Mol Microbiol, 1995 Jun, 16(5), 955 - 67 Exploring the Mycoplasma capricolum genome: a minimal cell reveals its physiology; Bork P et al.; We report on the analysis of 214kb of the parasitic eubacterium Mycoplasma capricolum sequenced by genomic walking techniques . The 287 putative proteins detected to date represent about half of the estimated total number of 500 predicted for this organism . A large fraction of these (75%) can be assigned a likely function as a result of similarity searches . Several important features of the functional organization of this small genome are already apparent . Among these are (i) the expected relatively large number of enzymes involved in metabolic transport and activation, for efficient use of host cell nutrients; (ii) the presence of anabolic enzymes; (iii) the unexpected diversity of enzymes involved in DNA replication and repair; and (iv) a sizeable number of orthologues (82 so far) in Escherichia coli . This survey is beginning to provide a detailed view of how M . capricolum manages to maintain essential cellular processes with a genome much smaller than that of its bacterial relatives. J Biochem (Tokyo), 1995 May, 117(5), 933 - 5 sn-glycerol-1-phosphate dehydrogenase in Methanobacterium thermoautotrophicum: key enzyme in biosynthesis of the enantiomeric glycerophosphate backbone of ether phospholipids of archaebacteria; Nishihara M et al.; One of the most characteristic features of archaebacterial ether phospholipids is the enantiomeric configuration of their glycerophosphate backbone (sn-glycerol-1-phosphate), that is the mirror image of the structure of the eubacterial or eukaryotic counterpart . The enzyme that forms glycerophosphate of this configuration was found for the first time in a cell-free extract of the methanogen, Methanobacterium thermoautotrophicum, and was identified as sn-glycerol-1-phosphate:NAD+ oxidoreductase (sn-glycerol-1-phosphate dehydrogenase) after partial purification . Because sn-glycerol-1-phosphate has been found to be a precursor of ether lipids of this organism, sn-glycerol-1-phosphate dehydrogenase is a key enzyme in the biosynthesis of the enantiomeric ether lipids of methanogens. Bioessays, 1995 May, 17(5), 457 - 64 A model for repair of radiation-induced DNA double-strand breaks in the extreme radiophile Deinococcus radiodurans; Minton KW et al.; The bacterium Deinococcus (formerly Micrococcus) radiodurans and other members of the eubacterial family Deinococaceae are extremely resistant to ionizing radiation and many other agents that damage DNA . Stationary phase D . radiodurans exposed to 1.0-1.5 Mrad gamma-irradiation sustains > 120 DNA double-strand breaks (dsbs) per chromosome; these dsbs are mended over a period of hours with 100% survival and virtually no mutagenesis . This contrasts with nearly all other organisms in which just a few ionizing radiation induced-dsbs per chromosome are lethal . In this article we present an hypothesis that resistance of D . radiodurans to ionizing radiation and its ability to mend radiation-induced dsbs are due to a special form of redundancy wherein chromosomes exist in pairs, linked to each other by thousands of four-stranded (Holliday) junctions . Thus, a dsb is not a lethal event because the identical undamaged duplex is nearby, providing an accurate repair template . As addressed in this article, much of what is known about D . radiodurans suggests that it is particularly suited for this proposed novel form of DNA repair. J Mol Evol, 1995 May, 40(5), 531 - 6 An operational RNA code for amino acids and variations in critical nucleotide sequences in evolution; Schimmel P; An operational RNA code relates specific amino acids to sequences/structures in RNA hairpin helices which reconstruct the seven-base-pair acceptor stems of transfer RNAs . These RNA oligonucleotides are aminoacylated by aminoacyl tRNA synthetases . The specificity and efficiency of aminoacylation are generally determined by three or four nucleotides which are near the site of amino acid attachment . These specificity-determining nucleotides include the so-called "discriminator base" and one or two base pairs within the first four base pairs of the helix . With three examples considered here, nucleotide sequence variations between the eubacterial E . coli tRNA acceptor stems and their human cytoplasmic and mitochondrial counterparts are shown to include changes of some of the nucleotides known to be essential for aminoacylation by the cognate E . coli enzymes . If the general locations of the specificity-determining nucleotides are the same in E . coli and human RNAs, these RNA sequence variations imply a similar covariation in sequences/structures of the E . coli and human tRNA synthetases . These covariations would reflect the integral relationship between the operational RNA code and the design and evolution of tRNA synthetases. J Mol Evol, 1995 May, 40(5), 476 - 81 Divergence of glutamate and glutamine aminoacylation pathways: providing the evolutionary rationale for mischarging; Rogers KC et al.; Aminoacyl-tRNA for protein synthesis is produced through the action of a family of enzymes called aminoacyl-tRNA synthetases . A general rule is that there is one aminoacyl-tRNA synthetase for each of the standard 20 amino acids found in all cells . This is not universal, however, as a majority of prokaryotic organisms and eukaryotic organelles lack the enzyme glutaminyl-tRNA synthetase, which is responsible for forming Gln-tRNAGln in eukaryotes and in Gram-negative eubacteria . Instead, in organisms lacking glutaminyl-tRNA synthetase, Gln-tRNAGln is provided by misacylation of tRNAGln with glutamate by glutamyl-tRNA synthetase, followed by the conversion of tRNA-bound glutamate to glutamine by the enzyme Glu-tRNAGln amidotransferase . The fact that two different pathways exist for charging glutamine tRNA indicates that ancestral prokaryotic and eukaryotic organisms evolved different cellular mechanisms for incorporating glutamine into proteins . Here, we explore the basis for diverging pathways for aminoacylation of glutamine tRNA . We propose that stable retention of glutaminyl-tRNA synthetase in prokaryotic organisms following a horizontal gene transfer event from eukaryotic organisms (Lamour et al . 