Eubacterium

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-like genome with greatly reduced sequence complexity . This 35 kb DNA circle resembles the plastid DNA of non-photosynthetic plants, encoding almost exclusively components involved in gene expression . The complete gene map described here includes genes for duplicated large and small subunit rRNAs, 25 species of tRNA, three subunits of a eubacterial RNA polymerase, 17 ribosomal proteins, and a translation elongation factor . In addition, it codes for an unusual member of the Clp family of chaperones, as well as an open reading frame of unknown function found in red algal plastids . Transcription is polycistronic . This plastid-like DNA molecule is conserved in several genera of apicomplexans and is conjectured to have been acquired by an early progenitor of the Phylum by secondary endosymbiosis . The function of the organelle (plastid) carrying this DNA remains obscure, but appears to be specified by genes transferred to the nucleus.

Biochemistry, 1996 Aug 6, 35(31), 9995 - 10003
A eubacterial Mycobacterium tuberculosis tRNA synthetase is eukaryote-like and resistant to a eubacterial-specific antisynthetase drug; Sassanfar M et al.; We report here the cloning and primary structure of Mycobacterium tuberculosis isoleucyl-tRNA synthetase . The predicted 1035-amino acid protein is significantly more similar in sequence to eukaryote cytoplasmic than to other eubacterial isoleucyl-tRNA synthetases . This similarity correlates with the enzyme being resistant to pseudomonic acid A, a potent inhibitor of Escherichia coli and other eubacterial isoleucyl-tRNA synthetases, but not of eukaryote cytoplasmic enzymes . Consistent with its eukaryote-like features, and unlike E . coli isoleucyl-tRNA synthetase, the M . tuberculosis enzyme charged yeast isoleucine tRNA . In spite of these eukaryote-like features, M . tuberculosis isoleucyl-tRNA synthetase exhibited highly specific cross-species aminoacylation, as demonstrated by its ability to complement isoleucyl-tRNA synthetase-deficient mutants of E . coli . When introduced into a pseudomonic acid-sensitive wild-type strain of E . coli, the M . tuberculosis enzyme conferred trans-dominant resistance to the drug . The results demonstrate that the sequence of a tRNA synthetase could have predictive value with respect to the interaction of that synthetase with a specific inhibitor . The results also demonstrate that mobilization of a pathogen's gene for a drug-resistant protein target can spread resistance to other, normally drug-sensitive pathogens infecting the same host.

Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8230 - 5
The primary structures of the Archaeon Halobacterium salinarium blue light receptor sensory rhodopsin II and its transducer, a methyl-accepting protein; Zhang W et al.; Recently, a large family of transducer proteins in the Archaeon Halobacterium salinarium was identified . On the basis of the comparison of the predicted structural domains of these transducers, three distinct subfamilies of transducers were proposed . Here we report isolation, complete gene sequences, and analysis of the encoded primary structures of transducer gene htrII, a member of family B, and its blue light receptor gene (sopII) of sensory rhodopsin II (SRII) . The start codon ATG of the 714-bp sopII gene is one nucleotide beyond the termination codon TGA of the 2298-bp htrII gene . The deduced protein sequence of HtrII predicts a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm . HtrII has a common feature with HtrI, the sensory rhodopsin I transducer; like HtrI, HtrII possesses a hydrophilic loop structure just after the second transmembrane segment . The C-terminal 299 residues (765 amino acid residues total) of HtrII show strong homology to the signaling and methylation domain of eubacterial transducer Tsr . The hydropathy plot of the primary structure of SRII indicates seven membrane-spanning alpha-helical segments, a characteristic feature of retinylidene proteins ("rhodopsins") from a widespread family of photoactive pigments . SRII shows high identity with SRI (42%), bacteriorhodopsin (BR) (32%), and halorhodopsin (24%) . The crucial positions for retinal binding sites in these proteins are nearly identical, with the exception of Met-118 (numbering according to the mature BR sequence), which is replaced by Val in SRII . In BR, residues Asp-85 and Asp-96 are crucial in proton pumping . In SRII, the position corresponding to Asp-85 in BR is conserved, but the corresponding position of Asp-96 is replaced by an aromatic Tyr . Coexpression of the htrII and sopII genes restores SRII phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI and insertion in htrII/sopII regions . This paper describes the first example that both HtrI and HtrII exist in the same halobacterial cell, confirming that different sensory rhodopsins SRI and SRII in the same organism have their own distinct transducers.

Int J Pept Protein Res, 1996 Aug, 48(2), 167 - 73
Structure-activity studies on C-terminal hirudin peptides containing sulfated tyrosine residues; Muramatsu R et al.; To clarify the role of the negative charge of the C-terminal region of hirudin, we chemically synthesized the C-terminal peptide of hirudin variant-1 (HV-1), HV-1-(54-65), and its analogs, {E61Y,E62Y}HV-1-(54-65) and {E62Y}HV-1-(54-65), and then sulfated the Tyr residue(s) in these peptides by both enzymic and chemical methods . Enzymic O-sulfation of Tyr residues in the peptides by use of sulfotransferase isolated from Eubacterium A-44 allowed us to produce four kinds of the sulfated peptide, whose C-terminal sequences were -PEY(SO3H)YLQ, -PYY(SO3H)YLQ, -PYYY(SO3H)LQ and -PYY(SO3H)Y(SO3H)LQ . On the other hand, all Tyr residues in the peptides were successfully sulfated by chemical reaction with N,N'-dicyclohexylcarbodiimide in the presence of sulfuric acid . Based on the analysis of structure-activity relationships of these sulfated peptides for thrombin inhibition, the Tyr62 and Tyr63 bisulfated peptide GDFEEIPEY(SO3H)Y(SO3H)LQ was found to be the most potent inhibitor of thrombin among the products tested . No increase in potency was observed by further substitution of Glu61 with Tyr(SO3H) . The inhibitory activity by substitution with Tyr(SO3H) at position 63 was greater than that obtained by the substitution at position 62.

J Clin Microbiol, 1996 Aug, 34(8), 1922 - 5
Phylogenetic analysis of pathogen-related oral spirochetes; Choi BK et al.; Recently, Riviere et al . reported as yet uncultivable invasive oral spirochetes that cross-reacted with monoclonal antibodies (MAbs) specific for Treponema pallidum (G . R . Riviere, K . S . Elliot, D . F . Adams, L . G . Simonson, L . B . Forgas, A . M . Nilius, and S . A . Lukehart, J . Periodontol . 63:131-136, 1992; G . R . Riviere, M . A . Wagoner, S . A . Baker-Zander, K . S . Weisz, D . F . Adams, L . Simonson, and S . A . Lukehart, N . Engl . J . Med . 325:539-543, 1991; G . R . Riviere, K . S . Weisz, D . F . Adams, and D . D . Thomas, Infect . Immun . 59:3377-3380, 1991; G . R . Riviere, K . S . Weisz, L . G . Simonson, and S . A . Lukehart, Infect . Immun . 59:2653-2657, 1991) . In an attempt to phylogenetically analyze these pathogen-related oral spirochetes, we used immunomagnetic separation, combined with comparative sequence analysis of 16S rRNA genes amplified in vitro by the PCR . The bacteria were immunomagnetically enriched from subgingival plaque samples of patients with rapidly progressive periodontitis by using MAb H9-2 specific for the 37-kDa endoflagellum sheath protein of T . pallidum . After PCR amplification with universal eubacterial primers 16S rRNA gene fragments were cloned into Escherichia coli . A total of 20 randomly selected recombinants were analyzed by sequencing about 200 to 300 bases of the 500-bp inserts . All the spirochetal 16S rRNA sequences clustered to previously described, as yet uncultivable cluster 7 treponemes of group I (B . K . Choi, B . J . Paster, F . E . Dewhirst, and U . B . Gobel, Infect . Immun . 62:1889-1895, 1994) . With a sequence similarity of 96.4% the most closely related cultivable treponeme was Treponema vincentii, which also belongs to the group I treponemes . Subsequent immunological analysis of cultured treponemes with MAb H9-2 revealed that only T . vincentii strains showed specific immunofluorescence or a characteristic 37-kDa band in immunoblots . We therefore conclude that pathogen-related oral spirochetes constitute a heterogeneous population of treponemes comprising T . vincentii and T . vincentii-related organisms that have common epitopes cross-reacting with MAb H9-2.

Appl Environ Microbiol, 1996 Aug, 62(8), 3017 - 22
Development of techniques to genetically manipulate members of the genera Cytophaga, Flavobacterium, Flexibacter, and Sporocytophaga; McBride MJ et al.; The Bacteroides-Cytophaga-Flavobacterium branch of the eubacterial phylogenetic tree contains a diverse group of bacterial species . Techniques for the genetic manipulation of Bacteroides spp . are well developed (A . A . Salyers, N . B . Shoemaker, and E . P . Guthrie, Crit . Rev . Microbiol . 14:49-71, 1987) . Recently we developed techniques to genetically manipulate the gliding bacterium Cytophaga johnsonae (M . J . McBride and M . J . Kempf, J . Bacteriol . 178:583-590, 1996) . We now demonstrate that some of these techniques allow genetic manipulation of a number of environmentally or medically significant bacteria in this group . The Bacteroides transposon Tn4351 was introduced into Cytophaga hutchinsonii, Cytophaga succinicans, Flavobacterium meningosepticum, Flexibacter canadensis, Flexibacter sp . strain FS1, and Sporocytophaga myxococcoides by conjugation . Tn4351 integrated itself into the host chromosomes and conferred erythromycin resistance . We isolated several auxotrophic mutants of Flavobacterium meningosepticum following Tn4351 mutagenesis . The C . johnsonae-Escherichia coli shuttle vector pCP11 functioned in C . succinicans but not in the other bacteria . pLYL03 did not replicate in any of these bacteria and should function as a convenient suicide vector . The identification of a system of gene transfer, a selectable marker, a suicide vector, and a transposon that functions in these diverse bacteria allows genetic manipulations to be performed.

J Biol Chem, 1996 Jul 26, 271(30), 17692 - 6
Archaebacterial DNA polymerases tightly bind uracil-containing DNA; Lasken RS et al.; We show that archaebacterial DNA polymerases are strongly inhibited by the presence of small amounts of uracil-containing DNA . Inhibition appears to be competitive, with the DNA polymerase exhibiting approximately 6500-fold greater affinity for binding the inhibitor than a DNase I-activated DNA substrate . All six archaebacterial DNA polymerases tested were inhibited, while no eubacterial, eukaryotic, or bacteriophage enzymes showed this effect . Only a small inhibition resulted when uracil was present as the deoxynucleoside triphosphate, dUTP . The rate of DNA synthesis was reduced by approximately 40% when dUTP was used in place of dTTP for archaebacterial DNA polymerases . Furthermore, an incorporated dUMP served as a productive 3'-primer terminus for subsequent elongation . In contrast, the presence of an oligonucleotide containing as little as a single dUrd residue was extremely inhibitory to DNA polymerase activity on other primer-template DNA.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7749 - 54
The root of the universal tree and the origin of eukaryotes based on elongation factor phylogeny; Baldauf SL et al.; The genes for the protein synthesis elongation factors Tu (EF-Tu) and G (EF-G) are the products of an ancient gene duplication, which appears to predate the divergence of all extant organismal lineages . Thus, it should be possible to root a universal phylogeny based on either protein using the second protein as an outgroup . This approach was originally taken independently with two separate gene duplication pairs, (i) the regulatory and catalytic subunits of the proton ATPases and (ii) the protein synthesis elongation factors EF-Tu and EF-G . Questions about the orthology of the ATPase genes have obscured the former results, and the elongation factor data have been criticized for inadequate taxonomic representation and alignment errors . We have expanded the latter analysis using a broad representation of taxa from all three domains of life . All phylogenetic methods used strongly place the root of the universal tree between two highly distinct groups, the archaeons/eukaryotes and the eubacteria . We also find that a combined data set of EF-Tu and EF-G sequences favors placement of the eukaryotes within the Archaea, as the sister group to the Crenarchaeota . This relationship is supported by bootstrap values of 60-89% with various distance and maximum likelihood methods, while unweighted parsimony gives 58% support for archaeal monophyly.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2648 - 51
Asn-tRNA in Lactobacillus bulgaricus is formed by asparaginylation of tRNA and not by transamidation of Asp-tRNA; Kim SI et al.; In many organisms (e.g., gram-positive eubacteria) Gin-tRNA is not formed by direct glutaminylation of tRNAGln but by a specific transamidation of Glu-tRNAGln . We wondered whether a similar transamidation pathway also operates in the formation of Asn-tRNA in these organisms . Therefore we tested in S-100 preparations of Lactobacillus bulgaricus, a gram-positive eubacterium, for the conversion by an amidotransferase of {14C}Asp-tRNA to {14C}Asn-tRNA . As no transamidation was observed, we searched for genes for asparaginyl-tRNA synthetase (AsnRS) . Two DNA fragments (from different locations of the L.bulgaricus chromosome) were found each containing an ORF whose sequence resembled that of the Escherichia coli asnS gene . The derived amino acid sequences of the two ORFs (432 amino acids) were the same and 41% identical with E.coli AsnRS . When one of the ORFs was expressed in E.coli, it complemented the temperature sensitivity of an E.coli asnS mutant . S-100 preparations of this transformant showed increased charging of unfractionated L.bulgaricus tRNA with asparagine . Deletion of the 3'-terminal region of the L.bulgaricus AsnRS gene led to loss of its complementation and aminoacylation properties . This indicates that L.bulgaricus contains a functional AsnRS . Thus, the transamidation pathway operates only for Gin-tRNAGln formation in this organism, and possibly in all gram-positive eubacteria.

Nature, 1996 Jul 4, 382(6586), 90 - 3
A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease; Ceska TA et al.; THE 5'-exonucleases are enzymes that are essential for DNA replication and repair . As well as their exonucleolytic action, removing nucleotides from the 5'-end of nucleic acid molecules such as Okazaki fragments, many 5'-3'-exonucleases have been shown to possess endonucleolytic activities . T5 5'-3'-exonuclease shares many similarities with the amino terminal of eubacterial DNA polymerases, although, unlike eubacteria, phages such as T5, T4 and T7 express polymerase and 5'-exonuclease proteins from separate genes . Here we report the 2.5-A crystal structure of the phage T5 5'-exonuclease, which reveals a helical arch for binding DNA . We propose a model consistent with a threading mechanism in which single-stranded DNA could slide through the arch, which is formed by two helices, one containing positively charged, and the other hydrophobic, residues . The active site is at the base of the arch, and contains two metal-binding sites.

Mol Microbiol, 1996 Jul, 21(2), 313 - 9
FtsZ ring: the eubacterial division apparatus conserved in archaebacteria; Wang X et al.; FtsZ is a tubulin-like protein that is essential for cell division in eubacteria . It functions by forming a ring at the division site that directs septation . The archaebacteria constitute a kingdom of life separate from eubacteria and eukaryotes . Like eubacteria, archaebacteria are prokaryotes, although they are phylogenetically closer to eukaryotes . Here it is shown that archaebacteria also possess FtsZ and that it is biochemically similar to eubacterial FtsZs . Significantly, FtsZ from the archaebacterium Haloferax volcanii is a GTPase that is localized to a ring that coincides with the division constriction . These results indicate that the FtsZ ring was part of the division apparatus of a common prokaryotic ancestor that was retained by both eubacteria and archaebacteria.

Biochem Mol Biol Int, 1996 Jul, 39(4), 697 - 702
A phylogenetic study of Rubrobacter radiotolerans by sequence analysis of the 16S ribosomal RNA gene; Kausar J et al.; Partial sequence of the 16S ribosomal RNA gene of an extremely high radiotolerant bacterium, Rubrobacter radiotolerans (reclassified from Arthrobacter radiotolerans by chemical characteristics) was determined by PCR-amplification from a small amount of heat-lysed biomass, followed by direct sequencing of the PCR product . The sequence was aligned with seven species of Arthrobacter and also with representatives of various other bacterial groups . R . radiotolerans was confirmed to be out of the Arthrobacter group justifying the reclassification . Moreover, it has an individual position among the selected representatives being closer to the eubacterial groups.

Antonie Van Leeuwenhoek, 1996 Jul, 70(1), 21 - 9
The dcr gene family of Desulfovibrio: implications from the sequence of dcrH and phylogenetic comparison with other mcp genes; Deckers HM et al.; Desulfovibrio vulgaris Hildenborough contains a family of genes for methyl-accepting chemotaxis proteins (MCPs) . Here we report the complete sequence of the gene for Desulfovibrio chemoreceptor H (dcrH) . The deduced amino acid sequence of DcrH protein, which has an enlarged N-terminal, ligand binding domain, indicates a structure similar to that of other MCPs . Comparison of the sequences for DcrA, determined earlier, and DcrH indicated that similarity is essentially limited to the C-terminal excitation region . The dcr gene family differs, in this respect, from mcp gene families in other eubacteria (e.g . Escherichia coli and Bacillus subtilis), where MCPs share significant homology throughout their C-terminal signal transduction domains . This may point to an ancient evolutionary origin of the dcr gene family, which is widely distributed throughout the genus Desulfovibrio . The evolutionary origin of mcp genes was traced by comparing nucleotide sequences for the excitation region that is common to all MCPs . Phylogenetic analysis of sequences for thirty mcp genes from nine eubacterial and one archaebacterial species suggested that multiplication of mcp genes has occurred at least twice since the eubacteria diverged from the archaebacteria.

Int J Syst Bacteriol, 1996 Jul, 46(3), 669 - 74
Comparative analysis of 16S and 23S rRNA sequences of Listeria species; Sallen B et al.; In order to establish the taxonomic value of 16S rRNA and 23S rRNA for distinguishing Listeria species, the complete 23S rRNA sequences for all Listeria species were determined by using the type strains . We designed and experimentally validated a universal 23S rRNA sequencing method, which included PCR amplification of the rDNA gene and direct cycle sequencing of the amplicon with eubacterial primers . The results of our sequence comparison indicated that the genus Listeria can be divided into two subgroups; one subgroup is composed of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri, whereas the other subgroup includes Listeria grayi subsp . grayi and Listeria grayi subsp . murrayi . A phylogenetic analysis revealed that these species diverged recently . These results are consistent with 16S rRNA sequence analysis data . For application purposes, one 16S rRNA region that can be sued to distinguish each Listeria species except L . Monocytogenes and L . innocua has been described . In this study we found four 23S rRNA signature regions which, when used in combination, can be used to distinguish the species.

J Bacteriol, 1996 Jul, 178(14), 4089 - 98
A NusG-like protein from Thermotoga maritima binds to DNA and RNA; Liao D et al.; The NusG-like protein from Thermotoga maritima was expressed in Escherichia coli and purified to homogeneity . Purified T . maritima NusG exhibited a generalized, non-sequence-specific and highly cooperative DNA and RNA binding activity . The complexes formed between nucleic acid and T . maritima NusG were unable to penetrate a polyacrylamide or agarose gel . The affinity of the protein for DNA was highest in buffers containing about 50 mM salt . The DNA-protein complexes could not be stained with ethidium bromide, were resistant to digestion by TaqI endonuclease, were able to be transcribed in vitro by T . maritima RNA polymerase, and contained a minimum of about 30 to 40 monomers of NusG per kb of duplex DNA . The protein had comparable affinities for duplex DNA and RNA but a lower affinity for single-stranded DNA . Electron microscopy showed that the DNA in the complex is condensed within a large structure that resembles the complex between DNA and histone-like protein Hcl from Chlamydia trachomatis . Neither the wild-type T . maritima nusG gene nor a deletion derivative more similar to the E . coli gene was able to substitute for the essential E . coli nusG . Two variants of the NusG protein were constructed, expressed, and purified: one contains only the entire 171-amino-acid insertion that is unique to T . maritima NusG, and the other has only the sequences present in NusG homologs from E . coli and other eubacteria . Both variants exhibited similar DNA and RNA binding behavior, although their apparent affinities were 5- to 10-fold lower than that of the wild-type T . maritima NusG.

Mol Biol Evol, 1996 Jul, 13(6), 873 - 82
Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids; Delwiche CF et al.; Previous work has shown that molecular phylogenies of plastids, cyanobacteria, and proteobacteria based on the rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) genes rbcL and rbcS are incongruent with molecular phylogenies based on other genes and are also incompatible with structural and biochemical information . Although it has been much speculated that this is the consequence of a single horizontal gene transfer (of a proteobacterial or mitochondrial rubisco operon into plastids of rhodophytic and chromophytic algae), neither this hypothesis nor the alternative hypothesis of ancient gene duplication have been examined in detail . We have conducted phylogenetic analyses of all available bacterial rbcL sequences, and representative plastid sequences, in order to explore these alternative hypothesis and fully examine the complexity of rubisco gene evolution . The rbcL phylogeny reveals a surprising number of gene relationships that are fundamentally incongruent with organismal relationships as inferred from multiple lines of other molecular evidence . On the order of six horizontal gene transfers are implied by the form I (L8S8) rbcL phylogeny, two between cyanobacteria and proteobacteria, one between proteobacteria and plastids, and three within proteobacteria . Alternatively, a single ancient duplication of the form I rubisco operon, followed by repeated and pervasive differential loss of one operon or the other, would account for much of this incongruity . In all probability, the rubisco operon has undergone multiple events of both horizontal gene transfer and gene duplication in different lineages.

Arch Microbiol, 1996 Jul, 166(1), 58 - 63
Identification of two classes of transcriptional regulator genes in the cyanobacterium Synechococcus sp . strain PCC 7942; Anandan S et al.; We designed a strategy to isolate and characterize response regulator genes from the cyanobacterium Synechococcus sp . strain PCC 7942 based on the premise that cyanobacterial response regulators would bear strong similarity to their counterparts from other eubacteria . Two response regulator genes, srrA and srrB, were isolated from Synechococcus and found to encode proteins similar to the OmpR subclass of response regulators . Disruption of either gene by insertional mutagenesis did not produce an obvious phenotype and did not affect the accumulation of psbAII mRNA under high-light conditions, indicating that these gene products are not involved in mediating the well characterized standard- to high-light transition response of photosystem II genes in this cyanobacterium . Analysis of the chromosomal region adjacent to srrA revealed the presence of another presumptive transcriptional activator gene . This gene, named lrrA, belongs to the lysR family . Attempts to disrupt lrrA or an adjacent ORF (orfG) were not successful, suggesting that these genes are important for the growth of Synechococcus.

Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6726 - 30
An archaebacterial homologue of the essential eubacterial cell division protein FtsZ; Baumann P et al.; Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes, . respectively) . Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria . Currently, little is known about the archaeal cell division apparatus . In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation . Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton . By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP . Furthermore, through screening an expression library of P . woesei genomic DNA, we have cloned the gene encoding this protein . Sequence comparisons reveal that the P . woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins . Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins . These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.

Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6431 - 6
Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin; Eisenreich W et al.; A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield . The incorporation of {U-13C6}glucose, {1-13C}glucose, and {1,2-13C2}acetate into this diterpene was analyzed by NMR spectroscopy . Label from {1,2-13C2}acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system . Label from {1-13C}glucose and {U-13C6}glucose was efficiently incorporated into both the taxane ring system and the acetyl groups . The four isoprenoid moieties of the diterpene showed identical labeling patterns . The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with {U-13C6}glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor . The labeling patterns are inconsistent with the mevalonate pathway . The taxoid data share important features with the alternative pathway of isoprenoid biosynthesis operating in certain eubacteria Rohmer, M., Knani, M., Simonin, P., Sutter, B . & Sahm, H . (1993) Biochem . J . 295, 517-524}.

