Nitrogen fixation by symbiotic and free-living spirochetes.

Spirochetes from termite hindguts and freshwater sediments possessed homologs of a nitrogenase gene (nifH) and exhibited nitrogenase activity, a previously unrecognized metabolic capability in spirochetes. Fixation of 15-dinitrogen was demonstrated with termite gut Treponema ZAS-9 and free-living Spirochaeta aurantia. Homologs of nifH were also present in human oral and bovine ruminal treponemes. Results implicate spirochetes in the nitrogen nutrition of termites, whose food is typically low in nitrogen, and in global nitrogen cycling. These results also proffer spirochetes as a likely origin of certain nifHs observed in termite guts and other environments that were not previously attributable to known microbes.

The RDP-II (Ribosomal Database Project).

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [Nucleic Acids Res. (2000), 28, 173-174], continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 8.0 (June 1, 2000) consisted of 16 277 aligned prokaryotic small subunit (SSU) rRNA sequences while the number of eukaryotic and mitochondrial SSU rRNA sequences in aligned form remained at 2055 and 1503, respectively. The number of prokaryotic SSU rRNA sequences more than doubled from the previous release 14 months earlier, and approximately 75% are longer than 899 bp. An RDP-II mirror site in Japan is now available (http://wdcm.nig.ac.jp/RDP/html/index.h tml). RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment polymorphism experiments. The RDP-II email address for questions and comments has been changed from curator@cme.msu.edu to rdpstaff@msu.edu.

The RDP (Ribosomal Database Project) continues.

The Ribosomal Database Project (RDP-II), previously described by Maidak et al., continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 7.1 (September 17, 1999) included more than 10 700 small subunit rRNA sequences. More than 850 type strain sequences were identified and added to the prokaryotic alignment, bringing the total number of type sequences to 3324 representing 2460 different species. Availability of an RDP-II mirror site in Japan is also near completion. RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/ ). Analysis services include rRNA probe checking, approx-i-mate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment length polymorphism (T-RFLP) experiments.

Phylogenetic diversity of termite gut spirochaetes.

A molecular phylogenetic analysis was done of not-yet-cultured spirochaetes inhabiting the gut of the termite, Reticulitermes flavipes (Kollar). Ninety-eight clones of near-full-length spirochaetal 16S rDNA genes were classified by ARDRA pattern and by partial sequencing. All clones grouped within the genus Treponema, and at least 21 new species of Treponema were recognized within R. flavipes alone. Analysis of 190 additional clones from guts of Coptotermes formosanus Shiraki and Zootermopsis angusticollis (Hagen), as well as published data on clones from Cryptotermes domesticus (Haviland), Mastotermes darwiniensis Froggatt, Nasutitermes lujae (Wasmann) and Reticulitermes speratus(Kolbe), revealed a similar level of novel treponemal phylogenetic diversity in these representatives of five of the seven termite families. None of the clones was closely related (i.e. all bore < or = 91% sequence similarity) to any previously recognized treponeme. The data also revealed the existence of two major phylogenetic groups of treponemes: one containing all of the currently known isolates of Treponema and a large number of phylotypes from the human gingival crevice, but only a minority of the termite gut spirochaete clones; another containing the majority of termite spirochaete clones and two Spirochaeta (S. caldaria and S. stenostrepta), which, although free living, group within the genus Treponema on the basis of 16S rRNA sequence. Signature nucleotides that almost perfectly distinguished the latter group, herein referred to as the 'termite cluster', occurred at the following (E. coli numbering) positions: 289-G x C-311; A at 812; and an inserted nucleotide at 1273. The emerging picture is that the long-recognized and striking morphological diversity of termite gut spirochaetes is paralleled by their phylogenetic diversity and may reflect substantial physiological diversity as well.

A new version of the RDP (Ribosomal Database Project).

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [ Nucleic Acids Res. (1997), 25, 109-111], is now hosted by the Center for Microbial Ecology at Michigan State University. RDP-II is a curated database that offers ribosomal RNA (rRNA) nucleotide sequence data in aligned and unaligned forms, analysis services, and associated computer programs. During the past two years, data alignments have been updated and now include >9700 small subunit rRNA sequences. The recent development of an ObjectStore database will provide more rapid updating of data, better data accuracy and increased user access. RDP-II includes phylogenetically ordered alignments of rRNA sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software programs for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (ftp.cme.msu. edu) and WWW (http://www.cme.msu.edu/RDP). The WWW server provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree. Additional utilities also exist at RDP-II, including distance matrix, T-RFLP, and a Java-based viewer of the phylogenetic trees that can be used to create subtrees.

Isolation of the PufX protein from Rhodobacter capsulatus and Rhodobacter sphaeroides: evidence for its interaction with the alpha-polypeptide of the core light-harvesting complex.