1994) was dependent on the evolving pool of glutamate and glutamine tRNAs in the organisms that acquired glutaminyl-tRNA synthetase by this mechanism . This model also addresses several unusual aspects of aminoacylation by glutamyl- and glutaminyl-tRNA synthetases that have been observed. Curr Microbiol, 1995 May, 30(5), 259 - 63 Expression in Escherichia coli and characterization of a bile acid-inducible 3 alpha-hydroxysteroid dehydrogenase from Eubacterium sp . strain VPI 12708; Mallonee DH et al.; We have previously cloned and sequenced three members of a bile acid-inducible gene family from Eubacterium sp . strain VPI 12708 that encode 27,000-M(r) polypeptides . Two copies of these genes (baiA1 and baiA3) are identical, while the third copy (baiA2) encodes a polypeptide sharing 92% amino acid identity with the baiA1 and baiA3 gene products . We have overexpressed the baiA1 gene in Escherichia coli and analyzed the expressed activity . Thin-layer chromatography of 14C-labeled bile acid products from reactions using cell-free extracts revealed a 3 alpha-hydroxysteroid dehydrogenase activity for the BaiA1 protein . The BaiA1 protein could utilize both NAD+ and NADP+, and the preferred steroid substrate was the cholyl-coenzyme A conjugate rather than free cholic acid . These results show that the BaiA proteins are novel 3 alpha-hydroxysteroid dehydrogenases. J Bacteriol, 1995 May, 177(10), 2858 - 62 An anticodon sequence mutant of Escherichia coli initiator tRNA: possible importance of a newly acquired base modification next to the anticodon on its activity in initiation; Mangroo D et al.; Initiator tRNAs from eubacteria and chloroplasts lack a base modification next to the anticodon . This is in contrast to virtually all other tRNAs from these sources . We show that a mutant Escherichia coli initiator tRNA which has an anticodon sequence change from CAU to CUA now has a 2-methylthio-N6-(delta 2-isopentenyl)adenosine (ms2i6A) modification, produced by posttranscriptional modification of A, next to the anticodon . This newly acquired base modification may be important for the function of the mutant tRNA in initiation . In a miaA mutant strain of E . coli defective in biosynthesis of ms2i6A, the mutant initiator tRNA is 10- to 12-fold less active in initiation . The mutant tRNA is aminoacylated and formylated normally in the miaA strain . Thus, the absence of the base modification affects the activity of the mutant tRNA at a step subsequent to its formylation. Mol Biol Evol, 1995 May, 12(3), 481 - 93 The concerted evolution of 5S ribosomal genes linked to the repeat units of other multigene families; Drouin G et al.; We review all instances in which the nuclear 5S rRNA genes of fungi, protist, nematode, and arthropod species have been reported to be linked to the tandemly repeated units of the rDNA, trans-spliced leader, and histone multigene families . The evolution of these gene arrangements is analyzed by mapping them to independently derived phylogenies . These analyses show that 5S rRNA genes have repeatedly become linked to diverse tandemly repeated gene families and that such linkages have also been subsequently inverted or lost in some species . These variable gene linkages are probably the result of stochastic gains and losses of variant repeat units, where functional 5S rRNA had transposed, by the mechanisms which are responsible for the concerted evolution of tandemly repeated multigene families . We discuss the possible mechanisms of 5S rRNA gene transposition and suggest that the characteristics of their promoter elements, transcription, and termination signals may allow functional copies of these genes to be fortuitously transposed through an RNA intermediate . We also review the evidence which shows that the linked 5S rRNA gene copies are transcribed . We conclude that the observed patterns of 5S rRNA gene linkages to the repeat units of other tandemly repeated multigene families have likely arisen due to fortuitous recombination events and are unlikely to represent the remnants of an eubacterial-like arrangement of rDNA operons or to have been established due to selective pressures. J Gen Virol, 1995 May, 76 ( Pt 5), 1099 - 107 Identification and properties of the largest subunit of the DNA-dependent RNA polymerase of fish lymphocystis disease virus: dramatic difference in the domain organization in the family Iridoviridae; Muller M et al.; Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes . The amino acid sequences of the known largest subunits of DdRPs from different species contain highly conserved regions . Oligonucleotide primers, deduced from two conserved domains (RQP{T/S}LH and NADFDGDE) were used for detecting the corresponding gene of fish lymphocystis disease virus (FLCDV), a member of the family Iridoviridae, which replicates in the cytoplasm of infected cells of flatfish . The gene coding for the largest subunit of the DdRP was identified using a PCR-derived probe . The screening of the complete EcoRI gene library of the viral genome led to the identification of the gene locus of the largest subunit of the DdRP within the