Characterization of a novel fucose-regulated promoter (PfcsK) suitable for gene essentiality and antibacterial mode-of-action studies in Streptococcus pneumoniae

Abstract

The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P(fcsK), fucose-dependent growth of the S. pneumoniae (P(fcsK)::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.

FIG. 1.

Genetic organization of the S. pneumoniae putative fucose (fcs) gene cluster and DNA sequence of its promoter (P_fcsK). (A) The open reading frames and directions of transcription of the 11 genes in the putative fucose cluster are indicated: fcsR (fucose repressor protein), fcsK (fuculose kinase), fcsA (fuculose-1-phosphate aldolase), fcsU (fucose operon protein and putative oxidoreductase), EIIA-fcs to EIID-fcs (enzyme IIA to IID components of the phosphotransferase-phosphoenolpyruvate sugar uptake system), fcsY (hypothetical protein), fcsL (putative fucose-lectin binding protein), and fcsI (l-fucose isomerase). (B) DNA sequence of the intergenic fcsR-fcsK region. The translational start sites of the fcsR and fcsK genes, which are divergently transcribed, are indicated by bold arrows. The open reading frames have been boxed. Putative ribosome binding sites (RBS) and extended −10 and −10 and −35 promoter sequences are highlighted. The 5′-fluorescently tagged primer fcsKpe, used to determine the 5′ end of the fcsK transcript and hence the putative location of the P_fcsK promoter, is shown. For the primer extension reaction, 15 μg of DNase-treated RNA (isolated from S. pneumoniae R6 cells grown in a semidefined medium in the presence of 0.2% glucose and 1% fucose) was denatured together with 5 pmol of fcsKpe primer and reverse transcribed to cDNA. Samples were separated with a Perkin-Elmer ABI 377XL sequencer electrophoresis set, and the size of the primer extension product (196 ± 2 nucleotides) was determined with Perkin-Elmer ABI Prism Genescan Analysis 2.1 software. The transcriptional initiation site (+1) upstream of the fcsK gene, predicted by primer extension analysis, is indicated. A palindromic sequence showing similarity to the catabolite repression element (CRE) sequence of B. subtilis (16), determined with the MAST software program, is boxed.

FIG. 2.

Effects of different sugars on the induction of the fuculose kinase (fcsK) gene measured by quantitative, real-time SybrGreen RT-PCR. S. pneumoniae R6 was grown statically at 37°C in AGCH-YE medium and supplemented with different test sugars. Total RNA was extracted from late-logarithmic-phase-grown S. pneumoniae cells by using the Bio 101 FastRNA kit (Vista, Calif.) following glass bead cell disruption and a hot phenol lysis step (5). DNase-treated RNA was reverse transcribed to cDNA with a First Strand synthesis kit (Invitrogen). Relative levels of bacterial transcripts in each sample were quantified by PCR following SybrGreen dye incorporation (SybrGreen PCR core reagent kit; Applied Biosystems, Perkin-Elmer), and products were detected in real time with the 7700 sequence detection system (Applied Biosystems) as described previously (22, 35, 37). Template primers used in the PCRs are available on request. The quantity of cDNA estimated was normalized to a housekeeping gene, era. Changes in steady-state levels of fcsK mRNA in each sample were expressed relative to the uninduced control (0.2% glucose).

FIG. 3.

