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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

Alignment of putative S. pneumoniae R6 PBP promoters. (A) The alignment shows the conserved sequences located upstream of the six PBP loci. The −35 regions marked in orange; the extended −10 region is shown in green within the gray area. Experimentally determined transcriptional start points are shown in blue. The first translation start codons (ATG) are shown in red. (B) The WebLogo48,49 representation of the position weight matrices derived from the sequences depicted in (A) is shown.
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f3: Alignment of putative S. pneumoniae R6 PBP promoters. (A) The alignment shows the conserved sequences located upstream of the six PBP loci. The −35 regions marked in orange; the extended −10 region is shown in green within the gray area. Experimentally determined transcriptional start points are shown in blue. The first translation start codons (ATG) are shown in red. (B) The WebLogo48,49 representation of the position weight matrices derived from the sequences depicted in (A) is shown.

Mentions: Based on the mapping results described above, we aligned the promoter regions of PBPs loci and analyzed the sequences (Fig. 3A). At a distance 6–8 nt upstream of the transcriptional start site, almost all sequences exhibit an extended −10 promoter element (5′-TRTGNTATAAT-3′), which was originally identified in Bacillus subtilis,56 and which is common in S. pneumoniae.57 The extended −10 element 5′-TRTG-3′ is located 1 nt upstream of the −10 hexamer, which consists of the 6 nt TATAAT and is absolutely essential for transcription. The 5′-TG-3′ motif of the extended −10 promoters is positioned at −15/−14 with respect to the transcriptional start site. The extended −10 element is highly conserved in all promoters of the PBP loci (Fig. 3B). In contrast, the −35 element is not well conserved and is difficult to define. Only the two promoters upstream of pbp1a and pbp1b contain, respectively, four and five matches to consensus −35 hexamer. However, in both cases, the length of the spacer between the promoter −10 and −35 elements is not optimal and contains 19 or 15 nt, respectively. For E. coli, it has been shown that the most frequent spacer region length is 17 nt.58 The promoter of pbp2a contains four matches to the consensus −35 hexamer sequence and has a 17 nt long spacer. The other three promoters displayed a poor match to the −35 hexamer with two or three matches only. Figure 3B depicts a DNA sequence logo generated from the alignment of pbp promoters from Fig. 3A. The best conserved promoter element is clearly the −10 sequence.


Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6
Alignment of putative S. pneumoniae R6 PBP promoters. (A) The alignment shows the conserved sequences located upstream of the six PBP loci. The −35 regions marked in orange; the extended −10 region is shown in green within the gray area. Experimentally determined transcriptional start points are shown in blue. The first translation start codons (ATG) are shown in red. (B) The WebLogo48,49 representation of the position weight matrices derived from the sequences depicted in (A) is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Alignment of putative S. pneumoniae R6 PBP promoters. (A) The alignment shows the conserved sequences located upstream of the six PBP loci. The −35 regions marked in orange; the extended −10 region is shown in green within the gray area. Experimentally determined transcriptional start points are shown in blue. The first translation start codons (ATG) are shown in red. (B) The WebLogo48,49 representation of the position weight matrices derived from the sequences depicted in (A) is shown.
Mentions: Based on the mapping results described above, we aligned the promoter regions of PBPs loci and analyzed the sequences (Fig. 3A). At a distance 6–8 nt upstream of the transcriptional start site, almost all sequences exhibit an extended −10 promoter element (5′-TRTGNTATAAT-3′), which was originally identified in Bacillus subtilis,56 and which is common in S. pneumoniae.57 The extended −10 element 5′-TRTG-3′ is located 1 nt upstream of the −10 hexamer, which consists of the 6 nt TATAAT and is absolutely essential for transcription. The 5′-TG-3′ motif of the extended −10 promoters is positioned at −15/−14 with respect to the transcriptional start site. The extended −10 element is highly conserved in all promoters of the PBP loci (Fig. 3B). In contrast, the −35 element is not well conserved and is difficult to define. Only the two promoters upstream of pbp1a and pbp1b contain, respectively, four and five matches to consensus −35 hexamer. However, in both cases, the length of the spacer between the promoter −10 and −35 elements is not optimal and contains 19 or 15 nt, respectively. For E. coli, it has been shown that the most frequent spacer region length is 17 nt.58 The promoter of pbp2a contains four matches to the consensus −35 hexamer sequence and has a 17 nt long spacer. The other three promoters displayed a poor match to the −35 hexamer with two or three matches only. Figure 3B depicts a DNA sequence logo generated from the alignment of pbp promoters from Fig. 3A. The best conserved promoter element is clearly the −10 sequence.

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