EcoRI DNA fragment B (12.4 kbp, 0.034 to 0.165 map units) . The nucleotide sequence of a part (8334 bp) of the EcoRI DNA fragment B was determined and a large ORF on the lower strand (ATG = 5787; TAA = 2190) was detected which encodes a protein of 1199 amino acids . Comparison of the amino acid sequences of the largest subunits of the DdRP (RPO1) of FLCDV and Chilo iridescent virus (CIV) revealed a dramatic difference in their domain organization . Unlike the 1051 aa RPO1 of CIV, which lacks the C-terminal domain conserved in eukaryotic, eubacterial and other viral RNA polymerases, the 1199 aa RPO1 of FLCDV is fully collinear with its cellular and viral homologues . Despite this difference, comparative analysis of the amino acid sequences of viral and cellular RNA polymerases suggests a common origin for the largest RNA polymerase subunits of FLCDV and CIV. Biochem Mol Biol Int, 1995 May, 36(1), 123 - 35 Glyceraldehyde-3-phosphate dehydrogenase in the hyperthermophilic archaeon Sulfolobus solfataricus: characterization and significance in glucose metabolism; Russo AD et al.; Glyceraldehyde-3-phosphate dehydrogenase in the archaeon Sulfolobus solfataricus (SsGAPD) has been purified 232 fold with an overall recovery of about 25% . The enzyme is a homomeric tetramer with an M(r) of 41 kDa/subunit . It utilizes either NAD+ or NADP+ as coenzyme but its affinity for the latter is about 50 fold higher . SsGAPD activity is maximum at 87 degrees C . In the range 45-87 degrees C the Arrhenius plot is linear and the activation energy is 55 kJ/mol . The enzyme is thermostable, with a half-life of 45 min at 87 degrees C . The primary structure of SsGAPD shows 35% identity with that of other archaeal GAPDs . Its N-domain shows sequence motifs typical of the dinucleotide binding proteins while the catalytic C-terminal region contains a cysteine residue (C140), required for catalysis, that is conserved in all the archaeal, eukaryal and bacterial GAPDs . These remarks suggest that archaeal GAPDs show a convergent molecular evolution to the eukaryal and eubacterial enzymes in the catalytic region. Yeast, 1995 Apr 30, 11(5), 459 - 65 Nucleotide sequence and chromosomal localization of the gene encoding the Old Yellow Enzyme from Kluyveromyces lactis; Miranda M et al.; A 6.6 kb genomic DNA fragment from the yeast Kluyveromyces lactis was isolated . Sequence analysis of this fragment revealed the presence of two incomplete open reading frames (ORFs) in one strand, one coding for the carboxyl terminus of the plasma membrane H(+)-ATPase and the other for the amino terminus of an unidentified product . In the complementary strand, a full-length ORF which encodes for a protein homologous to the yeast NADPH-dependent Old Yellow Enzyme was found . The deduced amino acid sequence of this ORF predicts a protein of 398 residues with 84% similarity in its full length to OYE1 from Saccharomyces carlsbergensis and OYE2 from Saccharomyces cerevisiae . In addition, an internal region showed considerable similarity to the bile acid-inducible polypeptide from Eubacterium sp., to the NADH oxidase from Thermoanaerobium brockii, to the trimethylamino dehydrogenase from bacterium W3A1 and to the estrogen-binding protein from Candida albicans, suggesting a functional or structural relationship between them . Inactivation of the KYE1 (Kluyveromyces Yellow Enzyme) gene by deletion of 0.6 kb fragment between positions +358 and +936 produced viable cells with a slight increase in their generation time . Haploid cells carrying the disrupted allele showed one-third of the NADPH oxidase activity, compared to wild-type cells . Southern blotting analysis of digested DNA and chromosomes separated by contour-clamped homogeneous electric field electrophoresis from K . lactis indicated that this is a single-copy gene and it is localized on chromosome II, whose molecular size has been estimated to be approximately 1.3 Mb. Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 3749 - 53 A prokaryotic origin for light-dependent chlorophyll biosynthesis of plants; Suzuki JY et al.; Flowering plants require light for chlorophyll synthesis . Early studies indicated that the dependence on light for greening stemmed in part from the light-dependent reduction of the chlorophyll intermediate protochlorophyllide to the product chlorophyllide . Light-dependent reduction of protochlorophyllide by flowering plants is contrasted by the ability of nonflowering plants, algae, and photosynthetic bacteria to reduce protochlorophyllide and, hence, synthesize (bacterio) chlorophyll in the dark . In this report, we functionally complemented a light-independent protochlorophyllide reductase mutant of the eubacterium Rhodobacter capsulatus with an expression library composed of genomic DNA from the cyanobacterium Synechocystis sp . PCC 6803 . The complemented R . capsulatus strain is capable of synthesizing bacteriochlorophyll in the light, thereby indicating that a chlorophyll biosynthesis enzyme can function in the bacteriochlorophyll biosynthetic pathway . However, under dark growth conditions the complemented R . capsulatus strain fails to synthesize bacteriochlorophyll and instead accumulates protochlorophyllide . Sequence analysis demonstrates that the complementing Synechocystis genomic DNA fragment exhibits a high degree of sequence identity (53-56%) with light-dependent protochlorophyllide reductase enzymes found in plants . The observation that a plant-type, light-dependent protochlorophyllide reductase enzyme exists in a cyanobacterium indicates that light-dependent protochlorophyllide reductase evolved before the advent of eukaryotic photosynthesis . As such, this enzyme did not arise to