Fucose induction and sucrose repression of P_fcsK in an S. pneumoniae (P_fcsK::luxAB) reporter strain. (A) Construction of a P_fcsK::luxAB transcriptional reporter fusion. A genetic map shows the organization of the P_fcsK::luxAB reporter cassette following integration into the chromosome of S. pneumoniae R6. Details of construction of the luxAB reporter strain in S. pneumoniae are available on request. Briefly, the cassette contains the promoter region of the fuculose kinase gene (P_fcsK) amplified from S. pneumoniae R6 and fused to a promoterless luxAB reporter gene from Vibrio harveyi (13). Transcriptional terminators of two large rRNA operons, TT1 and TT2, were amplified from S. pneumoniae R6 and introduced to flank the reporter fusion and prevent local transcriptional interference. The erythromycin resistance marker (ermAM) of Enterococcus faecalis was amplified from pAMβ1 for selection (21). The cassette was flanked by regions of the β-galactosidase (bgaA) structural gene (36). The construct was integrated into S. pneumoniae R6 at the bgaA locus by transformation (33). Erythromycin-resistant transformants were selected (5 μg of erythromycin/ml), and successful construction of the P_fcsK::luxAB reporter strain in single copy in the chromosome was confirmed by both diagnostic PCR and DNA sequencing. Arrows indicate the directions of transcription of the genes. Lollipop structures represent the transcriptional terminators. (B) Titration range of luciferase activity in the S. pneumoniae (P_fcsK::luxAB) reporter strain following growth in the presence of sucrose and fucose. To study the regulation of P_fcsK in S. pneumoniae, the S. pneumoniae (P_fcsK::luxAB) transcriptional reporter fusion strain was grown to late logarithmic phase (OD₆₅₀ of about 0.6) in AGCH-YE medium containing different concentrations of sucrose and fucose. To measure luciferase activity, bacterial cells (250 μl) were transferred to a microtiter plate and 2 μl of n-decyl aldehyde substrate (Sigma) was added. Light output from the reaction was counted for 2 s with a MicroLumat LB96P luminometer (EG & G Berthold). The relative light units were calculated as the light output per OD₆₅₀ unit per milliliter of culture (3, 13).

FIG. 4.

Fucose-dependent growth of an S. pneumoniae (P_fcsK::def1) regulatable strain. (A) An S. pneumoniae def1 regulatable strain (FD) was constructed by placing def1 under the control of the P_fcsK inducible promoter in the chromosome of S. pneumoniae. Briefly, by using a three-piece PCR strategy of overlapping primers (33), a promoter replacement cassette was constructed containing the P_fcsK promoter, transcriptional terminators (TT1 and TT2) located 5′ of the P_fcsK promoter, and an independent erythromycin resistance marker (ermAM) (21) and flanked by DNA sequences of the gene immediately upstream of def1 and the start of the def1 open reading frame. The construct was integrated into S. pneumoniae R6 at the def1 locus by transformation (33). An erythromycin-resistant mutant of P_fcsK::def1 (FD) was recovered in the presence of added fucose, and both diagnostic PCR and DNA sequencing confirmed its chromosomal organization. (B) The effect of fucose on growth of an S. pneumoniae (P_fcsK::def1) regulatable strain. S. pneumoniae strains were grown statically at 37°C in AGCH-YE medium containing 0.8% (wt/vol) sucrose and l-fucose at 0.1% (•), 0.05% (▪), or 0.025% (▴) (wt/vol). Growth experiments were performed in triplicate in a microtiter plate format with a SpectraMax250 spectrophotometer (Molecular Devices) as described previously (37). (C) Western blot analysis of PDF levels in S. pneumoniae (P_fcsK::def1) FD and R6. S. pneumoniae R6 (lanes 6 and 8) and S. pneumoniae (P_fcsK::def1) FD (lanes 7 and 9) were grown in AGCH-YE medium plus 0.8% (wt/vol) sucrose and fucose at 0.8% (lanes 6 and 7) or 0.1% (lanes 8 and 9) (wt/vol) final concentrations. Mid-logarithmic-phase cultures (OD₆₅₀ of approximately 0.15) were resuspended in sterile distilled water to an equivalent of an OD₆₅₀ of 4 (path length, 1 cm), and total cell lysates were prepared as described previously (30). Ten microliters of total protein samples (lanes 6 to 9) and S. pneumoniae PDF protein standards in 50-, 10-, 2-, 0.4-, and 0.08-ng amounts (lanes 1 to 5, respectively) were loaded onto each lane of a 10% NuPAGE Bis-Tris resolving gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (37). Western blotting was performed as described previously (37), and protein samples were probed with rabbit polyclonal antiserum raised against S. pneumoniae PDF (Covance Research Products) (diluted 1/1,000) and anti-rabbit horseradish peroxidase (Sigma) as secondary antibody (diluted 1/10,000).