Biochemistry, 1996 Jun 18, 35(24), 7781 - 6
Identification of S-(2,3-dihydroxypropyl)cystein in a macrophage-activating lipopeptide from Mycoplasma fermentans; Muhlradt PF et al.; Mycoplasmas are capable of stimulating monocytes and macrophages to release cytokines, prostaglandins, and nitric oxide . The aim of this study was to characterize the chemical nature of the previously isolated {Muhlradt, P . F., & Frisch, M . (1994) Infect . Immun . 62, 3801-3807} macrophage-stimulating material "MDHM" from Mycoplasma fermentans . Mycoplasmas were delipidated, and MDHM activity was extracted with octyl glucoside and further purified by reversed-phase HPLC . Macrophage-stimulating activity was monitored by nitric oxide release from peritoneal macrophages from C3H/HeJ endotoxin low responder mice . HPLC-purified MDHM was rechromatographed on an analytic scale RP 18 column before and after proteinase K treatment . Proteinase treatment did not diminish biological activity but shifted MDHM elution toward higher lipophilicity, suggesting that the macrophage-stimulating activity might reside in the lipopeptide moiety of a lipoprotein . Proteinase K-treated MDHM was hydrolyzed, amino groups were dansylated, and the dansylated material was isolated by HPLC . Dansylated S-(2,3-dihydroxypropyl)cystein (glycerylcystein thioether), typical for Braun's murein lipoprotein, and Dns-Gly and Dns-Thr were identified by tandem mass spectrometry . These amino acids were isolated from biologically active but not from the neighboring inactive HPLC fractions . IR spectra from proteinase K-treated, HPLC-purified MDHM and those from the synthetic lipopeptide {2,3-bis(palmitoyloxy)-(2-RS)-propyl}-N-palmitoyl-(R)-CysSerSer AsnAla were very similar . The data, taken together, indicate that lipoproteins of a nature previously detected in eubacteria are expressed in M . fermentans and that at least one of these lipoproteins and a lipopeptide derived from it constitute the macrophage-activating principle MDHM from these mycoplasmas.

Gene, 1996 Jun 12, 172(1), 105 - 9
An unusual gene arrangement for the putative chromosome replication origin and circadian expression of dnaN in Synechococcus sp . strain PCC 7942; Liu Y et al.; In eubacteria, the clustering of DnaA boxes around the dnaN (beta subunit of DNA polymerase III) and dnaA genes usually defines the chromosome replication origin (oriC) . In this study, the dnaN locus from the cyanobacterium Synechococcus sp . strain PCC 7942 was sequenced . The gene order in this region is cbbZp-dnaN-orf288-purL-purF which contrasts with other eubacteria . A cluster of eleven DnaA boxes (consensus sequence: TTTTCCACA) was found in the intergenic region between dnaN and cbbZp . We also found a 41-bp sequence within this region that is 80% identical to the proposed oriC of Streptomyces coelicolor . Therefore, we propose that this intergenic region may serve as an oriC in Synechococcus . Using bacterial luciferase as a reporter, we also showed that dnaN is rhythmically expressed, suggesting that DNA replication could be under circadian control in this organism.

Clin Oral Implants Res, 1996 Jun, 7(2), 90 - 5
Bacterial colonization on internal surfaces of Brånemark system implant components; Persson LG et al.; The aim of the present study was to examine the microbiota on the internal surface of the components of 28 Branemark implants in 10 partially edentulous patients who had been treated with 1 fixed partial prostheses each . The prostheses had been in function for 1 to 8 years . The fixed prostheses were checked for mobility and removed . The abutment screws were loosened and classified as stable, easily removed or loose . Then, bacterial samples were obtained from the various internal surfaces of the implant system . Estimation and identification of the most predominant species was performed on the blood agar plates . Identification was based on Gram reaction, oxygen sensitivity and biochemical tests . Internal surfaces of different components of the Branemark implants, after varying periods of function in the oral cavity, consistently harboured a heterogeneous and primarily anaerobic microbiota . The individual samples showed a great variation . No relation could be seen between type and length of abutment, abutment stability, bone loss and type and number of microorganisms found in the samples . The flora consisted mainly of facultative and anaerobic streptococci, Gram-positive anaerobic rods such as Propionibacterium, Eubacterium and Actinomyces species and Gram-negative anaerobic rods including Fusobacterium, Prevotella and Porphyromonas species . There are reasons to suggest that this presence of bacteria is the result of (i) a contamination of the fixture and abutment components during the 1st and/or 2nd stage of implant installation and/or (ii) a transmission of microorganisms from the oral environment during function subsequent to bridge installation.

Oral Microbiol Immunol, 1996 Jun, 11(3), 199 - 202
The establishment of a community of oral bacteria that controls the growth of Candida albicans in a chemostat; Basson NJ et al.; The purpose of this study was to establish and identify a community of oral bacteria that controls the growth of Candida albicans in the chemostat . The chemostat was operated under glucose-limiting conditions at a dilution rate of 0.05 h-1 and inoculated with a yeast-free suspension of a tongue scraping . After a steady state had been reached, it was inoculated with C . albicans to establish the yeast and determine whether its growth could be contained . The steady-state community comprised the species Streptococcus sanguis, Streptococcus sobrinus, Streptococcus mitis, Lactobacillus casei, Eubacterium saburreum, Veillonella dispar and Fusobacterium nucleatum . Bacteroides gracilus and Haemophilus segnis were also detected but infrequently . Yeast growth was suppressed and yeast cells were lost at the same rate as the theoretical washout rate . It is concluded that this mixed community of oral bacteria can be used to identify the parameters that maintain the equilibrium between oral bacteria and C . albicans in the oral cavity.

J Biochem (Tokyo), 1996 Jun, 119(6), 1124 - 30
Roles of the conserved cytoplasmic region and non-conserved carboxy-terminal region of SecE in Escherichia coli protein translocase; Kontinen VP et al.; SecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 amino acid residues . Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained . The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues . The conservation of positively charged residues corresponding to Arg80 and Lys81 is also substantial . We deleted or replaced these residues to assess their roles in the SecE function . Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function . Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function . These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function . A chimeric SecE possessing the cytoplasmic region of the E . coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG . Although a Leu to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function . The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE . Based on these results, the SecE function in the translocase is discussed.

J Lipid Res, 1996 Jun, 37(6), 1258 - 67
Expression and characterization of a C24 bile acid 7 alpha-dehydratase from Eubacterium sp . strain VPI 12708 in Escherichia coli; Dawson JA et al.; The intestinal bacterium Eubacterium sp . strain VPI 12708 has been shown to have a bile acid 7 alpha/7 beta-dehydroxylation pathway . A large bile acid inducible (bai) operon encoding at least 9 open reading frames has been cloned and sequenced from this bacterium . The baiE gene from this operon has been subcloned and expressed in E . coli and found to encode a bile acid 7 alpha-dehydratase (BA7 alpha D) . The purified BA7 alpha D was shown to have a calculated subunit mass of 19 kD and a relative native molecular weight of 36,000 . The Km and Vmax for 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid was 0.16 mM and 0.48 nmol/min per mg protein, respectively . Of the substrates tested, the BA7 alpha D used only 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid and 7 alpha-hydroxy-3-oxo-4-cholenoic acid as substrates . A molecular modeling program (SYBYL) was used to calculate the energy differences between the various intermediates in the 7 alpha-dehydroxylation pathway . A marked energy difference (-9.4 kcal/mol) was observed between 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid and 12 alpha-hydroxy-3-oxo-4,6-choldienoic acid, possibly accounting for the apparent irreversibility of the bile acid 7 alpha-dehydratase reaction under our experimental conditions . No significant amino acid sequence homologies were found between BA7 alpha D and other proteins in the data base; however, BA7 alpha D does contain a lipocalin signature sequence, possibly indicating a bile acid binding domain . The bile acid 7 alpha-dehydratase appears to be a unique enzyme in the bacterial bile acid 7 alpha-dehydroxylation pathway.

Poult Sci, 1996 Jun, 75(6), 743 - 5
Effect of orally administered Eubacterium coprostanoligenes ATCC 51222 on plasma cholesterol concentration in laying hens; Li L et al.; Thirty normocholesterolemic laying hens were used to investigate the effect of oral administration of Eubacterium coprostanoligenes on plasma cholesterol concentrations . Hens were divided randomly into three treatment groups (active, inactive, and control) with 10 hens in each group . The active group received 0.5 mL of E . coprostanoligenes suspension (approximately 2 x 10(7) cells per milliliter) daily for 4 wk; the inactive group received the same dosage of killed (boiled) bacterial suspension; and the control group received no supplemental bacteria . After bacterial feeding, the coprostanol to cholesterol ratio in feces of the active group was significantly greater than ratios of the inactive and control groups, indicating that E . coprostanoligenes was colonized in the intestine of hens and was converting intestinal cholesterol to coprostanol . Plasma cholesterol concentrations, however, were not affected by the bacterial treatment.

RNA, 1996 Jun, 2(6), 551 - 63
Domain structure of the ribozyme from eubacterial ribonuclease P; Loria A et al.; Large RNAs can be composed of discrete domains that fold independently . One such "folding domain" has been identified previously in the ribozyme from Bacillus subtilis ribonuclease P (denoted P RNA) . This domain contains roughly one-third of all residues . Folding of an RNA construct consisting of the remaining two-thirds of B . subtilis P RNA was examined by Fe(II)-EDTA hydroxyl radical protection . This molecule folds into the proper higher-order structure under identical conditions as the full-length P RNA, suggesting the presence of a second folding domain in B . subtilis P RNA . Folding analysis of the Escherichia coli P RNA by hydroxyl radical protection shows that this P RNA is completely folded at 5-6 mM Mg2+ . In order to analyze the structural organization of folding domains in E . coli P RNA, constructs were designed based on the domain structure of B . subtilis P RNA . Fe(II)-EDTA protection indicates that E . coli P RNA also contains two folding domains . Despite the significant differences at the secondary structure level, both P RNAs appear to converge structurally at the folding domain level . The pre-tRNA substrate, localized in previous studies, may bind across the folding domains with the acceptor stem/3'CCA contacting the domain including the active site and the T stem-loop contacting the other . Because all eubacterial P RNAs share considerable homology in secondary structure to either B . subtilis or E . coli P RNA, these results suggest that this domain structure may be applicable for most, if not all, eubacterial P RNAs . Identification of folding domains should be valuable in dissecting structure-function relationship of large RNAs.

J Bacteriol, 1996 Jun, 178(11), 3221 - 31
Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system; Brian P et al.; Streptomycete antibiotic synthesis is coupled to morphological differentiation such that antibiotics are produced as a colony sporulates . Streptomyces coelicolor produces several structurally and genetically distinct antibiotics . The S . coelicolor absA locus was defined by four UV-induced mutations that globally blocked antibiotic biosynthesis without blocking morphological differentiation . We show that the absA locus encodes a putative eubacterial two-component sensor kinase-response regulator system . All four mutations lie within a single open reading frame, designated absA1, which is predicted to encode a sensor histidine kinase . A second gene downstream of absA1, absA2, is predicted to encode the cognate response regulator . In marked contrast to the antibiotic-deficient phenotype of the previously described absA mutants, the phenotype caused by disruption mutations in the absA locus is precocious hyperproduction of the antibiotics actinorhodin and undecylprodigiosin . Precocious hyperproduction of these antibiotics is correlated with premature expression of XylE activity in a transcriptional fusion to an actinorhodin biosynthetic gene . We propose that the absA locus encodes a signal transduction mechanism that negatively regulates synthesis of the multiple antibiotics produced by S . coelicolor.

Gene, 1996 May 24, 171(1), 95 - 8
The unique organization of the rpoB region of Spiroplasma citri: a restriction and modification system gene is adjacent to rpoB; Laigret F et al.; A 6.5-kb DNA fragment containing the gene (rpoB) encoding the RNA polymerase (RNAP) beta subunit, from the mollicute Spiroplasma citri (Sc), was cloned and sequenced . The classical eubacterial organization, with the genes (rplK, A, J and L) encoding ribosomal proteins L11, L1, L10 and L12 located immediately upstream from rpoB, was not found in the Sc DNA . Instead, an open reading frame (hsdS) potentially encoding a component of a type I restriction and modification system was identified upstream from rpoB, and sequences showing similarities with insertion elements were found between hsdS and rpoB.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 427 - 31
The primary structure of rat ribosomal protein L14; Chan YL et al.; The amino acid sequence of the rat 60S ribosomal subunit protein L14 was deduced from the sequence of nucleotides in two recombinant cDNAs . Ribosomal protein L14 has 213 amino acids (the NH2-terminal methionine is removed after translation of the mRNA); the molecular weight is 23,193 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 6 to 8 copies of the L14 gene . The mRNA for the protein is about 800 nucleotides in length . Rat L14 is related to a number of previously unidentified ribosomal proteins from other eukaryotes but not to any from archaebacteria or eubacteria . The carboxyl-terminal amino acid sequences of rat L14 and of chicken histone H1 are related . Mutation of the Drosophila melanogaster homolog of rat L14 causes a severe Minute phenotype.

Biochem J, 1996 May 15, 316 ( Pt 1), 73 - 80
Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehyde 3-phosphate non-mevalonate pathway in the green alga Scenedesmus obliquus; Schwender J et al.; Isoprenoid biosynthesis was investigated in the green alga Scenedesmus obliquus grown heterotrophically on 13C-labelled glucose and acetate . Several isoprenoid compounds were isolated and investigated by 13C-NMR spectroscopy . According to the 13C-labelling pattern indicated by the 13C-NMR spectra, the biosynthesis of all plastidic isoprenoids investigated (prenyl side-chains of chlorophylls and plastoquinone-9, and the carotenoids beta-carotene and lutein), as well as of the non-plastidic cytoplasmic sterols, does not proceed via the classical acetate/mevalonate pathway (which leads from acetyl-CoA via mevalonate to isopentenyl diphosphate), but via the novel glyceraldehyde 3-phosphate/pyruvate route recently detected in eubacteria . Formation of isopentenyl diphosphate involves the condensation of a C2 unit derived from pyruvate decarboxylation with glyceraldehyde 3-phosphate and a transposition yielding the branched C5 skeleton of isoprenic units.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4649 - 54
Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins; Zhang W et al.; Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features . We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers . This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI . On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H . salinarium: (i) a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; (ii) a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains; and (iii) a cytoplasmic domain without hydrophobic transmembrane segments . We fractionated the halobacterial cell lysate into soluble and membrane fractions and localized different halobacterial methyl-accepting taxis proteins in both fractions.

Trends Biochem Sci, 1996 May, 21(5), 166 - 71
The origin of the eukaryotic cell; Gupta RS et al.; Molecular sequence data are beginning to provide important insights into the evolutionary origin of eukaryotic cells . Global phylogenies of numerous protein sequences indicate that the eukaryotic cell nucleus is a chimera, which has received major contributions from both a Gram-negative eubacterium and an archaebacterium . Recent studies also indicate that the formation of the nuclear envelope and the endoplasmic reticulum was accompanied by duplication of genes for the molecular chaperone proteins (e.g . hsp70, hsp90), which facilitate protein transport across membranes . Based on these observations, it is suggested that the ancestral eukaryotic cell arose by a unique endosymbiotic event involving engulfment of an eocyte archaebacterium by a Gram-negative eubacterial host.

RNA, 1996 May, 2(5), 473 - 82
Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli; Mandal N et al.; The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria . We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo . We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo . For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA . Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect . The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation . The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site . The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.

EMBO J, 1996 May 1, 15(9), 2138 - 49
Transfer of rps19 to the nucleus involves the gain of an RNP-binding motif which may functionally replace RPS13 in Arabidopsis mitochondria; Sanchez H et al.; The discovery of disrupted rps19 genes in Arabidopsis mitochondria prompted speculation about the transfer to the nuclear compartment . We here describe the functional gene transfer of rps19 into the nucleus of Arabidopsis . Molecular cloning and sequence analysis of rps19 show that the nuclear gene encodes a long N-terminal extension . Import studies of the precursor protein indicate that only a small part of this extension is cleaved off during import . The larger part of the extension, which shows high similarity to conserved RNA-binding domains of the RNP-CS type, became part of the S19 protein . In the Escherichia coli ribosome S19 forms an RNA-binding complex as heterodimer with S13 . By using immuno-analysis and import studies we show that a eubacterial-like S13 protein is absent from Arabidopsis mitochondria, and is not substituted by either a chloroplastic or a cytosolic homologue of this ribosomal protein . We therefore propose that either a highly diverged or missing RPS13 has been functionally replaced by an RNP domain that most likely derived from a glycine-rich RNA-binding protein . These results represent the first case of a functional replacement of a ribosomal protein by a common RNA-binding domain and offer a new view on the flexibility of biological systems in using well-adapted functional domains for different jobs.

Bioessays, 1996 May, 18(5), 421 - 5
Simple sequences and the expanding genome; Hancock JM; Recent analysis of the contribution of replication slippage to genome evolution shows that it has played a significant role in all species from eubacteria to humans . The overall level of repetition in genomes is related to genome size and to the degree of repetition that can be measured within individual ribosomal RNA genes, suggesting that the entire genome accepts simple sequences in a concerted manner when its size increases . Although coding sequences accept simple sequences much less readily than non-coding sequences, they accept some repeats, particularly (CAG)n, preferentially . This may have consequences for the evolution of the genes involved in trinucleotide expansion diseases and the transcriptional networks of which they may form a part.

J Cell Biol, 1996 May, 133(3), 495 - 505
Native 3D structure of eukaryotic 80s ribosome: morphological homology with E . coli 70S ribosome; Verschoor A et al.; A three-dimensional reconstruction of the eukaryotic 80S monosome from a frozen-hydrated electron microscopic preparation reveals the native structure of this macromolecular complex . The new structure, at 38A resolution, shows a marked resemblance to the structure determined for the E . coli 70S ribosome (Frank, J., A . Verschoor, Y . Li, J . Zhu, R.K . Lata, M . Radermacher, P . Penczek, R . Grassucci, R.K . Agrawal, and Srivastava . 1996b . In press; Frank, J., J . Zhu, P . Penczek, Y . Li, S . Srivastava ., A . Verschoor, M . Radermacher, R . Grassucci, R.K . Lata, and R . Agrawal . 1995 . Nature (Lond.).376:441-444.) limited to a comparable resolution, but with a number of eukaryotic elaborations superimposed . Although considerably greater size and intricacy of the features is seen in the morphology of the large subunit (60S vs 50S), the most striking differences are in the small subunit morphology (40S vs 30S): the extended beak and crest features of the head, the back lobes, and the feet . However, the structure underlying these extra features appears to be remarkably similar in form to the 30S portion of the 70S structure . The intersubunit space also appears to be strongly conserved, as might be expected from the degree of functional conservation of the ribosome among kingdoms (Eukarya, Eubacteria, and Archaea) . The internal organization of the 80S structure appears as an armature or core of high-density material for each subunit, with the two cores linked by a single bridge between the platform region of the 40S subunit and the region below the presumed peptidyltransferase center of the 60S subunit . This may be equated with a close contact of the 18S and 28S rRNAs in the translational domain centered on the upper subunit:subunit interface.

J Bacteriol, 1996 May, 178(10), 2934 - 40
Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation; Foissac X et al.; Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri . According to the S . citri strain used, spiralin shows polymorphism in its electrophoretic mobility . The spiralin gene sequences of eight S . citri strains were determined by direct sequencing of the PCR-amplified genes . All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long . The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities . In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins . When S . citri strains were grown in the presence of {3H}palmitic acid, at least 10 proteins, including spiralin, became labeled . In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected . Formic acid hydrolysis of the {3H}palmitic acid-labeled spiralins of four representative S . citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence . On fragment was {3H}palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used . Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 Da) . These results suggest that spiralin polymorphism in S . citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.

J Bacteriol, 1996 May, 178(9), 2564 - 71
Cloning and analysis of sodC, encoding the copper-zinc superoxide dismutase of Escherichia coli; Imlay KR et al.; Benov and Fridovich recently reported the existence of a copper- and zinc-containing superoxide dismutase (CuZnSOD) in Escherichia coli (L . T . Benov and I . Fridovich, J . Biol . Chem . 269:25310-25314,1994) . We have used the N-terminal protein sequence to isolate the gene encoding this enzyme . The gene, denoted sodC, is located at 37.1 min on the chromosome, adjacent to lhr and sodB . A monocistronic transcript of sodC accumulates only in stationary phase . The presence of a conventional leader sequence is consistent with physical data indicating that the E . coli enzyme, like other bacterial CuZnSODs, is secreted into the periplasm . Because superoxide cannot cross membranes, this localization indicates that the enzyme has evolved to defend periplasmic biomolecules against an extracytoplasmic superoxide source . Neither the source nor the target of the superoxide is known . Although once considered an exclusively eukaryotic enzyme, CuZnSOD has now been found in species that span three subdivisions of the purple bacteria . The bacterial CuZnSODs are more homologous to one another than to the eukaryotic enzymes, but active-site residues and structural motifs are clearly shared by both families of enzymes . The use of copper and an invariant disulfide bond suggest that the ancestral gene of present-day CuZnSODs evolved in an aerobic environment, long after the evolutionary split between the eukaryotes and the eubacteria . If so, a CuZnSOD gene must have been transferred laterally between members of these domains . The eukaryotic SODs most closely resemble that of Caulobacter crescentus, a relatively close descendant of the mitochondrial ancestor, suggesting that sodC may have entered the eukaryotes during the establishment of mitochondria.

FEMS Microbiol Lett, 1996 Apr 15, 138(1), 1 - 10
Regulation and organization of the groE and dnaK operons in Eubacteria; Segal R et al.; groEL and dnaK are the most highly conserved protein-coding genes known . Most groEL operons and several dnaK and dnaJ operons contain a highly conserved inverted repeat (IR) sequence in their regulatory region . So far, this IR has been found only as part of the groE, dnaK and dnaJ operons and genes . In most cases, the IR is part of the operon transcript, and is involved in the regulation of expression at both the DNA and mRNA levels . A detailed analysis of groE and dnaK operons indicates that the organization of the groE operons is highly conserved . They contain only the groES and groEL genes and always in the same order . In contrast, the organization of the dnaK operons has changed during evolution: genes have been added and deleted from it, and the gene order within the operon is variable.

Microb Drug Resist, 1996 Spring, 2(1), 17 - 23
Are S-layer glycoproteins and lipopolysaccharides related?
Schaffer C, Wugeditsch T, Neuninger C, Messner P.
Several glycan structures of S-layer glycoproteins of gram-positive eubacteria were compared with the principal structural organization of O-antigens of lipopolysaccharides of gram-negative eubacteria . Further, activated intermediates of the biosynthetic pathway of S-layer glycans were compared with activated intermediates of the route of assembly of lipopolysaccharide O-antigens . As a result, at least structural similarities between both types of molecules have been clearly observed . More detailed studies of the assembly of S-layer glycans are required to unambiguously demonstrate the extent to which the biosynthetic pathways of both molecules are related.

Microb Drug Resist, 1996 Spring, 2(1), 1 - 8
Periplasm, periplasmic spaces, and their relation to bacterial wall structure: novel secretion of selected periplasmic proteins from Pseudomonas aeruginosa; Beveridge TJ et al.; A brief overview of thin sections of cryopreserved walls from select eubacteria will be presented to suggest that all bacteria have functional periplasms, but that these are not necessarily confined to a periplasmic space such as found in typical gram-negative bacteria . Pseudomonas aeruginosa contains many components in its periplasmic space, some of which are required for infection . Throughout its growth cycle, P . aeruginosa blebs-off membrane vesicles that can possess DNA, endotoxin, phospholipase, protease, hemolysin, alkaline phosphatase, and autolysin, each of which must have a molecular phase that resides in the periplasm . These membrane packets make good delivery systems to convey these components to other bacteria and, possibly, tissue . Aminoglycoside antibiotics, such as gentamicin, produce a serious perturbation on the bacterium's surface (separate from the ribosomal effect), which contributes to the killing of the microorganism . Antibiotics such as this increase the size and number of the membrane blebs, which could contribute to septic shock of patients under drug therapy.

Appl Environ Microbiol, 1996 Apr, 62(4), 1405 - 15
Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments; Teske A et al.; The sulfate-reducing bacterial populations of a stratified marine water column, Mariager Fjord, Denmark, were investigated by molecular and culture-dependent approaches in parallel . Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA and DNA encoding rRNA (rDNA) isolated from the water column indicated specific bacterial populations in different water column layers and revealed a highly differentiated pattern of rRNA- and rDNA-derived PCR amplificates, probably reflecting active and resting bacterial populations . Hybridization of DGGE patterns with rRNA probes indicated the increased presence and activity (by at least 1 order of magnitude) of sulfate-reducing bacteria within and below the chemocline . Parallel to this molecular approach, an approach involving most-probable-number (MPN) counts was used, and it found a similar distribution of cultivable sulfate-reducing bacteria in the water column of Mariager Fjord, Approximately 25 cells and 250 cells per ml above and below the chemocline, respectively, were found . Desulfovibrio- and Desulfobulbus-related strains occurred in the oxic zone . DGGE bands from MPN cultures were sequenced and compared with those obtained from nucleic acids extracted from water column samples . The MPN isolates were phylogenetically affiliated with sulfate-reducing delta subdivision proteobacteria (members of the genera Desulfovibrio, Desulfobulbus, and Desulfobacter), whereas the molecular isolates constituted an independent lineage of the delta subdivision proteobacteria . DGGE of PCR-amplified nucleic acids with general eubacterial PCR primers conceptually revealed the general bacterial population, whereas the use of culture media allowed cultivable sulfate-reducing bacteria to be selected . A parallel study of Mariager Fjord biogeochemistry, bacterial activity, and bacterial counts complementing this investigation has been presented elsewhere (N.B . Ramsing, H . Fossing, T . G . Ferdelman, F . Andersen, and B . Thamdrup, Appl . Environ.

