Science, 1997 Jun 6, 276(5318), 1568 - 71 Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene; Maymo-Gatell X et al.; Tetrachloroethene is a prominent groundwater pollutant that can be reductively dechlorinated by mixed anaerobic microbial populations to the nontoxic product ethene . Strain 195, a coccoid bacterium that dechlorinates tetrachloroethene to ethene, was isolated and characterized . Growth of strain 195 with H2 and tetrachloroethene as the electron donor and acceptor pair required extracts from mixed microbial cultures . Growth of strain 195 was resistant to ampicillin and vancomycin; its cell wall did not react with a peptidoglycan-specific lectin and its ultrastructure resembled S-layers of Archaea . Analysis of the 16S ribosomal DNA sequence of strain 195 indicated that it is a eubacterium without close affiliation to any known groups. Plant Cell, 1997 Jun, 9(6), 925 - 45 Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas; Cerutti H et al.; The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants . In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s) . Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells . Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I . Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila . By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation. DNA Cell Biol, 1997 Jun, 16(6), 787 - 95 Kinesin light chain in a eubacterium; Celerin M et al.; A eubacterial homolog of a kinesin light chain gene has been isolated and characterized from the cyanobacterium Plectonema boryanum . Although the eubacterial and eukaryotic kinesin light chains are highly similar in amino acid sequence, the eubacterial sequence differs in several distinguishing structural features, including the absence of a putative PEST domain and the presence of additional highly conserved imperfect tandem repeats . Two soluble kinesin light chain antigens have been identified from whole-cell lysates by immunoblot analysis . Attempts to identify a canonical kinesin heavy-chain gene or protein were unsuccessful, suggesting that a kinesin heavy chain may be absent or unnecessary for kinesin light-chain function in this eubacterium . Our findings establish that certain basal elements of eukaryotic cellular transport appear to be resident in eubacteria . We discuss the possibility that the eukaryotic kinesin light chain was acquired by lateral gene transfer. Nature, 1997 May 29, 387(6632), 493 - 7 An ancestral mitochondrial DNA resembling a eubacterial genome in miniature; Lang BF et al.; Mitochondria, organelles specialized in energy conservation reactions in eukaryotic cells, have evolved from eubacteria-like endosymbionts whose closest known relatives are the rickettsial group of alpha-proteobacteria . Because characterized mitochondrial genomes vary markedly in structure, it has been impossible to infer from them the initial form of the proto-mitochondrial genome . This would require the identification of minimally derived mitochondrial DNAs that better reflect the ancestral state . Here we describe such a primitive mitochondrial genome, in the freshwater protozoon Reclinomonas americana . This protist displays ultrastructural characteristics that ally it with the retortamonads, a protozoan group that lacks mitochondria . R . americana mtDNA (69,034 base pairs) contains the largest collection of genes (97) so far identified in any mtDNA, including genes for 5S ribosomal RNA, the RNA component of RNase P, and at least 18 proteins not previously known to be encoded in mitochondria . Most surprising are four genes specifying a multisubunit, eubacterial-type RNA polymerase . Features of gene content together with eubacterial characteristics of genome organization and expression not found before in mitochondrial genomes indicate that R . americana mtDNA more closely resembles the ancestral proto-mitochondrial genome than any other mtDNA investigated to date. J Biol Chem, 1997 May 16, 272(20), 12905 - 8 Evidence for proton transfer from Glu-46 to the chromophore during the photocycle of photoactive yellow protein; Imamoto Y et al.; Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins . To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes . For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at -40 degrees C . The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R . A., Hoff, W . D., and Hellingwerf, K . J . (1995) Biochemistry 34, 12669-12672) were observed at 1482, 1437, and 1163 cm-1 for PYP . However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm-1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM . A 1736 cm-1 band was observed for PYP, but the corresponding band for PYPM was not . Because it disappeared in the Glu-46 --> Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46 . These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM. EMBO J, 1997 May 15, 16(10), 2927 - 36 Factor requirements for transcription in the Archaeon Sulfolobus shibatae; Qureshi SA et al.; Archaea (archaebacteria) constitute a domain of life that is distinct from Bacteria (eubacteria) and Eucarya (eukaryotes) . Although archaeal cells share many morphological features with eubacteria, their transcriptional apparatus is more akin to eukaryotic RNA polymerases I, II and III than it is to eubacterial transcription systems . Thus, in addition to possessing a 10 subunit RNA polymerase and a homologue of the TATA-binding protein (TBP), Archaea possess a polypeptide termed TFB that is homologous to eukaryotic TFIIB . Here, we investigate the factor requirements for transcription of several promoters of the archaeon Sulfolobus shibatae and its associated virus SSV . Through in vitro transcription and immunodepletion, we demonstrate that S . shibatae TBP, TFB and RNA polymerase are not complexed tightly with one another and that each is required for efficient transcription of all promoters tested . Furthermore, full transcription is restored by supplementing respective depleted extracts with recombinant TBP or TFB, indicating that TBP-associated factors or TFB-associated factors are not required . Indeed, gel-filtration suggests that Sulfolobus TBP and TFB are not associated stably with other proteins . Finally, all promoters analysed are transcribed accurately and efficiently in an in vitro system comprising recombinant TBP and TFB, together with essentially homogeneous preparation of RNA polymerase . Transcription in Archaea is therefore fundamentally homologous to that in eukaryotes, although factor requirements appear to be much less complex. Z Naturforsch {C}, 1997 May-Jun, 52(5-6), 287 - 91 Synthesis of 4-aza-5,6-dimethylbenzimidazole and biosynthetic preparation of 4- and 7-aza-5,6-dimethylbenzimidazolylcobamide; Renz P et al.; We report on the preparation of 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide . These vitamin B12-analogs were required as reference compounds for comparison with a corrinoid previously isolated in small amounts for Eubacterium limosum grown in the presence of 4(5)-aminoimidazole . 4(7)-Aza-5,6-dimethylbenzimidazole was synthesized from N-1-benzyl-4-nitroimidazole which was reduced to N-1-benzyl-4-aminoimidazole and condensed with 1-dimethylamino-2-methylbutan-3-one to yield N-1-benzyl-4-aza-5,6-dimethylbenzimidazole . The benzyl group of this compound was split off by catalytic hydrogenation to form 4(7)-aza-5,6-dimethylbenzimidazole . 4(7)-Aza-5,6-dimethylbenzimidazole was transformed by a growing culture of Propionibacterium shermanii into 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide . Both vitamin B12-analogs were almost as active as Vitamin B12 in a growth test with the vitamin B12-dependent Escherichia coli-mutant DSM 4261. J Cell Sci, 1997 May, 110 ( Pt 10), 1141 - 5 Histidine kinases in signal transduction pathways of eukaryotes; Loomis WF et al.; Autophosphorylating histidine kinases are an ancient conserved family of enzymes that are found in eubacteria, archaebacteria and eukaryotes . They are activated by a wide range of extracellular signals and transfer phosphate moieties to aspartates found in response regulators . Recent studies have shown that such two-component signal transduction pathways mediate osmoregulation in Saccharomyces cerevisiae, Dictyostelium discoideum and Neurospora crassa . Moreover, they play pivotal roles in responses of Arabidopsis thaliana to ethylene and cytokinin . A transmembrane histidine kinase encoded by dhkA accumulates when Dictyostelium cells aggregate during development . Activation of DhkA results in the inhibition of its response regulator, RegA, which is a cAMP phosphodiesterase that regulates the cAMP dependent protein kinase PKA . When PKA is activated late in the differentiation of prespore cells, they encapsulate into spores . There is evidence that this two-component system participates in a feedback loop linked to PKA in prestalk cells such that the signal to initiate encapsulation is rapidly amplified . Such signal transduction pathways can be expected to be found in a variety of eukaryotic differentiations since they are rapidly reversible and can integrate disparate signals. J Clin Periodontol, 1997 May, 24(5), 346 - 53 Protein degradation by Prevotella intermedia and Actinomyces meyeri supports the growth of non-protein-cleaving oral bacteria in serum; Jansen HJ et al.; The proteolytic activities of oral bacteria are thought to play an important role in the aetiology of dental abscesses . Bacteria-derived proteases may contribute to tissue destruction, and are likely to impair host defence by degrading immunoglobulins and complement . Degraded periodontal tissue and tissue fluid are likely to constitute essential sources of nutrients in the abscess . Tissue fluid, which is derived from serum, is rich in protein and poor in carbohydrate, suggesting that breakdown of protein and fermentation of amino acids is a crucial step to generate energy for growth of the microflora . The number of oral bacterial species that perform hydrolytic cleavage of protein into polypeptides, the first step in protein degradation, is relatively small compared to the large majority of peptidase-producing species . In this study, we therefore investigated the growth-promoting effect of proteinase-producing species like Prevotella intermedia and Actinomyces meyeri on the growth of some non-proteinase producing bacteria in mixed cultures . We used serum as a substitute for the supposed natural substrate of the abscess microflora . The breakdown of serum proteins was investigated using capillary electrophoresis . Poor growth was found in mono- and mixed cultures of non-proteinase producing species Eubacterium lentum, Fusobacterium nucleatum . Peptostreptococcus micros, and Streptococcus intermedius . The presence of P . intermedia in mixed cultures strongly enhanced growth of these 4 species, according to the hypothesis that the growth of the mixed cultures was peptide-limited . The enhanced growth of P . intermedia in pronase-digested serum indicated peptide-limited growth of this organism in serum, despite its production of proteinase . We found that growth of monocultures of Actinomyces meyeri was poor . In contrast, A . meyeri grew well in mixed cultures and its presence stimulated growth of F . nucleatum and P . micros, suggesting a synergistic relationship . The growth of mono- and mixed cultures was investigated using one representative strain of each species . Thus, there is a small risk of having selected unique strains . Proteinase inhibitors reduced the growth of Porphyromonas gingivalis, Prevotella nigrescens, and P . intermedia in trypticase peptone-yeast extract medium with, and without, IgG . Our study indicated that proteinase-producing organisms play a key role in mixed cultures of oral bacteria in human serum by providing polypeptides for growth . This may explain their association with dental abscesses. Curr Genet, 1997 May, 31(5), 430 - 8 Multiple recruitment of class-I aldolase to chloroplasts and eubacterial origin of eukaryotic class-II aldolases revealed by cDNAs from Euglena gracilis; Plaumann M et al.; The photosynthetic protist Euglena gracilis is one of few organisms known to possess both class-I and class-II fructose-1,6-bisphosphate aldolases (FBA) . We have isolated cDNA clones encoding the precursor of chloroplast class-I FBA and cytosolic class-II FBA from Euglena . Chloroplast class-I FBA is encoded as a single subunit rather than as a polyprotein, its deduced transit peptide of 139 amino acids possesses structural motifs neccessary for precursor import across Euglena's three outer chloroplast membranes . Evolutionary analyses reveal that the class-I FBA of Euglena was recruited to the chloroplast independently from the chloroplast class-I FBA of chlorophytes and may derive from the cytosolic homologue of the secondary chlorophytic endosymbiont . Two distinct subfamilies of class-II FBA genes are shown to exist in eubacteria, which can be traced to an ancient gene duplication which occurred in the common ancestor of contemporary gram-positive and proteobacterial lineages . Subsequent duplications involving eubacterial class-II FBA genes resulted in functional specialization of the encoded products for substrates other than fructose-1,6-bisphosphate . Class-II FBA genes of Euglena and ascomycetes are shown to be of eubacterial origin, having been acquired via endosymbiotic gene transfer, probably from the antecedants of mitochondria . The data provide evidence for the chimaeric nature of eukaryotic genomes. J Bacteriol, 1997 May, 179(10), 3222 - 31 The Streptomyces galP1 promoter has a novel RNA polymerase recognition sequence and is transcribed by a new form of RNA polymerase in vitro; Brawner ME et al.; We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro . Base substitutions were introduced throughout the galP1 promoter region, and bases at positions -34, -36, and -11 with respect to the transcription start site were shown to be required for promoter function . These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described . The -35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity . By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces . Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro . DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site. Proteins, 1997 May, 28(1), 72 - 82 An evolutionary treasure: unification of a broad set of amidohydrolases related to urease; Holm L et al.; The recent determination of the three-dimensional structure of urease revealed striking similarities of enzyme architecture to adenosine deaminase and phosphotriesterase, evidence of a distant evolutionary relationship that had gone undetected by one-dimensional sequence comparisons . Here, based on an analysis of conservation patterns in three dimensions, we report the discovery of the same active-site architecture in an even larger set of enzymes involved primarily in nucleotide metabolism . As a consequence, we predict the three-dimensional fold and details of the active site architecture for dihydroorotases, allantoinases, hydantoinases, AMP-, adenine and cytosine deaminases, imidazolonepropionase, aryldialkylphosphatase, chlorohydrolases, formylmethanofuran dehydrogenases, and proteins involved in animal neuronal development . Two member families are common to archaea, eubacteria, and eukaryota . Thirteen other functions supported by the same structural motif and conserved chemical mechanism apparently represent later adaptations for different substrate specificities in different cellular contexts. J Bacteriol, 1997 May, 179(9), 2938 - 43 Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum; Eberhardt S et al.; Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum . The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast . Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases . The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli . The ribC gene of M . thermoautotrophicum specifies a protein of 153 amino acid residues . The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii . The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12) . The amino acid sequence predicted by the ribC gene of M . thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast . Most notably, the M . thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases . The protein of M . thermoautotrophicum can be expressed efficiently in a recombinant E . coli strain . The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C . In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions . The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate . The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria. J Mol Evol, 1997 May, 44(5), 528 - 41 Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms; Brendel V et al.; Protein sequences with similarities to Escherichia coli RecA were compared across the major kingdoms of eubacteria, archaebacteria, and eukaryotes . The archaeal sequences branch monophyletically and are most closely related to the eukaryotic paralogous Rad51 and Dmc1 groups . A multiple alignment of the sequences suggests a modular structure of RecA-like proteins consisting of distinct segments, some of which are conserved only within subgroups of sequences . The eukaryotic and archaeal sequences share an N-terminal domain which may play a role in interactions with other factors and nucleic acids . Several positions in the alignment blocks are highly conserved within the eubacteria as one group and within the eukaryotes and archaebacteria as a second group, but compared between the groups these positions display nonconservative amino acid substitutions . Conservation within the RecA-like core domain identifies possible key residues involved in ATP-induced conformational changes . We propose that RecA-like proteins derive evolutionarily from an assortment of independent domains and that the functional homologs of RecA in noneubacteria comprise an array of RecA-like proteins acting in series or cooperatively. Arch Microbiol, 1997 May, 167(5), 302 - 9 Phylogeny and molecular fingerprinting of green sulfur bacteria; Overmann J et al.; The 16S rDNA sequences of nine strains of green sulfur bacteria (Chlorobiaceae) were determined and compared to the four known sequences of Chlorobiaceae and to sequences representative for all eubacterial phyla . The sequences of the Chlorobiaceae strains were consistent with the secondary structure model proposed earlier for Chlorobium vibrioforme strain 6030 . Similarity values > 90.1% and Knuc values < 0.11 indicate a close phylogenetic relatedness among the green sulfur bacteria . As a group, these bacteria represent an isolated branch within the eubacterial radiation . In Chlorobiaceae, a similar morphology does not always reflect a close phylogenetic relatedness . While ternary fission is a morphological trait of phylogenetic significance, gas vesicle formation occurs also in distantly related species . Pigment composition is not an indicator of phylogenetic relatedness since very closely related species contain different bacteriochlorophylls and carotenoids . Two different molecular fingerprinting techniques for the rapid differentiation of Chlorobiaceae species were investigated . The 16S rDNA fragments of several species could not be separated by denaturing gradient gel electrophoresis . In contrast, all strains investigated during the present work gave distinct banding patterns when dispersed repetitive DNA sequences were used as targets in PCR . The latter technique is, therefore, well suited for the rapid screening of isolated pure cultures of green sulfur bacteria. Biochemistry, 1997 Apr 15, 36(15), 4471 - 9 Electron transfer proteins from the haloalkaliphilic archaeon Natronobacterium pharaonis: possible components of the respiratory chain include cytochrome bc and a terminal oxidase cytochrome ba3; Scharf B et al.; Natronobacterium pharaonis, an aerobic haloalkaliphilic archaebacterium, expresses high concentrations of redox proteins as do alkaliphilic eubacteria . The first redox protein characterized from N . pharaonis was halocyanin {Scharf, B., & Engelhard, M . (1993) Biochemistry 32, 12894-12900}, a small blue copper protein . It is a peripheral membrane protein and is conjectured to function in a manner similar to plastocyanin . In the present work, the respiratory chain is further elucidated and the purification and characterization of the most abundant components cytochrome bc and cytochrome ba3 from the membrane fraction are described . The cytochrome bc complex consists of a 14 and an 18 kDa subunit in a 1:1 ratio, with heme c bound to the larger polypeptide . An Fe-S subunit similar to that found in eukaryotic bc complexes has not yet been identified . The second membrane complex carries two different heme groups of the ba3-type as well as copper . It contains two subunits of 36 and 40 kDa . This cytochrome ba3 binds carbon monoxide, a feature common to terminal oxidases . There is no spectroscopic evidence for a second terminal oxidase; hence, under the growth conditions chosen the respiratory chain of N . pharaonis appears to be unbranched . In addition to these cytochromes, a succinate dehydrogenase which is solubilized from the membrane by detergents was isolated . A cytochrome c which was isolated from the cytosol has an unusually high molecular weight and a redox potential of -142 mV . A second cytosolic protein, ferredoxin, was purified to homogeneity . A comparison of the redox potentials of the isolated proteins with those obtained from the native membrane allows the construction of a possible electron transfer chain. Mol Microbiol, 1997 Apr, 24(1), 1 - 6 DnaA initiator--also a transcription factor; Messer W et al.; The replication-initiator protein DnaA is ubiquitous in the eubacterial world . It binds to an asymmetric 9 bp consensus DNA sequence, the DnaA box . Besides its primary function as an initiator, it acts as a transcription factor that represses or activates several genes, or terminates transcription, depending on the location and arrangement of DnaA boxes. Gene, 1997 Apr 1, 188(2), 239 - 46 Gene cloning and sequencing, and enzyme purification of the malate synthase of Streptomyces arenae; Huttner S et al.; Streptomyces arenae is able to grow on acetate or ethanol as the sole carbon source . The metabolic pathway used for gluconeogenesis from C2 compounds in streptomycetes has not yet been characterized . In the course of a sequencing project we identified the gene for malate synthase (aceB), a key enzyme in the glyoxylate cycle in S . arenae . The gene was cloned and sequenced . The open reading frame of 1632 bp codes for a potential protein of 61.360 kDa . A comparison with the sequences of malate synthase from other organisms shows that the phylogenetic distance to the E . coli aceB gene is no closer than that to genes from plants or fungi . Malate synthase activity was detected in cell extracts from S . arenae . Its dependence on media conditions and on the growth phase was investigated . A purification procedure was established which allows a 188-fold enrichment of the enzyme . The molecular weight of the monomer determined by SDS PAGE confirms the weight calculated from the gene sequence . However, the holoenzyme appears to be dimeric as shown by gel filtration . All other known malate synthases from eubacteria are monomeric, while those of fungi or plants are oligomeric (di-, tri-, tetra- or octameric) . The apparent Km value for glyoxylate is significantly higher than that of the malate synthases of all other species published so far . The enzyme is inactive at pH values of 7 and below; the strain cannot grow on ethanol or acetate as the sole carbon source at media pH values of 7 or below. Gene, 1997 Apr 1, 188(2), 221 - 8 Glycolytic enzyme operon of Borrelia burgdorferi: characterization and evolutionary implications; Gebbia JA et al.; The genes encoding three enzymes of the glycolytic pathway have been identified and sequenced completely in Borrelia burgdorferi sensu stricto and partially in B . hermsii . They are clustered on the chromosome into an operon with a single putative promoter and are arranged downstream of this promoter in the following order: gapdh (glyceraldehyde-3-phosphate dehydrogenase), pgk (phosphoglycerate kinase), and tpi (triosephosphate isomerase) . gapdh and pgk are separated by 19 bp of intergenic sequence and pgk and tpi are separated by only 1 bp . Each of the three genes contains a putative RBS 6-7 bp upstream of each respective translational (ATG) start codon . The deduced protein encoded by gapdh consists of 335 amino acids (aa) with a predicted MW of 36,400, that of pgk is 393 aa (MW of 42,156) and that of tpi is 290 aa (MW of 27,683) . The aa sequences of each of the three enzymes share 58.4% (GAPDH), 52.8% (PGK) and 46.1% (TPI) identity with respective enzymes from other prokaryotic organisms . Phylogenetic analyses based on these universal and conserved proteins support the hypothesis that spirochetes are an ancient and distinct eubacterial phylum. Biotechniques, 1997 Apr, 22(4), 700 - 4 Quantitative analysis of 16S rDNA using competitive PCR and the QPCR System 5000; Blok