, and Pb 2؉ caused the release of CadC from DNA in gel retardation assays. DNase I footprinting measurements showed that the CadC protein specifically associated with and protected a region of operator/promoter DNA from nucleotide positions ؊7 to ؉14 relative to the start point of mRNA synthesis. Runoff transcription assays with the operator/promoter region of DNA (plus the first 69 nucleotides of the cadC gene) and purified E. coli RNA polymerase gave an mRNA product of the predicted size. Added CadC protein inhibited transcription in vitro.The cadmium resistance system of Staphylococcus aureus plasmid pI258 was identified by Novick and Roth (9) and subsequently mapped by deletion analysis (8). The cadA system results in reduced net uptake of Cd 2ϩ because of increased energy-dependent efflux of the toxic cation (22). Cloning and DNA sequence analysis identified two genes called cadC and cadA (10). The cadA gene product is a P-type ATPase, a member of the large family of related cation transport ATPases found in plant and animal cells as well as in microbes (16, While the properties of CadA satisfactorily explain cadmium resistance, the role of cadC and the nature of the trans-acting component of the carefully regulated CadA system (3, 25) remain unclear. The cadC gene is involved in full cadmium resistance; cadC can be provided in trans and need not be present on the same plasmid as cadA (26). The inducible nature of transcription was established, and the initiation site for cad mRNA synthesis was identified by reverse transcriptase primer extension (25) to a position 28 nucleotides upstream of the ATG start codon of the cadC gene (Fig. 1). Operon-length mRNA of 2.6 kb, including both cadC and cadA, was identified by Northern blot DNA-RNA hybridization (25). Canonical Ϫ10 and Ϫ35 Escherichia coli-like RNA polymerase recognition sites (Fig. 1) were identified in the sequence (25). A 7-nucleotide inverted repeat segment, including the first nucleotides transcribed into mRNA, was proposed as the operator site for binding of a then-elusive trans-acting DNA-binding regulatory protein (25).Cation-inducible gene function was studied by measurements of Cd 2ϩ efflux by whole cells (25) and subcellular membranes (21) and through induction of the reporter genes ␤-lactamase (25) and luciferase (3). However, the presumed transacting regulatory protein was not identified (25). Because of the need for cadC in gene regulation (21) and the sequence homologies (1, 26) among the predicted CadC protein product, the ArsR regulatory protein (12,14,15,23) for the arsenic resistance system, and the SmtB regulatory protein for cyanobacterial metallothionein synthesis (6), we undertook a direct test of the hypothesis that CadC is the regulatory protein for cadA operon transcription. By in vitro assays with CadC protein and the appropriate operator/promoter DNA, CadC has been shown to be a DNA-binding, negatively acting regulatory protein. MATERIALS AND METHODSAbbreviations. DTT, dithiothreitol; IPTG, isopropyl-␤-D-thiogalactopyranosid...

The characteristics of the degradation of cellulose, soluble starch, and glucose in the acidogenic phase and the effects of the substrate loading rate and biological solids retention time on the methanogenic phase of anaerobic digestion were investigated. The results obtained from continuous experiments using laboratory-scale anaerobic chemostat reactors elucidated the true rate-limiting step of anaerobic digestion. The specific rate of substrate utilization decreased in the following order: glucose, soluble starch, acetic acid, and cellulose. The rate of the hydrolysis of cellulose was so low that this was shown to be the rate-limiting step in overall anaerobic digestion. Among methanogenic bacteria Methanosarcina would provide a higher substrate utilization rate than Methanothrix, and the maximum allowable substrate loading rate in the methanogenic phase was 11.2 g acetic acid/L day.

In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 produces low levels of nitrous oxide (N 2 O) even under aerobic conditions. We compared the denitrification activity of strain TR2 with those of various denitrifiers in an artificial medium that was derived from piggery wastewater. Strain TR2 exhibited strong denitrification activity and produced little N 2 O under all conditions tested. Its growth rate under denitrifying conditions was near comparable to that under aerobic conditions, showing a sharp contrast to the lower growth rates of other denitrifiers under denitrifying conditions. Strain TR2 was tolerant to toxic nitrite, even utilizing it as a good denitrification substrate. When both nitrite and N 2 O were present, strain TR2 reduced N 2 O in preference to nitrite as the denitrification substrate. This bacterial strain was readily able to adapt to denitrifying conditions by expressing the denitrification genes for cytochrome cd 1 nitrite reductase (NiR) (nirS) and nitrous oxide reductase (NoS) (nosZ). Interestingly, nosZ was constitutively expressed even under nondenitrifying, aerobic conditions, consistent with our finding that strain TR2 preferred N 2 O to nitrite. These properties of strain TR2 concerning denitrification are in sharp contrast to those of well-characterized denitrifiers. These results demonstrate that some bacterial species, such as strain TR2, have adopted a strategy for survival by preferring denitrification to oxygen respiration. The bacterium was also shown to contain the potential to reduce N 2 O emissions when applied to sewage disposal fields.Wastewater treatment processes produce one of the major greenhouse effect gases, nitrous oxide (N 2 O) (7,25,30). The global warming potential of N 2 O relative to that of carbon dioxide (CO 2 ) is 298 for a 100-year time horizon, and its concentration in the atmosphere continues to increase by about 0.26% per year (9). Nitrogen removal in wastewater treatment plants is essentially based on the activities of nitrifying and denitrifying microorganisms, both of which are inhabitants of activated sludge. Nitrifying bacteria aerobically oxidize ammonium to nitrite (NO 2 Ϫ ) and nitrate (NO 3 Ϫ ), which are then reduced anaerobically by denitrifying bacteria to gaseous nitrogen forms, such as N 2 O and dinitrogen (N 2 ). It has long been known that N 2 O can be produced during both nitrification and denitrification processes of wastewater treatment (3, 19, 23), but the cause of N 2 O emission during the nitrification process was not clear. We recently showed, however, using activated sludge grown under conditions that mimicked a piggery wastewater disposal, that N 2 O emission during the nitrification process depends on denitrification by ammonia-oxidizing bacteria (Nitrosomonas) (18). On the other hand, it is believed that denitrifying bacteria produce N 2 O as a byproduct when anaerobiosis is insufficient during the denitrification process, because N 2 O reductase is the enzyme that is most sensitive to oxygen (6). Pigg...

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