== The SuperScript II RNase H reverse transcriptase kit (Invitrogen) was used to reverse transcribe total RNA. translation start codon. Transcripts initiated at thenapCpromoter that were extended across thenapM-napAboundary were detected by reverse transcription-PCR, confirming that the sixnapgenes can be cotranscribed as a single operon. Real-time PCR experiments confirmed thatnapoperon expression is regulated at the level of mRNA transcription by at least two mechanisms: nitrate induction and sulfate repression. We speculate that three almost perfect inverted-repeat sequences located upstream of the transcription start site might be binding sites for one or more proteins of the CRP/FNR family of transcription factors that mediate nitrate induction and sulfate repression of nitrate reduction byD. desulfuricans. Many different types of bacteria can adapt from aerobic to anaerobic growth, but when both oxygen and an alternative electron acceptor are available, they use oxygen preferentially. Similarly, during anaerobic growth, enteric bacteria use a powerful oxidizing agent, such as nitrate, in preference to nitrite or fumarate. This has led to a widespread assumption that when more than one potential Rabbit Polyclonal to PMS2 source for energy is available, bacterial regulatory mechanisms ensure that the thermodynamically most favorable electron acceptors are used first (1,14,29,36). This raises the fascinating question of whether bacteria normally associated with environments in which a less-powerful electron acceptor, such a Eribulin sulfate, is abundant preferentially use a thermodynamically more favorable electron acceptor, such as nitrate. There are many examples in the literature of sulfate-reducing bacteria that are also able to reduce nitrate (14,16,17,27,31,38), but this capacity is absent from the two strains ofDesulfovibrio vulgarisfor which complete genome sequence data are available (12). There is little agreement concerning how nitrate reduction is regulated, even in strains ofD. desulfuricans. Baumann and Denk (2) showed that nitrate reduction occurs only in the absence of sulfate, and others subsequently reported similar conclusions (7,17). In contrast, two groups reported that forD. desulfuricansstrain Essex, the presence of sulfate is essential for nitrate reduction to occur (14,27). However, nitrate reduction was prevented at sulfate concentrations typically used in media to propagate sulfate-reducing bacteria. In contrast, McCready et al. (16) proposed that nitrate reduction is inhibited rather than genetically repressed by sulfide generated as a product of sulfate reduction, a proposal that has since been confirmed (7). At very Eribulin low concentrations, sulfate was immediately consumed, so nitrate reduction was not inhibited. They also proposed, but did Eribulin not prove, that sulfate switches off nitrate reduction. Consequently, it is still undetermined whether sulfate represses the expression of theD. desulfuricansnitrate reductase operon. We recently reported the sequence of thenapoperon (for nitrate reduction in the periplasm) and its upstream regulatory region ofD. desulfuricansstrain 27774 (15). This information has provided for the first time an opportunity to investigate at the RNA level how expression of the nitrate reductase operon is regulated in a sulfate-reducing bacterium. In the present study, we first report results of experiments designed to determine whether nitrate or sulfate are reduced preferentially byD. desulfuricansstrain 27774 and compare growth yields on nitrate, nitrite, and sulfate. We confirm that sulfide strongly inhibits nitrate reduction by this strain. We then report results of experiments designed to determine whether the six genes of theD. desulfuricans27774napoperon are cotranscribed and therefore whether they form a single operon. Finally we demonstrate thatnapoperon transcription is induced by nitrate but repressed by sulfate and propose a possible mechanism for this regulation. == MATERIALS AND METHODS == == Bacterial strains and growth media. == D. desulfuricanssubsp.desulfuricansDSM 6949 (ATCC 27774) from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, was cultured in the following four media: Postgate B; Postgate C; Postgate N, which is a modified sulfate-free Postgate C liquid medium containing 14.7 mM nitrate or 5 mM nitrite; and Postgate SN, which is a modified medium containing 14.7 mM nitrate and 3.5 mM sulfate (22). The cultures were grown in sealed serum bottles (pregassed with N2), inoculated with 10% (vol/vol) of a 48-h-old culture, and incubated at 30C. The purity of the nitrate-grown culture was checked microscopically using the Gram stain by checking for inability of the grown culture to form colonies on nutrient agar during aerobic growth and for formation of a black FeS deposit when subcultured in Postgate B medium.Desulfovibrio vulgarisstrain Hildenborough (NCIMB 8303) was also used in this study as a negative control for the growth experiments, since it lacks the periplasmic nitrate reductase operon. The bacteria for the growth yield experiments were grown in a modified Postgate zero medium that was supplemented with sulfate, nitrate, and nitrite at the.