fulfill a function necessitated either by the endosymbiotic evolution of the chloroplast or by multicellularity; rather, it evolved to fulfill a fundamentally cell-autonomous role. J Mol Biol, 1995 Apr 21, 248(1), 180 - 9 Model structure of the Omp alpha rod, a parallel four-stranded coiled coil from the hyperthermophilic eubacterium Thermotoga maritima; Lupas A et al.; Omp alpha is an outer-membrane protein that spans the periplasmic space of the hyperthermophilic eubacterium Thermotoga maritima . The molecule contains a globular head with an apparent diameter of 8 nm and a rod-shaped tail of 40 nm length . The sequence of the globular domain is homologous to a conserved region of cell wall-bound proteins and probably attaches Omp alpha to the peptidoglycan . The sequence of the rod domain resembles that of coiled coil proteins and ends in a transmembrane segment that anchors Omp alpha to the outer membrane . We have analysed Omp alpha by scanning transmission electron microscopy (STEM) and by statistical sequence analysis methods . The Omp alpha rod is a tetramer with an unusual periodicity of hydrophobic residues close to 3.6 that differs from the 3.5 periodicity of canonical coiled coils . This is due to periodic omissions of three residues in the heptad repeat pattern ("stutters") whose effect is to locally distort the packing of hydrophobic layers in the core of the coiled coil . Residues in position alpha are shifted to occupy a position halfway between positions alpha and d (x layers) and residues in positions d and e are shifted so that both participate in core packing interactions (da layers) . Such distorted layers are frequently found in helical bundles and are characteristic of helices that do not undergo supercoiling . The only homo-oligomeric coiled coil of known structure which contains x and da layers is the three-stranded coiled coil of influenza haemagglutinin . Using geometric constraints derived from this structure, we have built a model for the Omp alpha rod in which the helices have a crossing angle of less than 15 degrees and maintain a residual degree of supercoiling with a pitch of approximately 40 nm . Our analysis of distorted layers in the hydrophobic core of coiled coils and helical bundles shows that stutters must not be viewed as discontinuities but rather as a departure from the canonical "knobs-into-holes" packing that allows helices to interact at a low angle without supercoiling . Although stutters have been considered to weaken helical interactions, their occurrence in a rigid, highly thermostable coiled coil indicates that this may not be generally true . Our analysis also indicates that skips and stutters are two different conventions for describing the same underlying structural feature. Biochim Biophys Acta, 1995 Apr 4, 1261(2), 257 - 64 Analysis of the primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves by protein and cDNA sequencing indicates a eukaryotic origin of its gene; Schmidt M et al.; The primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves was identified by protein and cDNA sequencing and compared to the deduced amino acid sequence of a cDNA for the cytosolic isozyme . The mature cytosolic and chloroplast isozyme proteins share 64% amino acid sequence identity . The cDNA for the chloroplast isozyme codes for a precursor protein consisting of an N-terminal transit peptide of Mr 4351 and a mature subunit of Mr 27,282 . Southern blot analysis indicates that the two rye isozymes are encoded by two independent single genes . Amino acid residues or sequence regions of basic functional relevance in known triosephosphate isomerases are strictly conserved in the chloroplast isozyme . The chloroplast isozyme contains 6 cysteine residues, instead of 4 in the cytosolic isozyme . A cysteine at position 143 of the chloroplast isozyme appears to be modified . Phylogenetic trees constructed on the basis of sequence comparisons for triosephosphate isomerases from different species of all major taxonomic groups indicate that the chloroplast isozyme is much more closely related to eukaryotic cytosolic enzymes than to eubacterial enzymes . The results indicate that the nuclear gene for the chloroplast isozyme originated with that for the cytosolic isozyme through duplication of an ancestral eukaryotic gene, rather than through gene transfer from a prokaryotic endosymbiont. Infect Immun, 1995 Apr, 63(4), 1270 - 7 Bacterial evasion of host immune defense: Yersinia enterocolitica encodes a suppressor for tumor necrosis factor alpha expression; Beuscher HU et al.; The ability of the enteropathogenic Yersinia enterocolitica to survive and proliferate in host tissue depends on a 70-kb plasmid known to encode a number of released Yersinia outer proteins that act as virulence factors by inducing cytotoxicity and inhibiting phagocytosis . This study demonstrates that one of the Yersinia outer proteins, the 41-kDa YopB, suppresses the production of tumor necrosis factor alpha (TNF-alpha), a macrophage-derived cytokine with central roles in the regulation of immune and inflammatory responses to infection . This conclusion is based on several lines of evidence . First, in macrophage cultures, suppression of TNF-alpha mRNA expression was induced by culture supernatant (CS+) of plasmid-bearing yersiniae, the effect which was blocked by anti-YopB antiserum . Second, suppression of TNF-alpha production, but not of interleukin-1 (IL-1) and IL-6, was induced by purified YopB . Third, in Yersinia-infected mice, no increase in TNF-alpha mRNA expression was observed in Peyer's patches, the primary site of bacterial invasion, compared with IL-1 (alpha and beta) mRNA . Finally, administration of