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.

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Single stranded DNA-targets from asymmetric polymerase chain reaction (PCR) of a sequence of the gram positive, spore forming bacterium Clostridium acetobutylicum were detected by square-wave voltammetry after labeling with osmium tetroxide bipyridine and hybridization with DNA capture probes immobilized on gold electrodes. The asymmetric PCR, performed with a 10-fold excess of the forward-primer, was used without any further purification for hybridization with protective strands and covalent labeling with osmium tetroxide bipyridine. Square-wave voltammetric signals of 20 nmol/L targets were significantly higher at 50 8C compared with 23 8C hybridization temperature. A fully noncomplementary protective strand yielded thoroughly modified targets unable for further hybridization. Coupling this with thermal discrimination opens new opportunities for sequence specific DNA detection.

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The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent twocomponent regulatory system. We analyzed the expression patterns of phoPR in P i -limited chemostat cultures and in response to P i pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external P i concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under P i -limiting growth conditions compared to growth with an excess of P i . At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.