Transcription of the pst operon of Clostridium acetobutylicum is dependent on phosphate concentration and pH

Abstract

The pst operon of Clostridium acetobutylicum ATCC 824 comprises five genes, pstS, pstC, pstA, pstB, and phoU, and shows a gene architecture identical to that of Escherichia coli. Deduced proteins are predicted to represent a high-affinity phosphate-specific ABC (ATP-binding cassette) transport system (Pst) and a protein homologous to PhoU, a negative phosphate regulon regulator. We analyzed the expression patterns of the pst operon in P(i)-limited chemostat cultures during acid production at pH 5.8 or solvent production at pH 4.5 and in response to P(i) pulses. Specific mRNA transcripts were found only when external P(i) concentrations had dropped below 0.2 mM. Two specific transcripts were detected, a 4.7-kb polycistronic mRNA spanning the whole operon and a quantitatively dominating 1.2-kb mRNA representing the first gene, pstS. The mRNA levels clearly differed depending on the external pH. The amounts of the full-length mRNA detected were about two times higher at pH 5.8 than at pH 4.5. The level of pstS mRNA increased by a factor of at least 8 at pH 5.8 compared to pH 4.5 results. Primer extension experiments revealed only one putative transcription start point 80 nucleotides upstream of pstS. Thus, additional regulatory sites are proposed in the promoter region, integrating two different extracellular signals, namely, depletion of inorganic phosphate and the pH of the environment. After phosphate pulses were applied to a phosphate-limited chemostat we observed faster phosphate consumption at pH 5.8 than at pH 4.5, although higher optical densities were recorded at pH 4.5.

FIG. 1.

Effect of phosphate pulses (10 mM) on growth and phosphate consumption of C. acetobutylicum in a phosphate-limited chemostat. The results for the 72-h time period after the pulse in a representative experiment are shown. Basically the same results were obtained in five independent experiments. Line 1, OD at pH 4.5; line 2, OD at pH 5.8; line 3, phosphate concentrations at pH 4.5; line 4, phosphate concentrations at pH 5.8, a temperature of 37°C, and dilution rate of 0.075 h⁻¹.

FIG. 2.

Comparison of the pst operons of C. acetobutylicum, E. coli, and B. subtilis. Boxes symbolize relative locations and sizes of the corresponding genes. Identical illustrations indicate proteins of equal functions, and numbers in parentheses represent amino acid residues. Percent values inform about the identity and similarity of the polypeptides of C. acetobutylicum (Cac) to their equivalents in E. coli (Eco) or B. subtilis (Bsu). In B. subtilis, the ATP-binding protein consists of two different subunits, PstBA and PstBB.

FIG. 3.

Hydropathicity plot of PstC (A) and PstA (B) of C. acetobutylicum. The x coordinates correspond to amino acid numbers; the y coordinates show hydrophobic (positive) and hydrophilic (negative) characteristics (20).

FIG. 4.

Gene architecture and mRNA transcripts of the pst operon of C. acetobutylicum. Gray bars below the genes indicate the relative locations of probes used in Northern blot hybridizations. Arrows symbolize extension of the two detected mRNA species starting 80 nt upstream of pstS.

FIG. 5.

Mapping of the transcription start point of the pst operon of C. acetobutylicum. (A) The product of a primer extension reaction using IRD800-labeled oligonucleotide yqgG-PE-2 (PE) was run on a polyacrylamide gel alongside with the corresponding sequencing reactions (T, G, C, A) generated with the same primer. (B) RT-PCR products based on pstS-specific cDNA as the template (upper photo) compared with PCR products generated with plasmid DNA (pUM8) as positive controls (lower photo). DNA fragments were separated electrophoretically in a 1.5% (wt/vol) agarose gel and visualized by ethidium bromide staining. “Forward” primers (lanes 1 to 5, yqgG-8, yqgG-3, yqgG-9, yqgG-10, and yqgG-4; lanes M, marker) were separately combined with yqgG-2 as “reverse” primer. (C) Locations of “forward” primers within the DNA region spanning the transcription start point of the pstS operon (gray arrows above the DNA sequence) as well as the identified transcription start point (gray circle) are highlighted. The dashed arrow below the DNA sequence symbolizes mRNA and the bold ATG the start codon of pstS.

FIG. 6.

Induction of the pst operon of C. acetobutylicum under conditions of phosphate limitation. (A) Northern blot hybridization of total RNA (20 μg/lane) of cells from a chemostat culture (pH 4.5) after a P_i pulse (10 mM) with a DIG-labeled pstB probe. External phosphate concentrations are given below the lanes. (B) RT-PCR analyses of pstS and of the ptb gene (encoding phosphotransbutyrylase, constitutively expressed under the experimental conditions) as a positive control. Bars represent relative signal intensities. The maximal signal intensity was set as 100%.

FIG. 7.

Transcript levels of the pst operon of C. acetobutylicum are dependent on the pH. The pH in a phosphate-limited chemostat was increased from 4.5 to 5.8 at time 0. At different time points (2 h, 7 h, 30 h, 72 h) the levels of the products butanol (filled squares) and butyrate (open squares) were determined (A) and Northern blot analysis of total RNA (10 μg per lane) with a DIG-labeled pstS probe was performed (B).

FIG. 8.

Comparison of transcript levels of the pst operon in C. acetobutylicum cells grown at steady state in a phosphate-limited chemostat at pH 4.5 or pH 5.8. The different RNA amounts used for a semiquantitative Northern blot analysis are given in micrograms below the lanes. A pstS fragment was used as a probe.

FIG. 9.

A hairpin structure in the intergenic region between pstS and pstC. UAG represents the stop codon of pstS gene and AUG the start codon of pstC gene; the putative ribosome binding site of pstC is given in bold letters.

FIG. 10.

Putative regulatory sites in the promoter region of the pst operon of C. acetobutylicum. The transcription start point of the pst operon is marked by a gray circle and +1. The deduced −10 and −35 promoter sequences are boxed. Bold arrowheads above the sequence indicate direct 11-bp repeats and arrowheads below the sequence an inverted repeat. Thick bars over sequences represent those exactly matching the B. subtilis Pho box motif, and thin bars over sequences represent those of similar motifs in which two nucleotides are changed. The ribosome binding site of pstS is underlined, and the start codon is printed in bold letters.