anti-YopB antiserum to mice prior to infection with Y . enterocolitica increased TNF activity levels in Peyer's patches and coincided with a reduction in bacterial growth . The results thus provide direct evidence for a secreted eubacterial virulence factor that mediates selective suppression of TNF-alpha production . Although suppression of this single cytokine response is probably not sufficient to facilitate survival of the infecting organisms, the results suggest that suppression of TNF-alpha production by YopB significantly contributes to the evasion of Y . enterocolitica from antibacterial host defense. J Investig Med, 1995 Apr, 43(2), 170 - 7 Effect of H2-receptor antagonists on bile acid metabolism; Shindo K et al.; BACKGROUND: Several reports have been presented concerning pronounced overgrowth of bacteria in gastric juices of patients treated with H2-receptor antagonists . However, there has been no report concerning influence of H2-receptor antagonists on jejunal flora . Thus, to investigate the influence and its effect on bile acid metabolism, this study was performed: 1) to examine whether patients with gastric ulcers who have been treated with H2-receptor antagonists have positive bile acid breath tests due to bacterial overgrowth in their jejuna; 2) to verify that these bacteria, isolated and identified, have deconjugation ability; and 3) to determine whether the changes in the gastric pH are related to bacterial overgrowth . METHODS: The methods used were detection of deconjugation of bile acids in early phase by a bile acid breath test using 5 muCi of oral glycine-1-14C labeled glycocholate, aspiration of jejunal fluids by a double lumen tube with a rubber cover on the tip, and examination of deconjugation ability by thin layer chromatography . RESULTS: Expired breath samples from all 18 patients after administration of H2-receptor antagonists showed a significant increase in 14CO2 specific activity compared with those before administration of H2-receptor antagonist and the normal controls, and bacterial overgrowth was found in the jejunal fluid of the patients after administration of H2-receptor antagonist . The administration of tetracycline to the 18 patients reduced the 14CO2 specific activity significantly . The following species were identified in the jejunal fluid samples obtained from the patients: Escherichia coli, Candida albicans, Pseudomonas aeruginosa, enterococcus, Lactobacillus bifidus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Eubacterium lentum, and Eubacterium parvum . All of the species identified except for Escherichia coli, Pseudomonas aeruginosa, and Candida albicans deconjugated bile acids . There were significant correlations between the 14CO2 activity and gastric pH before and after administration of H2-receptor antagonist, respectively . CONCLUSIONS: Patients with gastric ulcers who were treated with H2-receptor antagonists have increased bile acid deconjugation due to bacterial overgrowth in their jejuna containing species that can deconjugate bile acids . The bacterial overgrowth is probably associated with a shift to neutral pH in the gastric juice caused by the H2-receptor antagonists. J Bacteriol, 1995 Apr, 177(8), 2188 - 93 Similarity of "core" structures in two different glycans of tyrosine-linked eubacterial S-layer glycoproteins; Messner P et al.; Previously, the repeating-unit structure of the S-layer glycoprotein from the eubacterium Bacillus alvei CCM 2051 has been determined to be {-->3)-beta-D-Galp-(1-->4)-{alpha-D-Glcp-(1-->6)-}-beta-D-ManpNAc- (1-->}n (E . Altman, J.-R . Brisson, P . Messner, and U . B . Sleytr, Biochem . Cell Biol . 69:72-78, 1991) . Nuclear magnetic resonance spectroscopic reexamination of this glycan reveals that the O-antigen-like domain of the polysaccharide is {see text} connected with the S-layer polypeptide through the "core" structure -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-R hap-(1-->3)-beta-D-Galp-(1-->O)-Tyr . Except for the substitution in position 4 of the nonreducing rhamnose with the modified glyceric acid phosphate residue GroA-2-->OPO2-->4-beta-D-ManpNAc-(1-->, this core is identical to the core of the tyrosine-linked glycan from the S-layer glycoprotein of Thermoanaerobacter thermohydrosulfuricus L111-69 (K . Bock, J . Schuster-Kolbe, E . Altman, G . Allmaier, B . Stahl, R . Christian, U . B . Sleytr, and P . Messner, J . Biol . Chem . 269:7137-7144, 1994). Oral Microbiol Immunol, 1995 Apr, 10(2), 87 - 92 Detection of tet(M) and tet(O) using the polymerase chain reaction in bacteria isolated from patients with periodontal disease; Olsvik B et al.; The polymerase chain reaction was used to examine 114 tetracycline-resistant anaerobic and facultative anaerobic bacterial isolates from patients with periodontal disease for the tet(M) and tet(O) genes . A 740-base-pair fragment of the tet(M) gene was amplified from 84 of 114 isolates, and a 519-base-pair fragment of the tet(O) gene was amplified from 13 streptococcal isolates . Six of 7 tetracycline-resistant isolates of Veillonella spp . and tetracycline-resistant isolates of Eubacterium spp . (n = 3), Eubacterium saburreum (n = 1), Streptococcus intermedius (n = 5) and Gemella morbillorum (n = 2) all harbored the tet(M) gene . The tet(M) and tet(O) negative as well as selected positive isolates were tested for the tet(K) and tet(L) genes using DNA probes . All isolates of Staphylococcus spp . (n = 11) hybridized with the tet(K) probe . None of the isolates tested hybridized with the probe for tet(L) . This is the first report of the tet(M) gene in the facultative bacterium G . morbillorum and in E . saburreum. J Appl Bacteriol, 1995 Apr, 78(4), 394 - 401 Restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal DNA of human Capnocytophaga; Wilson MJ et al.; The confusion in the taxonomic status of the genus Capnocytophaga has made identification of strains and studies on the role of this genus in infectious diseases equivocal . In this study 33 strains of Capnocytophaga including reference strains and various clinical isolates, were studied using RFLP analysis of 16S ribosomal RNA genes . The 16S ribosomal RNA (rRNA) gene sequences from whole cell suspensions and isolated genomic DNA samples were amplified by the polymerase chain reaction (PCR) using eubacterial specific primers . PCR products were purified and characterized by single digestions with 12 restriction endonucleases . Five of these, BanI, CfoI, HaeIII, HphI and RsaII were found to discriminate reproducibly between strains, and restriction patterns (ribotypes) produced by these were analysed to clarify the classification of Capnocytophaga strains . Dendrograms inferring similarities were derived from these data by the UPGMA method . This analysis produced three major clusters of strains, each of which was associated with a previously proposed species type strain: C . gingivalis, C . sputigena and C . ochracea . The results support the division of Capnocytophaga into three species and demonstrate that, despite the heterogeneity of this genus, the modified ribotyping method provides a simple, rapid and reproducible way to identify Capnocytophaga strains. Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2441 - 5 Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications; Brown JR et al.; Universal trees based on sequences of single gene homologs cannot be rooted . Iwabe et al . {Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S . & Miyata, T . (1989) Proc . Natl . Acad . Sci . USA 86, 9355-9359} circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things . Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups . Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential . In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes . An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius) . In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis . Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases . All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al . As well, there was strong support for the monophyly (sensu Hennig) of Archaea . The valyl-tRNA synthetase gene from Tr . vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an endosymbiotic bacterium. J Mol Biol, 1995 Mar 10, 246(5), 563 - 71 A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli; Kumar A et al.; Most of the sigma (transcriptional initiation specificity) subunits of RNA polymerase, from a wide range of eubacteria, show strong elements of amino acid sequence similarity . There is evidence that two of the "conserved" regions, 2.4 and 4.2, are involved in recognition of the consensus DNA sequences centred near -10 and -35, respectively, which define promoter sites for the initiation of transcription . Since all the alternative sigma subunits of the above type function by binding to a common core polymerase enzyme in a given bacterium, it can be predicted that a hybrid sigma might be functional, and if so should permit RNA polymerase to initiate only at a correspondingly hybrid promoter . To test these predictions, a hybrid gene encoding the amino-proximal 529 amino acids of the major Escherichia coli sigma protein, sigma 70 (including region 2.4) followed by the last 82 amino acids of the heat-shock sigma protein, sigma 32 (including region 4.2) was constructed and fused to Plac on a plasmid . Major-consensus, heat-shock and hybrid promoters were fused to a chloramphenicol acetyl transferase (CAT) reporter gene on a compatible plasmid . CAT assays showed that, as predicted, a promoter with a "heat-shock" -35 consensus and a "major" -10 consensus sequence (PHM) required Plac-dependent production of the hybrid sigma (sigma 70-32) for activity in vivo . PHM then became a strong promoter . The hybrid sigma gene has potential advantages over its parents for structure-function studies. Appl Environ Microbiol, 1995 Mar, 61(3), 1141 - 3 Comparison of methods of DNA extraction from stream sediments; Leff LG et al.; In Upper Three Runs Creek (Aiken, S.C.) and many other environments, less than 1% of bacteria visible microscopically can be cultured . Exploitation of molecular biology techniques has led to development of new methods, such as extraction of nucleic acids from soils or sediments, to study the dominant, nonculturable bacteria . The purpose of this study was to compare three published methods of DNA extraction that fall into two general categories: those in which cells are lysed in sediments (the Ogram and Tsai and methods {A . Ogram, G . S . Sayler, and T . Barkay, J . Microbiol . Methods 7:57-66, 1987; Y . L . Tsai and B . H . Olson, Appl . Environ . Microbiol . 57:1070-1074, 1991}) and those in which cells are removed from sediments prior to lysis (the Jacobsen method {C . S . Jacobsen and O . S . Rasmussen; Appl . Environ . Microbiol . 58:2458-2462, 1992}) . DNA yield varied with extraction method; the Ogram method had a significantly higher yield than the other methods . However, DNA extracted via the Ogram method was badly sheared and contained a smaller proportion of eubacterial DNA . The Tsai method was less time consuming than the other methods, but DNA samples were of lower purity . If DNA purity is of paramount concern (as would be the case if PCR was to be performed) and quantity is not important, the Jacobsen method is recommended because of the low concentration of contaminants . If DNA is to be used directly in DNA-DNA hybridizations, the Ogram method is recommended since it gives maximal yields.