Appl Environ Microbiol, 1996 Apr, 62(4), 1391 - 404
Distribution of bacterial populations in a stratified fjord (Mariager Fjord, Denmark) quantified by in situ hybridization and related to chemical gradients in the water column; Ramsing NB et al.; The vertical distribution of major and intermediate electron acceptors and donors was measured in a shallow stratified fjord . Peaks of zero valence sulfur, Mn(IV), and Fe(III) were observed in the chemocline separating oxic surface waters from sulfidic and anoxic bottom waters . The vertical fluxes of electron acceptors and donors (principally O2 and H2S) balanced within 5%; however, the zones of oxygen reduction and sulfide oxidation were clearly separated . The pathway of electron transfer between O2 and H2S was not apparent from the distribution of sulfur, nitrogen, or metal compounds investigated . The chemical zonation was related to bacterial populations as detected by ethidium bromide (EtBr) staining and by in situ hybridization with fluorescent oligonucleotide probes of increasing specificity . About half of all EtBr-stained cells were detectable with a general oligonucleotide probe for all eubacteria when digital image analysis algorithms were used to improve sensitivity . Both EtBr staining and hybridization indicated a surprisingly uniform distribution of bacteria throughout the water column . However, the average cell size and staining intensity as well as the abundance of different morphotypes changed markedly within the chemocline . The constant overall cell counts thus concealed pronounced population shifts within the water column . Cells stained with a delta 385 probe (presumably sulfate-reducing bacteria) were detected at the chemocline at about 5 x 10(4) cells per ml, and this concentration increased to 2 x 10(5) cells per ml beneath the chemocline . A long slim rod-shaped bacterium was found in large numbers in the oxic part of the chemocline, whereas large ellipsoid cells dominated at greater depth . Application of selective probes for known genera of sulfate-reducing bacteria gave only low cell counts, and thus it was not possible to identify the dominant morphotypes of the sulfate-reducing community.

Micron, 1996 Apr, 27(2), 141 - 56
Biotechnology and biomimetic with crystalline bacterial cell surface layers (S-layers); Sara M et al.; Crystalline bacterial cell surface layers (S-layers) are the outermost cell envelope component of many eubacteria and archaeobacteria . S-layers are composed of a single protein or glycoprotein species and exhibit oblique, square or hexagonal lattice symmetry . Pores passing through these monomolecular arrays show identical size and morphology, and functional groups are aligned in well-defined positions and orientations . Due to these unique features, S-layers have broad application potential in biotechnology including functioning as biomimetic membranes . Presently, S-layers are used (i) for the production of isoporous ultrafiltration membranes with very well defined molecular sieving and adsorption properties, (ii) as matrices for the controlled immobilization of biologically active macromolecules (e.g., enzymes, antibodies, ligands) as required for biosensors, affinity membranes and affinity microparticles as well as for solid phase assays, (iii) as stabilizing structures for Langmuir-Blodgett films and liposomes and (iv) as carriers and adjuvants for weakly immunogenic antigens and haptens.

J Biochem (Tokyo), 1996 Apr, 119(4), 811 - 6
Characterization of a B . subtilis minor isoleucine tRNA deduced from tDNA having a methionine anticodon CAT; Matsugi J et al.; Bacillus subtilis, which belongs to Gram-positive eubacteria, has been predicted to have a minor isoleucine tRNA transcribed from the gene possessing the CAT anticodon, which corresponds to methionine . We isolated this tRNA and determined its sequence including modified nucleotides . Modified nucleotide analyses using TLC, UV, and FAB mass spectroscopy revealed that the first letter of the anticodon is modified to lysidine {4-amino-2-(N6-lysino)-1-beta-d-ribofuranosyl pyrimidine} . As a result, this tRNA agrees with the minor one predicted from the DNA sequence and is thought to decode the isoleucine codon AUA.

J Bacteriol, 1996 Apr, 178(8), 2314 - 9
Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall; Wang X et al.; The ftsZ gene is required for cell division in Escherichia coli and Bacillus subtilis . In these organisms, FtsZ is located in a ring at the leading edge of the septum . This ring is thought to be responsible for invagination of the septum, either causing invagination of the cytoplasmic membrane or activating septum-specific peptidoglycan biosynthesis . In this paper, we report that the cell division gene ftsZ is present in two mycoplasma species, Mycoplasma pulmonis and Acholeplasma laidlawii, which are eubacterial organisms lacking a cell wall . Sequencing of the ftsZ homolog from M . pulmonis revealed that it was highly homologous to other known FtsZ proteins . The M . pulmonis ftsZ gene was overexpressed, and the purified M . pulmonis FtsZ bound GTP . Using antisera raised against this purified protein, we could demonstrate that it was expressed in M . pulmonis . Expression of the M . pulmonis ftsZ gene in E . coli inhibited cell division, leading to filamentation, which could be suppressed by increasing expression of the E . coli ftsZ gene . The implications of these results for the role of ftsZ in cell division are discussed.

J Bacteriol, 1996 Apr, 178(7), 1881 - 94
Evolutionary conservation of RecA genes in relation to protein structure and function; Karlin S et al.; Functional and structural regions inferred from the Escherichia coli R ecA protein crystal structure and mutation studies are evaluated in terms of evolutionary conservation across 63 RecA eubacterial sequences . Two paramount segments invariant in specific amino acids correspond to the ATP-binding A site and the functionally unassigned segment from residues 145 to 149 immediately carboxyl to the ATP hydrolysis B site . Not only are residues 145 to 149 conserved individually, but also all three-dimensional structural neighbors of these residues are invariant, strongly attesting to the functional or structural importance of this segment . The conservation of charged residues at the monomer-monomer interface, emphasizing basic residues on one surface and acidic residues on the other, suggests that RecA monomer polymerization is substantially mediated by electrostatic interactions . Different patterns of conservation also allow determination of regions proposed to interact with DNA, of LexA binding sites, and of filament-filament contact regions . Amino acid conservation is also compared with activities and properties of certain RecA protein mutants . Arginine 243 and its strongly cationic structural environment are proposed as the major site of competition for DNA and LexA binding to RecA . The conserved acidic and glycine residues of the disordered loop L1 and its proximity to the RecA acidic monomer interface suggest its involvement in monomer-monomer interactions rather than DNA binding . The conservation of various RecA positions and regions suggests a model for RecA-double-stranded DNA interaction and other functional and structural assignments.

J Biol Chem, 1996 Mar 15, 271(11), 6252 - 9
Photolyase of Myxococcus xanthus, a Gram-negative eubacterium, is more similar to photolyases found in Archaea and "higher" eukaryotes than to photolyases of other eubacteria; O'Connor KA et al.; We report the identification of the gene encoding a DNA photolyase (phrA) from the Gram-negative eubacterium Myxococcus xanthus . The deduced amino acid sequence of M . xanthus photolyase indicates that the protein contains 401 amino acids (Mr 45,071) . By comparison of the amino acid and DNA sequences with those of other known photolyases, it has been found that it is more similar to the deduced amino acid sequences of the photolyases of "higher" eukaryotes than to the photolyases of other eubacteria . Recombinant plasmids carrying M . xanthus phrA rescue the photoreactivation activity of an irradiated strain of Escherichia coli with a deletion in phrA . This rescue is light-dependent.

J Mol Biol, 1996 Mar 15, 256(5), 829 - 37
Regulatory region C of the E . coli heat shock transcription factor, sigma32, constitutes a DnaK binding site and is conserved among eubacteria; McCarty JS et al.; The E . coli heat shock response is regulated at the transcriptional level through stress-dependent controls of the heat shock promoter-specific sigma32 subunit of RNA polymerase . A key aspect of this regulation, the sensing of stress and transmission of this information to sigma32, involves the chaperone system formed by the DnaK, DnaJ and GrpE heat shock proteins . This system mediates stress- dependent controls of levels and activity of sigma32 which rely, at least in part, on direct association of DnaK and DnaJ with sigma32 . We identified DnaK binding sites within the sigma32 sequence by probing a cellulose-bound peptide library scanning sigma32 . Two sites with high affinity for DnaK, containing the motifs RKLFFNLR and LRNWRIVK, were located centrally and peripherally, respectively, to the region C of sigma32, previously implicated genetically in chaperone-dependent control of sigma32 levels . Cloning and sequencing of rpoH homologs from five Gram-negative proteobacteria revealed that region C, including the DnaK binding motif central to it, is highly conserved among sigma32 homologs but missing in the other sigma factors . We propose that binding of DnaK to region C is central to a conserved regulatory mechanism allowing the sensing of stress by the heat shock gene transcription machinery.

FEBS Lett, 1996 Mar 11, 382(1-2), 73 - 8
Evidence for trans-cis isomerization of the p-coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein; Kort R et al.; Analysis of the chromophore p-coumaric acid, extracted from the ground state and the long-lived blue-shifted photocycle intermediate of photoactive yellow protein, shows that the chromophore is reversibly converted from the trans to the cis configuration, while progressing through the photocycle . The detection of the trans and cis isomers was carried out by high performance capillary zone electrophoresis and further substantiated by 1H NMR spectroscopy . The data presented here establish the photo-isomerization of the vinyl double bond in the chromophore as the photochemical basis for the photocycle of photoactive yellow protein, a eubacterial photosensory protein . A similar isomerization process occurs in the structurally very different sensory rhodopsins, offering an explanation for the strong spectroscopic similarities between photoactive yellow protein and the sensory rhodopsins . This is the first demonstration of light-induced isomerization of a chromophore double bond as the photochemical basis for photosensing in the domain of Bacteria.

Biochim Biophys Acta, 1996 Mar 7, 1293(1), 106 - 12
Archaeal elongation factor 1 beta is a dimer . Primary structure, molecular and biochemical properties; Raimo G et al.; The elongation factor 1 beta (EF-1 beta), that in eukarya and archaea promotes the replacement of GDP by GTP on the elongation factor 1 alpha x GDP complex, was purified to homogeneity from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1 beta) . Its primary structure was established by sequenced Edman degradation of the entire protein or its proteolytic peptides . The molecular weight of SsEF-1 beta was estimated as about 10000 or 20000 under denaturing or native conditions respectively; this finding suggests that the native protein exists as a dimer . The peptide chain of SsEF-1 beta is much shorter than that of its eukaryotic analogues and homology is found only at their C-terminal region; no homology exists between SsEF-1 beta and eubacterial EF-Ts . At 50 degrees C, at a concentration of SsEF-1 beta 5-fold higher than that of SsEF-1 alpha x {3H}GDP the rate of the exchange of {3H}GDP for GTP becomes about 160-fold faster . An analysis of the values of the energetic parameters indicates that in the presence of SsEF-1 beta the GDP/GTP exchange is entropically favoured . At 100 degrees C the half-life of SsEF-1 beta is about 4 h.

Int Endod J, 1996 Mar, 29(2), 69 - 75
Associations of endodontic symptoms and signs with particular combinations of specific bacteria; Gomes BP et al.; Significant associations have been reported between (a) specific bacterial species isolated from root canals and (b) between individual bacterial species and endodontic symptoms and signs . The prime objective of this study was to determine whether particular combinations of specific bacteria are associated with individual endodontic symptoms and signs . Seventy root canals were investigated microbiologically taking care to maintain the viability of obligate anaerobes, which accounted for 64% of the total species isolated, including Peptostreptococcus micros, Prevotella melaninogenica, Prevotella oralis, Eubacterium aerofaciens, Eubacterium lentum, Fusobacterium nucleatum, Prevotella buccae and Prevotella intermedia . Significant associations were found between individual clinical features and the following pairs of species: (a) pain (37 cases) and Peptostreptococcus spp./Prevotella spp., Peptostreptococcus spp./Prevotella melaninogenica, Pstr . micros/Prev . melaninogenica (all P < 0.01); (b) swelling (23 cases) and Pstr . micros/Prevotella spp . (P < 0.01); (c) 'Wet' canal (57 cases) and Prevotella spp./Eubacterium spp . (P < 0.01), Peptostreptococcus spp./Eubacterium spp . (P < 0.05) . Thus data from this investigation suggests that statistically significant associations exist between individual endodontic symptoms and signs and particular combinations of specific bacteria.

Microbiologia, 1996 Mar, 12(1), 17 - 28
Ancestral relationships of the major eukaryotic lineages; Sogin ML et al.; Molecular systematics has revolutionized our understanding of microbial evolution . Phylogenetic frameworks relating all organisms in this biosphere can be inferred from comparisons of slowly evolving molecules such as the small and large subunit ribosomal RNAs . Unlike today's text book standard, the "Five Kingdoms" (plants, animals, fungi, protists and bacteria), molecular studies define three primary lines of descent (Eukaryotes, Eubacteria, and Archaebacteria) . Within the Eukaryotes, the "higher" kingdoms (Fungi, Plantae, and Animalia) are joined by at least two novel complex evolutionary assemblages, the "Alveolates" (ciliates, dinoflagellates and apicomplexans) and the "Stramenopiles" (diatoms, oomycetes, labyrinthulids, brown algae and chrysophytes) . The separation of these eukaryotic groups (described as the eukaryotic "crown") occurred approximately 10(9) years ago and was preceded by a succession of earlier diverging protist lineages, some as ancient as the separation of the prokaryotic domains . The molecular phylogenies suggest that multiple endosymbiotic events introduced plastids into discrete eukaryotic lineages.

J Clin Microbiol, 1996 Mar, 34(3), 537 - 42
Molecular analysis of microflora associated with dentoalveolar abscesses; Dymock D et al.; The microflora associated with three dentoalveolar abscesses was determined by cultural and molecular methods . 16S rRNA genes were randomly amplified by means of conserved eubacterial primers and cloned . Restriction fragment length polymorphism analysis of the clones and amplified genes encoding 16S rRNA from the cultured bacteria was used to detect putative unculturable bacteria . Clones representative of five predominant groups of uncultured organisms were sequenced . Two were identified as Porphyromonas gingivalis and Prevotella oris, and one was found to be closely related to Peptostreptococcus micros . The remaining two clones did not correspond to known, previously sequenced organisms . One was related to Zoogloea ramigera, a species of aerobic waterborne organisms, while the other was distantly related to the genus Prevotella . This study has demonstrated the possibility of the characterization of microflora associated with human infection by molecular methods without the inherent biases of culture.

Plasmid, 1996 Mar, 35(2), 141 - 4
Complete nucleotide sequence of the Sulfolobus islandicus multicopy plasmid pRN1; Keeling PJ et al.; The complete sequence of the 5350-bp plasmid pRN1 from the crenarchaeote Sulfolobus islandicus has been determined . This plasmid is the first to be sequenced from this group of thermoacidophilic archaebacteria (Archaea) and its high copy number and wide host range make it a good candidate for a cloning vector . pRN1 contains several open reading frames, including one that spans over half the plasmid and has significant similarity to the helicase domain of viral primase proteins . Directly upstream of this putative primase is a homologue of Cop, a family of small proteins from promiscuous eubacterial plasmids which control copy number by repressing the expression of the replication initiation protein . In eubacterial plasmids cop is found upstream of the replication initiator protein . The location of a cop homologue upstream of a primase-like gene in pRN1 suggests that it controls DNA replication in a manner similar to these eubacterial plasmids, but does so using a mixture of components from plasmids and viruses.

Gene, 1996 Feb 22, 169(1), 97 - 100
Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli; De La Vega FM et al.; We have cloned and determined the nucleotide (nt) sequences of the genes encoding peptidyl-tRNA hydrolase (Pth) homologues of Salmonella typhi (St) and the Lyme disease spirochaete, Borrelia burgdorferi (Bb) . We also completed the nt sequence of a pth homologous gene contained in a Chlamydia trachomatis (Ct) clone identified in the databanks . The open reading frames (ORFs) of the Pth homologues encode putative polypeptides of 194 (St), 188 (Bb) and 194 (Ct) amino acids exhibiting significant identity with Escherichia coli (Ec) Pth . Together with the products of two previously unidentified ORFs from Bacillus subtilis and Saccharomyces cerevisiae, and the recently recognized Haemophilus influenzae and Mycoplasma genitalium pth genes, these seven putative polypeptides and the Ec Pth form a group of homologous basic proteins spanning eubacteria and eukaryota which can be defined by at least three conserved regions . Previously known Ec pth mutations were located in highly conserved residues.

Gene, 1996 Feb 22, 169(1), 101 - 3
Cloning of the Mycoplasma capricolum gene encoding peptide-chain release factor; Inagaki Y et al.; In Mycoplasma capricolum (Mc), a relative of Gram+ eubacteria with a high genomic A + T-content, the UGA codon is assigned to Trp instead of being a stop codon . We previously showed the lack of peptide-chain release factor (RF) activity in vitro responding to the UGA codon in this bacterium {Inagaki et al., Nucleic Acids Res . 21 (1993) 1335-1338} . To obtain more information on the translation termination mechanism of Mc, we isolated and sequenced the gene encoding RF . The deduced amino-acid sequence has no RF-2-specific + 1 frameshift site and shows 50 and 36% identity to Escherichia coli RF-1 and RF-2, respectively . We conclude that this gene encodes the putative RF-1 which would possess the conserved 'five-domain' structure of RF family found in various organisms.

Nucleic Acids Res, 1996 Feb 15, 24(4), 648 - 54
Sequences homologous to yeast mitochondrial and bacteriophage T3 and T7 RNA polymerases are widespread throughout the eukaryotic lineage; Cermakian N et al.; Although mitochondria and chloroplasts are considered to be descendants of eubacteria-like endo- symbionts, the mitochondrial RNA polymerase of yeast is a nucleus-encoded, single-subunit enzyme homologous to bacteriophage T3 and T7 RNA polymerases, rather than a multi-component, eubacterial-type alpha 2 beta beta' enzyme, as encoded in chloroplast DNA . To broaden our knowledge of the mitochondrial transcriptional apparatus, we have used a polymerase chain reaction (PCR) approach designed to amplify an internal portion of phage T3/T7-like RNA polymerase genes . Using this strategy, we have recovered sequences homologous to yeast mitochondrial and phage T3/T7 RNA polymerases from a phylogenetically broad range of multicellular and unicellular eukaryotes . These organisms display diverse patterns of mitochondrial genome organization and expression, and include species that separated from the main eukaryotic line early in the evolution of this lineage . In certain cases, we can deduce that PCR-amplified sequences, some of which contain small introns, are localized in nuclear DNA . We infer that the T3/T7-like RNA polymerase sequences reported here are likely derived from genes encoding the mitochondrial RNA polymerase in the organisms in which they occur, suggesting a phage T3/T7-like RNA polymerase was recruited to act in transcription in the mitochondrion at an early stage in the evolution of this organelle.

J Mol Biol, 1996 Feb 9, 255(5), 688 - 701
The mitochondrial DNA of Allomyces macrogynus: the complete genomic sequence from an ancestral fungus; Paquin B et al.; We have determined the complete nucleotide sequence of the circular mitochondrial DNA (mtDNA) of the chytridiomycete fungus, Allomyces macrogynus (57,473 bp; A + T content 60.5%) . The identified genes that are typical for most fungal mitochondria include those for the large (rnl) and small subunit (rns) ribosomal RNAs, a complete set of 25 tRNAs, three ATPase subunits (atp6, atp8 and atp9), apocytochrome b(cob), three subunits of the cytochrome oxidase complex (cox1, cox2 and cox3), and seven subunits of the NADH dehydrogenase complex (nad1, nad2, nad3, nad4, nad4L, nad5 and nad6) . A total of 28 introns of both groups are found, some of which contain open reading frames (ORFs) coding for potential endonucleases (group I) or reverse-transcriptases (group II) . All mitochondrial genes are transcribed from the same DNA strand, as is the case in many other eufungi . Particular features of the A . macrogynus mtDNA include: (1) the first documented case of a fungal mitochondrial ribosomal protein gene (rps3) that is clearly identified by similarity with bacterial homologues; (2) four unique ORFs; (3) the presence of an insert in the atp6 gene that may have been acquired by interspecific transfer; (4) more than 67 short, highly structured and conserved DNA elements inserted in intergenic spacers, introns, and variable regions of the rnl and rns genes: these elements are unusually G + C rich; (5) rRNA structures that resemble more closely those of eubacteria than their counterparts in other fungal mitochondria . The high degree of conservation of the A . macrogynus mitochondrial rRNA secondary structures, the existence of a mitochondrial rps3 gene (common to protist but unique in fungal mtDNAs), and phylogenetic relationships inferred from highly conserved protein genes, demonstrate consistently the ancestral character of this fungal mitochondrial genome.

Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1071 - 6
Archaeal-eubacterial mergers in the origin of Eukarya: phylogenetic classification of life; Margulis L; A symbiosis-based phylogeny leads to a consistent, useful classification system for all life . "Kingdoms" and "Domains" are replaced by biological names for the most inclusive taxa: Prokarya (bacteria) and Eukarya (symbiosis-derived nucleated organisms) . The earliest Eukarya, anaerobic mastigotes, hypothetically originated from permanent whole-cell fusion between members of Archaea (e.g., Thermoplasma-like organisms) and of Eubacteria (e.g., Spirochaeta-like organisms) . Molecular biology, life-history, and fossil record evidence support the reunification of bacteria as Prokarya while subdividing Eukarya into uniquely defined subtaxa: Protoctista, Animalia, Fungi, and Plantae.

Gene, 1996 Feb 2, 168(1), 87 - 92
The amino-acid sequence similarity of plant glutamate dehydrogenase to the extremophilic archaeal enzyme conforms to its stress-related function; Syntichaki KM et al.; A cDNA clone encoding grapevine (Vitis vinifera L . cv Sultanina) NAD(H)-glutamate dehydrogenase (GDH) was isolated from a cDNA expression library by immunoscreening with a polyclonal antibody raised against grapevine GDH . Nucleotide sequence analysis revealed an open reading frame (ORF) encoding a precursor protein of 411 amino acids (aa) with a calculated molecular mass of 44.517 kDa . The deduced aa sequence showed relatively higher homology to GDH from archaebacteria species, than to those from eukaryotes and eubacteria . This resemblance indicated a functional and/or evolutionary relationship in this class of enzymes which might be relevant to the stress-related function of plant GDH . We have shown that the bacterially produced plant GDH was thermostable.

Gene, 1996 Feb 2, 168(1), 77 - 80
Characterization of genes encoding topoisomerase IV of Mycoplasma genitalium; Bailey CC et al.; A type-II toposiomerase (Topo-IV) encoded by the parC and parE genes in Escherichia coli and Salmonella typhimurium is thought to be involved in cell septation and in the decatenation of newly replicated chromosomes . We have identified parC and parE homologs in the pleomorphic, wall-less organism Mycoplasma genitalium . Since the mechanics of cell septation in conventional eubacterial species is believed to be mediated by cell-wall constituents, there is no clear understanding of what coordinates that process in wall-less species . The presence of par genes in this bacterium, which has the smallest genome of any free-living organism, suggests that Topo-IV has been evolutionarily conserved because of an essential role in mediating cell division.