HJ et al.; A method for precise and accurate quantification of 16S rDNA has been developed that uses competitive PCR and the QPCR System 5000 . The method is based on co-amplification of 16S rDNA sequences, along with an internal standard sequence, using only one set of conserved eubacterial primers . Co-amplified PCR products are rapidly identified and quantified by measuring the electrochemiluminescent signals from specific oligonucleotide reporter probes that are directed against a hypervariable 16S rDNA sequence . Because in the exponential phase of amplification the different target sequences and the internal standard sequence are amplified with the same efficiency, unknown amounts of a target sequence in a sample can be inferred by extrapolating against a standard curve that is generated for the internal standard sequence . This method provides a rapid, nonradioactive and reliable way to simultaneously quantify different specific 16S rDNA targets that are present in low numbers, and may thus be suitable for enumeration of specific target microorganisms in environmental samples. Int J Syst Bacteriol, 1997 Apr, 47(2), 446 - 52 Rickettsia peacockii sp . nov., a new species infecting wood ticks, Dermacentor andersoni, in western Montana; Niebylski ML et al.; Rickettsia peacockii, a new species of spotted fever group rickettsiae, was identified from Rocky Mountain wood ticks (Dermacentor andersoni) collected in the Sapphire Mountain Range on the eastern side of Bitterroot Valley, Montana . DNA from R . peacockii SkalkahoT (T = type strain) in naturally infected tick tissue was amplified by a PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA), rickettsial citrate synthase, and 190-kDa surface antigen (rOmpA) genes . Partial 16S rDNA and rOmpA gene sequences exhibited levels of similarity of 99.7 and 93.2%, respectively, with the sequences of the spotted fever agent Rickettsia rickettsii R . By using Gimenez staining, fluorescent antibody tests, a PCR assay, and a restriction fragment length polymorphism analysis, 76 of 115 female ticks (minimal field infection rate, 66.1%) collected between 1992 and 1995 were found to be infected . The organism is passed transstadially and transovarially (minimal vertical transmission rate, 73.3%), and infections are localized in ovarial tissues . Attempts to cultivate R . peacockii were unsuccessful. J Bacteriol, 1997 Apr, 179(8), 2632 - 40 Gene duplications in evolution of archaeal family B DNA polymerases; Edgell DR et al.; All archaeal DNA-dependent DNA polymerases sequenced to date are homologous to family B DNA polymerases from eukaryotes and eubacteria . Presently, representatives of the euryarchaeote division of archaea appear to have a single family B DNA polymerase, whereas two crenarchaeotes, Pyrodictium occultum and Sulfolobus solfataricus, each possess two family B DNA polymerases . We have found the gene for yet a third family B DNA polymerase, designated B3, in the crenarchaeote S . solfataricus P2 . The encoded protein is highly divergent at the amino acid level from the previously characterized family B polymerases in S . solfataricus P2 and contains a number of nonconserved amino acid substitutions in catalytic domains . We have cloned and sequenced the ortholog of this gene from the closely related Sulfolobus shibatae . It is also highly divergent from other archaeal family B DNA polymerases and, surprisingly, from the S . solfataricus B3 ortholog . Phylogenetic analysis using all available archaeal family B DNA polymerases suggests that the S . solfataricus P2 B3 and S . shibatae B3 paralogs are related to one of the two DNA polymerases of P . occultum . These sequences are members of a group which includes all euryarchaeote family B homologs, while the remaining crenarchaeote sequences form another distinct group . Archaeal family B DNA polymerases together constitute a monophyletic subfamily whose evolution has been characterized by a number of gene duplication events. Appl Environ Microbiol, 1997 Apr, 63(4), 1557 - 63 Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria; Lange JL et al.; Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment . In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria . Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger) . Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH . DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry . The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry . In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding . With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding . Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust . This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment. Nucleic Acids Res, 1997 Apr 1, 25(7), 1369 - 74 Characterization of an ATP-dependent DNA ligase encoded by Haemophilus influenzae; Cheng C et al.; We report that Haemophilus influenzae encodes a 268 amino acid ATP-dependent DNA ligase . The specificity of Haemophilus DNA ligase was investigated using recombinant protein produced in Escherichia coli . The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km = 0.2 microM) . Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP . Haemophilus ligase reacted with ATP in the absence of DNA substrate to form a covalent ligase-adenylate intermediate . This nucleotidyl transferase reaction required a divalent cation and was specific for ATP . The Haemophilus enzyme is the first example of an ATP-dependent DNA ligase encoded by a eubacterial genome . It is also the smallest member of the covalent nucleotidyl transferase superfamily, which includes the bacteriophage and eukaryotic ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. Biochim Biophys Acta, 1997 Mar 20, 1351(1-2), 31 - 6 A new sigma factor homolog in a cyanobacterium: cloning, sequencing, and light-responsive transcripts of rpoD2 from Microcystis aeruginosa K-81; Asayama M et al.; We isolated an rpoD2 gene encoding the potential sigma factor of RNA polymerase from the cyanobacterium Microcystis aeruginosa K-81, which can perform photosynthesis . The deduced amino acid sequence of RpoD2 (sigmaA2) exhibits extensive homology to other eubacterial RpoD proteins . This gene possessed multiple 5'-end transcripts, expressed specifically under light (P(L)), dark (P(D)), or constitutively light/dark (P(C)) conditions during exponential cell growth. J Mol Biol, 1997 Mar 14, 266(5), 939 - 49 A survey of polypeptide deformylase function throughout the eubacterial lineage; Mazel D et al.; N-terminal formylation of ribosome-synthesized polypeptides is assumed to be among the most conserved features that distinguish the eubacterial line of descent from other living phyla . In order to assess the ancientness of this trait, def genes encoding polypeptide deformylase were characterized from four eubacterial species, Lactococcus lactis, Bacillus subtilis, Calothrix PCC7601 and Thermotoga maritima, taking advantage of the conditional viability of the def mutants of Escherichia coli . Altogether, eight sequences of polypeptide deformylase have been obtained from all the eubacterial sources which were investigated, either through systematic genome sequence analysis or through genetic screening, yielding a highly homologous family . A gene putatively encoding Met-tRNAi formyltransferase, fmt, was found downstream of the deformylase gene except in L . lactis, Mycoplasma genitalium, Calothrix PCC7601 and T . maritima . These results argue strongly for the ancestral character of N-terminal formylation in eubacteria . Most of the wide deviations of amino acid usage observed in def- and fmt-encoded proteins among species is best accounted for by the nucleotide composition of genomes . Furthermore, the species of origin of each protein appears to be more recognizable than its function, considering only its amino acid composition. J Biol Chem, 1997 Mar 7, 272(10), 6382 - 7 Molecular identification of a novel protein that regulates biogenesis of photosystem I, a membrane protein complex; Bartsevich VV et al.; Photosystem I (PSI) is a multisubunit pigment-protein complex in the thylakoid membranes of cyanobacteria and chloroplasts . BP26, a random photosynthesis-deficient mutant strain of the cyanobacterium Synechocystis 6803 has a severely reduced PSI content, whereas its photosystem II is present in normal amount . The BP26 mutant is complemented by a novel gene, btpA, that encodes a 30-kDa protein . In this strain, a missense mutation in the btpA gene, resulting in the replacement of a valine by a glycine residue, significantly affects the accumulation of the PSI reaction center proteins, PsaA and PsaB . Northern blot analysis revealed that the steady-state levels of the transcripts from the psaAB operon, encoding these proteins, remain unaltered in the mutant strain . Hence the BtpA protein regulates a post-transcriptional process during the life cycle of the PSI protein complex such as 1) translation of the psaAB mRNA, 2) assembly of the PsaA/PsaB polypeptides and their associated cofactors into a functional complex, or 3) degradation of the protein complex . Close relatives of the BtpA protein have been found in nonphotosynthetic organisms, viz . the archaebacterium Methanococcus jannaschii, the eubacterium Escherichia coli, and the nematode, Caenorhabditis elegans, suggesting that these proteins may regulate biogenesis of other protein complexes in these evolutionarily distant organisms. Mol Biol Rep, 1997 Mar, 24(1-2), 125 - 31 Eubacterial proteasomes; Lupas A et al.; Proteasomes are large, multisubunit proteases with highly conserved structures . The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependent pathway of protein degradation . The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria . Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure. Microbiol Mol Biol Rev, 1997 Mar, 61(1), 1 - 16 Extrachromosomal DNA in the Apicomplexa; Wilson RJ et al.; Malaria and related apicomplexan parasites have two highly conserved organellar genomes: one is of plastid (pl) origin, and the other is mitochondrial (mt) . The organization of both organellar DNA molecules from the human malaria parasite Plasmodium falciparum has been determined, and they have been shown to be tightly packed with genes . The 35-kb circular DNA is the smallest known vestigial plastid genome and is presumed to be functional . All but two of its recognized genes are involved with genetic expression: one of the two encodes a member of the clp family of molecular chaperones, and the other encodes a conserved protein of unknown function found both in algal plastids and in eubacterial genomes . The possible evolutionary source and intracellular location of the plDNA are discussed . The 6-kb tandemly repeated mt genome is the smallest known and codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two bizarrely fragmented rRNAs . The organization of the mt genome differs somewhat among genera . The mtDNA sequence provides information not otherwise available about the structure of apicomplexan cytochrome b as well as the unusually fragmented rRNAs . The malarial mtDNA has a phage-like replication mechanism and undergoes extensive recombination like the mtDNA of some other lower eukaryotes. J Bacteriol, 1997 Mar, 179(5), 1734 - 47 Molecular systematic studies of eubacteria, using sigma70-type sigma factors of group 1 and group 2; Gruber TM et al.; Sigma factors of the sigma70 family were used as a phylogenetic tool to compare evolutionary relationships among eubacteria . Several new sigma factor genes were cloned and sequenced to increase the variety of available sequences . Forty-two group 1 sigma factor sequences of various species were analyzed with the help of a distance matrix method to establish a phylogenetic tree . The tree derived by using sigma factors yielded subdivisions, including low-G+C and high-G+C gram-positive bacteria, cyanobacteria, and the alpha, beta, gamma, and delta subdivisions of proteobacteria, consistent with major bacterial groups found in trees derived from analyses with other molecules . However, some groupings (e.g., the chlamydiae, mycoplasmas, and green sulfur bacteria) are found in different positions than for trees obtained by using other molecular markers . A direct comparison to the most extensively used molecule in systematic studies, small-subunit rRNA, was made by deriving trees from essentially the same species set and using similar phylogenetic methods . Differences and similarities based on the two markers are discussed . Additionally, 31 group 2 sigma factors were analyzed in combination