Using mutant strains of Rhodobacter capsulatus and Rhodobacter sphaeroides in which the pufX gene had been deleted, it was possible to identify by HPLC membrane protein components present in pufX+ cells but absent in pufX- cells. In parallel preparations, membrane proteins soluble in chloroform/methanol containing ammonium acetate were first extracted from lyophilized membrane fractions of the pufX+ cells and separated from pigments and larger protein material by gel-filtration chromatography. Protein-containing fractions were examined by HPLC, and several peaks were collected from pufX+ material that were not present in pufX- material. From N-terminal amino acid sequencing, the PufX protein of Rb. capsulatus was identified, and from positive interaction with a PufX protein antibody, the Rb. sphaeroides PufX protein was identified. Although overall yields were very small, sufficient quantities of these proteins were isolated to evaluate their effect on the reconstitution of the core light-havesting antenna (LH1) and its subunit complex. From the behavior of the PufX protein and the alpha-polypeptide of LH1 on HPLC, qualitative evidence was obtained that the two proteins have a high affinity for each other. In reconstitution assays with bacteriochlorophyll (Bchl) and the LH1 alpha- and beta-polypeptides of Rb. capsulatus, the PufX protein of Rb. capsulatus was inhibitory to LH1 formation at low concentration. A similar inhibition was exhibited by Rb. sphaeroides PufX protein for reconstitution of LH1 with Bchl and the LH1 alpha- and beta-polypeptides of Rb. sphaeroides. In both cases, the ratios of concentrations of the PufX protein to the alpha-polypeptide causing 50% inhibition were approximately 0.5. Formation of the heterologous (alpha beta) subunit-type complex formed with Bchl and the alpha- and beta-polypeptides of LH1 of Rb. capsulatus was also inhibited by low concentrations of the Rb. capsulatus PufX protein (approximately 50% inhibition at PufX:alpha-polypeptide ratios = 0.5). However, neither PufX protein inhibited formation of a homologous (beta beta) subunit-type complex, which indicates that the PufX proteins do not interact with the beta-polypeptides.

arfI and arfII, two genes encoding alpha-L-arabinofuranosidases in Cytophaga xylanolytica.

arfI encoded the 57.7-kDa subunit of Cytophaga xylanolytica arabinofuranosidase I (ArfI). arfII encoded a 59.2-kDa subunit of ArfII. Products of both cloned genes liberated arabinose from arabinan and arabinoxylan. The deduced amino acid sequences of ArfI and ArfII revealed numerous regions that were identical to each other and to regions of homologous proteins from Bacteroides ovatus, Bacillus subtilis, and Clostridium stercorarium.

Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes.

Rhodobacter capsulatus puhA mutant strains containing either a nonpolar, translationally in-frame deletion or a polar insertion of an antibiotic resistance cartridge were constructed and evaluated for their photosynthetic growth properties, absorption spectroscopy profiles, and chromatophore protein compositions. Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction center (RC) and light-harvesting 1 (LH1) complexes. The translationally in-frame puhA deletion strains were restored to the parental strain phenotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not. Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described above. The strain containing the nonpolar deletion of orf 214 exhibited severely impaired photosynthetic growth properties and had greatly reduced levels of the RC and LH1 complexes. Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restored photosynthetic growth capability, as well as the RC and LH1 complexes. The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 complexes, and complementation in trans with orf 214 only marginally improved these deficiencies. These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puhA superoperon is necessary for optimal expression of these new photosynthesis genes.

Mutation of the Ser2 codon of the light-harvesting B870 alpha polypeptide of Rhodobacter capsulatus partially suppresses the pufX phenotype.

The exact function of the pufX gene product of Rhodobacter capsulatus is uncertain, but deletion of the pufX gene renders cells incapable of phototrophic growth on a minimal medium, and photosynthetic electron transfer is impaired in vitro. However, suppressor mutants that are able to grow phototropically are readily isolated. Two such suppressor mutants were characterized as to their phototrophic growth properties, their fluorescence at different incident light intensities, the integrity of their chromatophores, and their abilities to generate a transmembrane potential. We found that the photosynthetic apparatus in the suppressor mutants was less stable than that of the pseudo-wild-type and primary mutant strains and that the suppressor mutants used light energy less efficiently than the pseudo-wild-type strain. Therefore, the suppressor strains are more precisely designated partial suppressor mutants. The locations and sequences of the suppressor mutations were determined, and both were found to change the second codon of the pufA gene. It is hypothesized that the serine residue specified by this codon is important in interactions between the B870 alpha protein and other membrane-bound polypeptides and that suppressor mutations at this position partially compensate for loss of the PufX protein. A model is proposed for the function of the PufX protein.

Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus.

By deletion of the pufX gene of Rhodobacter capsulatus from a plasmid carrying the puf operon and complementation of a chromosomal puf operon deletion, we created pufX mutants and used them to characterize possible functions of the pufX gene product. The pufX mutants were incapable of photosynthetic growth in a minimal medium, or in a rich medium at low light intensities, although second-site mutations suppressed this phenotype. Measurements made in vitro with intact and solubilized chromatophore preparations indicated that the individual complexes of the photosynthetic unit seemed to function normally, but electron transfer from the reaction center to the cytochrome b/c1 complex was impaired. The structures of the photosynthetic apparatus of pseudo-wild type and mutant strains were evaluated using absorption spectroscopy and electron microscopy. The pufX mutants had intracytoplasmic membrane invaginations about 50% larger in diameter than those of the pseudo-wild type and higher levels of B870 light-harvesting complex. It is concluded that the PufX protein plays an important role in the structure of the functional photosynthetic unit, and its absence results in loss of efficient electron transfer from the QB site of the reaction center to the Qz site of the cytochrome b/c1 complex.

Formation of functional inter-species hybrid photosynthetic complexes in Rhodobacter capsulatus.

A Rhodobacter capsulatus mutant strain deficient in all pigment-binding peptides and hence incapable of photosynthetic growth was genetically complemented with a plasmid-borne copy of the Rhodobacter sphaeroides puf operon. Hybrid reaction centers composed of R. sphaeroides L and M and R. capsulatus H subunits assembled in vivo, and host cells were photosynthetically competent. Light-harvesting complex B875, also encoded by the R. sphaeroides puf operon, was present along with the hybrid reaction center. These cells emitted fluorescence, however, indicating an impairment in energy transduction.