity within the sodC upstream region between B. abortus and B. melitensis, and compared to B. suis biovars 1 and 4, these two bacteria contained 3 fewer nucleotides at the location where the nucleotide insertion was detected in B. neotomae and B. suis biovar 2 (Fig. 3). In addition, the nucleotide 48 upstream to the start codon was G in B. abortus and B. melitensis, but it was A in B. suis.


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)

Multiple alignment of nucleotide sequences of the 5′ flanking region of sodC start codon from B. abortus, B. suis biovars 4 (strain 40) or 1 (strain 1330) and 2 (strain Thomsen), and B. neotomae.Alignment was performed with the Clustal method of the MegAlign program of LaserGene software. Gaps are indicated by dashes. The nucleotides differing between B. suis and B. abortus or B. neotomae are in bold and underlined. Note: B. melitensis nucleotide sequences are identical to those of B. abortus shown above.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991346&req=5

pone-0014112-g003: Multiple alignment of nucleotide sequences of the 5′ flanking region of sodC start codon from B. abortus, B. suis biovars 4 (strain 40) or 1 (strain 1330) and 2 (strain Thomsen), and B. neotomae.Alignment was performed with the Clustal method of the MegAlign program of LaserGene software. Gaps are indicated by dashes. The nucleotides differing between B. suis and B. abortus or B. neotomae are in bold and underlined. Note: B. melitensis nucleotide sequences are identical to those of B. abortus shown above.
Mentions: In order to verify if the low level of SOD expression in B. neotomae is because of mutations within the coding or the upstream regions of sodC gene, we first performed PCR amplification using B. neotomae genomic DNA as template and a primer-pair designed based on the known sodC gene sequence from B. abortus. The PCR amplification resulted in a 575 bp product, the expected size in the presence of a complete sodC gene. Nucleotide sequence of the amplified product showed 99.7% identity with the corresponding region from B. suis and 99.5% identity with that from B. abortus or B. melitensis. The B. neotomae sequence differed in 2 and 3 nucleotides from that of B. suis and B. abortus or B. melitensis, respectively. All the nucleotide differences were located within the open reading frame of the sodC gene and only one of the differed nucleotides caused a change in the deduced amino acid sequence, a F→V change at amino acid position 52 of the precursor polypeptide (data not shown). We then amplified a 350 bp region flanking the 5′ end of the start codon from the B. neotomae DNA. Nucleotide sequence analysis of this region revealed that B. neotomae differed in 2 nucleotides from that of the B. suis strain 1330 (biovar 1) sequence (GenBank accession no. NC_009504), one was a substitution of A with G and the other was an insertion of A (Fig. 3). Both the changes were within the 138 bp region upstream to the start codon that was previously shown to contain the sodC promoter element in B. abortus [16]. We also PCR amplified and sequenced the upstream region from strains belonging to B. suis biovars 2 (strain Thomsen) and 4 (strain 40). While the B. suis biovar 4 sequence was 100% identical with that of B. suis biovar 1, the sequence from B. suis biovar 2 differed in just 1 nucleotide, an insertion of A at the same location as that found in the B. neotomae sequence. Computer analysis of nucleotide sequences reported in the databases indicated 100% identity within the sodC upstream region between B. abortus and B. melitensis, and compared to B. suis biovars 1 and 4, these two bacteria contained 3 fewer nucleotides at the location where the nucleotide insertion was detected in B. neotomae and B. suis biovar 2 (Fig. 3). In addition, the nucleotide 48 upstream to the start codon was G in B. abortus and B. melitensis, but it was A in B. suis.

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