(ABSTRACT TRUNCATED AT 250 WORDS) Lett Appl Microbiol, 1995 Mar, 20(3), 137 - 40 Hypocholesterolemic effect of Eubacterium coprostanoligenes ATCC 51222 in rabbits; Li L et al.; Recently, a unique bacterium, Eubacterium coprostanoligenes ATCC 51222, that reduces cholesterol to coprostanol was isolated . Because coprostanol is absorbed poorly, it was hypothesized that oral administration of Eu . coprostanoligenes might decrease cholesterol concentration in blood because the micro-organisms will decrease the absorption of endogenous and dietary cholesterol by conversion to coprostanol . To test the hypothesis, three adult New Zealand White rabbits received 4 ml of Eu . coprostanoligenes suspension (ca 2 x 10(7) cells ml-1) daily per os for 10 d; three other adult New Zealand White rabbits received the same dosage of boiled bacterial suspension . Plasma cholesterol concentration of experimental rabbits (183.3 +/- 11.0 mg dl-1, mean +/- S.E.) was significantly lower (P < 0.001) than that of controls (248.8 +/- 12.3 mg dl-1, mean +/- S.E.) . The coprostanol-to-cholesterol ratios in contents of digestive tracts of experimental rabbits were greater than those of controls . The data indicate that oral administration of Eu . coprostanoligenes caused a significant hypocholesterolemic effect in rabbits and that this effect can be explained by the conversion of cholesterol to coprostanol in the intestine. J Mol Evol, 1995 Mar, 40(3), 273 - 9 Molecular classification of living organisms; Saccone C et al.; Recent studies in molecular evolution have generated strong conflicts in opinion as to how world living organisms should be classified . The traditional classification of life into five kingdom has been challenged by the molecular analysis carried out mostly on rRNA sequences, which supported the division of the extant living organisms into three major groups: Archaebacteria, Eubacteria, and Eukaryota . As to the problem of placing the root of the tree of life, the analysis carried out on a few genes has provided discrepant results . In order to measure the genetic distances between species, we have carried out an evolutionary analysis of the glutamine synthetase genes, which previously have been revealed to be good molecular clocks, and of the small and large rRNA genes . All data demonstrate that archaebacteria are more closely related to eubacteria than to eukaryota, thus supporting the classical division of living organisms into two main superkingdoms, Prokaryota and Eukaryota. Mol Biol Evol, 1995 Mar, 12(2), 189 - 97 Glutamine synthetase gene evolution in bacteria; Pesole G et al.; The evolution of the prokaryotic glutamine synthase (GS) genes, namely the GSI and GSII isoforms, has been investigated using the second codon positions, which have previously proven to behave as a good molecular clock . Our data confirm the early divergence between prokaryotic and eukaryotic GSII before the splitting between plants and animals . The phylogenetic tree of the GSI isoforms shows Archaebacteria to be more closely related to Eubacteria than to Eukaryotes . This finding is confirmed by the phylogenetic analysis carried out on both large and small subunits of rRNA . However, differently from the rRNA analyses, Crenarchaeota and Euryarchaeota Archaebacteria, as well as high- and low-GC gram-positive bacteria, appear to be polyphyletic . We provide evidence that the observed polyphyly of Archaebacteria might be only apparent, resulting from a gene duplication event preceding the split between Archaebacteria and Eubacteria and followed by the retention of only one isoform in the extant lineages . Both gram-negative bacteria and high-GC gram-positive bacteria, which appear closely related, have GS activity regulated by an adenylylation/deadenylylation mechanism . A lateral gene transfer from Archaebacteria to low-GC eubacteria is invoked to explain the observed polyphyly of gram-positive bacteria. J Bacteriol, 1995 Mar, 177(6), 1614 - 9 A gene encoding a putative membrane protein homologous to the major facilitator superfamily of transporters maps upstream of the beta-glycosidase gene in the archaeon Sulfolobus solfataricus; Prisco A et al.; We have identified a gene encoding a putative membrane protein homologous to the major facilitator superfamily, mapping upstream of the lacS gene in Sulfolobus solfataricus . Permeases from this family mediate secondary transport and are widely distributed among eubacteria and eukaryotes; the finding of an archaeal member suggests that this mechanism of transport evolved before the divergence of the three living domains . We also report a transcriptional mapping of the gene cluster. Nucleic Acids Res, 1995 Feb 25, 23(4), 565 - 70 Novel protein families in archaean genomes; Ouzonis C et al.; In a quest for novel functions in archaea, all archaean hypothetical open reading frames (ORFs), as annotated in the Swiss-Prot protein sequence database, were used to search the latest databases for the identification of characterized homologues . Of the 95 hypothetical archaean ORFs, 25 were found to be homologous to another hypothetical archaean ORF, while 36 were homologous to non-archaean proteins, of which as many as 30 were homologous to a characterized protein family . Thus the level of sequence similarity in this set reaches 64%, while the level of function assignment is only 32% . Of the ORFs with predicted functions, 12 homologies are reported here for the first time and represent nine new functions and one gene duplication at an acetyl-coA synthetase locus . The novel functions include components of the transcriptional