Limits...
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

Activity of the PBP promoters in S. pneumoniae R6 strain during growth in C + Y medium. Growth curves and β-galactosidase activities of one representative experiment are shown. Growth was monitored by nephelometry (N, nephelo units). β-galactosidase activities were determined in 30 min intervals and are given in nmol nitrophenol produced per min and mg of protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036317&req=5

f4: Activity of the PBP promoters in S. pneumoniae R6 strain during growth in C + Y medium. Growth curves and β-galactosidase activities of one representative experiment are shown. Growth was monitored by nephelometry (N, nephelo units). β-galactosidase activities were determined in 30 min intervals and are given in nmol nitrophenol produced per min and mg of protein.

Mentions: To evaluate pbp promoter activity, the integrative promoter probe system pPP2 was used.42 PBP promoter fragments were cloned upstream of the promoterless E. coli β-galactosidase LacZ gene. In the next step, the reporter constructs were stably integrated into the S. pneumoniae R6 genome by homologous recombination at the bgaA locus encoding the endogenous β-galactosidase. Upon integration by double crossover, most of the BgaA gene was deleted and the background β-galactosidase level was eliminated. As controls, the RP200 and RKL44 strains were used, which carry a promoterless-lacZ and a PvegW-lacZ fusion, respectively. The vegW promoter served as a nonregulated control for regulation studies in S. pneumoniae R6 derivatives. PvegW is a derivative of PvegII and was constructed by reducing the spacing between the −10 and the −35 region from 18 to 17 nt, thereby deleting a G.42 Subsequently, β-galactosidase activities were determined throughout the growth of the strains in C + Y medium. As shown in Fig. 4, all PBPs promoters are constitutively expressed and highly active during the exponential growth and early stationary growth phase as well, similar to the constitutive promoter PvegW, which showed a β-galactosidase activity of 200 U throughout; the promoterless control construct gave values below 1 U (data not shown). All data were validated by the Student's t-test implying no growth phase-specific regulation of PBP expression in the sensitive R6 strain. Interestingly, the individual expression activities of the PBP promoters varied. Lowest promoter activities were observed for PBP3 and PBP1b and the strongest promoter was PBP1a, leading to the following hierarchy of the PBP promoter activities PBP3 < PBP1b < PBP2x = PBP2a < PBP2b < PBP1a (Fig. 4). Promoter activities were also determined in cells grown in BHI medium since this medium is easy to prepare and the measurements of β-galactosidase activities provide stable results. Consistently, the same relative expression patterns were observed, but the levels of expression were ranging between 2.1- and 2.8-fold higher than in C + Y medium (data not shown). The expression of PvegW-lacZ also increased to the same degree (400 U). A higher expression of a variety of promoters in cells grown in BHI medium has been reported45; the reason for this phenomenon is unclear.


Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6
Activity of the PBP promoters in S. pneumoniae R6 strain during growth in C + Y medium. Growth curves and β-galactosidase activities of one representative experiment are shown. Growth was monitored by nephelometry (N, nephelo units). β-galactosidase activities were determined in 30 min intervals and are given in nmol nitrophenol produced per min and mg of protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Activity of the PBP promoters in S. pneumoniae R6 strain during growth in C + Y medium. Growth curves and β-galactosidase activities of one representative experiment are shown. Growth was monitored by nephelometry (N, nephelo units). β-galactosidase activities were determined in 30 min intervals and are given in nmol nitrophenol produced per min and mg of protein.
Mentions: To evaluate pbp promoter activity, the integrative promoter probe system pPP2 was used.42 PBP promoter fragments were cloned upstream of the promoterless E. coli β-galactosidase LacZ gene. In the next step, the reporter constructs were stably integrated into the S. pneumoniae R6 genome by homologous recombination at the bgaA locus encoding the endogenous β-galactosidase. Upon integration by double crossover, most of the BgaA gene was deleted and the background β-galactosidase level was eliminated. As controls, the RP200 and RKL44 strains were used, which carry a promoterless-lacZ and a PvegW-lacZ fusion, respectively. The vegW promoter served as a nonregulated control for regulation studies in S. pneumoniae R6 derivatives. PvegW is a derivative of PvegII and was constructed by reducing the spacing between the −10 and the −35 region from 18 to 17 nt, thereby deleting a G.42 Subsequently, β-galactosidase activities were determined throughout the growth of the strains in C + Y medium. As shown in Fig. 4, all PBPs promoters are constitutively expressed and highly active during the exponential growth and early stationary growth phase as well, similar to the constitutive promoter PvegW, which showed a β-galactosidase activity of 200 U throughout; the promoterless control construct gave values below 1 U (data not shown). All data were validated by the Student's t-test implying no growth phase-specific regulation of PBP expression in the sensitive R6 strain. Interestingly, the individual expression activities of the PBP promoters varied. Lowest promoter activities were observed for PBP3 and PBP1b and the strongest promoter was PBP1a, leading to the following hierarchy of the PBP promoter activities PBP3 < PBP1b < PBP2x = PBP2a < PBP2b < PBP1a (Fig. 4). Promoter activities were also determined in cells grown in BHI medium since this medium is easy to prepare and the measurements of β-galactosidase activities provide stable results. Consistently, the same relative expression patterns were observed, but the levels of expression were ranging between 2.1- and 2.8-fold higher than in C + Y medium (data not shown). The expression of PvegW-lacZ also increased to the same degree (400 U). A higher expression of a variety of promoters in cells grown in BHI medium has been reported45; the reason for this phenomenon is unclear.

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 &beta;-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 &minus;10 region is highly conserved and complies with a &sigma;A-type promoter consensus sequence. In contrast, the &minus;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 &sim;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