Mol Microbiol, 1996 Feb, 19(3), 417 - 28
Heat-shock and general stress response in Bacillus subtilis; Hecker M et al.; The induction of stress proteins is an important component of the adaptional network of a non-growing cell of Bacillus subtilis . A diverse range of stresses such as heat shock, salt stress, ethanol, starvation for oxygen or nutrients etc . induce the same set of proteins, called general stress proteins . Although the adaptive functions of these proteins are largely unknown, they are proposed to provide general and rather non-specific protection of the cell under these adverse conditions . In addition to these non-specific general stress proteins, all extracellular signals induce a set of specific stress proteins that may confer specific protection against a particular stress factor . In B . subtilis at least three different classes of heat-inducible genes can be defined by their common regulatory characteristics: Class I genes, as exemplified by the dnaK and groE operons, are most efficiently induced by heat stress . Their expression involves a sigma A-dependent promoter, an inverted repeat (called the CIRCE element) highly conserved among eubacteria, and probably a repressor interacting with the CIRCE element . The majority of general stress genes (class II, more than 40) are induced at sigma B-dependent promoters by different growth-inhibiting conditions . The activation of sigma B by stress or starvation is the crucial event in the induction of this large stress regulon . Only a few genes, including Ion, clpC, clpP, and ftsH, can respond to different stress factors independently of sigma B or CIRCE (class III) . Stress induction of these genes occurs at promoters presumably recognized by sigma A and probably involves additional regulatory elements which remain to be defined.

Mol Phylogenet Evol, 1996 Feb, 5(1), 50 - 77
Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families; Hewett-Emmett D et al.; The carbonic anhydrases (CA) catalyze with high efficiency the reversible hydration of carbon dioxide, a reaction underlying many diverse physiological processes in animals, plants, archaebacteria, and eubacteria . We examined the evolutionary history and functional convergence of the CAs encoded by members of three independent CA gene families (alpha-CA, beta-CA and gamma-CA) . Surprisingly, the six mammalian alpha-CA isozymes of defined function and tissue expression are evolving more rapidly than four mammalian alpha-CA-related proteins of unknown function . We have identified and included several previously unrecognized CA homologues present in the sequence databases, many of which are the fruits of genome project sequencing and expressed cDNA studies . We examined alpha-CA active site evolution and the putative beta-CA and gamma-CA active sites . We found support for the "introns late" hypothesis by analysis of alpha-CA intron locations . The view that alpha-CAs would be restricted to the animal kingdom and plant green algae (Chlamydomonas), the beta-CAs to plants and eubacteria, and the gamma-CAs to archaebacteria and eubacteria is breaking down . The plant Arabidopsis has homologues of all three families.

Plant Mol Biol, 1996 Feb, 30(3), 479 - 92
Characterization of a plastid-specific HSP90 homologue: identification of a cDNA sequence, phylogenetic descendence and analysis of its mRNA and protein expression; Schmitz G et al.; The isolation of cDNAs is described which encode the complete sequence of a precursor protein for a HSP90 homologue consisting of an N-terminal transit peptide of 5850 Da and a mature protein (cpHSP82) of 82 260 Da, located in the plastids of rye leaves (Secale cereale) . Hybridization analysis indicated the presence of a single gene in the DNA of rye and a transcript size of 2.8 kb . A phylogenetic tree constructed on the basis of sequence comparisons for HSP90 homologues from different species and compartments indicated that the plastidic HSP82 from rye was more closely related to an eubacterial protein than to HSP90 homologues of the cytosol or ER from both plants and animals . The results suggest that during chloroplast evolution the gene for cpHSP82 was transferred to the nucleus from a prokaryotic endosymbiont . Immunoblots with specific antibodies and Percoll gradient-purified organelles confirmed the location of cpHSP82 in chloroplasts or non-green plastids . In green rye leaves cpHSP82 was constitutively expressed and equally distributed among tissues of different age . The expression of cpHSP82 was enhanced within 2 h by exposure to 42 degrees C . The cpHSP82 transcript and protein were much more strongly expressed in non-green tissues, such as etiolated, 70S ribosome-deficient 32 degrees C-grown, or herbicide-bleached, than in normal green leaves . Also chromoplasts from the pericarp of tomato fruits contained high levels of a HSP90 polypeptide while a photosynthetic protein, the large subunit of ribulose-1,5-bisphosphate carboxylase was largely degraded during ripening.

Plant Mol Biol, 1996 Feb, 30(3), 419 - 26
A second and differentially expressed glutamyl-tRNA reductase gene from Arabidopsis thaliana; Kumar AM et al.; 5-Aminolevulinic acid (ALA) is the universal precursor of tetrapyrroles (e.g., chlorophylls and hemes) . In the chloroplasts of plants and in several eubacterial species ALA is formed in a two-step process known as the C5 pathway . In the first step, glutamyl-tRNA reductase (GluTR), converts glutamate of glutamyl-tRNA to glutamate 1-semialdehyde (GSA) which is rearranged to ALA by glutamate 1-semialdehyde-2,1-aminomutase (GSA-A) in the second step . Since ALA formation is a limiting step in chlorophyll biosynthesis, GluTR, which is encoded by the HEMA gene in Arabidopsis thaliana plays a vital role in that biosynthesis . Here we report the occurrence of a second functional HEMA gene (HEMA2) in A . thaliana . This gene was isolated by screening a genomic library with a probe from HEMA1 . The nucleotide sequence of the cDNA and the corresponding genomic DNA indicates that the Arabidopsis HEMA2 gene contains two short introns (285 bp and 159 bp) . The deduced amino acid sequence predicts a HEMA2 protein of 530 amino acids with 79% identity to the HEMA1-encoded GluTR . The 5'-flanking sequence of the HEMA2 gene includes several motifs (e.g., GT-1 boxes, GATA motifs) similar to light-responsive regulatory elements found in light-inducible genes . Unlike the HEMA1 transcript, which is present in all parts of the plant, HEMA2 is expressed in low levels in roots and flowers . The presence of a second functional HEMA gene in Arabidopsis raises the possibility that two C5 pathways exist in chloroplasts.

RNA, 1996 Feb, 2(2), 134 - 45
Structural features of the large subunit rRNA expressed in Plasmodium falciparum sporozoites that distinguish it from the asexually expressed subunit rRNA; Rogers MJ et al.; The developmentally regulated transcription of at least two distinct sets of nuclear-encoded ribosomal RNAs is detected in Plasmodium species . The identification of functional differences between the two sets of rRNAs is of interest . To facilitate the search for such differences, we have identified the 5.8S and 28S rRNAs from Plasmodium falciparum that are expressed in the sporozoite stage (S gene) of the parasites' life cycle in the mosquito host and compare them to transcripts expressed in the red blood cells (A gene) of the vertebrate host . This completes the first set of A- and S-type nuclear-encoded rRNA genes for a Plasmodium species . Analysis of the predicted secondary structures of the two units reveals the majority of differences between the A- and S-type genes occur in regions previously known to be variable . However, the predicted secondary structure of both 28S rRNAs indicates 11 positions within conserved areas that are not typical of eucaryotic rRNAs . Although the A-type gene resembles almost all eucaryotes, being atypical in only 4 of the 11 positions, the S gene is variant in 8 of the 11 positions . In three of these positions, the S-type gene resembles the consensus nucleotides for the 23S rRNA from Eubacteria and/or Archaea . A few differences occur in regions associated with ribosome function, in particular the GTPase site where the S-type differs in a base pair and loop from all known sequences . Further, the identification of compensatory changes at conserved points of interactions between the 5.8S-28S rRNAs indicates that transcripts from A- and S-units should not be interchangeable.

Arch Microbiol, 1996 Feb, 165(2), 141 - 7
Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport; Dobrindt U et al.; Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions . The oxygen-sensitive enzyme was partially reactivated under reducing conditions, resulting in a maximal activity of 19.8 μmol H2 oxidized min-1 (mg protein)-1 with benzyl viologen as electron acceptor and an apparent Km value for H2 of 341 μM . The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively . SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio . The native protein contained 15.6 +/- 1.7 mol Fe, 11.4 +/- 1.4 mol S2-, and 0.6 mol Ni per mol enzyme . The hydrogenase coupled with viologen dyes, but not with other various artificial electron carriers, FAD, FMN, or NAD(P)+ . The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound uptake hydrogenases . Washed membranes catalyzed a H2-dependent cytochrome b reduction at a rate of 0.18 nmol min-1 (mg protein)-1.

Curr Microbiol, 1996 Feb, 32(2), 95 - 100
DNA-binding activities in Streptococcus gordonii: identification of a receptor-nickase and a histonelike protein; Lunsford RD et al.; Extraction of Streptococcus gordonii cells with the mild chaotropic agent, LiCl, drastically decreased DNA transforming ability, had little effect on viability, and released both DNA nicking and binding activities . Both activities were Mg2+ and Ca2+ independent and were not competence specific . Southwestern blot analysis of the extract identified putative surface proteins of 56 kDa and 68 kDa in strain Challis and Wicky, respectively . Extracts also contained a 10-kDa DNA-binding protein, designated HSgo, that belongs to the eubacterial histonelike class of proteins.

Curr Microbiol, 1996 Feb, 32(2), 77 - 84
Identification and discrimination of oral asaccharolytic Eubacterium spp . by pyrolysis mass spectrometry and artificial neural networks; Goodacre R et al.; Curie-point pyrolysis mass spectra were obtained from 29 oral asaccharolytic Eubacterium strains and 6 abscess isolates previously identified as Peptostreptococcus heliotrinreducens . Pyrolysis mass spectrometry (PyMS) with cluster analysis was able to clarify the taxonomic position of this group of organisms . Artificial neural networks (ANNS) were then trained by supervised learning (with the back-propagation algorithm) to recognize the strains from their pyrolysis mass spectra; all Eubacterium strains were correctly identified, and the abscess isolates were identified as un-named Eubacterium taxon C2 and were distinct from the type strain of P . heliotrinreducens . These results demonstrate that the combination of PyMS and ANNs provides a rapid and accurate identification technique.

J Bacteriol, 1996 Feb, 178(3), 619 - 24
Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase; Hahn FM et al.; Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway . A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus . The open reading frame was cloned into an Escherichia coli expression vector . The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate . Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R . capsulatus . More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli) . The R . capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme.

Science, 1996 Jan 26, 271(5248), 470 - 7
Determining divergence times of the major kingdoms of living organisms with a protein clock; Doolittle RF et al.; Amino acid sequence data from 57 different enzymes were used to determine the divergence times of the major biological groupings . Deuterostomes and protostomes split about 670 million years ago and plants, animals, and fungi last shared a common ancestor about a billion years ago . With regard to these protein sequences, plants are slightly more similar to animals than are the fungi . In contrast, phylogenetic analysis of the same sequences indicates that fungi and animals shared a common ancestor more recently than either did with plants, the greater difference resulting from the fungal lineage changing faster than the animal and plant lines over the last 965 million years . The major protist lineages have been changing at a somewhat faster rate than other eukaryotes and split off about 1230 million years ago . If the rate of change has been approximately constant, then prokaryotes and eukaryotes last shared a common ancestor about 2 billion years ago, archaebacterial sequences being measurably more similar to eukaryotic ones than are eubacterial ones.

Eur J Biochem, 1996 Jan 15, 235(1-2), 297 - 303
The RNA component of mitochondrial ribonuclease P from Aspergillus nidulans; Lee YC et al.; Several RNA molecules that copurified with Aspergillus nidulans mitochondrial ribonuclease (RNase) P were identified {Lee, Y C., Lee, B . J., Hwang, D . S . & Kang, H . S . (1996) Eur J . Biochem . 235, 289-296}, and their partial sequences were determined . Using an oligonucleotide probe, we cloned and mapped the gene encoding this putative RNA component of RNase P (RNase P-RNA), situated between URFA3 (unidentified reading frame A3) and cobA (apocytochrome b) genes in the mitochondrial genome of A . nidulans . The gene is extremely (A+T)-rich and contains two regions of sequence similarity conserved among the known mitochondrial RNase P-RNAs and the eubacterial RNase P-RNAs . The determination of 5' and 3' termini by primer extension and sequencing indicated that the length of the RNA transcript is 232 nucleotides . Northern-blot analysis revealed that its only subcellular location was the mitochondria . Two RNase P-RNA fragments of 110 nucleotides and 80 nucleotides, each containing one of the two conserved regions, could be recovered from the nuclease-treated enzyme without significant loss of activity . The sizes of these fragments appeared to be the minimum lengths required for the vitro activity of the enzyme.

FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 259 - 64
Primary structure of a cytosolic malate dehydrogenase of the amitochondriate eukaryote, Trichomonas vaginalis; Markos A et al.; The nucleotide sequence of a gene coding for a 37 kDa subunit of a cytosolic malate dehydrogenase of Trichomonas vaginalis was established . The sequence of a gDNA clone and a cDNA clone, which lacked seven amino-terminal codons, were identical, indicating an absence of introns from the gene . Cell fractionation combined with sequencing of peptide fragments of the purified enzyme showed that the gene codes for an expressed cytosolic enzyme . The derived amino acid sequence was closely related to cytosolic malate dehydrogenases from animals and plants and from the eubacteria Thermus aquaticus and Mycobacterium leprae and was more distant from the enzyme of mitochondria and from Escherichia coli and certain other eubacteria . In phylogenetic reconstructions this enzyme shared a most recent common ancestor with other cytosolic enzymes.

Mol Gen Genet, 1996 Jan 15, 250(1), 50 - 8
Transcriptional regulation of the iron-responsive sigma factor gene pbrA; Sexton R et al.; In response to the intracellular iron concentration Pseudomonas fluorescens M114 coordinately regulates the production of pseudobactin M114, its cognate receptor PbuA, and a casein protease . Transcriptional initiation of this coordinate iron-stress response requires the sigma factor PbrA . PbrA is a member of the ECF (Extracytoplasmic function) subgroup of the sigma 70 family of eubacterial RNA polymerase sigma factors . Regulatory studies of the pbrA gene utilising promoter-lacZ transcriptional fusions demonstrate that expression of pbrA dictates the cellular response to iron . pbrA is transcribed in all phases of iron-limited growth but maximally at late-logarithmic to stationary phase . pbrA expression is independent of autoregulatory control but is strictly repressed in iron-rich conditions in a Fur-dependent fashion . Constitutive expression of pbrA from an inducible tac promoter permits the induction of PbrA-dependent transcription and pseudobactin M114 biosynthesis in high-iron conditions . A PbrA consensus sequences was derived from significant DNA sequence homologies observed within the "-25 bp" and "-16 bp" regions conserved among all PbrA-dependent promoters . The predicted PbrA target promoter consensus is homologous for the promoter recognition sites for other environmentally responsive ECF sigma factors.

Biochemistry, 1996 Jan 9, 35(1), 315 - 20
Hydrolysis of dihydrouridine and related compounds; House CH et al.; Dihydrouridine is absent from the tRNA of almost all hyperthermophiles and most Archaea but is ubiquitous in the tRNA of Eubacteria and Eukaryotes . In order to investigate whether this could be due to instability, the rate of ring opening of dihydrouridine was measured between 25 and 120 degrees C . The dihydrouridine ring is stable at 25 degrees C, but the half-life at 100 degrees C and pH 7 is 9.1 h, which is comparable to the doubling time of hyperthermophiles . This suggests an explanation for the absence of dihydrouridine from the tRNA of hyperthermophiles . The rates of ring opening of dihydrouracil, dihydrothymine, and 1-N-methyldihydrouracil were measured at 100 degrees C and pH 6-9, as were the equilibrium constants for ring closure of the ureido acids to the dihydrouracils . The pH rate profiles for ring opening and ring closing were calculated from the data . Possible roles for dihydrouracils in the pre-RNA world are discussed.

Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 166 - 70
Evidence that two present-day components needed for the genetic code appeared after nucleated cells separated from eubacteria; Ribas de Pouplana L et al.; The trinucleotide/amino acid relationships of the present-day genetic code are established by the amino-acylation reactions of tRNA synthetases, whereby each of 20 specific amino acids is attached to its cognate tRNAs, which bear anticodon trinucleotides . Because of its universality, the appearance of the modern genetic code is thought to predate the separation of prokaryotic and eukaryotic organisms in the universal phylogenetic tree . In the light of new sequence information, we present here a phylogenetic analysis that shows an unusual picture for tyrosyl- and tryptophanyl-tRNA synthetases . Ij particular, the eukaryotic tyrosyl- and tryptophanyl-tRNA synthetases are more related to each other than to their respective prokaryotic counterparts . In contrast, each of the other 18 eukaryotic synthetases is more related to its prokaryotic counterpart than to any eukaryotic synthetase specific for a different amino acid . Our results raise the possibility that present day tyrosyl- and tryptophanyl-tRNA synthetases appeared after the separation of nucleated cells from eubacteria . The results have implications for the development of the genetic code.

Annu Rev Genet, 1996, 30, 79 - 107
Bacterial diversity based on type II DNA topoisomerase genes; Huang WM; Type II DNA topoisomerases are essential and ubiquitous DNA metabolic enzymes that alter DNA topology . Eubacteria have two indispensable type II DNA topoisomerases, DNA gyrase encoded by gyrB and gyrA and topoisomerase IV encoded by parE and parC . These genes belong to a single family whose members span both eukaryotes and prokaryotes . The highly conserved motifs in these genes provide a rationale for the design of universal primers used in the polymerase chain reaction in order to systematically generate a data set suitable for bacterial diversity studies at the macro-diversity level, as well as at the micro-diversity level displaying individual species and isolates . This family of genes is the subject of intensive biochemical and genetic analyses, which provide an opportunity for comprehensive understanding of sequence conservation and variability and their relationship to function . These genes are ideally suited for microbial identification and biodiversity analyses.

Med Dosw Mikrobiol, 1996, 48(1-2), 49 - 54
{Occurrence of non-spore forming anaerobic bacteria in the upper airways of patients with chronic obstructive pulmonary disease}; Jassem E et al.; The aim of this study was to assess the occurrence of anaerobic non-sporeforming bacteria upper airways flora in the exacerbation of COPD . Sputum from 35 COPD patients was sent to the laboratory in sterile, filled with CO2 containers and cultured under anaerobic conditions . In all patients bacteriological tests were positive . The most common anaerobic bacteria were as follows: Peptostreptococcus - in 28 pts, Fusobacterium - in 27 pts, Bacteroides - in 26 pts and Prevotella in 24 . Less common were: Propionibacterium, Actinomyces, Eubacterium, Porphyromonas and Peptococcus . The susceptibility to most common antimicrobial agents was evaluated . All anaerobic bacteria show a significant sensitivity to amoxicillin either with clavulanic acid or ampicillin with sulbactam . Gram-positive rods were resistant to metronidazole and tinidazole.

Annu Rev Microbiol, 1996, 50, 25 - 57
Molecular biology of mycoplasmas; Dybvig K et al.; Although mycoplasmas lack cell walls, they are in many respects similar to the gram-positive bacteria with which they share a common ancestor . The molecular biology of mycoplasmas is intriguing because the chromosome is uniquely small (< 600 kb in some species) and extremely A-T rich (as high as 75 mol% in some species) . Perhaps to accommodate DNA with a lower G + C content, most mycoplasmas do not have the "universal" genetic code . In these species, TGA is not a stop codon; instead it encodes tryptophan at a frequency 10 times greater than TGG, the usual codon for this amino acid . Because of the presence of TGA codons, the translation of mycoplasmal proteins terminates prematurely when cloned genes are expressed in other eubacteria, such as Escherichia coli . Many mycoplasmas possess strikingly dynamic chromosomes in which high-frequency changes result from errors in DNA repair or replication and from highly active recombination systems . Often, high-frequency changes in the mycoplasmal chromosome are associated with antigenic and phase variation, which regulate the production of factors critical to disease pathogenesis.

Annu Rev Biochem, 1996, 65, 801 - 47
Structure and functions of the 20S and 26S proteasomes; Coux O et al.; The proteasome is an essential component of the ATP-dependent proteolytic pathway in eukaryotic cells and is responsible for the degradation of most cellular proteins . The 20S (700-kDa) proteasome contains multiple peptidase activities that function through a new type of proteolytic mechanism involving a threonine active site . The 26S (2000-kDa) complex, which degrades ubiquitinated proteins, contains in addition to the 20S proteasome a 19S regulatory complex composed of multiple ATPases and components necessary for binding protein substrates . The proteasome has been highly conserved during eukaryotic evolution, and simpler forms are even found in archaebacteria and eubacteria . Major advances have been achieved recently in our knowledge about the molecular organization of the 20S and 19S particles, their subunits, the proteasome's role in MHC-class 1 antigen presentation, and regulators of its activities . This article focuses on recent progress concerning the biochemical mechanisms and intracellular functions of the 20S and 26S proteasomes.

Steroids, 1996 Jan, 61(1), 33 - 40
Mechanism of cholesterol reduction to coprostanol by Eubacterium coprostanoligenes ATCC 51222; Ren D et al.; The mechanism of reduction of cholesterol to coprostanol by Eubacterium coprostanoligenes ATCC 51222 was studied by incubating the bacterium with a mixture of alpha- and beta-isomers of {4-3H,4-14C}cholesterol . Coprostanol, isolated after incubation of {4-3H,4-14C}cholesterol in a growth medium under anaerobic conditions, retained 97% of the tritium originally present in cholesterol . The majority of this tritium (64%), however, was in the C-6 position in coprostanol, which showed that the conversion of cholesterol into coprostanol by E . coprostanoligenes involved the intermediate formation of 4-cholesten-3-one followed by the reduction of the latter to coprostanol . In resting cell assays in which washed bacterial cells were incubated with micellar cholesterol in phosphate buffer at 37 degrees C, both 4-cholesten-3-one and coprostanone were produced in addition to coprostanol . Furthermore, 5-cholesten-3-one, 4-cholesten-3-one, and coprostanone were converted efficiently to coprostanol by E . coprostanoligenes . These results support the hypothesis that the major pathway for reduction of cholesterol by E . coprostanoligenes involves the intermediate formation of 4-cholesten-3-one followed by reduction of the latter to coprostanol through coprostanone as an intermediate.

SAAS Bull Biochem Biotechnol, 1996, 9, 57 - 62
Regulation of the cellulolytic activity of Eubacterium cellulosolvens 5494: a review; Anderson KL et al.; Eubacterium cellulosolvens 5494 is a cellulolytic gram positive bacterium isolated from the rumen . Substrate specific regulation has not been previously demonstrated in any members of this genera . However, we have recently found that E . cellulosolvens regulates some of its membrane proteins . Growth on different substrates, including cellulose and cellobiose, gave different SDS-PAGE profiles of proteins from the membrane fraction . Using scanning electron microscopy, we also found that growth of E . cellulosolvens on cellulose induces an ultrastructural complex that is not present when grown on any other substrate . Further study revealed that this ultrastructure was subsequently lost when an alternative substrate was made available to cellulose growing cells . We also found that cellulose, cellobiose, and maltose utilization are inhibited in the presence of a glucose analog, indicating glucose is the preferred substrate.

Int J Urol, 1996 Jan, 3(1), 31 - 4
Reduction of oxalate content of foods by the oxalate degrading bacterium, Eubacterium lentum WYH-1; Ito H et al.; BACKGROUND: Urinary oxalate may contribute far more than urinary calcium to the pathogenesis of urinary calculi . Urinary oxalate may be reduced by restricting the intake of foods high in oxalate . The oxalate content foods might be reduced by oxalate-degrading bacteria . The purpose of this experiment was to reduce the oxalate content of foods with an oxalate-degrading bacterium which was isolated from the feces of Japanese male . METHODS: An artificial intestinal juice was prepared by modifying Rogosa medium . An infusion of black tea was prepared from a commercial tea bag . The oxalate-degrading bacteria used were Eubacterium lentum WYH-1 which were have isolated . To 5 ml of the above oxalate-containing artificial intestinal juice and infusion of black tea, 0.5 ml of the bacterial culture was added and incubated anaerobically at 37 degrees C . Oxalic acid in the supernatant of the culture medium was assayed by high-performance liquid chromatography . RESULTS: In 24 hours, 1 x 10(6) cells/ml of Eubacterium lentum WYH-1 decomposed 100% of 1 mg/ml oxalate in the artificial intestinal juice . The oxalate in the black tea infusion (1 mg/mL) was also decomposed completely within 48 hours by 1 x 10(7) cells/mL of bacteria . CONCLUSION: Eubacterium lentum WYH-1 was able to efficiently decompose the oxalate in foods.

