Several linear megaplasmids were detected in the facultatively lithoautotrophic Gram-positive bacterium Nocardia opaca The wild-type strain M R l l contains, in addition to the cccDNA plasmids pHG31-a and pHG31-b, the linear plasmids pHG201(270 kb), pHG202 (400 kb) and pHG203 (420 kb). The wild-type strain MR22 contains, in addition to the cccDNA plasmid pHG33, the linear plasmids pHG204 (180 kb), pHG205 (280 kb) and pHG206 (510 kb). After preparation of DNA from cells embedded in agarose, the linear plasmids were demonstrated by pulsed-field electrophoresis. By means of DNA probes for genes of soluble hydrogenase and ribulose-bisphosphate carboxylase, the conjugative plasmids pHG201 and pHG205 were shown to be the carriers of the genetic information for these enzymes. A restriction map of pHG201 for the enzymes AsnI, SpeI, XbaI is presented.

As described previously, in Rhodococcus sp. (formerly Nocardia opaca) strains MRll and MR22, the ability to grow as an aerobic hydrogen bacterium (the Aut character) is located on giant conjugative linear plasmids -on pHG201 (270 kb) in strain MRll and on pHG205 (280 kb) in strain MR22. In an autotrophic transconjugant originating from MR22 a smaller plasmid, pHG207 (225 kb), was detected and shown to be a recombination product of the wild-type plasmids pHG204 and pHG205. A donor carrying pHG207 as the sole plasmid transferred the Aut marker at a 1000-fold frequency compared to the wild-type plasmid pHG205. Analysis of the plasmid ends revealed that plasmid pHG207 carries proteins at both ends; the proteins are linked to the 5' ends of the strands. The cloned end fragments of about 2 kb were sequenced and found to contain highly homologous sequences within the terminal 583 bp (left end part) and 560 bp (right end part). Several potential reading frames were detected, but database searching gave no indication about possibIe functions.

The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus M R l l into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7-4 kbp Asnl fragment of the linear plasmid pHG2Ol via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hoxF, -U, -Y, -H, -Wand ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hoxF, -U, -Y and -H encode the subunits a, 7, 6 and B, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the / 3 subunit. Finally, ORF7 encodes a protein which has similarities to CAMP-and cGMP-binding protein kinases, but its function is not known. ORFI, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus M R l l indicated that the hydrogenase genes are under control of a like promoter located at the right end of ORFI and are even transcribed under heterotrophic conditions a t a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHYl harbouring the 7.4 kbp Asnl fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.Keywords : Rhodococcus opacus MR11, NAD-reducing hydrogenase, box genes INTRODUCTIONRhodococcus opacus strain M R l l (formerly Nocardia opaca 1 b) is a Gram-positive, facultative chemolithoautotrophic bacterium which can grow on carbon dioxide and gaseous H, as the sole carbon and energy sources. Physiologically, it belongs to the knallgas bacteria, a group which is composed of phylogenetically diverse organisms. For the activation of H,, all knallgas bacteria contain hydrogenases, which are of two basic types : the membrane-bound hydrogenases (MBHs) , which are coupled to the electron transfer chain and are The GenBank accession number for the nucleotide sequence reported in this paper is U70364.not capable of reducing NAD, and the soluble hydrogenases (SHs), which are localized in the cytoplasm and catalyse the transfer of electrons directly to NAD (Aragno & Schlegel, 1992;Schneider et al., 1984a;Zaborosch et al., 1989; Schink & Schlegel, 1979). The MBHs belong to the more common group of NiFehydrogenases consisting of one large and one small subunit, whereas the SHs belong to a family of less abundant multimeric NiFe-hydrogenases (Friedrich & Schwartz, 1993 ;Wu & Mandrand, 1993). The majority of the knallgas bacteria contain only the MBH, but a few, ...

The telomers of several linear plasmids o f Rhodococcus opacus (formerly Nocardia opaca) were studied. The plasmids pHG201, pHG204 and pHG205 carry proteins bound to their ends, as shown by gel retardation experiments. A sequence hybridizing with the terminal sequence of pHG207, a recombinant linear plasmid consisting of the left part o f pHG204 and the right part of pHG205, which was analysed in a previous study by the authors, could be detected in all linear plasmids of the wild-type R. opacus strains MR11 and MR22. However, only pHG204 and pHG206 carry terminal inverted repeats (TIRs) like pHG207. Cloning and sequencing of the terminal fragment of pHG204 revealed a nearly perfect TIR of 1016 bp. In contrast, the termini of pHG2Ol and pHG205 share little homology. Sequence analysis of the two end fragments of pHG201 revealed a similarity of only 65% within the terminal 34/32 bp and a perfect TIR of only 3 bp. The results support the assumption that long TlRs are not absolutely necessary for replication and maintenance of linear plasmids.

Five new strains of yellow-pigmented, gram-negative, motile, hydrogen-oxidizing bacteria were isolated; each served as a host for simultaneously isolated bacteriophages. These isolates and two additional strains were compared with other gram-negative, hydrogen-oxidizing bacteria with respect to morphology; nutritional and biochemical properties; growth parameters; cytochrome content; pigment production; susceptibility to bacteriophages, bacteriostatic agents, and antibiotics; deoxyribonucleic acid base composition; and deoxyribonucleic aciddeoxyribonucleic acid homology. Six of the strains were characterized by a high degree of interstrain similarity and were found to be related to Pseudomonas flaua. However, due to basic differences between these strains and P. fZaua, the former are regarded as comprising a new species for which, because of its moderate relationship to P. flava, the name Pseudomonas pseudoflava is proposed. The type strain of P. pseudoflava, GA3, has been deposited with the Deutsche Sammlung von Mikroorganismen in Gottingen under the number DSM 1034.The facultatively autotrophic hydrogen-oxidizing bacteria-commonly called hydrogen bacteria-are a taxonomically heterogeneous group among the chemolithoautotrophs. Their physiology has recently been reviewed by Schlegel (30). In the summer and autumn of 1973, about 80 strains of hydrogen bacteria were isolated and tested for ability to serve as hosts for simultaneously isolated bacteriophages. Among these, only a few strains of polarly flagellated bacteria forming yellow colonies were found to be susceptible to the bacteriophages. These host bacteria had a high interstrain similarity and were considered to be related to Pseudomonas flaua (15).Further studies were aimed at characterizing the six host bacteria, evaluating their similarity and delineating them from other yellow-pigmented hydrogen bacteria, and determining their taxonomic niche. For direct comparison with these strains, several type strains and some other strains of hydrogen bacteria, especially the yellow-pigmented ones, were included in this study. Morphological features; nutritional and biochemical properties; growth parameters; the cytochromes; pigmentation; and susceptibility to bacteriophages, bacteriostatic agents, and antibiotics were investigated. In addition, the serological relationships to other gram-negative hydrogen bacteria and the deoxyribonucleic acid (DNA) base compositions and DNA-DNA homologies of these strains were studied.As a result of these studies, it was concluded that the six new isolates belong to a new species, for which we propose the name Pseudomonas pseudoflava (pseu.do