with the group 1 proteins in order to detect functional groupings of these alternative sigma factors . The data suggest that promoters recognized by the major vegetative sigma factors of eubacteria will contain sequence motifs and spacing very similar to those for the sigma70 sigma factors of Escherichia coli. J Theor Biol, 1997 Feb 7, 184(3), 339 - 44 The detection of nucleotide sequences with strong similarity to hormone responsive elements in the genome of eubacteria and archaebacteria and their possible relation to similar sequences present in the mitochondrial genome; Hatzoglou E et al.; To account for the presence of nucleotide sequences in mitochondria with similarity to the Hormone Response Elements (HREs) of the nuclear genomes of man, rat and mouse, the genomes of several procaryotes have been screened for the presence of the sequences AGAACA NNN TGTTCT and GGTACA NNN TGTTCT, which represent perfect palindromic and consensus class I HREs, respectively, and for the sequence AGGTCA NNN TGACCT, which represents class II HRE . In many of the examined procaryotes, eubacteria and archaebacteria, almost perfect palindromic class I HREs and perfect or almost perfect class II half palindromic HREs have been detected in various genes, some of which encode proteins involved in energy metabolism, in replication and in transcription control . These findings support the hypothesis that the similar sequences found in mitochondria, potentially involved in hormonal regulation of respiratory enzyme biosynthesis, were introduced into eucaryotic cell by the procaryotic endosymbionts. Curr Genet, 1997 Feb, 31(2), 144 - 57 Isolation and characterization of rad51 orthologs from Coprinus cinereus and Lycopersicon esculentum, and phylogenetic analysis of eukaryotic recA homologs; Stassen NY et al.; In eubacteria, the recA gene has long been recognized as essential for homologous recombination and DNA repair . Recent work has identified recA homologs in archaebacteria and eukaryotes, thus emphasizing the universal role this gene plays in DNA metabolism . We have isolated and characterized two new recA homologs, one from the basidiomycete Coprinus cinereus and the other from the angiosperm Lycopersicon esculentum . Like the RAD51 gene of Saccharomyces cerevisiae, the Coprinus gene is highly induced by gamma irradiation and during meiosis . Phylogenetic analyses of eukarotic recA homologs reveal a gene duplication early in eukaryotic evolution which gave rise to two putatively monophyletic groups of recA-like genes . One group of 11 characterized genes, designated the rad51 group, is orthologous to the Saccharomyces RAD51 gene and also contains the Coprinus and Lycopersicon genes . The other group of seven genes, designated the dmc1 group, is orthologous to the Saccharomyces DMC1 gene . Sequence comparisons and phylogenetic analysis reveal extensive lineage- and gene-specific differences in rates of RecA protein evolution . Dmc1 consistently evolves faster than Rad51, and fungal proteins of both types, especially those of Saccharomyces, change rapidly, particularly in comparison to the slowly evolving vertebrate proteins . The Drosophila Rad51 protein has undergone remarkably rapid sequence divergence. J Biol Chem, 1997 Jan 24, 272(4), 2046 - 9 Inhibition of Plasmodium falciparum protein synthesis . Targeting the plastid-like organelle with thiostrepton; McConkey GA et al.; The human malaria parasite Plasmodium falciparum has two extrachromosomal DNAs associated with organelles whose function is unclear . Both genomes encode ribosomal RNAs (rRNAs) that are distinct from the nuclear-encoded rRNAs . Secondary structure analysis of all the P . falciparum rRNAs indicates that only the large subunit (LSU) rRNA encoded by the plastid-like genome is the target for thiostrepton . Indeed we find that thiostrepton inhibits growth of the parasite in the micromolar range which is 10-fold below concentrations with observable effects on total protein synthesis . We have further examined selective effects of thiostrepton on the plastid function by comparing differential effects of the drug on cytoplasmic and organellar encoded transcripts . Treatment with either thiostrepton or rifampin, an inhibitor of organellar and eubacterial RNA polymerase, both showed disappearance of organellar-encoded RNA transcripts within 6 h of treatment while transcripts of a nuclear-encoded mRNA remained constant for at least 8 h of treatment . Hence, we show a selective effect on organelle function that is suggestive of interference in the protein synthesis apparatus of the plastid . Sensitivity of P . falciparum to thiostrepton confirms that the plastid-like genome is essential for the erythrocytic cycle and presents a novel therapeutic site for this class of antibiotics. FEMS Microbiol Lett, 1997 Jan 15, 146(2), 271 - 8 Rapid identification and typing of Staphylococcus aureus by nested PCR amplified ribosomal DNA spacer region; Saruta K et al.; We designed a polymerase chain reaction (PCR) assay for rapid detection of prokaryotic 16S-23S spacer regions . This PCR assay consisted of nested DNA amplifications . The first-step PCR was able to detect the general presence of eubacteriales with a unified set of universal primers . The universal primers were selected from highly conserved regions in 16S and 23S ribosomal RNA (rRNA) genes and amplified DNAs from all 62 different species of bacteria tested . In the second-step PCR, the identification primers could detect four important bacterial species through amplification of the rRNA spacer regions between the 16S-23S rRNA genes . For Staphylococcus aureus, intraspecies variation in spacer amplification products was observed with S . aureus specific primers . We suggest that the nested PCR assay could be used as a novel method for the identification and typing in epidemiological studies of S . aureus. FEBS Lett, 1997 Jan 6, 400(3), 271 - 4 Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway; Lichtenthaler HK et al.; Isopentenyl diphosphate (IPP) is the biological C5 precursor of isoprenoids . By labeling experiments using {1-(13)C}glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast-bound isoprenoids (beta-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga . The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway. Cell Mol Life Sci, 1997 Jan, 53(1), 34 - 50 Phylogenetic relationship of organisms obtained by ribosomal protein comparison; Muller EC et al.; The evolutionary relationships of ribosomal proteins from eubacteria, archaea, eukaryotes, chloroplasts and mitochondria were examined by their degree of conservation, their structural and functional properties and by the occurrence of conserved structural elements . The structural domains formed by the different protein families were studied . The occurrence of monophyletic groups was investigated for each protein family within the archaea . Phylogenetic trees were constructed between these organisms and together with the homologous sequences of the other phylogenetic domains . All organisms belonging to the archaea clearly formed a monophyletic group . The conserved sequence motifs were checked for the potential to form similar secondary structural elements. Genes Cells, 1997 Jan, 2(1), 81 - 94 A nested set of C-terminal deletions of the alpha subunit of Escherichia coli RNA polymerase define regions concerned with assembly, proteolysis, stabilization and transcriptional activation in vivo; Zou C et al.; BACKGROUND: The alpha subunit of eubacterial RNA polymerase comprises an N-terminal assembly domain and a mobile C-terminal domain which provides an activation contact site for class I transcription activators . One particular C-terminal alpha mutant, rpoA341, impairs the response of Escherichia coli RNA polymerase to several activators, including MelR . RESULTS: The in vivo properties of a set of C-terminally truncated alpha variants were investigated . Derivatives of 230 amino acids or longer were assembled into functional RNA polymerase . However, derivatives greater than 271 residues in length were sensitised to proteolysis near K271 . Deletion of only 13 C-terminal amino acids impaired the response to CRP at a class I promoter whereas the complete removal of the alpha C-terminal domain did not prevent complementation of MelR-dependent PmelAB activity in the rpoA341 mutant . CONCLUSIONS: Our results refine the C-terminal limit of the alpha assembly domain to between residues 221 and 230 . The 13 extreme C-terminal amino acids are exposed in the holoenzyme and participate in the protection of an otherwise proteolytically sensitive bond near K271 . Their presence is also essential for transcription activation at class I CRP-dependent promoters . The rpoA341-mediated substitution, K271E, does not define an activation contact site for MelR. J Basic Microbiol, 1997, 37(1), 3 - 9 Copurification of ribosomal protein S2 and DNA-dependent RNA polymerase from heat-shocked cells of Bacillus subtilis; Buttner K et al.; The DNA-dependent RNA polymerases from heat-shocked and vegetatively grown cells of Bacillus subtilis were isolated and compared . The RNA polymerase from non-stressed cells had the well known alpha, beta, beta' and sigma composition of eubacterial RNA polymerases . The RNA polymerase from heat-shocked cells exhibited one additional band shown by SDS-PAGE . N-terminal sequencing of the first 16 amino acids of the associated protein demonstrated its identity with the ribosomal protein S2. Glycoconj J, 1997 Jan, 14(1), 3 - 11 Bacterial glycoproteins; Messner P; Glycoproteins are a diverse group of complex macromolecules that are present in virtually all forms of life . Their presence in prokaryotes, however, has been demonstrated, and accepted, only recently . Bacterial glycoproteins have been identified in many archaeobacteria and in eubacteria . They comprise a wide range of different cell envelope components such as membrane-associated glycoproteins, surface-associated glycoproteins and crystalline surface layers (S-layers), as well as secreted glycoproteins and exoenzymes . Even their occurrence in the cytoplasm cannot yet be ruled out . This minireview tries to cover the whole subject as completely as possible and refers to available information on presence, structure, biosynthesis, and molecular biology of bacterial glycoproteins. J Mol Evol, 1997, 44 Suppl 1, S23 - 7 The reason for as well as the consequence of the Cambrian explosion in animal evolution; Ohno S; The first 1 billion years of our 4.5-billion-year-old planet were extremely violent, characterized by constant meteorite bombardment . Therefore, it is with a great surprise that we note that cellular life flourished 3.5 billion years ago . It appears that the cellular life came into being as soon as the earth's environment became hospitable . Because the main ingredient of the Archean sea was sodium bicarbonate, neither archeobacteria nor eubacteria but rather photosynthesizing organisms dominated-initially, prokaryotic cyanobacteria, soon joined by eukaryotic blue-green algae . These consumers of carbon dioxide were also releasers of molecular oxygen . The toil of 3 billion years by these releasers of molecular oxygen finally triggered the Cambrian animal explosion . With exceptions of two animal phyla, Porifera and Coelenterata, which amde slightly earlier appearances, nearly all other extant animal phyla sprang into almost simultaneous existence within 6 to 10 million years . The notion of the Cambrian pananimalia genome was advanced to explain various evolutionary consequences of this Cambrian explosion. J Exp Biol, 1997 Jan, 200 ( Pt 2), 203 - 16 Animal plasma membrane energization by chemiosmotic H+ V-ATPases; Harvey WR et al.; Proton-motive forces are thought to be less important than sodium-motive forces in energizing animal membranes . On the supply side, proton-motive forces across mitochondrial inner membranes are well-known energizers of ATP synthesis, catalyzed by F-type ATP synthases . However, on the demand side, proton-motive forces, generated from ATP by V-ATPases, are not widely accepted as energizers of animal membranes; instead, sodium-motive forces, generated by P-ATPases, are thought to predominate . During the 1980s, Anraku, Nelson, Forgac and others showed that proton-motive forces from H+ V-ATPases energize endomembranes of all eukaryotic cells; in most cases, chloride ions accompany the protons and the output compartment is acidified . Unexpectedly, numerous examples of animal plasma membrane energization by proton-motive forces are now appearing . In many insect