J Dent, 1996 Jan-Mar, 24(1-2), 47 - 55
Clinical significance of dental root canal microflora; Gomes BP et al.; OBJECTIVES: Previous work by this group has shown that a significant association exists between pain and the presence of either Prevotella or Peptostreptococcus spp . in dental root canals . The aim of this study was to examine a more extensive series of canals microbiologically, to determine whether any other particular endodontic symptoms or clinical signs showed specific associations with individual bacterial species . METHODS: Seventy root canals were examined microbiologically and clinical data collected to investigate in detail such associations . RESULTS: Of the canals studied, 37 were associated with pain, 49 with tenderness to percussion, 23 with swelling, six with purulent exudate and 57 presented with wet root canals . Anaerobes were isolated from 70.3% of painful canals and from 29.7% of pain-free canals . Significant associations were found between (a) pain and either Prevotella spp . or peptostreptococci, both with P < 0.01; (b) tenderness to percussion and Prevotella spp . (P < 0.01) or anaerobes (P < 0.05); (c) swelling and Eubacterium spp . (P < 0.01), or with Prevotella spp . or Pstr . micros, both with P < 0.05; (d) purulent exudate and any one of F . necrophorum (P < 0.01), Prev . loescheii, Streptoccoccus constellatus or Bacteroides spp . (each P < 0.05); (e) wet canal and facultative anaerobes (P < 0.01), and any one of the genera of Eubacterium, Peptostreptococcus, Prevotella or Propionibacterium (each P < 0.05) . CONCLUSION: It was concluded that several different endodontic clinical signs and symptoms are significantly associated with specific bacterial species.

Plant Mol Biol, 1996 Jan, 30(1), 213 - 8
Transaldolase genes from the cyanobacteria Anabaena variabilis and Synechocystis sp . PCC 6803: comparison with other eubacterial and eukaryotic homologues; Kohler U et al.; We have sequenced and analysed the transaldolase (tal) genes from two cyanobacteria, Anabaena variabilis (ATCC 29413) and Synechocystis sp . PCC 6803, which are filamentous heterocyst-forming and unicellular organisms, respectively . The deduced amino acid sequences of the two cyanobacterial tal genes are 78% identical and are highly homologous to both eubacterial and eukaryotic transaldolases (Escherichia coli, two yeasts, and man) with values ranging from 54 to 60% amino acid identity . In contrast, the transaldolase homologous sequences from the cyanobacterium Nostoc sp . ATCC 29133, from Mycobacterium leprae, and the partial sequence from the higher plant Arabidopsis thaliana have a much lower degree of homology with each other and relative to the sequences mentioned above . These data indicate three different types of transaldolases.

Adv Biochem Eng Biotechnol, 1996, 53, 119 - 78
Microbial carotenoids; Johnson EA et al.; Carotenoids occur universally in photosynthetic organisms but sporadically in nonphotosynthetic bacteria and eukaryotes . The primordial carotenogenic organisms were cyanobacteria and eubacteria that carried out anoxygenic photosynthesis . The phylogeny of carotenogenic organisms is evaluated to describe groups of organisms which could serve as sources of carotenoids . Terrestrial plants, green algae, and red algae acquired stable endosymbionts (probably cyanobacteria) and have a predictable complement of carotenoids compared to prokaryotes, other algae, and higher fungi which have a more diverse array of pigments . Although carotenoids are not synthesized by animals, they are becoming known for their important role in protecting against damage by singlet oxygen and preventing chronic diseases in humans . The growth of aquaculture during the past decade as well as the biological roles of carotenoids in human disease will increase the demand for carotenoids . Microbial synthesis offers a promising method for production of carotenoids.

Int J Syst Bacteriol, 1996 Jan, 46(1), 348 - 51
The phylogeny of Methanopyrus kandleri; Rivera MC et al.; The phylogenetic position of Methanopyrus kandleri has been difficult to determine because reconstructions of phylogenetic trees based on rRNA sequences have been ambiguous . The most probable trees determined by most algorithms place the genus Methanopyrus at the base of a group that includes the halobacteria and the methanogens and their relatives, although occasionally some algorithms place this genus near the eocytes (the hyperthermophilic, sulfur-metabolizing prokaryotes), suggesting that it may belong to this lineage . In order to resolve the phylogeny of the genus Methanopyrus, we determined the sequence of an informative region of elongation factor 1-alpha that contains an 11-amino-acid insertion in eocytes and eukaryotes which is replaced by a 4-amino-acid insertion in methanogens, halobacteria, and eubacteria . On the basis of the results of our elongation factor 1-alpha gene analysis, we concluded that the genus Methanopyrus diverged from the eocyte branch before the eukaryotic and eocyte lineages separated and therefore is not an eocyte.

Int J Syst Bacteriol, 1996 Jan, 46(1), 31 - 4
Eubacterium minutum sp . nov., isolated from human periodontal pockets; Poco SE Jr et al.; We describe Eubacterium minutum sp . nov., which was isolated from human periodontal pockets . This new species was established on the basis of DNA-DNA hybridization data . The guanine-plus-cytosine content of its DNA is 38 to 40 mol% . The results of differential biochemical and enzymatic tests are described . The type strain of this species is strain M-6.

Curr Microbiol, 1996 Jan, 32(1), 38 - 42
Selective acylation of plasma membrane proteins of Mycoplasma mycoides subsp . mycoides SC, the contagious bovine pleuropneumonia agent; Jan G et al.; The plasma membrane of Mycoplasma mycoides subsp . mycoides SC (strain KH3J) contains over 160 polypeptides with apparent molecular masses ranging from 14 to 125 kDa and isoelectric point values (pIs) from 5 to 9 . In vivo labeling with {14C}-fatty acids revealed about 35 acylated polypeptides including the two major components p42 and p65 and displaying pIs between 5.5 and 9.0, with a majority between 6.5 and 8 . The amphiphilic nature of most of these acyl proteins was confirmed by Triton X-114 phase partitioning . Gas-liquid chromatography analyses showed that the order of preference for protein acylation was 16:0 > 18:2c > 18:1c > 18:0 > 14:0, with 16:0 being the major O-ester-bound fatty acyl chain and 18:2c the major N-linked chain . The presence of S-glycerylcysteine and a ratio of {O-ester-bound acyl chains + N-linked chains}/O-ester bound chains of approximately 1.2 in M . mycoides subsp . mycoides SC membrane proteins are consistent with a lipid modification similar to that occurring in lipoproteins of Gram-negative eubacteria that contain an N-terminal acyl S-glycerylcysteine.

J Bacteriol, 1996 Jan, 178(1), 19 - 23
3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli; Bischoff KM et al.; Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases . We report here the characterization of an HMG-CoA reductase from the third domain, the archaea . HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H . volcanii . Subsequently, a portion of the H . volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7 . While no HMG-CoA reductase activity was detectable following expression in E . coli, activity could be recovered after extracts were exposed to 3 M KCl . Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P . mevalonii HMG-CoA reductase . Activity was optimal at pH 7.3 . Kms were 66 microM (NADPH) and 60 microM {(S)-HMG-CoA} . (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA . H . volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde {optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM {(R,S)-mevaldehyde}} and the oxidative acylation of mevaldehyde {optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM {(R,S)-mevaldehyde}} . These properties are comparable to those of hamster and P . mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism.

Gene, 1995 Dec 29, 167(1-2), 297 - 302
Isolation and characterization of a cDNA encoding a new type of human transcription elongation factor S-II; Umehara T et al.; We report the isolation of a cDNA encoding a new type of transcription factor S-II, termed h-SII-T1, from a human library . The mRNA corresponding to the clone is highly expressed in testis and ovary . Comparison of the deduced amino acid (aa) sequence with those of other S-II molecules shows that (i) the C-terminal zinc finger (Zf) domain is highly conserved, and (ii) the central segment is most similar to that of the rat testis-specific S-II . Further analyses of the hS-II-T1 aa sequence indicate that its N-terminal sequence exhibits similarity to eubacterial sigma 54 . The significance of tissue-specific S-II molecules for the regulation of transcription elongation is discussed.

Gene, 1995 Dec 29, 167(1-2), 137 - 40
The recA gene of Borrelia burgdorferi; Dew-Jager K et al.; The nucleotide sequence of the Borrelia burgdorferi (Bb) Sh-2-82 recA gene has been determined using PCR-based approaches without the construction of a genomic library . The gene should encode a protein of 365 amino acids which is highly homologous to other known RecA proteins . It represents a new homolog from a distinct phylogenetic branch of eubacteria . Although, previous reports concluded that recA is absent from Bb, the identification presented here conclusively shows its presence and reaffirms the ubiquity of RecA in prokaryotes.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12510 - 4
Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts between the RNase P RNA and pre-tRNA; Pan T et al.; A general method has been developed to analyze all 2' hydroxyl groups involved in tertiary interactions in RNA in a single experiment . This method involves comparing the activity of populations of circularly permuted RNAs that contain or lack potential hydrogen-bond donors at each position . The 2' hydroxyls of the pre-tRNA substrate identified as potential hydrogen bond donors in intermolecular interactions with the ribozyme from eubacterial RNase P (P RNA) are located in the T stem and T loop, acceptor stem, and 3' CCA regions . To locate the hydrogen-bond acceptors for one of those 2' hydroxyls in the P RNA, a phylogenetically conserved adenosine was mutated to a guanosine . When this mutant P RNA was used, increased cleavage activity of a single circularly permuted substrate within the population was observed . The cleavage efficiency (kcat/Km) of a singly 2'-deoxy-substituted substrate at this position in the T stem was also determined . For the wild-type P RNA, the catalytic efficiency was significantly decreased compared with that of the all-ribo substrate, consistent with the notion that this 2' hydroxyl plays an important role . For the P RNA mutant, no additional effect was found upon 2'-deoxy substitution . We propose that this particular 2' hydroxyl in the pre-tRNA interacts specifically with this adenosine in the P RNA . This method should be useful in examining the role of 2' hydroxyl groups in other RNA-RNA and RNA-protein complexes.

Chin Med Sci J, 1995 Dec, 10(4), 195 - 8
Taxonomic status of car bacillus based on the small subunit ribosomal RNA sequences; Wei Q et al.; In an attempt to identify the taxonomic relationship between CAR bacillus and other bacteria, the SSU rRNA gene sequences of two CAR bacillus strains, CBM and CBR isolated from mice and rats respectively were used in the present studies . The SSU rRNA gene sequences, approximately 1.5 kb in size amplified from genomic DNAs from both strains, were determined and 96.8% homologies were found to exist between them . Those sequences were aligned to most eubacteria with a computer search showing high homology with those of Flavobacter/Flexibacter species especially closed to Fx . sancti and Fv . ferrugineum . Phylogenetic analysis indicated that CAR bacillus belongs to a species close to Fx . sancti and Fv . ferrugineum subdivision.

Glycobiology, 1995 Dec, 5(8), 791 - 6
Glycan structure of a heptose-containing S-layer glycoprotein of Bacillus thermoaerophilus; Kosma P et al.; The characterization of the S-layer glycoprotein of Bacillus thermoaerophilus revealed unexpected novelties . The isolation and purification procedure had to be changed due to complete solubility in aqueous buffers of the constituting S-layer protomers . Upon degradation of the S-layer glycoprotein by pronase and purification of the products by gel filtration, ion-exchange chromatography, chromatofocusing and HPLC, one representative glycopeptide fraction was selected for further characterization . From the combined evidence of composition analysis, chemical degradation, NMR spectroscopy experiments and comparison with synthesized model substance, we propose the following repeating unit structure of the glycan chain: -->4)-alpha-L-Rhap-(1-->3)-beta-D-glycero-D-manno-Hepp-(1--> This is the first description of heptose residues occurring as a constituent of S-layer glycoproteins of gram-positive eubacteria.

Asian Pac J Allergy Immunol, 1995 Dec, 13(2), 129 - 37
Antitumor mechanisms of Eubacterium lentum and its components; Hatta M; In the present study, some antitumor mechanisms of Eubacterium lentum (TYH-11) and bacterial components having antitumor effects were investigated . E.lentum induced maximum NK cell activity in C3H/He mice on day 1 after injection (90.6% against 33.9% of control at E:T ratio 50:1) and the activity was kept at a level of 48.6% on day 7 . Tumoricidal peritoneal macrophages were induced 9 days after E.lentum injection into BALB/c mice (56.2% against 10.1% control at E:T ratio 10:1) . Tumoricidal macrophage activity persisted at the same level for at least 11 days . Cytotoxic T lymphocyte (CTL) activity was induced only in tumor bearing mice treated with E.lentum, 4 weeks after tumor inoculation . Antitumor activity was observed in the cell wall (CW) and membrane fractions (CM) of E.lentum . CW induced NK cell activity; the activity was transient while the kinetics of NK activity by CM showed 2 peaks, on day 1 and day 7 . Tumoricidal macrophages were induced by CW and the activity level was the same as that induced by whole body, while that induced by CM was at a lower level . Neither CW nor CM induced CTL in tumor bearing mice.

J Mol Evol, 1995 Dec, 41(6), 1038 - 47
The contribution of slippage-like processes to genome evolution; Hancock JM; Simple sequences present in long (> 30 kb) sequences representative of the single-copy genome of five species (Homo sapiens, Caenorhabditis elegans, Saccharomyces cerevisiae, E . coli, and Mycobacterium leprae) have been analyzed . A close relationship was observed between genome size and the overall level of sequence repetition . This suggested that the incorporation of simple sequences had accompanied increases of genome size during evolution . Densities of simple sequence motifs were higher in noncoding regions than in coding regions in eukaryotes but not in eubacteria . All five genomes showed very biased frequency distributions of simple sequence motifs in all species, particularly in eukaryotes where AAA and TTT predominated . Interspecific comparisons showed that noncoding sequences in eukaryotes showed highly significantly similar frequency distributions of simple sequence motifs but this was not true of coding sequences . ANOVA of the frequency distributions of simple sequence motifs indicated strong contributions from motif base composition and repeat unit length, but much of the variation remained unexplained by these parameters . The sequence composition of simple sequences therefore appears to reflect both underlying sequence biases in slippage-like processes and the action of selection . Frequency distributions of simple sequence motifs in coding sequences correlated weakly or not at all with those in noncoding sequences . Selection on coding sequences to eliminate undesirable sequences may therefore have been strong, particularly in the human lineage.

Plant Mol Biol, 1995 Dec, 29(5), 1057 - 70
Isolation, expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana; Kuhlman P et al.; We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu) . This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes . However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu . Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves . Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus . As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.

FASEB J, 1995 Dec, 9(15), 1559 - 69
The role of molecular chaperones in protein folding; Hendrick JP et al.; Folding of newly synthesized polypeptides in the crowded cellular environment requires the assistance of so-called molecular chaperone proteins . Chaperones of the Hsp70 class and their partner proteins interact with nascent polypeptide chains on ribosomes and prevent their premature (mis)folding at least until a domain capable of forming a stable structure is synthesized . For many proteins, completion of folding requires the subsequent interaction with one of the large oligomeric ring-shaped proteins of the chaperonin family, which is composed of the GroEL-like proteins in eubacteria, mitochondria, and chloroplasts, and the TRiC family in eukaryotic cytosol and archaea . These proteins bind partially folded polypeptide in their central cavity and promote folding by ATP-dependent cycles of release and rebinding . In these reactions, molecular chaperones interact predominantly with the hydrophobic surfaces exposed by nonnative polypeptides, thereby preventing incorrect folding and aggregation.

J Bacteriol, 1995 Dec, 177(23), 6958 - 65
Cloning and characterization of senC, a gene involved in both aerobic respiration and photosynthesis gene expression in Rhodobacter capsulatus; Buggy J et al.; The purple nonsulfur photosynthetic eubacterium Rhodobacter capsulatus is a versatile organism that can obtain cellular energy by several means, including the capture of light energy for photosynthesis as well as the use of light-independent respiration, in which molecular oxygen serves as a terminal electron acceptor . In this study, we have identified and characterized a novel gene, senC, mutations in which affect respiration as well as the induction of photosynthesis gene expression . The protein coded by senC exhibits 33% sequence identity to the yeast nucleus-encoded protein SCO1, which is thought to be a mitochondrion-associated cytochrome c oxidase assembly factor . Like yeast SCO1, SenC is required for optimal cytochrome c oxidase activity in aerobically grown R . capsulatus cells . We further show that senC is required for maximal induction from the puf and puh operons, which encode the structural polypeptides of the light-harvesting and reaction center complexes.

J Bacteriol, 1995 Dec, 177(23), 6881 - 93
Bacterial classifications derived from recA protein sequence comparisons; Karlin S et al.; RecA protein sequences from 62 eubacterial sources were compared with one another and relative to one archaebacterial RecA-like and a number of eukaryotic RecA-like sequences . Pairwise similarity scores were determined by a novel method based on significant segment pair alignment . The sequences of different species were grouped on the basis of mutually high similarity scores within groups and consistency of score ranges in comparison to other groups . Following this protocol, the gamma-proteobacteria can be subclassified into two major groups, those of mostly vertebrate hosts and those of mostly soil habitat . The alpha-proteobacterial sequences also divide into two distinct groups, whereas classification of the beta-proteobacteria is more complex . The gram-positive bacterial sequences split into three groups of low and three groups of high G+C genome content . However, neither the combined low-G+C-content nor the combined high-G+C-content group nor the aggregate of all gram-positive bacteria form homogeneous groups . The mycoplasma sequences score best with the Bacillus subtilis sequence, consistent with their presumed origin from a gram-positive ancestor . The eukaryotic RAD proteins generally show a single high-scoring segment pair with the proteobacterial RecA sequences around the ATP-binding domain . The bacteriophage T4 UvsX protein aligns best with RecA sequences on two segments disjoint from the ATP-binding domain . The distribution of the most highly conserved regions shared between RecA and noneubacterial RecA-like sequences suggests a mosaic character and evolution of RecA . The discussion considers some questions on the validity and consistency of bacterial classifications derived from RecA sequence comparisons.

Infect Immun, 1995 Dec, 63(12), 4584 - 8
Intergeneric coaggregation of oral Treponema spp . with Fusobacterium spp . and intrageneric coaggregation among Fusobacterium spp; Kolenbrander PE et al.; A total of 22 strains of Treponema spp . including members of all four named human oral species were tested for coaggregation with 7 strains of oral fusobacteria, 2 strains of nonoral fusobacteria, and 45 strains of other oral bacteria, which included actinobacilli, actinomyces, capnocytophagae, eubacteria, porphyromonads, prevotellae, selenomonads, streptococci, and veillonellae . None of the treponemes coaggregated with any of the latter 45 oral strains or with the two nonoral fusobacteria . All treponemes, eight Treponema denticola strains, eight T . socranskii strains, four oral pectinolytic treponemes, one T . pectinovorum strain, and one T . vincentii strain coaggregated with at least one strain of the fusobacteria tested as partners . The partners consisted of one strain of Fusobacterium periodonticum, five F . nucleatum strains including all four subspecies of F . nucleatum, and a strain of F . simiae obtained from the dental plaque of a monkey . In the more than 100 coaggregations observed, the fusobacterial partner was heat inactivated (85 degrees C for 30 min), while the treponemes were unaffected by the heat treatment . Furthermore, the fusobacteria were usually inactivated by proteinase K treatment, and the treponemes were not affected . Only the T . denticola coaggregations were inhibited by lactose and D-galactosamine . None were inhibited by any of 23 other different sugars or L-arginine . Intragenic coaggregations were seen among the subspecies of F . nucleatum and with F . periodonticum, and none were inhibited by any of the sugars tested or by L-arginine . No intrageneric coaggregations were observed among the treponemes . These data indicate that the human oral treponemes show a specificity for oral fusobacteria as coaggregation partners . Such cell-to cell contact may facilitate efficient metabolic communication and enhance the proliferation of each cell in the progressively more severe stages of periodontal disease.

Eur J Biochem, 1995 Nov 15, 234(1), 184 - 91
Purification and characterization of protein PB of betaine reductase and its relationship to the corresponding proteins glycine reductase and sarcosine reductase from Eubacterium acidaminophilum; Meyer M et al.; Simple complementation assay systems were developed for the substrate-specific proteins PB of glycine reductase, sarcosine reductase, and betaine reductase, in which acetyl phosphate was detected as the product in all three cases . The betaine-specific subunits of protein B (PB betaine) responsible for betaine reductase activity were purified to homogeneity from cells of Eubacterium acidaminophilum . The molecular masses of the two different subunits were 45 kDa and 48 kDa according to SDS/PAGE . The molecular mass of the native protein was about 200 kDa, indicating and alpha 2 beta 2 structure . The glycine-specific protein B (PB glycine) was partially purified and subunits of 47 kDa and 27 kDa were N-terminally sequenced . The latter subunits cross-reacted with antibodies raised against PB betaine and showed high sequence similarity to the 45-kDa and 48-kDa subunits of PB betaine, respectively . {2-14C}Glycine could be covalently coupled to the 47-kDa subunit by treatment with borohydride . By the same procedure, {2-14C}sarcosine labeled a protein of the same size . Like the sarcosine reductase activity, this protein was not present in glycine-grown cells, indicating its specific involvement in sarcosine metabolism . The labile viologen-dependent formate dehydrogenase purified with the respective PB proteins and could be tentatively assigned to a 95-kDa protein.

Biochem Biophys Res Commun, 1995 Nov 13, 216(2), 495 - 500
The identification and partial characterisation of a novel inducible extracellular thermostable esterase from the archaeon Sulfolobus shibatae; Huddleston S et al.; Extracellular esterases have so far only been reported in eubacteria, here we report the first identification and partial characterisation of a novel inducible extracellular esterase from the thermoacidophilic archaeon Sulfolobus shibatae . This esterase exhibits remarkable stability to both acid and heat . Esterase activity is induced by growth on a range of polyoxyethylenesorbitan (Tween) compounds as sole carbon source . Activity occurs over a wide temperature (25-99 degrees C) and pH (pH4.0-9.0) range and is optimal at 90 degrees C and pH6.0 . It exhibits high thermal stability, with a half-life of 20 min at 120 degrees C, and shows a transient thermal activation of 60% at 90 degrees C . The thermal inactivation of function occurs by first order kinetics, and after 120 min incubation at 120 degrees C 50% of activity still remains . It is able to hydrolyse mono- and diglycerides, but is unable to hydrolyse the triglycerides olive oil and triolein, which is indicative of an esterase and not a lipase.

Biochim Biophys Acta, 1995 Nov 7, 1264(2), 173 - 7
An archaeal gene upstream of grpE different from eubacterial counterparts; Macario AJ et al.; In some eubacteria with a dnaK locus in which grpE is close upstream of dnaK, grpE is preceded by an open reading frame (orf) believed to be a heat-shock gene . We also found an orf, orf16, upstream of grpE in the archaeon Methanosarcina mazei S-6, but this gene differs from the eubacterial counterpart: it is shorter, does not respond to a temperature upshift as heat-shock genes do, and the deduced protein Orf16, does not resemble the proteins coded by the eubacterial equivalents . orf16 is expressed monocistronically, with a transcription initiation site 24 bases upstream of the translation start codon, 22 bases downstream of a putative promoter identical to the consensus promoter for genes in methanogens . This initiation site is used by heat-shocked and non-heat-shocked cells in the two morphologic stages of M . mazei S-6 tested, i.e., packets and single cells . Three transcription termination sites were identified, one of which is detectable only in non-heat-shocked cells . Data from comparative analyses of the Orf16 deduced amino acid sequence and those of other known proteins, as well as the apparent biochemical characteristics of Orf16, suggest that the latter is a membrane molecule.

Gene, 1995 Nov 7, 165(1), 81 - 6
Characterization of the unlinked 16S rDNA and 23S-5S rRNA operon of Wolbachia pipientis, a prokaryotic parasite of insect gonads; Bensaadi-Merchermek N et al.; The rRNA-encoding genes (rDNAs) have been cloned and characterized from Wolbachia pipientis (Wp), the gonadial bacteria-like parasite of the mosquito Culex pipiens (Cp) and the moth Ephestia cautella (Ec) . In Wp from both insect species the rDNAs are organized in a way which appears to be very unusual . The rRNAs are encoded by two unlinked transcription units, each present in a single copy per genome . One contains the 16S rDNA only, while the other is an operon encoding both the 23S and 5S rDNAs . Each transcription unit contains two putative upstream promoters, and downstream a Rho-independent terminator . The 16S rDNA, as well as the 23S-5S rRNA operon are not linked to any tRNA-encoding sequence and lack the antitermination boxes which are usually present immediately downstream from eubacterial promoters of rDNAs . Wp infecting Ec and Cp are highly similar taking as criteria the rDNAs and their flanking sequences . However, it clearly appears that each insect species harbours a different and specific Wp strain, or even subspecies . Phylogenetic relationships deduced from the complete sequences of their rDNAs undoubtedly confirm that Wp from Cp and Ec belong to the alpha-group of Proteobacteria, and are closely related to the Rickettsia.

