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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.


Determination of transcriptional start sites of PBP genes using 5′-RACE. Electropherograms from the DNA sequencing reactions of the 5′-RACE PCR products are shown. The sequences of the RNA adaptors are shown as gray bars. The nucleotides complementary to the 5′ ends of mRNA are shown and the directions of the transcription are indicated by arrows. The first nucleotide represents the transcription initiation site. RACE, rapid amplification of cDNA ends.
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f2: Determination of transcriptional start sites of PBP genes using 5′-RACE. Electropherograms from the DNA sequencing reactions of the 5′-RACE PCR products are shown. The sequences of the RNA adaptors are shown as gray bars. The nucleotides complementary to the 5′ ends of mRNA are shown and the directions of the transcription are indicated by arrows. The first nucleotide represents the transcription initiation site. RACE, rapid amplification of cDNA ends.

Mentions: To determine the transcriptional start sites of the six pbp promoters, 5′-RACEs were performed using oligonucleotides located in the downstream genes of the predicted promoters (Fig. 1 and Table 2). RNA of the R6 strain was treated with and without the TAP enzyme, which converts the 5′-triphosphate of RNA to a monophosphate and thereby enables ligation of the RNA to an adapter oligonucleotide. The adaptor allows specific amplification of the 5′-end sequence subsequent to reverse transcription. Finally, the PCR fragments were sequenced and the start points were determined for the six transcripts that include the PBP genes (Fig. 2). The DNA sequence electropherograms of six PCR products showed high-quality sequences and thereby clearly identified the transcription start sites (Fig. 2). The transcriptional start of pbp1a is located 620 nt upstream from the pbp1a start codon and 27 nt upstream of the recU start codon, which is the first gene in operon (Fig. 1). For pbp1b, the transcription start was mapped to the adenine residue located 13 nt upstream of the ATG start codon. The pbp2a transcriptional start overlaps with the putative translational start codon, which implies that the mRNA is leaderless; no ribosomal binding site (RBS) was detected upstream of the start codon. The transcriptional start of pbp2x was mapped 1,286 nt upstream from the pbp2x start codon and is located only 3 nt upstream of the mraW start codon. Similarly, the transcriptional start of pbp3 was mapped as the adenine residue located 3 nt upstream of the pbp3 start codon. The spacer between the transcriptional and putative translational initiation sites for both, pbp2x (mraW) and pbp3, is also too short to accommodate an RBS. Finally, for pbp2b, the transcripts initiate at an adenine residue located 17 nt from the first putative start codon of pbp2b.


Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6
Determination of transcriptional start sites of PBP genes using 5′-RACE. Electropherograms from the DNA sequencing reactions of the 5′-RACE PCR products are shown. The sequences of the RNA adaptors are shown as gray bars. The nucleotides complementary to the 5′ ends of mRNA are shown and the directions of the transcription are indicated by arrows. The first nucleotide represents the transcription initiation site. RACE, rapid amplification of cDNA ends.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Determination of transcriptional start sites of PBP genes using 5′-RACE. Electropherograms from the DNA sequencing reactions of the 5′-RACE PCR products are shown. The sequences of the RNA adaptors are shown as gray bars. The nucleotides complementary to the 5′ ends of mRNA are shown and the directions of the transcription are indicated by arrows. The first nucleotide represents the transcription initiation site. RACE, rapid amplification of cDNA ends.
Mentions: To determine the transcriptional start sites of the six pbp promoters, 5′-RACEs were performed using oligonucleotides located in the downstream genes of the predicted promoters (Fig. 1 and Table 2). RNA of the R6 strain was treated with and without the TAP enzyme, which converts the 5′-triphosphate of RNA to a monophosphate and thereby enables ligation of the RNA to an adapter oligonucleotide. The adaptor allows specific amplification of the 5′-end sequence subsequent to reverse transcription. Finally, the PCR fragments were sequenced and the start points were determined for the six transcripts that include the PBP genes (Fig. 2). The DNA sequence electropherograms of six PCR products showed high-quality sequences and thereby clearly identified the transcription start sites (Fig. 2). The transcriptional start of pbp1a is located 620 nt upstream from the pbp1a start codon and 27 nt upstream of the recU start codon, which is the first gene in operon (Fig. 1). For pbp1b, the transcription start was mapped to the adenine residue located 13 nt upstream of the ATG start codon. The pbp2a transcriptional start overlaps with the putative translational start codon, which implies that the mRNA is leaderless; no ribosomal binding site (RBS) was detected upstream of the start codon. The transcriptional start of pbp2x was mapped 1,286 nt upstream from the pbp2x start codon and is located only 3 nt upstream of the mraW start codon. Similarly, the transcriptional start of pbp3 was mapped as the adenine residue located 3 nt upstream of the pbp3 start codon. The spacer between the transcriptional and putative translational initiation sites for both, pbp2x (mraW) and pbp3, is also too short to accommodate an RBS. Finally, for pbp2b, the transcripts initiate at an adenine residue located 17 nt from the first putative start codon of pbp2b.

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.