The hydBGDA genes, which encode the four subunits /?, y, S and a of the [Ni-Fe] hydrogenase from the archaeon Pymcoccus furiosus, have been isolated and sequenced using a PCWIPCR-based strategy. From the sequence analysis it appears that the four structural genes are tightly linked and organized in a single transcription unit. The hydD and hydA gene products are related to the small and the large subunits of several archaeal and eubacterial [Ni-Fe] hydrogenases with an overall degree of sequence relatedness ranging from 35 YO to 50% (identity +similarity). In particular, the amino acid sequence motifs involved in the accommodation of nickel and iron-sulfur clusters are conserved. In addition, the database search revealed that the hydB and hydG gene products are homologous to the asrA-and asrB-encoded subunits of the sulf ite reductase enzyme from Salmonella typhimurium . This is particularly interesting in view of the recent finding that the P. furiosus hydrogenase appears t o be a bifunctional enzyme endowed with both proton-and sulfurreducing activities.

Three differently metabolically engineered strains, 2 single PHA ) and Hup ) mutants and one double PHA ) /Hup ) mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H 2 photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H 2 recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup ) and the double PHA ) / Hup ) mutants, but not the single PHA ) mutant, exhibited increased rates of H 2 photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup ) mutant showed higher rates of H 2 production, but all 3 mutants sustained a longer-term H 2 photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H 2 evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H 2 photoproduction using actual waste-derived substrates.

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A search for pilin genes in a Bordetella pertussis (Bp) genomic library has led to the identification of several clones which hybridize to synthetic oligonucleotides with sequences derived from amino acid sequences of Bp fimbrial subunits. One of these clones (corresponding to a gene we have named fimX) contains an open reading frame encoding a protein with a molecular weight of about 20 kD and a sequence similar but not identical to the fimbrial subunit fim2 and to other fimbrial protein sequences. In this communication we present the cloning and nucleotide sequence of the fimX gene and its homology to the fim2 gene. A genomic analysis on the positional relationship between the two genes is also presented.