epithelia, H+ V-ATPases generate transmembrane voltages which secondarily drive sensory signalling, fluid secretion and even alkalization, rather than acidification . Plasma membranes of phagocytes and osteoclasts as well as polarized membranes of epithelia in vertebrate kidney, bladder and epididymis, even apical membranes of frog skin epithelial cells, are now known to be energized by proton-motive forces . The list of proton-energized animal plasma membranes grows daily and includes cancer cells . The localization of H+ V-ATPases either on endomembranes or on plasma membranes may reflect a key event in their evolution . Proton-motive ATPases, like the H+ A-ATPases in present-day archaebacteria, appear to be ancestors of both H+ F-ATP synthases and H+ V-ATPases . On the basis of a greater than 25% overall sequence identity and much higher identity in the nucleotide-binding and regulatory sites, Nelson and others have argued that the A and B subunits of V-ATPases, like the corresponding beta and alpha subunits of F-ATP synthases, derive from common 'A-ATPase-like' ancestral subunits . They postulate that oxygen, introduced into the earth's atmosphere by cyanobacteria, was a selective agent as these key subunits diverged during evolution . Forgac has focused the issue more sharply by showing that the catalytic 'A' subunit of H+ V-ATPases has tow key sulfhydryl residues that are proximal to each other in the tertiary structure; these residues form a disulfide bond under oxidizing conditions, thereby inactivating the enzyme . The corresponding beta subunit of H+ F-ATPases lacks such sulfhydryl residues . Perhaps because their plasma membranes are the site of oxygen-dependent ATP synthesis, which would select against their sulfhydryl-containing regulatory sites, eubacterial cells lack H+ V-ATPases . This retention of the regulatory cysteine residue in the active sites during evolution may explain why H+ V-ATPases . are commonly found in the reducing atmosphere of the cytoplasm, where they would be active, rather than in the putatively oxidizing atmosphere of many plasma membranes, where they would be inactive . It may also explain why animal plasma membrane H+ V-ATPases are commonly found in 'mitochondria-rich' cells . We suggest that the high oxygen affinity of cytochrome oxidase leads to localized reducing conditions near mitochondria which would allow H+ V-ATPases to remain active in plasma membranes of such cells . Moreover, this 'redox modulation mechanism' may obviate the need to evoke two types of enzyme to explain selective targeting of H+ V-ATPases to plasma membranes or endomembranes: membrane that contains a single form of H+ V-ATPase may cycle between the membranes of the cytoplasmic organelles and the cell surface, the enzyme being active only when reducing conditions remove the disulfide bonding restraint. Microbiology, 1997 Jan, 143 ( Pt 1), 55 - 61 Monoclonal antibodies against Streptococcus pneumoniae detect epitopes on eubacterial ribosomal proteins L7/L12 and on streptococcal elongation factor Ts; Kolberg J et al.; Two monoclonal antibodies (mAbs) designated 144,H-3 (IgG2a) and 218,C-5 (IgM) were produced after immunization of mice with two different heat-treated and sonicated pneumococcal strains . Western blotting, with solubilized proteins from different bacterial genera and from mammalian lymphocytes, showed that both mAbs reacted with a protein of approximately 12 kDa in all 66 strains of eubacteria examined, representing 27 different species . The 12 kDa protein was isolated by immunoaffinity chromatography . Subsequent preparative Western blotting enabled N-terminal amino acid sequence analysis by microsequencing . A high degree of amino acid sequence similarity with eubacterial ribosomal proteins L7/L12 was demonstrated . One of the mAbs (144,H-3) also cross-reacted in Western blotting with a 43 kDa protein, but only from streptococci . The 43 kDa protein carrying the common streptococcal epitope was isolated and sequenced in the N-terminal region . A high degree of amino acid sequence identity was found to elongation factor Ts from Escherichia coli. Genetics, 1997 Jan, 145(1), 97 - 110 A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression; Cerutti H et al.; We have constructed a dominant selectable marker for nuclear transformation of C . reinhardtii, composed of the coding sequence of the eubacterial aadA gene (conferring spectinomycin resistance) fused to the 5' and 3' untranslated regions of the endogenous RbcS2 gene . Spectinomycin-resistant transformants isolated by direct selection (1) contain the chimeric gene(s) stably integrated into the nuclear genome, (2) show cosegregation of the resistance phenotype with the introduced DNA, and (3) synthesize the expected mRNA and protein . Small linearized plasmids appeared to be inserted into the nuclear genome preferentially through their ends, with relatively few large deletions and/or rearrangements . Multiple copy transformants often integrated concatemers of transforming DNA . Our detailed analysis of the complex integration patterns of plasmid DNA in C . reinhardtii nuclear transformants should be useful for improving the technique of insertional mutagenesis . We also found that the spectinomycin-resistance phenotype was unstable in about half of the transformants . When maintained under nonselective conditions, neither the aadA mRNA nor the AadA protein were detected in these subclones . Moreover, since the integrated transforming DNA was not altered or lost expression of the RbcS2::aadA::RbcS2 gene(s) appears to be repressed . Measurements of transcriptional activity, mRNA accumulation, and mRNA stability suggest that expression of this chimeric gene(s) may also be affected by rapid RNA degradation, presumably due to defects in mRNA processing and, or nuclear export . Thus, both gene silencing and transcript instability, rather than biased codon usage, may explain the difficulties encountered in the expression of foreign genes in the nuclear genome of Chlamydomonas. Nucleic Acids Res, 1997 Jan 1, 25(1), 96 - 7 Compilation of 5S rRNA and 5S rRNA gene sequences; Specht T et al.; The compilation of 5S rRNA and 5S rRNA gene nucleotide sequences as of 30 September 1996, contains a total of 1661 primary structures of 5S rRNAs or their genes, which is an increase of 928 new sequence entries over the last compilation . It covers sequences from 54 archaea, 449 eubacteria, 34 plastids, nine mitochondria and 430 eukaryotes . The databank uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information . The taxonomic classification of organisms was totally updated . Now the database is also available via anonymous FTP or WWW. J Bacteriol, 1997 Jan, 179(2), 345 - 57 Sequencing of heat shock protein 70 (DnaK) homologs from Deinococcus proteolyticus and Thermomicrobium roseum and their integration in a protein-based phylogeny of prokaryotes; Gupta RS et al.; The 70-kDa heat shock protein (hsp70) sequences define one of the most conserved proteins known to date . The hsp70 genes from Deinococcus proteolyticus and Thermomicrobium roseum, which were chosen as representatives of two of the most deeply branching divisions in the 16S rRNA trees, were cloned and sequenced . hsp70 from both these species as well as Thermus aquaticus contained a large insert in the N-terminal quadrant, which has been observed before as a unique characteristic of gram-negative eubacteria and eukaryotes and is not found in any gram-positive bacteria or archaebacteria . Phylogenetic analysis of hsp70 sequences shows that all of the gram-negative eubacterial species examined to date (which includes members from the genera Deinococcus and Thermus, green nonsulfur bacteria, cyanobacteria, chlamydiae, spirochetes, and alpha-, beta-, and gamma-subdivisions of proteobacteria) form a monophyletic group (excluding eukaryotic homologs which are derived from this group via endosybitic means) strongly supported by the bootstrap scores . A closer affinity of the Deinococcus and Thermus species to the cyanobacteria than to the other available gram-negative sequences is also observed in the present work . In the hsp7O trees, D . proteolyticus and T . aquaticus were found to be the most deeply branching species within the gram-negative eubacteria . The hsp70 homologs from gram-positive bacteria branched separately from gram-negative bacteria and exhibited a closer relationship to and shared sequence signatures with the archaebacteria . A polyphyletic branching of archaebacteria within gram-positive bacteria is strongly favored by different phylogenetic methods . These observations differ from the rRNA-based phylogenies where both gram-negative and gram-positive species are indicated to be polyphyletic . While it remains unclear whether parts of the genome may have variant evolutionary histories, these results call into question the general validity of the currently favored three-domain dogma. Appl Environ Microbiol, 1997 Jan, 63(1), 169 - 77 Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068; Duffaud GD et al.; Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase {EC 3.2.1.78}), beta-mannosidase (beta-D-mannopyranoside hydrolase {EC 3.2.1.25}) and alpha-galactosidase (alpha-D-galactoside galactohydrolase {EC 3.2.1.22}) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media . The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa . The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C . The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts . The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits . The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C . The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C . These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T . neapolitana for nutritional purposes . The significance of such substrates in geothermal environments remains to be seen. J Biol Chem, 1996 Dec 13, 271(50), 31949 - 56 Spectral tuning, fluorescence, and photoactivity in hybrids of photoactive yellow protein, reconstituted with native or modified chromophores; Kroon AR et al.; Photoactive yellow proteins (PYPs) constitute a new class of eubacterial photoreceptors, containing a deprotonated thiol ester-linked 4-hydroxycinnamic acid chromophore . Interactions with the protein dramatically change the (photo)chemical properties of this cofactor . Here we describe the reconstitution of apoPYP with anhydrides of various chromophore analogues . The resulting hybrid PYPs, their acid-denatured states, and corresponding model compounds were characterized with respect to their absorption spectrum, pK for chromophore deprotonation, fluorescence quantum yield, and Stokes shift . Three factors contributing to the tuning of the absorption of the hybrid PYPs were quantified: (i) thiol ester bond formation, (ii) chromophore deprotonation, and (iii) specific chromophore-protein interactions . Analogues lacking the 4-hydroxy substituent lack both contributions (chromophore deprotonation and specific chromophore-protein interactions), confirming the importance of this substituent in optical tuning of PYP . Hydroxy and methoxy substituents in the 3- and/or 5-position do not disrupt strong interactions with the protein but increase their pK for protonation and the fluorescence quantum yield . Both deprotonation and binding to apoPYP strongly decrease the Stokes shift of chromophore fluorescence . Therefore, coupling of the chromophore to the apoprotein not only reduces the energy gap between its ground and excited state but also the extent of reorganization between these two states . Two of the PYP hybrids show photoactivity comparable with native PYP, although with retarded recovery of the initial state. Gene, 1996 Dec 5, 182(1-2), 221 - 3 Phylogenetic distribution of the global regulatory gene csrA among eubacteria; White D et al.; The gene csrA encodes a unique kind of global regulator, CsrA, which modulates glycolysis, gluconeogenesis, glycogen biosynthesis and glycogen catabolism in Escherichia coli . Southern hybridization and nucleotide sequencing data have revealed apparent csrA homologs within several families of the alpha and gamma subdivisions of the proteobacteria (purple bacteria) and in the Gram-positive bacterium Bacillus subtilis . Thus, the CsrA regulatory system appears widely distributed among eubacteria. J Mol Evol, 1996 Dec, 43(6), 672 - 7 Early evolutionary origin of the planktic foraminifera inferred from small subunit rDNA sequence comparisons; Wade CM et al.; Phylogenetic analysis of five partial planktic foraminiferal small subunit (SSU) ribosomal (r) DNA sequences with representatives of a diverse range of eukaryote, archaebacterial, and eubacterial taxa has revealed that the evolutionary origin of the foraminiferal lineage precedes the rapid eukaryote diversification represented by the "crown" of the eukaryotic tree and probably represents one of the earliest splits among extant free-living aerobic eukaryotes . The foraminiferal rDNA sequences could be clearly separated from known symbionts, commensals, and food organisms . All five species formed a single monophyletic group distinguished from the "crown" group by unique foraminiferal specific insertions as well as considerable nucleotide distance in aligned regions. Lett Appl Microbiol, 1996 Dec, 23(6), 421 - 5 The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid; Newbold CJ et al.; Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen . However, little is known about the factors affecting the lysis of archaea in rumen fluid . Bacterial lysis was followed from the release of acid-soluble 14C from 14C leucine-labelled bacteria . The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non-ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoa population or monofaunated with Polyplastron multivesiculatum or Entodinium spp . The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter, while lysis of the non-methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70% . Substantial lysis of Methanobrevibacter occurred in cell-free rumen fluid and this effect could be abolished by autoclaving . In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage-induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid. RNA, 1996 Dec, 2(12), 1306 - 10 Phylogenetic analysis of tmRNA secondary structure; Williams KP et al.; The bacterial tmRNA acts with dual tRNA-like and mRNA-like character to tag incomplete translation products for degradation . Comparative analysis of 17 tmRNA genes (including eight new sequences) has allowed us to deduce conserved features of the tmRNA secondary structure . Except in a segment that includes the first codon of the tag reading frame, tmRNA is highly structured, with four pseudoknots and a total of 11 conserved base pairing regions . The previously identified tRNA minihelix structure is connected by a long base paired region to a large structured domain composed of a pseudoknot, followed by the tag reading frame and a string of three rather similar pseudoknots . The conservation of numerous structural elements among diverse eubacterial species indicates that these elements have important function beyond simply forming an endonuclease-resistant link between the reading frame and the tRNA-like domain. Mol Microbiol, 1996 Dec, 22(5), 841 - 8 Introducing mutations into a chromosomal rRNA gene using a genetically modified eubacterial host with a single rRNA operon; Sander P et al.; Gene-inactivation techniques were employed to construct a eubacterial organism harbouring a single functional rRNA operon . This mutant of Mycobacterium smegmatis permits replacement of the single remaining rRNA operon with a homologous fragment from a vector-borne gene . By homologous recombination with the chromosome a plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, resulting in a homogeneous population of mutated ribosomes in the cell . As a first result we demonstrate that the single allelic knock-out strain allows for isolation of rRNA mutants with a drug-resistant phenotype, circumventing the problem of recessivity which prohibits the isolation of such mutants in organisms with multiple rRNA operons . Subsequently, by allelic exchange experiments, it was demonstrated that the rRNA mutation found indeed confers drug resistance in vivo . This system provides intriguing potential for the study of the structure and function of ribosomal RNAs. J Bacteriol, 1996 Dec, 178(24), 7212 - 20 The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA; Baggio L et al.; Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria . The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100 . Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins . Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B . thetaiotaomicron and E . coli gdhA+ transformants . Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E . coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed . Enzyme activity was restored if these E . coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region . Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity . Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity . Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B . thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful. J Bacteriol, 1996 Dec, 178(24), 7053 - 8 Sequencing and expression of a gene encoding a bile acid transporter from Eubacterium sp . strain VPI 12708; Mallonee DH et al.; Eubacterium sp . strain VPI 12708 expresses inducible bile acid 7alpha-dehydroxylation activity via a multistep pathway . The genes encoding several of the inducible proteins involved in the pathway have been previously mapped to a bile acid-inducible (bai) operon in Eubacterium sp . strain VPI 12708 . We now report the cloning, sequencing, and characterization of the baiG gene, which is part of the bai operon . The predicted amino acid sequence of the BaiG polypeptide shows significant homology to several membrane transport proteins, including sugar and antibiotic resistance transporters, which are members of the major facilitator superfamily . Hydrophilicity plots of BaiG show a high degree of similarity to class K and L TetA proteins from gram-positive bacteria, and, like these classes of TetA proteins, BaiG has 14 proposed transmembrane domains . The baiG gene was cloned into Escherichia coli and shown to confer an energy-dependent bile acid uptake activity . Primary bile acids were preferentially transported into E . coli cells expressing this gene, with at least sevenfold and fourfold increases in the uptake of cholic acid and chenodeoxycholic acid, respectively, over control reactions . Less transport activity was observed with cholylglycine, 7-oxocholic acid, and deoxycholic acid . The transport activity was inhibited by the proton ionophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and nigericin but not by the potassium ionophore valinomycin, suggesting that the transport is driven by the proton motive force across the cell membrane . In summary, we have cloned, sequenced, and expressed a bile acid-inducible bile acid transporter from Eubacterium sp . strain VPI 12708 . To our knowledge, this is the first report of the cloning and expression of a gene encoding a procaryotic bile acid transporter. J Bacteriol, 1996 Dec, 178(23), 6665 - 70 Mannitol, a novel bacterial compatible solute in Pseudomonas putida S12; Kets EP et al.; The aim of this study was to identify the compatible solutes accumulated by Pseudomonas putida S12 subjected to osmotic stress . In response to reduced water activity, P . putida S12 accumulated Nalpha-acetylglutaminylglutamine amide (NAGGN) simultaneously with a novel compatible solute identified as mannitol (using 13C- and 1H-nuclear magnetic resonance, liquid chromatography-mass spectroscopy and high-performance liquid chromatography methods) to maximum concentrations of 74 and 258 micromol g (dry weight) of cells(-1), respectively . The intracellular amounts of each solute varied with both the type and amount of osmolyte applied to induce osmotic stress in the medium . Both solutes were synthesized de novo . Addition of betaine to the medium resulted in accumulation of this compound and depletion of both NAGGN and mannitol . Mannitol and NAGGN were accumulated when sucrose instead of salts was used to reduce the medium water activity . Furthermore, both compatible solutes were accumulated when glucose was substituted by other carbon sources . However, the intracellular quantities of mannitol decreased when fructose, succinate, or lactate were applied as a carbon source . Mannitol was also raised to high intracellular concentrations by other salt-stressed Pseudomonas putida strains . This is the first study demonstrating a principal role for the de novo-synthesized polyol mannitol in osmoadaptation of a heterotrophic eubacterium. Arch Biochem Biophys, 1996 Dec 1, 336(1), 77 - 85 Eubacteria-type isocitrate dehydrogenase from an archaeon: cloning, sequencing, and expression of a gene encoding isocitrate dehydrogenase from a hyperthermophilic archaebacterium, Caldococcus noboribetus; Aoshima M et al.; A gene coding for isocitrate dehydrogenase (ICDH) was cloned from a hyperthermophilic archaebacterium, Caldococcus noboribetus, and sequenced . The gene was preceded by a promoter-like sequence and was followed by a terminator-like sequence . The deduced amino acid sequence of C . noboribetus ICDH showed high similarities to eubacterial ICDH . In particular, extremely high identity scores were found for ICDHs from Vibrio sp . (48.2%) and Escherichia coli (47.9%) . The gene was expressed in E . coli by connecting it with the T7 promoter . The molecular weight of the gene product was estimated to be 48,000, which is consistent with that calculated from the deduced amino acid sequence . The gene product showed NADP-dependent ICDH activity at 80 degrees C . While the host-derived ICDH was completely inactivated by treatment at 70 degrees C for 10 min, the ICDH from C . noboribetus showed much higher thermostability. Gene, 1996 Nov 28, 181(1-2), 213 - 7 Cloning, sequencing and characterization of the gene encoding a principal sigma factor homolog from the cyanobacterium Microcystis aeruginosa K-81; Asayama M et al.; We cloned and sequenced the rpoD1 gene of Microcystis aeruginosa K-81, a unicellular colony-forming cyanobacterium that can perform photosynthesis involving light-responsive gene expression . The deduced amino acid sequence of RpoD1 exhibited extensive homology to the other eubacterial principal sigma factors . Primer extension and Western blot analyses revealed that the rpoD1 gene, which encodes a principle sigma factor homolog, had two transcription start points, P1 and P2 . These transcripts, and the corresponding protein, constitutively appeared in M . aeruginosa, irrespective of light or dark conditions. EMBO J, 1996 Nov 15, 15(22), 6311 - 20 Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit; Erickson FL et al.; Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma . In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion . Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA . We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2 . The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides . In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met) . For eIF-2{gamma-K250R}, the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro . In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains . These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo. Plant Mol Biol, 1996 Nov, 32(3), 485 - 91 Higher-plant chloroplast and cytosolic fructose-1,6-bisphosphatase isoenzymes: origins via duplication rather than prokaryote-eukaryote divergence; Martin W et al.; Full-size cDNAs encoding the precursors of chloroplast fructose-1,6-bisphosphatase (FBP), sedoheptulose-1,7-bisphosphatase (SBP), and the small subunit of Rubisco (RbcS) from spinach were cloned . These cDNAs complete the set of homologous probes for all nuclear-encoded enzymes of the Calvin cycle from spinach (Spinacia oleracea L.) . FBP enzymes not only of higher plants but also of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a eubacterial origin of these eukaryotic nuclear genes . Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution . Both FBP and SBP of higher plant chloroplasts have acquired substrate specificity, i.e . have undergone functional specialization since their divergence from bifunctional FBP/SBP enzymes of free-living eubacteria. Plant Mol Biol, 1996 Nov, 32(3), 475 - 84 Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts; Flechner A et al.; A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach . Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T7 promoter . Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts . Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome . TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes . Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants. J Appl Bacteriol, 1996 Nov, 81(5), 545 - 52 Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle; Attwood GT et al.; The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates . The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p-nitroanilide (LPNA) . The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N-succinyl alanine alanine phenylalanine proline p-nitroanilide (NSAAPPPNA) and some LPNA activity . A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio-like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA . A Prevotella-like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA . The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N-succinyl phenylalanine p-nitroanilide . The contribution of these strains to proteolysis in the rumen is discussed. J Biol Chem, 1996 Nov 1, 271(44), 27969 - 74 Structural modules of the large subunits of RNA polymerase . Introducing archaebacterial and chloroplast split sites in the beta and beta' subunits of Escherichia coli RNA polymerase; Severinov K et al.; The beta and beta' subunits of Escherichia coli DNA-dependent RNA polymerase are highly conserved throughout eubacterial and eukaryotic kingdoms . However, in some archaebacteria and chloroplasts, the corresponding sequences are "split" into smaller polypeptides that are encoded by separate genes . To test if such split sites can be accommodated into E . coli RNA polymerase, subunit fragments encoded by the segments of E . coli rpoB and rpoC genes corresponding to archaebacterial and chloroplast split subunits were individually overexpressed . The purified fragments, when mixed in vitro with complementing intact RNA polymerase subunits, yielded an active enzyme capable of catalyzing the phosphodiester bond formation . Thus, the large subunits of eubacteria and eukaryotes are composed of independent structural modules corresponding to the smaller subunits of archaebacteria and chloroplasts. Appl Environ Microbiol, 1996 Nov, 62(11), 3978 - 84 Demethylation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by marine sulfate-reducing bacteria; van der Maarel MJ et al.; The initial step in the anaerobic degradation of the algal osmolyte dimethylsulfoniopropionate (DMSP) in anoxic marine sediments involves either a cleavage to dimethylsulfide and acrylate or a demethylation to 3-S-methylmercaptopropionate . Thus far, only one anaerobic bacterial strain has been shown to carry out the demethylation, namely, Desulfobacterium sp . strain PM4 . The aims of the present work were to study how common this property is among certain groups of anaerobic bacteria and to obtain information on the affinities for DMSP of DMSP-demethylating strains . Screening of several pure cultures of sulfate-reducing and acetogenic bacteria showed that Desulfobacterium vacuolatum DSM 3385 and Desulfobacterium niacini DSM 2059 are also able to demethylate DMSP; a very slow demethylation of DMSP was observed with a salt-tolerant strain of Eubacterium limosum . From a 10(5) dilution of intertidal sediment a new marine DMSP-demethylating sulfate-reducing bacterium (strain WN) was isolated . Strain WN was a short, gram-negative, nonmotile rod that grew on betaine, sarcosine, palmitate, H2 plus CO2, and several alcohols, organic acids, and amino acids . Extracts of betaine-grown cells had hydrogenase, formate dehydrogenase, and CO dehydrogenase activities but no alpha-ketoglutarate oxidoreductase activity, indicating the presence of the acetyl coenzyme A-CO dehydrogenase pathway . Analysis of the 16S rRNA gene sequence of strain WN revealed a close relationship with Desulfobacter hydrogenophilus, Desulfobacter latus, and Desulfobacula toluolica . Strain PM4 was shown to group with Desulfobacterium niacini . The K(m) of strain WN for DMSP, as derived from substrate progress curves in cell suspensions, was approximately 10 microM . A similar value was found for D . niacini PM4. J Urol, 1996 Nov, 156(5), 1843 - 5 Absence of bacterial DNA in the bladder of patients with interstitial cystitis; Haarala M et al.; PURPOSE: Although bacterial infection has been long considered a possible cause of interstitial cystitis (IC), no definitive proof for or against this hypothesis has been presented so far . We have used 16S rDNA bacterial polymerase chain reaction to study bladder biopsies and sterile urine samples from patients suffering from IC . This method is sensitive and detects all known eubacteria . MATERIALS AND METHODS: Bladder biopsies and sterile urine samples obtained by transabdominal puncture were studied from 11 patients with IC . As controls we studied 4 patients with other urological problems leading to partly similar symptoms and 5 healthy individuals . RESULTS: All samples from the IC patients were negative . One positive sample was obtained from a woman with a history of urinary tract infections who suffered from nonIC ulcerative cystitis . Her sterile urine sample yielded Lactobacillus acidophilus . CONCLUSION: These results indicate that an ongoing bacterial infection is not the cause of interstitial cystitis. J Biol Chem, 1996 Oct 25, 271(43), 27146 - 51 Endopolyphosphatases for long chain inorganic polyphosphate in yeast and mammals; Kumble KD et al.; Whereas exopolyphosphatases have been purified from yeast and a variety of bacteria, this is the first report characterizing endopolyphosphatases that act on long chain inorganic polyphosphate (polyP) . The activity from Saccharomyces cerevisiae, localized in vacuoles, has been purified to homogeneity from a strain that possesses vacuolar proteases . The endopolyphosphatase is a dimer of 35-kDa subunits . Distributive action on polyP750 produces shorter chains to a limit of about polyP60, as well as the more abundant release of polyP3; the Km for polyP750 is 185 nM . Endopolyphosphatases have been identified in a wide variety of sources, except for most eubacteria tested . The activity has been partially purified from rat and bovine brain where its abundance is about 10 times higher than in other tissues but less than 1/10 that of yeast; the limit product of digestion of the partially purified brain enzyme is polyP3. Emerg Infect Dis, 1996 Oct-Dec, 2(4), 307 - 19 Chlamydiae as pathogens: new species and new issues; Peeling RW et al.; The recognition of genital chlamydial infection as an important public health problem was made first by the recognition of its role in acute clinical syndromes, as well as in serious reproductive and ocular complications, and secondly by our awareness of its prevalence when diagnostic tests became widely accessible . The recent availability of effective single dose oral antimicrobial therapy and sensitive molecular amplification tests that allow the use of noninvasive specimens for diagnosis and screening is expected to have a major impact in reducing the prevalence of disease in the next decade . Clinical manifestations associated with Chlamydia pneumoniae infection continue to emerge beyond respiratory illness . In particular, its association with atherosclerosis deserves further investigation . Chlamydia pecorum, a pathogen of ruminants, was recently recognized as a new species . The continued application of molecular techniques will likely elucidate an expanding role for chlamydiae in human and animal diseases, delineate the phylogenetic relationships among chlamydial species and within the eubacteria domain, and provide tools for detection and control of chlamydial infections. J Investig Med, 1996 Oct, 44(8), 462 - 9 Alteration of bile acid metabolism by cimetidine in healthy humans; Shindo K et al.; BACKGROUND: To clarify an effect of cimetidine on bile acid metabolism, we evaluated whether an increased deconjugation of bile acids would occur in healthy humans who have received cimetidine . We examined: 1) whether healthy volunteers taking cimetidine would have positive bile acid breath tests because of bacterial overgrowth in the jejunum; 2) whether the isolated bacteria would exhibit deconjugation ability; and 3) whether a change in gastric pH was related to the bacterial overgrowth . METHODS: We evaluated 73 healthy Japanese volunteers; 53 of them received cimetidine and 20 did not . Deconjugation of bile acids was detected as 14CO2 specific activity of expired air measured by a bile acid breath test giving 5 muCi of oral glycine-1-(14)C labeled glycocholate . Aspiration of jejunal fluids was performed by a double lumen tube with a rubber cover on the tip, and deconjugation ability of bacteria was evaluated using thin layer chromotography . RESULTS: Samples of expired breath from the 53 healthy volunteers showed a significant increase in 14CO2 specific activity after the administration of cimetidine rather than before the administration of cimetidine . Bacterial over-growth was found in the jejunal fluid after the administration of cimetidine . The administration of tetracycline to 27 subjects significantly reduced the 14CO2 specific activity . The following species were identified in the jejunal fluid samples obtained from the subjects: enterococcus, Lactobacillus bifidus, Bacteroides vulgatus, B uniformis, Eubacterium lentum, E parvum, and Escherichia coli . Except for E coli, all of the bacterial species identified deconjugated bile acids . We observed a significant relationship between 14CO2's specific activity and gastric pH before and after administration of cimetidine, respectively . CONCLUSIONS: Healthy volunteers who received cimetidine showed an increased deconjugation of bile acid caused by overgrowth of bacteria in the jejunum, which can deconjugate bile acids . The bacterial overgrowth is probably associated with a shift to neutral pH in the gastric juice caused by cimetidine. J Biochem (Tokyo), 1996 Oct, 120(4), 752 - 8 The rpoD1 gene product is a principal sigma factor of RNA polymerase in Microcystis aeruginosa K-81; Asayama M et al.; We performed molecular characterization of the RpoD1 protein encoded by the rpoD1 gene isolated from a cyanobacterium, Microcystis aeruginosa K-81 . The deduced amino acid sequence (416 aa, 48,871 Da) of RpoD1 exhibited extensive similarity to those of proteins of the eubacterial RpoD family (Escherichia coli sigma 70 homologs) . We overproduced and purified RpoD1 (54 kDa) from E . coli . Biological and biochemical analyses suggested that RpoD1 has a function homologous to that of E . coli sigma 70 as follows: (i) the RpoD1 protein complemented an rpoD mutant of E . coli strain YN543 (rpoD285) and (ii) the heterologous RNA polymerase holoenzyme reconstituted from the E . coli core enzyme and recombinant RpoD1 was specifically transcribed from E . coli promoters . Furthermore, Western blot analysis with antiserum against Synechococcus sp . strain PCC 7942 RpoD1 (a principal sigma factor of the sigma 70 type) indicated that M . aeruginosa K-81 RpoD1 (sigma A1) is the principal sigma factor, which is a major component of the sigma subunit on exponential cell growth. Mol Microbiol, 1996 Oct, 22(1), 175 - 91 Organizational characteristics and information content of an archaeal genome: 156 kb of sequence from Sulfolobus solfataricus P2; Sensen CW et al.; We have initiated a project to sequence the 3 Mbp genome of the thermoacidophilic archaebacterium Sulfolobus solfataricus P2 . Cosmids were selected from a provisional set of minimally overlapping clones, subcloned in pUC18, and sequenced using a hybrid (random plus directed) strategy to give two blocks of contiguous unique sequence, respectively, 100,389 and 56,105 bp . These two contigs contain a total of 163 open reading frames (ORFs) in 26-29 putative operons; 56 ORFs could be identified with reasonable certainty . Clusters of ORFs potentially encode proteins of glycogen biosynthesis, oxidative decarboxylation of pyruvate, ATP-dependent transport across membranes, isoprenoid biosynthesis, protein synthesis, and ribosomes . Putative promoters occur upstream of most ORFs . Thirty per cent of the predicted strong and medium-strength promoters can initiate transcription at the start codon or within 10 nucleotides upstream, indicating a process of initial mRNA-ribosome contact unlike that of most eubacterial genes . A novel termination motif is proposed to account for 15 additional terminations . The two contigs differ in densities of ORFs, insertion elements and repeated sequences; together they contain two copies of the previously reported insertion sequence ISC 1217, five additional IS elements representing four novel types, four classes of long non-IS repeated sequences, and numerous short, perfect repeats. Microbiology, 1996 Oct, 142 ( Pt 10), 2887 - 95 The response of selected members of the archaea to the gram stain; Beveridge TJ et al.; Archaea possess a broader range of cell envelope structural formats than eubacteria and their cell walls do not contain peptidoglycan . Some archaea have only a single S-layer as their cell wall (e.g . Methanococcus jannaschii and Sulfolobus acidocaldarius), whereas others have multiple layers (e.g . Methanospirillum hungatei) . Sometimes there can also be a high proportion of tetraether lipids in membranes to make the envelope more resilient to environmental stress (e.g . Methanococcus jannaschii and Sulfolobus acidocaldarius grown at 70 degrees C) . Since the Gram reaction depends on both the structural format and the chemical composition of the cell envelope of eubacteria, it was important to determine if the same is true for archaea . Methanospirillum hungatei, Methanosarcina mazei, Methanobacterium formicicum, Methanococcus jannaschii and Sulfolobus acidocaldarius, chosen because of their different envelope formats and chemistries, were subjected to a Gram stain that can be used for transmission electron microscopy . In this staining regimen, the iodine is replaced by potassium trichloro(eta 2-ethylene)platinate(II) as the mordant, and the platinum of the new compound is the electron-scattering agent for electron microscopy . Of all these archaea, only Methanobacterium formicicum stained Gram-positive since its pseudomurein wall remained intact; the platinum compound formed large electron-dense aggregates with the crystal violet that were located in the vicinity of the cell wall and the plasma membrane . All but the terminal filament cells of Methanospirillum hungatei stained Gram-negative because the limiting porosity of its external sheath was so small that the Gram reagents could not enter the cells . The terminal cells of filaments stained Gram-positive because the staining reagents gained entry through the terminal plugs . All other archaea stained Gram-negative because their cell walls were so disrupted during staining that the crystal violet-platinum complex could not be retained by the cells . Methanococcus jannaschii was grown at both 50 degrees C and 70 degrees C so that the tetraether lipids in its plasma membrane could be increased from 20% (50 degrees C) to 45% (70 degrees C) of the total lipids; in both cases the cells stained Gram-negative. Int J Syst Bacteriol, 1996 Oct, 46(4), 1120 - 4 Eubacterium exiguum sp . nov., isolated from human oral lesions; Poco SE Jr et al.; Eubacterium exiguum sp . nov . is the name proposed for organisms formerly described as Eubacterium group S strains and similar bacteria isolated from various types of oral lesions . This new species was established on the basis of the results of DNA-DNA hybridization experiments and DNA base composition determinations (G + C contents, 60 to 64 mol%) . The results of an API ZYM analysis, Western blotting (immunoblotting) reactions, and phenotypic tests are also given . The type strain of E . exiguum is strain S-7. Int J Syst Bacteriol, 1996 Oct, 46(4), 957 - 9 Phylogeny of oral asaccharolytic Eubacterium species determined by 16S ribosomal DNA sequence comparison and proposal of Eubacterium infirmum sp . nov . and Eubacterium tardum sp . nov; Cheeseman SL et al.; 16S rRNA gene sequences of Eubacterium brachy, Eubacterium nodatum, Eubacterium saphenum, Eubacterium timidum, and two previously unnamed taxa were determined . The results of a phylogenetic analysis indicated that all of the strains sequenced belonged to a deep branch of the low-G+C-content gram-positive group . The levels of 16S ribosomal DNA sequence similarity between species were low, suggesting that a number of genera may be represented in this group . The representatives of the two unnamed taxa, which were isolated from patients with periodontitis, were clearly distinct from the previously described species, and, therefore, the following two new species are proposed: Eubacterium infirmum (type strain, NCTC 12940) and Eubacterium tardum (type strain, NCTC 12941). Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10653 - 6 Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases; Dang QD et al.; Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes . Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates . In these proteases, the catalytic activity is enhanced allosterically by Na+ binding . Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria . These proteases have no requirement for Na+ or other monovalent cations . The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin . The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone . Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases . To directly test this hypothesis, we have engineered the thrombin mutant Y225P . This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form . The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases . A strong correlation exists between residue 225 and the codon used for the active site S195 . Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon . It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor . We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement. J Bacteriol, 1996 Oct, 178(19), 5712 - 8 Molecular cloning and expression of the spsB gene encoding an essential type I signal peptidase from Staphylococcus aureus; Cregg KM et al.; The gene, spsB, encoding a type I signal peptidase has been cloned from the gram-positive eubacterium Staphylococcus aureus . The gene encodes a protein of 191 amino acid residues with a calculated molecular mass of 21,692 Da . Comparison of the protein sequence with those of known type I signal peptidases indicates conservation of amino acid residues known to be important or essential for catalytic activity . The enzyme has been expressed to high levels in Escherichia coli and has been demonstrated to possess enzymatic activity against E . coli preproteins in vivo . Experiments whereby the spsB gene was transferred to a plasmid that is temperature sensitive for replication indicate that spsB is an essential gene . We identified an open reading frame immediately upstream of the spsB gene which encodes a type I signal peptidase homolog of 174 amino acid residues with a calculated molecular mass of 20,146 Da that is predicted to be devoid of catalytic activity. Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9651 - 6 A common evolutionary origin for mitochondria and hydrogenosomes; Bui ET et al.; Trichomonads are among the earliest eukaryotes to diverge from the main line of eukaryotic descent . Keeping with their ancient nature, these facultative anaerobic protists lack two "hallmark" organelles found in most eukaryotes: mitochondria and peroxisomes . Trichomonads do, however, contain an unusual organelle involved in carbohydrate metabolism called the hydrogenosome . Like mitochondria, hydrogenosomes are double-membrane bounded organelles that produce ATP using pyruvate as the primary substrate . Hydrogenosomes are, however, markedly different from mitochondria as they lack DNA, cytochromes and the citric acid cycle . Instead, they contain enzymes typically found in anaerobic bacteria and are capable of producing molecular hydrogen . We show here that hydrogenosomes contain heat shock proteins, Hsp70, Hsp60, and Hsp10, with signature sequences that are conserved only in mitochondrial and alpha-Gram-negative purple bacterial Hsps . Biochemical analysis of hydrogenosomal Hsp60 shows that the mature protein isolated from the organelle lacks a short, N-terminal sequence, similar to that observed for most nuclear-encoded mitochondrial matrix proteins . Moreover, phylogenetic analyses of hydrogenosomal Hsp70, Hsp60, and Hsp10 show that these proteins branch within a monophyletic group composed exclusively of mitochondrial homologues . These data establish that mitochondria and hydrogenosomes have a common eubacterial ancestor and imply that the earliest-branching eukaryotes contained the endosymbiont that gave rise to mitochondria in higher eukaryotes. Microbiologia, 1996 Sep, 12(3), 405 - 10 Buffering capacity and membrane H+ conductance of Halobacterium halobium; Rius N et al.; Buffering capacity and membrane H+ conductance were measured in Halobacterium halobium suspensions in the light and in the dark over a wide range of external pH . The values of both variables for this archaeobacterium were significantly higher than those found for eubacteria in other reports . It appears from our results that the special chemical composition of the cell envelope and the movement of ions, mainly protons, may influence the magnitude of the buffering power and the H+ membrane conductance of these cells. Appl Environ Microbiol, 1996 Sep, 62(9), 3405 - 12 Bacterial diversity in a deep-subsurface clay environment; Boivin-Jahns V et al.; The presence of bacteria in a deep clay sediment was analyzed in a 20-m-long core horizontally drilled from a mine gallery at a depth of 224 m in the Boom clay formation (Mol, Belgium) . This clay deposit is the result of a marine sedimentary process that occurred 35 million years ago . Bacterial activities were estimated by measuring respiration on {14C}glucose . Using the same samples, universal primers for the genes coding for eubacterial 16S rRNA were used to amplify extracted DNA . PCR products were then cloned, sequenced, and analyzed by molecular phylogeny . Our data showed a decrease in bacterial densities as a function of distance from the gallery, with few bacteria detectable by culture at more than 80 cm from the gallery wall . PCR experiments showed the presence of bacteria in all samples, and phylogenetic analyses were then used to tentatively identify these organisms . Because of low bacterial densities in deep clay samples, direct counts and enumeration of viable bacteria on diverse culture media remained negative . All experiments, both cultures and PCR, demonstrated the difficulty of analyzing samples that contain only a few poorly active bacteria as it is difficult to avoid a small contamination by active bacteria during sampling . Since the porosity of the Boom clay formation is less than the expected size of bacteria, it is possible that some of the bacteria present in this 35-million-year-old deep clay deposit derive from cells initially trapped during the sedimentation process. Appl Environ Microbiol, 1996 Sep, 62(9), 3112 - 20 Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism; Lee DH et al.; We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem . Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations . A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains . The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains . The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured . In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community . The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study . The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems. Fertil Steril, 1996 Sep, 66(3), 463 - 7 Polymerase chain reaction-based detection of bacteria in semen; Jarvi K et al.; OBJECTIVE: To determine if the presently used bacterial detection techniques provide accurate and complete profiles of microorganisms found in human semen . DESIGN: Routine bacterial cultures and molecular biology techniques using polymerase chain reaction (PCR), with a universal eubacterial primer, cloning, then sequence analysis were used to detect bacteria (culturable or nonculturable) in the semen . SETTING: University and hospital-based research laboratory . PATIENTS: Thirty infertile men and nine semen donors, all with no symptoms of a urinary tract infection, donated semen for the study . INTERVENTIONS: None . MAIN OUTCOME MEASURES: Detection of bacteria using routine cultures and molecular biology techniques . RESULTS: Using PCR, we found > 10(4) bacteria/mL in the semen of 66% of the infertile asymptomatic men and 66% of the semen donors . This contrasts with our routine culture results which detected "significant" bacteriospermia in only 27% of the infertile men and in none of the preselected semen donors . From four of these semen specimens, DNA sequence analysis identified an average of nine different bacterial species per specimen, with close to 90% of the species being anaerobes . CONCLUSIONS: These data indicate that the present microbiologic detection methods underestimate the incidence of significant bacteriospermia, particularly anaerobic bacteria . The molecular biologic methods should help researchers confirm or refute the role of infection in male infertility. J Mol Biol, 1996 Aug 16, 261(2), 155 - 72 Complete gene map of the plastid-like DNA of the malaria parasite Plasmodium falciparum; Wilson RJ et al.; Malaria parasites, and other parasitic protists of the Phylum Apicomplexa, carry a plastid