Gene, 1995 Nov 7, 165(1), 51 - 6
Characterization and overexpression of the gene encoding Staphylococcus aureus DNA polymerase III; Pacitti DF et al.; The polC gene specifying DNA polymerase III (PolIII) of Staphylococcus aureus (Sa), was cloned with a novel strategy and found to contain a 4305-bp open reading frame (ORF) encoding a polypeptide of approx . 162 kDa . The 1435-codon ORF was engineered into an Escherichia coli (Ec) expression plasmid under the control of the lac promoter and its repressor . Derepression of Ec transformants carrying the recombinant (re-) vector generated high-level synthesis of active re-Sa PolIII . The re-PolIII was purified to > 98% homogeneity and was shown by N-terminal amino acid sequence analysis to be the bona fide product of the Sa polC ORF . The physical and catalytic properties of re-Sa PolIII and its responsiveness to inhibitors of the HPUra type were generally similar to those of Bacillus subtilis (Bs) PolIII . Comparative analysis of the primary structures of Sa PolIII, Bs PolIII and Mycoplasma pulmonis PolIII indicated strong conservation of essential catalytic domains and a novel zinc-finger motif . Comparison of the primary structures of Ec PolIII and these three Gram+ enzymes revealed a region of novel homology and reinforced the likelihood of a specific evolutionary relationship between PolIII of Gram+ and Gram- eubacteria . The polC gene mapped between omega 1074 {Tn551} and recA/ngr on the Sa NCTC 8325 genome.

Protein Eng, 1995 Nov, 8(11), 1171 - 5
The amino acid sequence required for 5' --> 3' exonuclease activity of Bacillus caldotenax DNA polymerase; Ishino Y et al.; We studied the 5' --> 3' exonuclease activity of Bacillus caldotenax DNA polymerase by site-directed mutagenesis . Among seven mutants constructed, two mutant DNA polymerases with an amino acid substitution of Gly184 --> Asp or Gly192 --> Asp were confirmed to be deficient in this exonuclease . The two positions corresponded to those of the Escherichia coli DNA polymerase I mutants defective in 5' --> 3' exonuclease, polA480ex and polA214 . These results provide experimental support for the proposed amino acid sequence essential for the 5' --> 3' exonuclease activity associated with eubacterial polymerase I-like DNA polymerases (family A), including E.coli and Thermus aquaticus.

Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1187 - 97
Structural and functional implications in the eubacterial ribosome as revealed by protein-rRNA and antibiotic contact sites; Wittmann-Liebold B et al.; Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2-iminothiolane as cross-linkers . Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, and L36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified . Further, the binding sites of puromycin and spiramycin were established at the peptide level in several proteins that were found to constitute the antibiotic-binding sites . Peptide stretches of puromycin binding were identified from proteins S7, S14, S18, L18, AND L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35 . Comparison of the RNA-peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides . Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E . coli ribosomes that were active . The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes . These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis . Incorporation studies with a mutant of HmaL2 with a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome . Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.

Genetika, 1995 Nov, 31(11), 1566 - 74
{A comparative computer analysis of thermodynamic parameters of transport RNA secondary structure}; Kuznetsov IB et al.; Thermodynamic parameters of the cloverleaf secondary structure of tRNAs of several major taxons (archaebacteria, eubacteria, eukaryotes, chloroplasts, and mitochondria) were subjected to computer analysis . The distribution of free-energy values was close to normal in all groups (except for the mitochondrial tRNA) . In the organisms existing under extreme environmental conditions, the stability of the tRNA secondary structure was higher . Comparative analysis of teh frequency of inverted question markquasi-complementary inverted question mark GU pairs in stems of randomly generated and real sequences demonstrated preferential fixation of such pairs in the contexts with the most favorable thermodynamics parameters . Noncanonic pairs in tRNA stems set limits on the presence of highly mutable CG dinucleotides . Moreover, the effect of noncanonic pairs other than GU on the frequency of such dinucleotides is far more pronounced than that of GU pairs.

Antimicrob Agents Chemother, 1995 Nov, 39(11), 2466 - 71
Intracellular accumulation of norfloxacin in Mycobacterium smegmatis; Corti S et al.; To evaluate the intracellular accumulation of norfloxacin in mycobacteria, two methods were used with Mycobacterium smegmatis . A radiometric method (K . V . Cundy, C . E . Fasching, K . E . Willard, and L . R . Peterson, J . Antimicrob . Chemother . 28:491-497, 1991) was used without great modification, but the fluorometric method (P . G . S . Mortimer and L . J . V . Piddock, J . Antimicrob . Chemother . 28:639-653, 1991) was changed considerably . Indeed, adsorption of the quinolone to the bacterial surface was characterized by measuring the level of accumulation of 0 degree C . Taking into account the adsorption, the pH of the washing buffer was increased from 7.0 to 9.0 to improve the desorption of norfloxacin from the cell surface . Both the fluorometric method, with the technical improvement, and the radiometric method could be used to estimate the intracellular accumulation of norfloxacin, which resulted from the difference between the whole uptake measured at 37 degrees C and the adsorption measured at 0 degrees C . A total of 35 ng of norfloxacin per mg of cells (dry weight) penetrated into the M . smegmatis cell, and the steady state was achieved in 5 min . Use of inhibitors of the proton motive force revealed that transport of norfloxacin was energy independent . Thus, the same mechanisms of quinolone accumulation that occur in eubacteria seem to occur in mycobacteria, at least in M . smegmatis.

Res Microbiol, 1995 Nov-Dec, 146(9), 739 - 50
Acylation and immunological properties of Mycoplasma gallisepticum membrane proteins; Jan G et al.; The acylation of Mycoplasma gallisepticum membrane proteins was studied by electrophoresis after in vivo labelling with different 14C-fatty acids and by chemical analysis . The immunological properties of these proteins were investigated by Western blotting and crossed immunoelectrophoresis . Among the ca . 200 membrane polypeptides resolved by two-dimensional electrophoresis, 35 components (including the major protein p67) were covalently modified with acyl chains . These acylated proteins displayed lower pls than average (5.0-7.4 vs . 5.0-9.0) and proved to be the major membrane protein antigens and immunogens of M . gallisepticum . The apparent selectivity of fatty acid incorporation into proteins was, as suggested by in vivo labelling: palmitic acid (16:0) > myristic acid (14:0) > oleic acid (18:1c) > stearic acid (18:0) > linoleic acid (18:2c) . However, the true order of selectivity, as revealed by chemical analysis, proved to be 18:2c > 16:0 > 18:1c > 18:0 > 14:0 . More specifically, palmitic acid was the major O-ester-bound fatty acid and linoleic acid the major amide-linked fatty acid . The observed average ratio {O-ester-bound + amide-linked acyl chains}/O-ester-bound chains approximately 1.4 and the presence of S-glycerylcysteine suggest that, in M . gallisepticum, membrane proteins are lipid-modified according to a mechanism identical to that depicted for lipoproteins of Gram-negative eubacteria.

Plant J, 1995 Nov, 8(5), 751 - 62
Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins; Stallmeyer B et al.; The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes . It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes . This paper reports the isolation of cnx1, a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA . The mapping data of this cDNA correlate well with the mapping position of the A . thaliana molybdenum co-factor locus chl6 . As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin . The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E . coli Moco protein MoeA, the C-terminal domain is homologous to the E . coli Moco proteins MoaB and MogA, respectively . These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E . coli, is performed by a single multifunctional protein in eukaryotes . In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis . These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1 . Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.

J Eukaryot Microbiol, 1995 Nov-Dec, 42(6), 696 - 701
Preliminary characterisation of chlorarachniophyte mitochondrial DNA; Gilson P et al.; The division Chlorarachniophyte comprises amoeboflagellate protists with complex chloroplasts derived from the endosymbiosis of a eukaryotic alga . Analysis of chlorarachniophyte chromosomal DNAs by pulsed-field gel electrophoresis revealed an apparently linear 36-kb chromosome that could not be ascribed to either the host or endosymbiont nuclei . A single eubacterial-like small subunit ribosomal RNA gene is encoded on this chromosome and phylogenetic analyses places this gene within a clade of mitochondrial genes from other eukaryotes . High resolution in situ hybridization demonstrates that transcripts of the small subunit ribosomal RNA gene encoded by the 36-kb chromosome are exclusively located in the mitochondria . The 36-kb chromosome thus likely represents a linear mitochondrial genome . Small amounts of an apparently dimeric (72 kb) form are also detectable in pulsed-field gel electrophoresis.

J Bacteriol, 1995 Nov, 177(22), 6527 - 35
Mycobacterium smegmatis dnaA region and autonomous replication activity; Rajagopalan M et al.; Two key elements that are thought to be required for replication initiation in eubacteria are the DnaA protein, a trans-acting factor, and the replication origin, a cis-acting element . As a first step in studying the replication initiation process in mycobacteria, we have isolated a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that contains the dnaA gene . Nucleotide sequence analysis of this region revealed homologies with the rpmH gene, which codes for the ribosomal protein L34, the dnaA gene, which codes for the replication initiator protein DnaA, and the 5' end of the dnaN gene, which codes for the beta subunit of DNA polymerase III . Further, we provide evidence that when cloned into pUC18, a plasmid that is nonreplicative in M . smegmatis, the DNA fragment containing the dnaA gene and its flanking regions rendered the former capable of autonomous replication in M . smegmatis . We suggest that the M . smegmatis chromosomal origin of replication is located within the 4-kb DNA fragment.

DNA Res, 1995 Oct 31, 2(5), 231 - 4
Ribonuclease-P RNA gene of the plastid chromosome from Cyanophora paradoxa; Shevelev EL et al.; The gene, rnpB, encoding the RNA portion of ribonuclease-P has been found in the cyanelle DNA of Cyanophora paradoxa . A secondary structure model for the cyanelle RNA fits into that for eubacterial Rapb-RNAs.

Proc R Soc Lond B Biol Sci, 1995 Oct 23, 262(1363), 87 - 93
Multiple origins of anaerobic ciliates with hydrogenosomes within the radiation of aerobic ciliates; Embley TM et al.; Some ciliates live anaerobically and lack mitochondria, but possess hydrogenosomes: organelles that contain hydrogenase and produce hydrogen . The origin of hydrogenosomes has been explained by two competing hypotheses: (i) they are biochemically modified mitochondria; or (ii) they are derived from endosymbiotic association(s) of ciliates and anaerobic eubacteria that possessed the hydrogenosome biochemistry . Phylogenetic analyses of representative aerobic, and anaerobic hydrogenosomal ciliates using host nuclear SSU rDNA sequences indicate a minimum of three, but more likely four, separate origins of hydrogenosomes . Whereas this does not refute either hypothesis, the implausibility of multiple convergent endosymbioses gives further support to the view that hydrogenosomes in ciliates derive from an existing organelle, which ultrastructural evidence suggests is the mitochondrion . Our results indicate a considerable potential for physiological-biochemical plasticity among a group of predominantly aerobic eucaryotes, and provide a phylogenetic framework to further refine and test hypotheses of the origins of the hydrogenosomal enzymes.

Mol Microbiol, 1995 Oct, 18(2), 283 - 93
Identification of mycoplasma membrane proteins by systematic Tn phoA mutagenesis of a recombinant library; Cleavinger CM et al.; Wall-less prokaryotes in the genus Mycoplasma include over 90 species of infectious agents whose pathogenicity for humans and other animals is currently being assessed . Molecular characterization of surface proteins is critical in this regard but is hampered by the lack of genetic systems in these organisms . We used TnphoA transposition to systematically mutagenize, in Escherichia coli, a genomic plasmid library constructed from Mycoplasma fermentans, a potential human pathogen . The strategy circumvented problems of expressing mycoplasma genes containing UGA (Trp) codons and relied on the construction of the vector pG7ZCW, designed to reduce TnphoA transposition into vector sequences . Functional phoA gene fusions directly identified genes encoding 19 putative membrane-associated proteins of M . fermentans . Sequences of fusion constructs defined three types of export sequence: (1) non-cleavable, membrane-spanning sequences, (2) signal peptides with signal peptidase (SPase) I-like cleavage sites, and (3) signal peptides with SPase II-like lipoprotein-cleavage sites which, like most other mycoplasmal lipoprotein signals analysed to date, differed from those in several Gram-negative and Gram-positive eubacteria in their lack of a Leu residue at the -3 position . Antibodies to synthetic peptides that were deduced from two fusions to predicted lipoproteins, identified corresponding amphiphilic membrane proteins of 57 kDa and 78 kDa expressed in the mycoplasma . The P57 sequence contained a proline-rich N-terminal region analogous to an adhesin of Mycoplasma gallisepticum . The P78 protein was identical to a serologically defined phase-variant surface lipoprotein . TnphoA mutagenesis provides an efficient means of systematically characterizing functionally diverse lipoproteins and other exported proteins in mycoplasmas.

Mol Microbiol, 1995 Oct, 18(1), 163 - 74
Transcriptional regulation of ferric citrate transport in Escherichia coli K-12 . Fecl belongs to a new subfamily of sigma 70-type factors that respond to extracytoplasmic stimuli; Angerer A et al.; Transcription of the ferric citrate transport system of Escherichia coli K-12 is repressed by Fe(2+)-Fur and activated by ferric citrate . Ferric citrate does not have to enter the cytoplasm; it initiates a signal transduction mechanism by binding to the outer membrane receptor FecA . Presumably, a conformational change is transmitted in a TonB-dependent manner to the FecR protein . FecR activates FecI, and FecI activates transcription of the fecABCDE transport genes . In this communication, FecI was isolated after cloning fecI downstream of an ideal ribosome-binding site . Overexpressed FecI formed inclusion bodies which were solubilized and purified in active form using a mild detergent . FecI, in conjunction with RNA polymerase core enzyme, directed transcription from the fecA promoter in an in vitro run-off transcription assay . Furthermore, FecI retarded the electrophoretic mobility of a specific 75 bp DNA fragment located upstream of fecA . An in vivo competition experiment between the fecA promoters of wild-type and mutant strains identified the nucleotide positions 2747, 2749, 2751 and 2753, located within the 75 bp fragment, as important for FecI-induced transcription . Mobility band shift of fecA promoter DNA caused by cell lysates required growth of cells in the presence of ferric citrate and expression of FecA, FecI and FecR . These data support the previous assignment of FecI, based on sequence homologies, to a new subfamily of eubacterial RNA polymerase sigma 70 factors that respond to extra-cytoplasmic stimuli and regulate extracytoplasmic functions.

Biol Chem Hoppe Seyler, 1995 Oct, 376(10), 595 - 601
7-Azabenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide, new vitamin B12-analogs synthesized from 4(5)-aminoimidazole by Eubacterium limosum; Endres B et al.; In anaerobic bacteria, glycine, formate, and the amide-N of glutamine are building blocks for the biosynthesis of the imidazole moiety of the vitamin B12-base 5,6-dimethylbenzimidazole . These building blocks are also used for the biosynthesis of the imidazole moiety of purine bases . Therefore we tested 4(5)-aminoimidazole, the base moiety of the purine nucleotide precursor 5-aminoimidazole ribonucleotide, for its putative function as precursor of 5,6-dimethylbenzimidazole . The anaerobic vitamin B12-producer Eubacterium limosum, grown in the presence of {2-13C}4(5)-aminoimidazole, synthesized nonlabeled vitamin B12, but also {2-13C}7-azabenzimidazolylcobamide and {2-13C}5,6-dimethyl-7-azabenzimidazolylcobamide . {2-13C}limidazole was used by E . limosum to form {2-13C}imidazolylcobamide . Simultaneously nonlabeled vitamin B12 was synthesized . This shows that 4(5)-aminoimidazole and imidazole are not intermediates in the biosynthesis of 5,6-dimethylbenzimidazole . However, 4(5)-aminoimidazole has obviously a structure similar to the structure of an as yet unknown precursor of the vitamin B12-base, and is therefore transformed into the aza analogs . In order to prepare a reference compound 4(5)-azabenzimidazole was added to a culture of Propionibacterium shermanii and to a culture of E . limosum, P . shermanil transformed this base mainly to 4-azabenzimidazolylcobamide, as determined by 1H NMR-spectroscopy (NOE experiment) . In contrast E . limosum produced mainly 7-azabenzimidazolylcobamide . The reason for this difference is discussed.

Microbiology, 1995 Oct, 141 ( Pt 10), 2519 - 28
The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control; van Wezel GP et al.; In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively . While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown . To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses . Replacement of the 5'-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation . The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site . The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in sigma hrdB, the principal sigma factor of S . coelicolor . Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S . coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain . However, tuf3 transcription was observed in cultures of S . coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response . Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S . coelicolor J1681, a strain deleted for bldA (which specifies a tRNA(Leu) for the rare leucine codon UUA) . In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.

RNA, 1995 Oct, 1(8), 807 - 14
G.U base pairing motifs in ribosomal RNA; Gautheret D et al.; An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs . In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G.U pairs . Approximately 120 such sites were identified and classified according to their secondary structure and sequence environment . Overall biases in the distribution of G.U pairs are consistent with previously proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions . However, multiple sites violate these rules and display highly conserved G.U pairs in orientations that could cause severe stacking problems . In addition, three motifs displaying a conserved G.U pair in a specific sequence/structure environment occur at an unusually high frequency . These motifs, of which two had not been reported before, involve sequences 5'UG3' 3'GA5' and 5'UG3' 3'GU5', as well as G.U pairs flanked by a bulge loop 3' of U . The possible structures and functions of these recurrent motifs are discussed.

Plant Physiol, 1995 Oct, 109(2), 619 - 25
Molecular cloning and characterization of the cDNA coding for the biotin-containing subunit of the chloroplastic acetyl-coenzyme A carboxylase; Choi JK et al.; We report the molecular cloning and sequence of the cDNA coding for the biotin-containing subunit of the chloroplastic acetylcoenzyme A (CoA) carboxylase (ACCase) of Arabidopsis thaliana (CAC1) . The 3' end of the CAC1 sequence, coding for a peptide of 94 amino acids, which includes a putative biotinylation motif, was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein . The resulting GST-CAC1 fusion protein was biotinylated in vivo, indicating that CAC1 codes for a biotin-containing protein . Antibodies generated to the GST-CAC1 protein reacted solely with the 38-kD biotin-containing polypeptide of Arabidopsis . Furthermore, these antibodies inhibited ACCase activity in extracts from Arabidopsis leaves . The deduced amino acid sequence of CAC1 has an apparent N-terminal chloroplast-targeting transit peptide . The CAC1 protein is coded by a single Arabidopsis gene, and its mRNA accumulates to the highest levels in organs that are undergoing rapid growth . The amino acid sequence of the CAC1 protein is most similar to the biotin carboxyl-carrier protein component of eubacterial ACCases . These characterizations identify CAC1 as the biotin-containing subunit of the plastidic, heteromeric ACCase of Arabidopsis . The results support the ancient origin of the two structurally distinct ACCases of plants.

Philos Trans R Soc Lond B Biol Sci, 1995 Sep 29, 349(1329), 235 - 40
The origins and evolution of eukaryotic proteins; Doolittle RF; The common ancestry of eukaryotes, archaebacteria and eubacteria is well demonstrated by amino acid sequence comparisons of numerous proteins that are common to all three groups . On the other hand, there are a few proteins, like ubiquitin, that are common to eukaryotes and archaebacteria and which have yet to be observed in eubacteria . Some proteins appear to be wholly restricted to eukaryotes; this is especially true of cytoskeletal proteins . Recently, actin has been found by crystallography to be homologous with an ATP-binding domain found in a heat shock protein and several other proteins common to all three urkingdoms . This observation is puzzling on several counts . Most cytoskeletal proteins like actin and tubulin are very slow changing and must have been so for a very long time . How is it, then, that no sequence resemblance can be discerned with their alledged prokaryotic antecedents? The question is addressed by considering two bacterial fts proteins which appear to be related to actin, on the one hand, and tubulin, on the other . One answer may be that the rate of change of these proteins changed dramatically at a key point in their history . Another possibility is that eukaryotes are much older than some of their other proteins indicate.

Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9122 - 6
A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution; Henze K et al.; Genes for glycolytic and Calvin-cycle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher eukaryotes derive from ancient gene duplications which occurred in eubacterial genomes; both were transferred to the nucleus during the course of endosymbiosis . We have cloned cDNAs encoding chloroplast and cytosolic GAPDH from the early-branching photosynthetic protist Euglena gracilis and have determined the structure of its nuclear gene for cytosolic GAPDH . The gene contains four introns which possess unusual secondary structures, do not obey the GT-AG rule, and are flanked by 2- to 3-bp direct repeats . A gene phylogeny for these sequences in the context of eubacterial homologues indicates that euglenozoa, like higher eukaryotes, have obtained their GAPDH genes from eubacteria via endosymbiotic (organelle-to-nucleus) gene transfer . The data further suggest that the early-branching protists Giardia lamblia and Entamoeba histolytica--which lack mitochondria--and portions of the trypanosome lineage have acquired GAPDH genes from eubacterial donors which did not ultimately give rise to contemporary membrane-bound organelles . Evidence that "cryptic" (possibly ephemeral) endosymbioses during evolution may have entailed successful gene transfer is preserved in protist nuclear gene sequences.

FEBS Lett, 1995 Sep 25, 372(2-3), 135 - 9
1H-NMR and photo-CIDNP spectroscopies show a possible role for Trp23 and Phe31 in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus; Consonni R et al.; Investigations were performed on recombinant ribonuclease P2 from Sulfolobus solfataricus, previously cloned and expressed in Escherichia coli {Fusi, P., Grisa, M., Mombelli, E., Consonni, R., Tortora, P . and Vanoni, M . (1995) Gene 154, 99-103} . NMR and photo-CIDNP spectroscopies showed that the enzyme possesses an aromatic cluster consisting of Phe5, Tyr7, Phe31 and Tyr33 while Trp23 is fully exposed to solvent . Phe31, Tyr33 and Trp23 are located within a triple stranded antiparallel beta-sheet, each one being part of an amino acid stretch matching consensus sequences for RNA binding . Phe31 and Trp23 are exposed to and specifically interact with a flavin dye used as a model ligand, with a topology reminiscent of that found in several eubacterial and eukariotic RNA-binding proteins.

Gene, 1995 Sep 22, 163(1), 75 - 9
Amplification and cloning of the Mycobacterium tuberculosis dnaA gene; Rajagopalan M et al.; To identify and subsequently clone the gene encoding the DnaA protein, degenerate oligodeoxyribonucleotide (oligo) primers targeted against two highly conserved domains of the eubacterial DnaA were used to amplify a 780-bp DNA region spanning the two primers from genomic DNA preparations of Mycobacterium tuberculosis (Mt), M . bovis (Mb) and M . avium (Ma) . Nucleotide (nt) sequences and deduced amino acid (aa) sequences of these fragments revealed homologies with each other and with the corresponding regions from other bacteria . Using an oligo specific to Mt dnaA as a probe, the Mt genomic DNA cosmid libraries propagated in Escherichia coli were screened and a cosmid DNA clone hybridizing with the oligo was identified . Furthermore, a 5-kb DNA fragment containing the Mt dnaA was subcloned into a pUC18 vector.

Proc Natl Acad Sci U S A, 1995 Sep 12, 92(19), 8630 - 4
Postseptational chromosome partitioning in bacteria; Sharpe ME et al.; Mutations in the spoIIIE gene prevent proper partitioning of one chromosome into the developing prespore during sporulation but have no overt effect on partitioning in vegetatively dividing cells . However, the expression of spoIIIE in vegetative cells and the occurrence of genes closely related to spoIIIE in a range of nonsporulating eubacteria suggested a more general function for the protein . Here we show that SpoIIIE protein is needed for optimal chromosome partitioning in vegetative cells of Bacillus subtilis when the normal tight coordination between septation and nucleoid partitioning is perturbed or when septum positioning is altered . A functional SpoIIIE protein allows cells to recover from a state in which their chromosome has been trapped by a closing septum . By analogy to its function during sporulation, we suggest that SpoIIIE facilitates partitioning by actively translocating the chromosome out of the septum . In addition to enhancing the fidelity of nucleoid partitioning, SpoIIIE also seems to be required for maximal resistance to antibiotics that interfere with DNA metabolism . The results have important implications for our understanding of the functions of genes involved in the primary partitioning machinery in bacteria and of how septum placement is controlled.

