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IM-2 is a butyrolactone autoregulator that controls production of blue pigment and nucleoside antibiotics in Streptomyces sp. strain FRI-5. An IM-2-specific receptor gene, farA, was cloned from strain FRI-5, and nucleotide sequencing revealed that the farA gene consists of 666 bp encoding a 221-amino-acid protein of 24.3 kDa with an NH 2 -terminal amino acid sequence identical to that of purified native receptor. Another gene, farX, encoding a homolog of AfsA of Streptomyces griseus, was present upstream of farA. The monocistronic nature of the farA transcript was shown by Northern blot hybridization, and the transcript level increased upon addition of IM-2. Recombinant FarA expressed in and purified from E. coli showed clear ligand specificity toward IM-2, with a dissociation constant (K d ) for [ 3 H]IM-2-C 5 of 18.2 nM. FarA showed high overall homology to BarA (virginiae butanolide receptor from S. virginiae) and ArpA (A-factor receptor from S. griseus). Sequence alignment of the three receptor proteins revealed that the NH 2 -terminal region containing a helix-turn-helix DNA binding motif was highly conserved. The DNA binding motif is common in procaryotic repressors of the TetR family, suggesting that all the Streptomyces autoregulator receptors may act as transcriptional repressors.butanolide] of Streptomyces sp. strain FRI-5 is one of the butyrolactone autoregulators of Streptomyces species and triggers production of blue pigment as well as the nucleoside antibiotics showdomycin and minimycin at a concentration of 0.6 ng/ml (6,17,27,34). Butyrolactone autoregulators have been regarded as a kind of Streptomyces hormone which switches on morphological differentiation, such as aerial mycelium formation, and/or physiological differentiation, such as the production of antibiotics. They have a 2,3-disubstituted ␥-butyrolactone skeleton in common, and the 10 butyrolactone autoregulators identified to date are classified into the following three groups, on the basis of their minor structural differences: (i) A-factor type, possessing the 1'-keto group, to which only A-factor of S. griseus belongs (13, 18); (ii) virginiae butanolide (VB) type, possessing the 1'-␣-hydroxyl group, to which VB-A through VB-E (14, 24, 33) and Gräfe's three factors (3) belong; and (iii) IM-2 type, possessing the 1'-␤-hydroxyl group, to which IM-2 and factor I (4) belong (Fig. 1).Although the three groups of butyrolactone autoregulators differ in only minor structural detail, almost no cross-reactivity was observed among the responsive strains; i.e., induction by VB of aerial mycelium formation or streptomycin production by A-factor in Streptomyces griseus was not observed (16), while virginiamycin production in Streptomyces virginiae triggered by VBs requires a 1.7 ϫ 10 4 -fold higher concentration of A-factor or IM-2 (7, 19). Blue pigment production in strain FRI-5 triggered by IM-2 requires 170-and 1.7 ϫ 10 5 -fold-higher concentrations of the corresponding VB-and A-factor-type compounds, respectively (our unpublished data). In addi...

We examined hydrogen production from a dairy cow waste slurry (13.4 g of volatile solids per liter) by batch cultures in a temperature range from 37 to 85 degrees C, using microflora naturally present within the slurry. Without the addition of seed bacteria, hydrogen was produced by simply incubating the slurry, using the microflora within the slurry. Interestingly, two peaks of fermentation temperatures for hydrogen production from the slurry were observed at 60 and 75 degrees C (392 and 248 ml H2 per liter of slurry, respectively). After the termination of the hydrogen evolution, the microflora cultured at 60 degrees C displayed hydrogen-consuming activity, but hydrogen-consuming activity of the microflora cultured at 75 degrees C was not detected, at least for 24 days. At both 60 and 75 degrees C, the main by-product was acetate, and the optimum pH of the slurry for hydrogen production was around neutral. Bacteria related to hydrogen-producing moderate and extreme thermophiles, Clostridium thermocellum and Caldanaerobacter subterraneus, were detected in the slurries cultured at 60 and 75 degrees C, respectively, by denaturing gradient gel electrophoresis analyses, using the V3 region of 16S rDNA.

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75 degrees C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.

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