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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.


Related in: MedlinePlus

Growth and pbp2x promoter activity in response to cefotaxime treatment. (A) Growth of S. pneumoniae R6 in BHI medium was followed by nephelometry (N). Cefotaxime was added to the exponentially growing cultures at the concentrations (μg/ml) at the time point as indicated by the arrow. (B) Activity of the pbp2x promoter at different time points after the addition of cefotaxime. 0 indicates the time immediately before the cefotaxime addition. β-galactosidase activities were measured after 30, 90, and 150 min and are given in nmol nitrophenol produced per min and mg of protein. Black bars: control, no cefotaxime; gray bars: 0.04 μg/ml cefotaxime; white bars: 0.4 μg/ml cefotaxime. The results are mean value ± SD of three independent experiments. SD, standard deviation.
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f5: Growth and pbp2x promoter activity in response to cefotaxime treatment. (A) Growth of S. pneumoniae R6 in BHI medium was followed by nephelometry (N). Cefotaxime was added to the exponentially growing cultures at the concentrations (μg/ml) at the time point as indicated by the arrow. (B) Activity of the pbp2x promoter at different time points after the addition of cefotaxime. 0 indicates the time immediately before the cefotaxime addition. β-galactosidase activities were measured after 30, 90, and 150 min and are given in nmol nitrophenol produced per min and mg of protein. Black bars: control, no cefotaxime; gray bars: 0.04 μg/ml cefotaxime; white bars: 0.4 μg/ml cefotaxime. The results are mean value ± SD of three independent experiments. SD, standard deviation.

Mentions: We tested whether the presence of cefotaxime in BHI medium affects the expression of PBP genes. Bacterial growth was inhibited already at a concentration of 0.01 μg/ml (0.5 × MIC) cefotaxime and the cells stopped growing at a cell density of N = 30 (Fig. 5A). The expression of PBP genes was determined at 30, 60, and 150 min after the addition of cefotaxime at a concentration of 0.04 μg/ml (2 × MIC). No significant effect on the expression of PBP genes was observed according to Student's t-test. The expression of pbp2x is shown as an example in Fig. 5B, and expression of all PBP genes at 2 × MIC cefotaxime are shown in Supplementary Fig. S1 (Supplementary Data are available online at www.liebertpub.com/mdr). The addition of cefotaxime at a concentration of 0.4 μg/ml (20 × MIC) did not affect the expression level of pbp2x (Fig. 5B), but R6 cells start to lyse two hours later. After 30 min growth in the presence of 0.4 μg/ml, only PBP2b could be labeled with BOCILLIN™FL, documenting that all other PBPs were acylated (not shown).


Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6
Growth and pbp2x promoter activity in response to cefotaxime treatment. (A) Growth of S. pneumoniae R6 in BHI medium was followed by nephelometry (N). Cefotaxime was added to the exponentially growing cultures at the concentrations (μg/ml) at the time point as indicated by the arrow. (B) Activity of the pbp2x promoter at different time points after the addition of cefotaxime. 0 indicates the time immediately before the cefotaxime addition. β-galactosidase activities were measured after 30, 90, and 150 min and are given in nmol nitrophenol produced per min and mg of protein. Black bars: control, no cefotaxime; gray bars: 0.04 μg/ml cefotaxime; white bars: 0.4 μg/ml cefotaxime. The results are mean value ± SD of three independent experiments. SD, standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5036317&req=5

f5: Growth and pbp2x promoter activity in response to cefotaxime treatment. (A) Growth of S. pneumoniae R6 in BHI medium was followed by nephelometry (N). Cefotaxime was added to the exponentially growing cultures at the concentrations (μg/ml) at the time point as indicated by the arrow. (B) Activity of the pbp2x promoter at different time points after the addition of cefotaxime. 0 indicates the time immediately before the cefotaxime addition. β-galactosidase activities were measured after 30, 90, and 150 min and are given in nmol nitrophenol produced per min and mg of protein. Black bars: control, no cefotaxime; gray bars: 0.04 μg/ml cefotaxime; white bars: 0.4 μg/ml cefotaxime. The results are mean value ± SD of three independent experiments. SD, standard deviation.
Mentions: We tested whether the presence of cefotaxime in BHI medium affects the expression of PBP genes. Bacterial growth was inhibited already at a concentration of 0.01 μg/ml (0.5 × MIC) cefotaxime and the cells stopped growing at a cell density of N = 30 (Fig. 5A). The expression of PBP genes was determined at 30, 60, and 150 min after the addition of cefotaxime at a concentration of 0.04 μg/ml (2 × MIC). No significant effect on the expression of PBP genes was observed according to Student's t-test. The expression of pbp2x is shown as an example in Fig. 5B, and expression of all PBP genes at 2 × MIC cefotaxime are shown in Supplementary Fig. S1 (Supplementary Data are available online at www.liebertpub.com/mdr). The addition of cefotaxime at a concentration of 0.4 μg/ml (20 × MIC) did not affect the expression level of pbp2x (Fig. 5B), but R6 cells start to lyse two hours later. After 30 min growth in the presence of 0.4 μg/ml, only PBP2b could be labeled with BOCILLIN™FL, documenting that all other PBPs were acylated (not shown).

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