Biol Pharm Bull, 1995 Sep, 18(9), 1175 - 8
Purification and characterization of a geniposide-hydrolyzing beta-glucosidase from Eubacterium sp . A-44, a strict anaerobe from human feces; Yang L et al.; A geniposide-hydrolyzing beta-glucosidase was discovered in Eubacterium sp . A-44, a human intestinal anaerobe . The enzyme was intracellularly distributed in the bacterium, and purified to homogeneity from the extract using Butyl-Toyopearl 650M, Sephacryl S-300, hydroxyapatite and chromatofocusing column chromatography . The enzyme was a single polypeptide chain with the molecular weight of 90 kDa and the N-terminal amino acid sequence initiated from methionine up to the 29th residue did not show more than 50% homology against known protein sequences . A broad substrate specificity was shown for the beta-glucosidase to hydrolyze aryl beta-D-glucosides (p-nitrophenyl beta-D-glucopyranoside-pNPG, esculin and salicin), alkyl beta-D-glucosides (geniposide and amygdalin) and cellobiose . The Km values (mM) for various beta-D-glucosides were 0.068 for geniposide, 0.10 for pNPG, 0.21 for esculin, 0.22 for salicin, 2.9 for amygdalin, and 0.91 for cellobiose . The pH optimum with pNPG and geniposide as the substrates was 6.0 . The enzyme was inhibited by sulfhydryl reagents, Cu2+, and nojirimycin bisulfite.

J Biochem (Tokyo), 1995 Sep, 118(3), 494 - 9
Nucleotide sequence of a principal sigma factor gene (hrdB) of Streptomyces griseus; Shinkawa H et al.; The hrdB homologue was isolated from a streptomycin-producing Streptomyces griseus 2247 strain, which is independent of A-factor . The nucleotide sequence of the cloned DNA fragment revealed the presence of an open reading frame (ORF) of 1,542bp, which predicted a primary product of 514 amino acids and Mr 56,100 . The N-terminal sequence of the purified HrdB protein of S . griseus was identical to the amino acid sequence deduced from the nucleotide sequence . The deduced amino acid sequence contains an inverted question markrpoD box inverted question mark conserved in the principal sigma factors of eubacteria, and shows high similarity to the hrdB products of S . coelicolor A3(2)(89.9%) and S . aureofaciens (88.1%) . The cloned gene encodes a principal sigma factor of S . griseus . The promoter region was identified by using a promoter-probe vector and by means of primer extensions experiments . The transcription start point is located 158-bp upstream of the initiation codon.

J Indian Med Assoc, 1995 Sep, 93(9), 333 - 5, 339
Non-sporing anaerobes in certain surgical group of patients; Chatterjee BD et al.; The magnitude of non-sparing anaerobic (NSA) infections has been defined in the postoperative wounds on colorectum in children (57.1%), general surgery (0%), abdominoperineal and uterocervical operations (11-45%) in gynaecologic and obstetrical cases and perforative peritonitis (25.8-32.3%) . Children below the 6 months age group bear less risk of acquiring NSA infection . Under certain situations, metronidazole combats NSA infections in a better way than other antibacterials . The bacteriology of NSA infections has been probed at the species level in the gynaecologic and obstetrical patients . The species of normal cervix (44.6%) are represented in wounds involving abdominal wall (11%), perineum (22.8%) and uterocervix (45.6%) to suggest endogenous infection . Out of the 22 species of NSA isolated, Peptostreptococcus anaerobius, Acidaminococcus fermentans and Peptococcus prevotii are the commonest . Others were Peptococcus niger, Gaffkya anaerobia, Lactobacillus bulgaricus, Actinomyces bovis, Bacteroides oralis, Fusobacterium gonidiaformans and the different species of peptococcus, peptostreptococcus, eubacterium, propionibacterium and fusobacterium . The weight of evidence indicated a pathogenic role of Peptostreptococcus anaerobius and Ruminococcus flavefaciens, in view of their heavy growth . The umbrella of antibacterials reduced Gram-positive anaerobic cocci from 40% to 16% . The facultative anaerobes Staph aureus, Staph epidermidis, Kl pneumoniae and proteus appeared as the exogenous agents of nosocomial infections.

Curr Genet, 1995 Sep, 28(4), 346 - 52
Characterization of the nuclear gene encoding chloroplast ribosomal protein S13 from Arabidopsis thaliana; Kumar R et al.; We have characterised a cDNA clone and a nuclear gene encoding the chloroplast 30 s ribosomal protein S13 from Arabidopsis thaliana . The identification is based on the high similarity of the predicted amino-acid sequence with eubacterial S13 protein sequences, and immunodetection of a 14.5-kDa chloroplast ribosomal polypeptide using antibodies raised against the polypeptide produced from part of the cDNA expressed in bacteria . The predicted amino-acid sequence contains an N-terminal extension which has several features characteristic of chloroplast transit peptides . Experiments suggest there is a single copy of this gene in A . thaliana and multiple copies in Brassica species . The origin of the mitochondrial S13 polypeptide in crucifers is also discussed.

Mol Cell Biol, 1995 Sep, 15(9), 5071 - 81
The duplicated Saccharomyces cerevisiae gene SSM1 encodes a eucaryotic homolog of the eubacterial and archaebacterial L1 ribosomal proteins; Petitjean A et al.; A previously unknown Saccharomyces cerevisiae gene, SSM1a, was isolated by screening for high-copy-number suppressors of thermosensitive mutations in the RNA14 gene, which encodes a component from the polyadenylation complex . The SSM1 a gene codes for a 217-amino-acid protein, Ssm1p, which is significantly homologous to eubacterial and archaebacterial ribosomal proteins of the L1 family . Comparison of the Ssm1p amino acid sequence with that of eucaryotic polypeptides with unknown functions reveals that Ssm1p is the prototype of a new eucaryotic protein family . Biochemical analysis shows that Ssm1p is a structural protein that forms part of the largest 60S ribosomal subunit, which does not exist in a pool of free proteins . SSM1 a is duplicated . The second gene copy, SSM1b, is functional and codes for an identical and functionally interchangeable Ssm1p protein . In wild-type cells, SSM1b transcripts accumulate to twice the level of SSM1a transcripts, suggesting that SSM1b is responsible for the majority of the Ssm1p pool . Haploid cells lacking both SSM1 genes are inviable, demonstrating that, in contrast with its Escherichia coli homolog, Ssm1p is an essential ribosomal protein . Deletion of the most expressed SSM1b gene leads to a severe decrease in the level of SSM1 transcript, associated with a reduced growth rate . Polysome profile analysis suggests that the primary defect caused by the depletion in Ssm1p is at the level of translation initiation.

J Clin Periodontol, 1995 Sep, 22(9), 709 - 17
Cellular source, activation and inhibition of dental plaque collagenase; Sorsa T et al.; Dental plaque is the major aetiological factor in periodontal diseases and contains several proteolytic enzymes . The origin of these proteinases is, however, poorly studied . This study was undertaken to characterize collagenase present in dental plaque of adult periodontitis patients . Vertebrate-type rather than bacterial-derived collagenase activity was detected in extracts of both supra- and subgingival dental plaque extracts of adult periodontitis patients . Dental plaque collagenase was found to exist predominantly in autoactive form . Dental plaque collagenase from periodontally healthy individuals existed in latent from . Latent dental plaque collagenase from periodontitis lesions could be activated by a 95 kD chymotrypsin-like proteinase from Treponema denticola and human leukocyte cathepsin G but not by human plasmin . Incubation of purified latent leukocyte collagenase with whole cells of Fusobacterium nucleatum, Eubacterium saburreum, Prevotella buccae and Porphyromonas gingivalis, however, did not result to the activation of the enzyme . Doxycycline in vitro inhibited dental plaque collagenase with an IC50-value of 20 microM . Dental plaque collagenase degraded more efficiently type I and II collagens than type III collagen . Western-blot analysis with specific anti-human neutrophil collagenase-antibody revealed that both in supra- and subgingival dental plaque extracts dental plaque collagenase had undergone proteolytic conversion from an 80 kD proform to a 58 kD active form which is associated with catalytic autoactivity as measured by functional collagenase assay . This reflects proteolytic activation of leukocyte collagenase in dental plaque probably by other proteases derived from potent periodontopathogenic bacteria such as T . denticola or other PMN proteases such as cathepsin G.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Evol, 1995 Sep, 41(3), 388 - 96
Primary structure and eubacterial relationships of the pyruvate:ferredoxin oxidoreductase of the amitochondriate eukaryote Trichomonas vaginalis; Hrdy I et al.; In the eukaryotic unicellular organism Trichomonas vaginalis a key step of energy metabolism, the oxidative decarboxylation of pyruvate with the formation of acetyl-CoA, is catalyzed by the iron-sulfur protein pyruvate:ferredoxin oxidoreductase (PFO) and not by the almost-ubiquitous pyruvate dehydrogenase multienzyme complex . This enzyme is localized in the hydrogenosome, an organelle bounded by a double membrane . PFO and its closely related homolog, pyruvate:flavodoxin oxidoreductase, are enzymes found in a number of archaebacteria and eubacteria . The presence of these enzymes in eukaryotes is restricted, however, to a few amitochondriate groups . To gain more insight into the evolutionary relationships of T . vaginalis PFO we determined the primary structure of its two genes (pfoA and pfoB) . The deduced amino acid sequences showed 95% positional identity . Motifs implicated in related enzymes in liganding the Fe-S centers and thiamine pyrophosphate were well conserved . The T . vaginalis PFOs were found to be homologous to eubacterial pyruvate:flavodoxin oxidoreductases and showed about 40% amino acid identity to these enzymes over their entire length . Lack of eubacterial PFO sequences precluded a comparison . pfoA and pfoB revealed a greater distance from related enzymes of Archaebacteria . The conceptual translation of the nucleotide sequences predicted an amino-terminal pentapeptide not present in the mature protein . This processed leader sequence was similar to but shorter than leader sequences noted in other hydrogenosomal proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1995 Sep 1, 131(2), 219 - 25
The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system; Hoffmann T et al.; The Gram-positive soil bacterium Bacillus subtilis, generally regarded as an aerobe, grows under strict anaerobic conditions using nitrate as an electron acceptor and should be designated as a facultative anaerobe . Growth experiments demonstrated a lag phase of 24 to 36 hours after the shift from aerobic, to the onset of anaerobic respiratory growth . Anaerobically adapted cells grew without further lag phase after their transfer to fresh anaerobic growth medium . The cells change their morphology from rods to longer filament-like structures when moved from aerobic to anaerobic respiratory growth conditions . Surprisingly, anaerobically grown B . subtilis lost the capacity for sporulation . An investigation of the molecular basis of the switch between aerobic and anaerobic growth was initiated by the cloning of the genes encoding the respiratory nitrate reductase from B . subtilis . Oligonucleotides deduced from conserved amino acid sequence regions of eubacterial respiratory nitrate reductases and related enzymes were used for the isolation of the genes . Four open reading frames with significant homology to the E . coli respiratory nitrate reductase operons (narGHIJ, narZYWV) were isolated and termed narGHJI . A chromosomal knock-out mutation of the B . subtilis nar operon totally abolished nitrate respiration.

EMBO J, 1995 Sep 1, 14(17), 4249 - 57
Phosphorylation in halobacterial signal transduction; Rudolph J et al.; Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya . Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world . Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides . The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively . The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation . Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of {gamma-32P}ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled . Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY . The mutant CheY(D53A) had no effect on phosphorylated CheA . The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli.

Mol Gen Genet, 1995 Aug 30, 248(4), 417 - 22
DNA supercoiling in a thermotolerant mutant of Escherichia coli; Friedman SM et al.; A spontaneously occurring, nalidixic acid-resistant (NalR), thermotolerant (T/r) mutant of Escherichia coli was isolated . Bacteriophage P1-mediated transduction showed that NalR mapped at or near gyr A, one of the two genes encoding DNA gyrase . Expression of gyrA+ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping in gyrA . Plasmid linking number measurements, made with DNA from cells grown at 37 degrees C or shifted to 48 degrees C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain . Each strain preferentially expressed two different proteins at 48 degrees C . The genetic and supercoiling data indicate that thermotolerance can arise from an alteration in DNA gyrase that lowers supercoiling . This eubacterial study, when coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.

Mol Gen Genet, 1995 Aug 21, 248(3), 341 - 50
Molecular characterization of the proline-1 (pro-1) locus of Neurospora crassa, which encodes delta 1-pyrroline-5-carboxylate reductase; Davis CR et al.; delta 1-pyrroline-5-carboxylate reductase (P5CR; {L-proline: NAD(P+) 5-oxidoreductase}; EC 1.5.1.2) catalyzes the final step in proline biosynthesis . We have shown that the proline-1 (pro-1) locus of Neurospora crassa encodes P5CR . The pro-1 gene was localized to a 3.2 kb region by complementation of (restoration of proline-independent growth to) a proline auxotroph carrying a recessive mutation at the pro-1 locus . The nucleotide sequence of this 3.2 kb region contains an open reading frame with coding capacity of 311 amino acids . The deduced polypeptide shows significant similarity to P5CR amino acid sequences . Similarity of N . crassa P5CR is greatest to that of the yeast, Saccharomyces cerevisiae, but is also strong to P5CR sequences from archaea, eubacteria, plants, and humans . In N . crassa, amino acid imbalance, including deficiency or excess of a single amino acid, such as histidine, induces expression of many amino acid biosynthetic genes that are under cross-pathway control, a general regulatory system analogous to general amino acid control in Saccharomyces . Although P5CR catalyzes the only committed step in proline biosynthesis, pro-1 expression was unaltered by histidine starvation and independent of CPC1, a positively acting transcription factor that mediates cross-pathway control in N . crassa.

J Mol Biol, 1995 Aug 18, 251(3), 378 - 89
The mitochondrial LSU rDNA of the brown alga Pylaiella littoralis reveals alpha-proteobacterial features and is split by four group IIB introns with an atypical phylogeny; Fontaine JM et al.; The mitochondrial DNA region coding for the large ribosomal RNA subunit from the brown alga Pylaiella littoralis (L.) Kjellm was sequenced . The LSU rRNA was folded into a secondary structure and aligned with homologous, mitochondrial and eubacterial sequences . Taking into account the primary and secondary structure levels, the mitochondrial LSU rRNA of P . littoralis shares more structural features with alpha-proteobacterial genes than do those of the green alga Prototheca wickerhamii and land plants . In phylogenetic trees, branches leading to brown algae, red algae, the protozoan Acanthamoeba castellanii and land plants, respectively, emerge approximately at the same time, as they do in nuclear-gene based phylogenies . This suggests that there is only one origin for the mitochondrial rRNA genes found in these lineages . The LSU rDNA is split by four group IIB introns . The first two introns each contain one open reading frame which encodes a reverse transcriptase-like protein . Comparison of their amino acid sequences with those of other reverse transcriptase-like genes contained in group II introns shows that these genes are more closely related to plastid and cyanobacterial homologous genes than to any known mitochondrial intronic reverse transcriptase.

Vet Rec, 1995 Aug 5, 137(6), 141 - 4
Clinical, and light and electron microscopical findings in sows with cystitis; Liebhold M et al.; The clinical findings, and urinary and morphological changes in the urinary bladder were investigated in 25 sows with a urinary tract infection . Eubacterium suis was isolated from 12 of the sows but not from the other 13 . The clinical signs did not always correlate with the morphological changes . The only clinical sign indicating the beginning of cystitis appeared to be a significant bacteriuria . Other urinary changes occurred later when the inflammatory processes were more severe . In contrast with cystitis due to other bacteria, infection with E suis frequently resulted in a macrohaematuria and urinary pH values above 8.0 . However, the light and electron microscopical findings in the bladder mucosa were similar in the sows with and without cystitis due to E suis . The transformation of goblet cells and the development of mucin cysts were probably due to the local bladder defence mechanisms . More severe lesions were observed with E suis infections, which resulted in changes in the ureterovesical junctions and in ascending renal infection and uraemia.

Mol Microbiol, 1995 Aug, 17(4), 737 - 46
Developmental control of the heat-shock stress regulon in Streptomyces coelicolor; Puglia AM et al.; In the differentiating eubacterium Streptomyces coelicolor, nutritional imbalances activate a developmental programme which involves the heat-shock stress regulon . In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S) . Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two-dimensional gels and analysed using advanced statistical methods . The responses of all heat-shock proteins (HSPs) were highly dependent upon growth phase, thus demonstrating that the four phases of growth were physiologically distinct . For many HSPs, the level of thermal induction attained were closely related to growth stage-determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements . Cluster analysis identified five groups of HSPs displaying similar kinetics of heat- and developmentally induced synthesis, probably reflecting the influence of major regulatory systems . Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.

Mol Microbiol, 1995 Aug, 17(4), 663 - 74
The dnaK operon of Streptomyces coelicolor encodes a novel heat-shock protein which binds to the promoter region of the operon; Bucca G et al.; Transcriptional studies have demonstrated that the dnaK gene of Streptomyces coelicolor A3(2) is contained within a 4.3 kb operon . The operon is transcribed from a single (transiently) heat-inducible promoter, dnaKp, that resembles the typical vegetative (sigma 70-recognized) eubacterial consensus promoter sequence . dnaK transcription was found to be heat-inducible at all stages of development in surface-grown cultures . In addition, at the normal growth temperature of 30 degrees C, dnaK transcript levels were shown to vary at different stages of development, being more abundant in young germinating cultures and in mycelium undergoing sporogenesis . The nucleotide sequence of the dnaK operon has been completed, revealing the gene organization 5'dnaK-grpE-dnaJ orfX . orfX represents a novel heat-shock gene . Its predicted product displays high similarity to the GlnR repressor proteins of Bacillus spp . and to the MerR family of eubacterial transcriptional regulators . The S . coelicolor OrfX protein has been over-produced in Escherichia coli, and DNA-binding experiments indicate that it interacts specifically with the dnaKp region, binding to three partially related inverted repeat sequences; they are centered at -75, -49 and +4, respectively, relative to the transcription start site of the operon . These results suggest that OrfX plays a direct role in the regulation of the dnaK operon.

Protein Eng, 1995 Aug, 8(8), 779 - 89
Structural basis for the extreme thermostability of D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: analysis based on homology modelling; Szilagyi A et al.; D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a hyperthermophilic eubacterium, Thermotoga maritima, is remarkably heat stable (Tm = 109 degrees C) . In this work, we have applied homology modelling to predict the 3-D structure of Th.maritima GAPDH to reveal the structural basis of thermostability . Three known GAPDH structures were used as reference proteins . First, the rough model of one subunit was constructed using the identified structurally conserved and variable regions of the reference proteins . The holoenzyme was assembled from four subunits and the NAD molecules . The structure was refined by energy minimization and molecular dynamics simulated annealing . No errors were detected in the refined model using the 3-D profile method . The model was compared with the structure of Bacillus stearothermophilus GAPDH to identify structural details underlying the increased thermostability . In all, 12 extra ion pairs per subunit were found at the protein surface . This seems to be the most important factor responsible for thermostability . Differences in the non-specific interactions, including hydration effects, were also found . Minor changes were detected in the secondary structure . The model predicts that a slight increase in alpha-helical propensities and helix-dipole interactions also contribute to increased stability, but to a lesser degree.

Jpn J Med Sci Biol, 1995 Aug, 48(4), 177 - 91
Semiquantitative analysis of periodontopathogens by gene amplification; Sumi Y et al.; Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, which are known as major causative organisms of periodontitis, were semiquantitatively identified by two-step polymerase chain reaction (PCR) . The sets of specific primers for A . actinomycetemcomitans and P . gingivalis were prepared on the basis of the nucleotide sequences of the lktA and the fimA genes, respectively . The set of universal primers for eubacteria was designed from the nucleotide sequence of a highly conserved region in the eubacterial 16S rRNA sequence . The number of bacteria detectable by one-step PCR assay was no fewer than 10(3) cells . Less than 10(3) bacterial cells were detectable by two-step PCR assay . Subgingival plaque samples from 37 sites of 16 patients were obtained with paperpoints and analyzed by two-step PCR assay . More than 2 x 10(6) bacterial cells were found in the subgingival plaque samples from all diseased sites . In contrast, the number of total bacteria in those from more than half of healthy sites estimated by PCR assay was less than 2 x 10(6) cells, suggesting that subgingival plaque in diseased sites consists of a relatively larger number of bacteria compared with the population in healthy sites . While A . actinomycetemcomitans was detected in both healthy and diseased sites, P . gingivalis was observed only in diseased sites . Both periodontopathic bacteria occupied a minor part (less than 0.1%) of the total subgingival plaque bacteria.

Int J Parasitol, 1995 Aug, 25(8), 929 - 38
An organelle-like small subunit ribosomal RNA gene from Babesia bovis: nucleotide sequence, secondary structure of the transcript and preliminary phylogenetic analysis; Gozar MM et al.; Investigations aimed at identifying the mitochondrial genome of Babesia bovis using the polymerase chain reaction (PCR) have established the existence of an organelle-like small subunit ribosomal RNA (SSU rRNA) gene in the parasite . The sequence, compiled from three main PCR products, was 1448 bp in length (including the primer regions), had a 73% A+T content and showed significant similarity (68% sequence identity) to the "organellar" SSU rRNA gene from Plasmodium falciparum . The proposed secondary structure of the transcript showed several features which were consistent with a eubacterial origin for the organelle-like gene . The presence of putative binding sites for streptomycin and tetracycline also supported an "organellar" location for the gene and suggested that the SSU rRNA transcript is functional in protein synthesis because tetracycline has anti-babesial activity . Phylogenetic analyses based on the conserved regions of the SSU-like rRNA genes from a wide variety of organisms showed only a weak association of the babesial sequence with its mitochondrial homologues and an even weaker association with the corresponding genes of plastid origin . The origin of this organelle-like gene in B . bovis therefore remains unresolved, as is the case for its homologue from P . falciparum.

Curr Genet, 1995 Aug, 28(3), 205 - 16
Mitochondrial transcription initiation: promoter structures and RNA polymerases; Tracy RL et al.; A diversity of promoter structures . It is evident that tremendous diversity exists between the modes of mitochondrial transcription initiation in the different eukaryotic kingdoms, at least in terms of promoter structures . Within vertebrates, a single promoter for each strand exists, which may be unidirectional or bidirectional . In fungi and plants, multiple promoters are found, and in each case, both the extent and the primary sequences of promoters are distinct . Promoter multiplicity in fungi, plants and trypanosomes reflects the larger genome size and scattering of genes relative to animals . However, the dual roles of certain promoters in transcription and replication, at least in yeast, raises the interesting question of how the relative amounts of RNA versus DNA synthesis are regulated, possibly via cis-elements downstream from the promoters . Mitochondrial RNA polymerases . With respect to mitochondrial RNA polymerases, characterization of human, mouse, Xenopus and yeast enzymes suggests a marked degree of conservation in their behavior and protein composition . In general, these systems consist of a relatively non-selective core enzyme, which itself is unable to recognize promoters, and at least one dissociable specificity factor, which confers selectivity to the core subunit . In most of these systems, components of the RNA polymerase have been shown to induce a conformational change in their respective promoters and have also been assigned the role of a primase in the replication of mtDNA . While studies of the yeast RNA polymerase have suggested it has both eubacterial (mtTFB) and bacteriophage (RPO41) origins, it is not yet clear whether these characteristics will be conserved in the mitochondrial RNA polymerases of all eukaryotes . mtTFA-mtTFB; conserved but dissimilar functions . With respect to transcription factors, mtTFA has been found in both vertebrates and yeast, and may be a ubiquitous protein in mitochondria . However, the divergence in non-HMG portions of the proteins, combined with differences in promoter structure, has apparently relegated mtTFA to alternative, or at least non-identical, physiological roles in vertebrates and fungi . The relative ease with which mtTFA can be purified (Fisher et al . 1991) suggests that, where present, it should be facile to detect . mtTFB may represent a eubacterial sigma factor adapted for interaction with the mitochondrial RNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)

FEMS Microbiol Rev, 1995 Aug, 17(1-2), 191 - 205
Holins: form and function in bacteriophage lysis; Young R et al.; During the lytic cycle of most bacteriophages, a phage-encoded peptidoglycan-degrading activity is elaborated . At least four entirely distinct types of enzymes fulfill this role and are given the generic name 'endolysin' . Endolysins characterized to date are synthesized without a signal sequence and thus accumulate fully folded and active in the cytosol during the vegetative phase . Small membrane proteins are required in order for endolysins to gain access to the peptidoglycan . Because the available data suggest that the membrane lesion formed by these proteins is stable and non-specific, these proteins have been given the designation 'holins' ('hole'-formers) . Analysis of the primary sequence suggests a simple membrane topology with two or more membrane-spanning helical domains and a highly charged, hydrophilic C-terminus . Comparison of the sequences of holins from phages of Gram-negative hosts suggests there are at least two major holin groups . Putative holin genes have also been found in bacteriophages of Gram-positive bacteria . Altogether, in phages of Eubacteria, 11 or more unrelated gene families which share the functional and structural characteristics of holins have been identified . Genetic and physiological analysis suggest that holins are primarily regulated at the level of function . Holin function is modulated in some cases by a second protein encoded by the holin gene . The primary regulation of holin function, however, appears to be intrinsic to the holin structure itself, since a missense allele of the S holin gene of phage lambda has been found which abolishes the normal delay that allows the vegetative phase to generate a useful number of progeny.

