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Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6

View Article: PubMed Central - PubMed

ABSTRACT

Penicillin-binding proteins (PBPs) are membrane-associated enzymes, which are involved in the last two steps of peptidoglycan biosynthesis, and some of them are key players in cell division. Furthermore, they are targets of β-lactams, the most widely used antibiotics. Nevertheless, very little is known about the expression and regulation of PBP genes. Using transcriptional mapping, we now determined the promoter regions of PBP genes from the laboratory strain Streptococcus pneumoniae R6 and examined the expression profile of these six promoters. The extended −10 region is highly conserved and complies with a σA-type promoter consensus sequence. In contrast, the −35 region is poorly conserved, indicating the possibility for differential PBP regulation. All PBP promoters were constitutively expressed and highly active during the exponential and early stationary growth phase. However, the individual expression of PBP promoters varied approximately fourfold, with pbp1a being the highest and pbp3 the lowest. Furthermore, the deletion of one nucleotide in the spacer region of the PBP3 promoter reduced pbp3 expression ∼10-fold. The addition of cefotaxime above the minimal inhibitory concentration (MIC) did not affect PBP expression in the penicillin-sensitive R6 strain. No evidence for regulation of S. pneumoniae PBP genes was obtained.

No MeSH data available.


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Effect of the mutation in the PBP3 promoter of S. pneumoniae 801 on PBP3 expression. (A) Comparison of the pbp3 promoter region between S. pneumoniae R6 and the 801 mutant. The −35 (orange) and extended −10 promoter (green within the gray area) regions are boldfaced. The transcriptional start point is shown in blue and the translation start codon of pbp3 is indicated in red. The deletion of one nucleotide in 801 is highlighted by the red box. (B) β-galactosidase activities expressed from the promoters pbp3R6 (gray) and pbp3R801 (black) in KP06 and KP09 strains. Strains were grown in C + Y medium. β-galactosidase activities were determined at four different time points and are given in nmol nitrophenol produced per min and mg of protein. The results are mean value ± SD of three independent experiments.
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f6: Effect of the mutation in the PBP3 promoter of S. pneumoniae 801 on PBP3 expression. (A) Comparison of the pbp3 promoter region between S. pneumoniae R6 and the 801 mutant. The −35 (orange) and extended −10 promoter (green within the gray area) regions are boldfaced. The transcriptional start point is shown in blue and the translation start codon of pbp3 is indicated in red. The deletion of one nucleotide in 801 is highlighted by the red box. (B) β-galactosidase activities expressed from the promoters pbp3R6 (gray) and pbp3R801 (black) in KP06 and KP09 strains. Strains were grown in C + Y medium. β-galactosidase activities were determined at four different time points and are given in nmol nitrophenol produced per min and mg of protein. The results are mean value ± SD of three independent experiments.

Mentions: While investigating the physiological role of PBP3 and its effect on cefotaxime resistance, Selakovitch-Chenu et al. used the laboratory strain 801, an R6 derivative, which produces a reduced amount of PBP3.43 They described that this strain carries a deletion of 1 nt in the upstream sequence of the PBP3 gene.60 We sequenced the promoter region of the pbp3 gene from mutant 801 to identify the position of this mutation within the promoter region defined in the present study. The deletion of 1 nt is located in the space region between the −35 and the extended −10 elements (Fig. 6A). It is well established that the length of the spacer region in E. coli is critical.61 Usually, the −35 sequence is located 17 nt upstream of the −10 element.58 In case of the 801 strain, the length of the PBP3 promoter spacer region is 16 nt. Therefore, we decided to evaluate the promoter activity and cloned this promoter in the plasmid pPP2. The resulting plasmid pPP3M was transformed into the S. pneumoniae R6 and the reporter construct was integrated into the genome by homologous recombination. Subsequently, β-galactosidase activities were measured during growth at four time points (Fig. 6B). The promoter activity of the mutated PBP3 was reduced 10-fold in comparison to wild-type promoter indicating that the deletion of 1 nt in the space region greatly affects the promoter strength, in agreement with the lower PBP3 protein production in strain 801.


Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6
Effect of the mutation in the PBP3 promoter of S. pneumoniae 801 on PBP3 expression. (A) Comparison of the pbp3 promoter region between S. pneumoniae R6 and the 801 mutant. The −35 (orange) and extended −10 promoter (green within the gray area) regions are boldfaced. The transcriptional start point is shown in blue and the translation start codon of pbp3 is indicated in red. The deletion of one nucleotide in 801 is highlighted by the red box. (B) β-galactosidase activities expressed from the promoters pbp3R6 (gray) and pbp3R801 (black) in KP06 and KP09 strains. Strains were grown in C + Y medium. β-galactosidase activities were determined at four different time points and are given in nmol nitrophenol produced per min and mg of protein. The results are mean value ± SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Effect of the mutation in the PBP3 promoter of S. pneumoniae 801 on PBP3 expression. (A) Comparison of the pbp3 promoter region between S. pneumoniae R6 and the 801 mutant. The −35 (orange) and extended −10 promoter (green within the gray area) regions are boldfaced. The transcriptional start point is shown in blue and the translation start codon of pbp3 is indicated in red. The deletion of one nucleotide in 801 is highlighted by the red box. (B) β-galactosidase activities expressed from the promoters pbp3R6 (gray) and pbp3R801 (black) in KP06 and KP09 strains. Strains were grown in C + Y medium. β-galactosidase activities were determined at four different time points and are given in nmol nitrophenol produced per min and mg of protein. The results are mean value ± SD of three independent experiments.
Mentions: While investigating the physiological role of PBP3 and its effect on cefotaxime resistance, Selakovitch-Chenu et al. used the laboratory strain 801, an R6 derivative, which produces a reduced amount of PBP3.43 They described that this strain carries a deletion of 1 nt in the upstream sequence of the PBP3 gene.60 We sequenced the promoter region of the pbp3 gene from mutant 801 to identify the position of this mutation within the promoter region defined in the present study. The deletion of 1 nt is located in the space region between the −35 and the extended −10 elements (Fig. 6A). It is well established that the length of the spacer region in E. coli is critical.61 Usually, the −35 sequence is located 17 nt upstream of the −10 element.58 In case of the 801 strain, the length of the PBP3 promoter spacer region is 16 nt. Therefore, we decided to evaluate the promoter activity and cloned this promoter in the plasmid pPP2. The resulting plasmid pPP3M was transformed into the S. pneumoniae R6 and the reporter construct was integrated into the genome by homologous recombination. Subsequently, β-galactosidase activities were measured during growth at four time points (Fig. 6B). The promoter activity of the mutated PBP3 was reduced 10-fold in comparison to wild-type promoter indicating that the deletion of 1 nt in the space region greatly affects the promoter strength, in agreement with the lower PBP3 protein production in strain 801.

View Article: PubMed Central - PubMed

ABSTRACT

Penicillin-binding proteins (PBPs) are membrane-associated enzymes, which are involved in the last two steps of peptidoglycan biosynthesis, and some of them are key players in cell division. Furthermore, they are targets of β-lactams, the most widely used antibiotics. Nevertheless, very little is known about the expression and regulation of PBP genes. Using transcriptional mapping, we now determined the promoter regions of PBP genes from the laboratory strain Streptococcus pneumoniae R6 and examined the expression profile of these six promoters. The extended −10 region is highly conserved and complies with a σA-type promoter consensus sequence. In contrast, the −35 region is poorly conserved, indicating the possibility for differential PBP regulation. All PBP promoters were constitutively expressed and highly active during the exponential and early stationary growth phase. However, the individual expression of PBP promoters varied approximately fourfold, with pbp1a being the highest and pbp3 the lowest. Furthermore, the deletion of one nucleotide in the spacer region of the PBP3 promoter reduced pbp3 expression ∼10-fold. The addition of cefotaxime above the minimal inhibitory concentration (MIC) did not affect PBP expression in the penicillin-sensitive R6 strain. No evidence for regulation of S. pneumoniae PBP genes was obtained.

No MeSH data available.


Related in: MedlinePlus