J Mol Evol, 1995 Aug, 41(2), 196 - 202
DNA sequence, structure, and phylogenetic relationship of the mitochondrial small-subunit rRNA from the red alga Chondrus crispus (Gigartinales rhodophytes); Leblanc C et al.; The entire nucleotide sequence containing the small-subunit ribosomal RNA gene (SSU rRNA) from the mitochondrial genome of Chondrus crispus was determined . To our knowledge, this is the first sequence of a mitochondrial 16S-like rRNA from a red alga . The length of this gene is 1,376 nucleotides . Its secondary structure was constructed and compared with other known secondary structures from eubacteria and from mitochondria of land plants, green and brown algae, and fungi . Phylogenetic trees were built upon SSU rRNA sequence alignment from mitochondria and eubacteria . The results show that rhodophytes and chromophytes provide additional links in the evolution of mitochondria between the green plant lineage and the "nonplant" lineages.

Arch Biochem Biophys, 1995 Aug 1, 321(1), 137 - 9
A thermolabile triosephosphate isomerase from the psychrophile Vibrio sp . strain ANT-300; Adler E et al.; We report the isolation of a gene encoding triosephosphate isomerase (TIM; EC 5.3.1.1) from Vibrio sp . strain ANT-300, a psychrophilic marine eubacterium that grows optimally at 7 degrees C . The deduced primary sequence of this isomerase is 50% identical to Escherichia coli TIM and 37% identical to the isomerase of the psychrotroph Moraxella sp . TA137 . Transformation with this gene allowed growth of a TIM-deficient E . coli strain on selective media, but only at temperatures below 30 degrees C . The temperature dependence of this complementation is likely to result from an intrinsic thermolability of the isomerase . Indeed, the TIM activity present in ANT-300 lysates is markedly heat-sensitive, with a half-life of inactivation of 520 s at 25 degrees C.

J Bacteriol, 1995 Aug, 177(15), 4245 - 51
Genetic structure of the dnaA region of the cyanobacterium Synechocystis sp . strain PCC6803; Richter S et al.; We have cloned and sequenced the dnaA region of Synechocystis sp . strain PCC6803, a bacterium with a light-dependent cell cycle . The dnaA gene product, DnaA, is the central factor for replication initiation in bacteria . The deduced amino acid sequence of the protein encoded by the cyanobacterial dnaA gene is 45% identical to DnaA of Bacillus subtilis and fits very well into the homology pattern of the known eubacterial DnaA proteins . The genetic environment of the Synechocystis sp . strain PCC6803 dnaA gene is completely different from the one in other eubacteria . An open reading frame of unknown function, orf134, was detected upstream of dnaA . The purT gene homolog encoding the glycinamide ribonucleotide transformylase T starts about 200 bp away from this open reading frame in the opposite direction . Downstream of the dnaA gene we detected the start of the psbDC operon, which codes for the photosystem II reaction center proteins D2 and CP43 that are involved in the positioning of chlorophyll a.

J Antibiot (Tokyo), 1995 Aug, 48(8), 787 - 92
The ripostatins, novel inhibitors of eubacterial RNA polymerase isolated from myxobacteria; Irschik H et al.; A new antibiotic, ripostatin, was isolated from the culture supernatant of the myxobacterium, sorangium cellulosum strain So ce377 . It is a macrocyclic lactone carbonic acid containing an unsubstituted phenyl ring in a side chain . The antibiotic acts especially on Staphylococcus aureus, but seems not to penetrate most bacteria . The MIC values are in the range of 1 microgram/ml . Ripostatin is an inhibitor of eubacterial RNA polymerase . It interferes with the initiation of RNA synthesis.

J Ind Microbiol, 1995 Aug, 15(2), 85 - 93
Use of polymerase chain reaction (PCR) fingerprinting to differentiate bacteria for microbial products screening; Hirsch CF et al.; PCR fingerprinting offers a practical molecular means to quickly and reliably differentiate bacteria for microbial products screening . A combination of low resolution and high resolution PCR fingerprinting provides a hierarchical system which allows the discrimination of bacteria at species and subspecies level within 7 h . DNA was extracted from cells by incubating them in water at 95 degrees C for 30 min . A sample of 1 microliter of the cell-free aqueous extract then was used as a source of template DNA in the PCR . The PCR products were separated by electrophoresis on an acrylamide gel and visualized by ethidium bromide staining . The band patterns generated for each different culture were unique, reproducible, and independent of cultivation conditions . Band patterns may be compared visually or by using imaging and pattern matching software . In our laboratory, bacteria such as actinomycetes, Gram-negative and Gram-positive soil eubacteria, and photosynthetic non-sulfur bacteria have been differentiated using PCR fingerprinting.

J Bacteriol, 1995 Aug, 177(15), 4252 - 60
Characterization of late gene promoters of Chlamydia trachomatis; Fahr MJ et al.; Chlamydiae possess an intracellular developmental cycle defined by the orderly interconversion of infectious, metabolically inactive elementary bodies and noninfectious, dividing reticulate bodies . Only a few stage-specific genes have been cloned and sequenced, including the late-stage cysteine-rich protein operon and two late-stage genes encoding histone-like proteins . The aims of this study were to identify additional late-stage genes of Chlamydia trachomatis, analyze the upstream DNA sequence of late genes, and determine the sigma factor requirement of late genes . Stage-specific RNA, made by chlamydiae isolated from host cells, was used to probe C . trachomatis genomic libraries . Two new late genes, designated ltuA and ltuB, were identified, cloned, and sequenced . The predicted peptides encoded by ltuA and ltuB do not bear strong homology to known proteins, and the function of the new late genes is not known . The 5' ends of the transcripts of ltuA, ltuB, the cysteine-rich protein operon, and the two histone-like genes (hctA and hctB) were mapped, and a consensus -10 promoter region of TATAAT was derived from their upstream DNA sequences . In vitro transcription from templates encoding the promoter regions of ltuA, ltuB, and hctA cloned into the transcription assay vector pUC19-spf was found to be strongly stimulated by the addition of recombinant chlamydial sigma 66, while transcription from the putative hctB promoter region cloned in pUC19-spf was not detected in either the presence or absence of added sigma 66 . These results suggest that the transcription of at least some chlamydial late-stage genes is dependent on sigma 66, which is homologous to the major sigma factors of other eubacteria.

J Mol Biol, 1995 Jul 28, 250(5), 587 - 94
Homology in structural organization between E . coli ClpAP protease and the eukaryotic 26 S proteasome; Kessel M et al.; Energy-dependent protein degradation is carried out by large multimeric protein complexes such as the proteasomes of eukaryotic and archaeal cells and the ATP-dependent proteases of eubacterial cells . Clp protease, a major multicomponent protease of Escherichia coli, consists of a proteolytic component, ClpP, in association with an ATP-hydrolyzing, chaperonin-like component, ClpA . To provide a structural basis for understanding the regulation and mechanism of action of Clp protease, we have used negative staining electron microscopy and image analysis to examine ClpA and ClpP separately, as well as active ClpAP complexes . Digitized images of ClpP and ClpA were analyzed using a novel algorithm designed to detect rotational symmetries . ClpP is composed of two rings of seven subunits superimposed in bipolar fashion along the axis of rotational symmetry . This structure is similar to that formed by the beta subunits of the eukaryotic and archaeal proteasomes . In the presence of MgATP, ClpA forms an oligomer with 6-fold symmetry when viewed en face . Side views of ClpA indicate that the subunits are bilobed with the respective domains forming two stacked rings . ClpAP complexes contain a tetradecamer of ClpP flanked at one or both ends with a hexamer of ClpA, resulting in a symmetry mismatch between the axially aligned molecules . Our findings demonstrate that, despite the lack of sequence similarity between ClpAP and proteasomes, these multimeric proteases nevertheless have a profound similarity in their underlying architecture that may reflect a common mechanism of action.

Biochim Biophys Acta, 1995 Jul 25, 1263(1), 86 - 8
The first nucleotide sequence of an archaeal elongation factor 1 beta gene; Arcari P et al.; An archaeal elongation factor 1 beta gene has been isolated for the first time from a Sulfolobus solfataricus genomic library . The sequenced clone (869 bp) contained two open reading frames, one coding for a protein made of 91 amino acid residues (SsEF-1 beta), the other one encoding a nonidentified product (ORF 115) . The amino acid sequences of segments at the N- and C-terminal of the translated SsEF-1 beta were identical to those determined for the native protein . Northern and Southern analyses showed that the SsEF-1 beta gene is represented in S . solfataricus by a unique sequence . Compared to eubacterial or eukaryal corresponding genes the SsEF-1 beta is much shorter.

J Mol Biol, 1995 Jul 7, 250(2), 191 - 201
The archaeal dnaK-dnaJ gene cluster: organization and expression in the methanogen Methanosarcina mazei; Clarens M et al.; The organization and expression of the first archeael dnaK-dnaJ gene cluster cloned and sequenced have been elucidated . The work focused on the methanogen Methanosarcina mazei strain S-6, but a survey of two other strains (JC3 and LYC) and species (Methanosarcina sp . JCV and Methanosarcina acetivorans) showed that the findings are pertinent to other mesophilic methanosarcinas as well . The organization and some expression features of the archaeal genes resemble eubacterial equivalents for which comparable sequence information is available . However, the archaeal genes also display characteristics that are distinct from those of eubacterial and eucaryotic homologs . dnaK and dnaJ are transcribed into monocistronic messages . The initiation site is the same for transcription under optimal cell-growth conditions, and under stress due to a temperature upshift . The two genes are expressed constitutively at lower levels than those observed after heat shock . The constitutive and post-heat-shock expression levels are higher for dnaK than for dnaJ . Both genes withstand heat shocks of at least one and a half hours without a decline in transcript levels . While the transcription termination signals are to some extent reminiscent of those of eubacteria, the initiation signals are not . These have archaeal characteristics, which resemble those of eukaryotes . The intergenic dnaK-dnaJ region contains inverted repeats . These have the potential to build firm stem-loops in the transcript and in single-stranded DNA.

J Appl Bacteriol, 1995 Jul, 79(1), 22 - 9
Identification of proteolytic rumen bacteria isolated from New Zealand cattle; Attwood GT et al.; The protease activities of 212 strains of rumen bacteria isolated from New Zealand cattle grazing pasture were measured . Thirty-seven per cent of strains had activity greater than or equal to the proteolytic rumen bacterium Prevotella ruminicola and 43 of these isolates were identified by morphology, carbon source utilization, Gram stain, biochemical tests and fermentation end-product analysis . Hierarchical Cluster Analysis showed that the strains formed four clusters: cluster A contained 26 strains and clustered with a reference strain of Streptococcus bovis; cluster C contained three strains and clustered with a reference strain of Butyrivibrio fibrisolvens, while clusters B (10 strains) and D (three strains) did not cluster with any of the remaining rumen bacterial type strains . Further tests identified strains of cluster B as Eubacterium budayi, while cluster D strains most closely resembled B . fibrisolvens and were described as B . fibrisolvens-like . An unclustered strain, C21a, was identified as P . ruminicola . The significance of these proteolytic bacterial populations is discussed in relation to protein breakdown in New Zealand ruminants.

Lett Appl Microbiol, 1995 Jul, 21(1), 34 - 7
Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum; Hodrova B et al.; The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum . More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation . These products were metabolized by bacteria in mixed cultures . In co-culture with the fungus M . elsdenii and E . limosum increased the extent of microcrystalline cellulose degradation by 10.12% and 7.96%, respectively . Biomass yield in co-cultures was increased by 89.9% and 59.4% for M . elsdenii and E . limosum . Y cellulose for fungus alone was 52.29 g dry matter mol-1 glucose . These values were 64.93 and 55.92 g mol-1 glucose unit in co-culture with M . elsdenii and E . limosum, respectively.

J Bacteriol, 1995 Jul, 177(14), 4166 - 70
Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance; Munoz R et al.; We report the cloning of the gyrB gene from Streptococcus pneumoniae 533 that carries the nov-1 allele . The gyrB gene codes for a protein homologous to the gyrase B subunit of archaebacteria and eubacteria . The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates of S . pneumoniae . This amino acid position is equivalent to Val-120 of Escherichia coli GyrB, a residue that lies inside the ATP-binding domain as revealed by the crystal structure of the protein.

Curr Biol, 1995 Jul 1, 5(7), 766 - 74
The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus; Tamura T et al.; BACKGROUND: The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation . The proteolytic core of the complex is formed by the 20S proteasome, a cylinder-shaped particle that in archaebacteria contains two different subunits (alpha and beta) and in eukaryotes contains fourteen different subunits (seven of the alpha-type and seven of the beta-type) . RESULTS: We have purified a 20S proteasome complex from the nocardioform actinomycete Rhodococcus sp . strain NI86/21 . The complex has an apparent relative molecular mass of 690 kD, and efficiently degrades the chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of 0.05% SDS . Purified preparations reveal the existence of four subunits, two of the alpha-type and two of the beta-type, the genes for which we have cloned and sequenced . Electron micrographs show that the complex has the four-ringed, cylinder-shaped appearance typical of proteasomes . CONCLUSIONS: The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life.

Trends Genet, 1995 Jul, 11(7), 279 - 83
Transcription: new insights from studies on Archaea; Baumann P et al.; Molecular-genetic analyses have revealed that the Archaea (archaebacteria) are phylogenetically distinct from both eukaryotes and eubacteria . Archaea lack nuclei and resemble eubacteria in morphology and genomic organization, but their molecular design shares many features with eukaryotes . Here, we review recent work that indicates that the archaeal transcriptional machinery is strikingly similar to the RNA polymerase I, II, and III systems of eukaryotic cell nuclei . These findings provide important insights into the evolution of the transcriptional apparatus.

Mol Microbiol, 1995 Jul, 17(1), 37 - 48
A new RNA polymerase sigma factor, sigma F, is required for the late stages of morphological differentiation in Streptomyces spp; Potuckova L et al.; A gene (sigF) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial sigma 70 family . Homologues were present in other Streptomyces spp., and that of the genetically well studied Streptomyces coelicolor A3(2) was also cloned . The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 (S . coelicolor) and 295 (S . aureofaciens) amino acid residues, both showing greatest similarity to sigma B of Bacillus subtilis . However, while sigma B is involved in stationary-phase gene expression and in the general stress response in B . subtilis, sigma F affects morphological differentiation in Streptomyces . Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype . Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense . sigma F is proposed to control the late stages of spore development in Streptomyces.

Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6077 - 81
Molecular cloning of the transcription factor TFIIB homolog from Sulfolobus shibatae; Qureshi SA et al.; The Archaea (archaebacteria) constitute a group of prokaryotes that are phylogenetically distinct from Eucarya (eukaryotes) and Bacteria (eubacteria) . Although Archaea possess only one RNA polymerase, evidence suggests that their transcriptional apparatus is similar to that of Eucarya . For example, Archaea contain a homolog of the TATA-binding protein which interacts with the TATA-box like A-box sequence upstream of many archaeal genes . Here, we report the cloning of a Sulfolobus shibatae gene that encodes a protein (transcription factor TFB) with striking homology to the eukaryotic basal transcription factor TFIIB . We show by primer extension analysis that transcription of the S . shibatae TFB gene initiates 27 bp downstream from a consensus A-box element . Significantly, S . shibatae TFB contains an N-terminal putative metal-binding region and two imperfect direct repeats--structural features that are well conserved in eukaryotic TFIIBs . This suggests that TFB may perform analogous functions in Archaea and Eucarya . Consistent with this, we demonstrate that S . shibatae TFB promotes the binding of S . shibatae TBP to the A-box element of the Sulfolobus 16S/23S rRNA gene . Finally, we show that S . shibatae TFB is significantly more related to TFB of the archaeon Pyrococcus woesei than it is to eukaryotic TFIIBs . These data suggest that TFB arose in the common archaeal/eukaryotic ancestor and that the lineages leading to P . woesei and S . shibatae separated after the divergence of the archaeal and eukaryotic lines of descent.

Biochim Biophys Acta, 1995 Jun 12, 1249(2), 145 - 54
Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp . VPI 12708 in Escherichia coli; Baron SF et al.; The intestinal microorganism Eubacterium sp . VPI 12708 synthesizes a bile acid-inducible NADH:flavin oxidoreductase (NADH:FOR) which presumably functions in the 7 alpha-dehydroxylation of cholic acid to deoxycholic acid . The baiH gene encoding NADH:FOR was subcloned into an IPTG-inducible expression vector, pBaiH2.2 . Escherichia coli DH5 alpha cells transformed with pBaiH2.2 expressed 10-fold higher levels of NADH:FOR upon induction with IPTG than did Eubacterium sp . VPI 12708 cells induced with cholic acid . The NADH:FOR produced by E . coli DH5 alpha(pBaiH2.2) was purified to > 95% electrophoretic homogeneity in three steps . The purified NADH:FOR was similar to that of Eubacterium sp . VPI 12708 in subunit and native M(r) (ca . 72,000 and 210,000, respectively), pH optimum, sensitivity to inhibitors, and electron acceptor specificity . It contained 1 mol of FAD, up to 2 mol of iron, and 1 mol of copper per mol of subunit . The enzyme reduced synthetic quinones, dyes, flavins, and O2 with NADH as the electron donor, but did not reduce disulfide compounds, various unsaturated bile acids, cytochrome c, physiological quinones, or cell fractions from Eubacterium sp . VPI 12708 . Addition of purified NADH:FOR to Eubacterium sp . VPI 12708 cell extracts altered the balance of oxidized and reduced bile acid intermediates produced during cholic acid 7 alpha-dehydroxylation, suggesting that the enzyme may regulate the cellular ratio of NAD to NADH.

Curr Genet, 1995 Jun, 28(1), 26 - 31
Cloning and analysis of the nuclear gene MRP-S9 encoding mitochondrial ribosomal protein S9 of Saccharomyces cerevisiae; Kotter P et al.; The Saccharomyces cerevisiae nuclear gene MRP-S9 was identified as part of the European effort in sequencing chromosome II . MRP-S9 encodes for a hydrophilic and basic protein of 278 amino acids with a molecular mass of 32 kDa . The C-terminal part (aa 153-278) of the MRP-S9 protein exhibits significant sequence similarity to members of the eubacterial and chloroplast S9 ribosomal-protein family . Cells disrupted in the chromosomal copy of MRP-S9 were unable to respire and displayed a characteristic phenotype of mutants with defects in mitochondrial protein synthesis as indicated by a loss of cytochrome c oxidase activity . Additionally, no activities of the gluconeogenetic enzymes, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, could be observed under conditions of glucose de-repression . The respiration-deficient phenotype could not be restored by transformation of the disruption strain with a wild-type copy of MRP-S9, indicating that MRP-S9 disruption led to rho- or rho0 cells . Sequence similarities of MRP-S9 to other members of the ribosomal S9-protein family and the phenotype of disrupted cells are consistent with an essential role of MRP-S9 is assembly and/or function of the 30s subunit of yeast mitochondrial ribosomes.

J Bacteriol, 1995 Jun, 177(11), 3027 - 35
DNA repair mutants of Rhodobacter sphaeroides; Mackenzie C et al.; The genome of the photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1 comprises two chromosomes and five endogenous plasmids and has a 65% G+C base composition . Because of these characteristics of genome architecture, as well as the physiological advantages that allow this organism to live in sunlight when in an anaerobic environment, the sensitivity of R . sphaeroides to UV radiation was compared with that of the more extensively studied bacterium Escherichia coli . R . sphaeroides was found to be more resistant, being killed at about 60% of the rate of E . coli . To begin to analyze the basis for this increased resistance, a derivative of R . sphaeroides, strain 2.4.1 delta S, which lacks the 42-kb plasmid, was mutagenized with a derivative of Tn5, and the transposon insertion mutants were screened for increased UV sensitivity (UVs) . Eight UVs strains were isolated, and the insertion sites were determined by contour-clamped homogeneous electric field pulsed-field gel electrophoresis . These mapped to at least five different locations in chromosome I . Preliminary analysis suggested that these mutants were deficient in the repair of DNA damage . This was confirmed for three loci by DNA sequence analysis, which showed the insertions to be within genes homologous to uvrA, uvrB, and uvrC, the subunits of the nuclease responsible for excising UV damage.

Mol Cell Biol, 1995 Jun, 15(6), 3003 - 11
Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA; Cerutti H et al.; The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance . The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart . Therefore, we examined whether dominant negative mutants of the E . coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii . Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination . These results strongly support the existence of a RecA-mediated recombination system in chloroplasts . We also found that the wild-type E . coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival . Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates . Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses . This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.

Mol Phylogenet Evol, 1995 Jun, 4(2), 110 - 28
Phylogenetic analysis of tufA sequences indicates a cyanobacterial origin of all plastids; Delwiche CF et al.; DNA sequences of the gene tufA, encoding elongation factor Tu, were determined from five cyanobacteria and 21 plastids . Three were full-length (ca . 1230 bp) sequences from cloned DNA, and 23 were partial (ca . 740 bp) sequences from PCR fragments . These sequences were aligned with sequences available from the literature, creating a data set of 56 tufA sequences of eubacterial or plastid origin . Phylogenetic analysis was performed on inferred amino acid sequences with parsimony and neighbor joining techniques, and on first and second position nucleotide sequences with maximum likelihood, and bootstrapping was performed with each method . Trees determined by the three methods were highly congruent with respect to well supported nodes . All examined plastids, including those of green and red algae, chromophytes, and Cyanophora paradoxa, cluster strongly with the cyanobacteria in all analyses . A cyanobacterial origin of all plastids confirms phylogenetic analyses of 16S rRNA and atpB sequences, but conflicts with those of rbcL and rbcS sequences . This discrepancy may be attributable to an ancient gene transfer of the rubisco operon in an ancestor of red algae and chromophytes . Maximum likelihood analysis also provides some support for a monophyletic origin of all plastids, while neighbor joining and parsimony analyses showed cyanobacteria and red, brown, and green plastid lineages as an unresolved polytomy . These tufA analyses also provide a broad perspective on eubacterial evolution and, in conjunction with published rRNA trees, point to at least two major radiations within eubacteria and their descendants: one of many eubacterial phyla, a second of cyanobacteria, and possibly a third radiation early in plastid evolution.

Int J Oral Maxillofac Surg, 1995 Jun, 24(3), 239 - 42
Factors affecting the occurrence of bacteremia associated with tooth extraction; Okabe K et al.; This study examined the factors that affect the occurrence of bacteremia associated with tooth extraction and the kinds of bacteria causing this bacteremia . Bacteremia was found in 132 (72.1%) of 183 patients who had one or more teeth extracted for various reasons . Bacteremia occurred more frequently when teeth were extracted because of inflammatory dental diseases . The occurrence of bacteremia also increased with the number of teeth extracted and the age of the patients . When the volume of blood lost during surgery was > 50 ml and the time required for the operation exceeded 100 min, the occurrence of bacteremia was also higher . Anaerobes were isolated from 104 (78.8%) of the 132 cases of bacteremia . Of the 187 isolates obtained, three (1.6%) were aerobes, 51 (27.3%) were facultative anaerobes (including microaerophils), and 133 (71.1%) were anaerobes . Among facultative anaerobes and microaerophils, the most frequently isolated bacterial genera were Lactobacillus (n = 15), Streptococcus (n = 13), and Staphylococcus (n = 12); and among anaerobes, Eubacterium (n = 40), Peptostreptococcus (n = 40), and Propionibacterium (n = 20).

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