and translational apparatus, such as ribosomal proteins, modification enzymes and a translation initiation factor . In addition, new enzymes are identified in archaea, such as cobyric acid synthase, dCTP deaminase and the first archaean homologues of a new subclass of ATP binding proteins found in fungi . Finally, it is shown that the putative laminin receptor family of eukaryotes and an archaean homologue belong to the previously characterized ribosomal protein family S2 from eubacteria . From the present and previous work, the major implication is that archaea seem to have a mode of expression of genetic information rather similar to eukaryotes, while eubacteria may have proceeded into unique ways of transcription and translation . In addition, with the detection of proteins in various metabolic and genetic processes in archaea, we can further predict the presence of additional proteins involved in these processes. J Biol Chem, 1995 Feb 24, 270(8), 3732 - 40 Altered monovinyl and divinyl protochlorophyllide pools in bchJ mutants of Rhodobacter capsulatus . Possible monovinyl substrate discrimination of light-independent protochlorophyllide reductase; Suzuki JY et al.; In land plants in particular, it has been well established that chlorophyll intermediates, Mg-protoporphyrin, Mg-protoporphyrin monomethylester, protochlorophyllide, and chlorophyllide occur as monovinyl and divinyl forms . The pool of monovinyl and divinyl intermediates differ according to species, age of tissue, and light regime . In this study, we investigated the monovinyl and divinyl characteristics of protochlorophyllide and chlorophyllide in the purple non-sulfur photosynthetic eubacterium Rhodobacter capsulatus . Our results indicate that mutations in genes known to completely block the reduction of protochlorophyllide to chlorophyllide (such as bchN, bchB, and bchL mutants), accumulate a pool of monovinyl and divinyl forms of protochlorophyllide just as observed in plants . However, we also observed that directed insertion and deletion mutations in bchJ, a gene located in the photosynthesis gene cluster, affected the ratio of monovinyl and divinyl protochlorophyllide . Specifically, bchJ-disrupted strains accumulate reduced levels of bacteriochlorophyll concomitant with the accumulation of divinyl protochlorophyllide . Mutants of bchJ in combination with a second mutation in bchL still produce a mixed pool of monovinyl and divinyl protochlorophyllide; however, the ratio of monovinyl to divinyl protochlorophyllide is skewed in favor of divinyl protochlorophyllide . These results thus identify bchJ as the first sequenced gene that affects the divinyl to monovinyl ratio of photopigment intermediates in any photosynthetic organism . In addition, the results of our study also suggest that light-independent protochlorophyllide reductase is discriminatory for a monovinyl substrate. J Biol Chem, 1995 Feb 24, 270(8), 3720 - 5 Ubiquitin in the prokaryote Anabaena variabilis; Durner J et al.; The ubiquitin-dependent pathway for protein degradation has been found to play a major role in controlling protein turnover in the cell . Ubiquitin is one of the most conserved proteins yet identified, and up until now it has been thought to be present only in eukaryotes and archaebacteria . This is the first report on the detection and purification of ubiquitin from a eubacterium, the cyanobacterium Anabaena variabilis . The purification procedure included a heat denaturing step, fractionated ammonium sulfate precipitation, two gel filtration runs (Sephadex G-50 and Superose 12), and a final hydroxylapatite chromatography . Comparisons with bovine ubiquitin showed a high similarity with respect to antigenicity to anti-ubiquitin (bovine), molecular mass (M(r) = 6,000), isoelectric point (pI 6.5), and NH2-terminal sequence . The existence of ubiquitin in A . variabilis was confirmed by Southern hybridization . In in vitro experiments both cyanobacterial and bovine ubiquitin were covalently attached to several target proteins from A . variabilis, respectively . Data are presented which suggest ubiquitination of dinitrogenase reductase, the Fe-protein subunit of nitrogenase . Our findings imply that ubiquitination equivalent to the eukaryotic system is instrumental in this organism. Biochem Biophys Res Commun, 1995 Feb 6, 207(1), 265 - 72 Expression of rat liver AdoHcy hydrolase in a Rhodobacter capsulatus ahcY mutant restores pigment formation and photosynthetic growth; Aksamit RR et al.; An amino acid alignment of fourteen S-adenosylhomocysteine hydrolases shows that sequences from six photosynthetic species and one species possibly derived from algae have an internal 36 to 41 amino acid sequence that is not present in hydrolase sequences from seven non-photosynthetic species . In the photosynthetic eubacterium Rhodobacter capsulatus, the StLB1 strain has a disrupted hydrolase gene, and hydrolase activity is not detectable . Photopigment synthesis and photosynthetic growth are significantly reduced in the StLB1 strain . Introduction of rat hydrolase cDNA into the StLB1 strain restored hydrolase activity, photopigment synthesis and photosynthetic growth . The results show that the 36 amino acid sequence of Rhodobacter capsulatus S-adenosylhomocysteine hydrolase does not have a photosynthesis specific function. J Mol Biol, 1995 Feb 3, 245(5), 461 - 6 Three-dimensional reconstruction of mammalian 40 S ribosomal subunit embedded in ice; Srivastava S et al.; A platform-like structure, which appears equivalent to the platform or lobe structure of the 30 S subunit of the eubacterial ribosome, is observed in the reconstruction of the small 40 S ribosomal subunit from images of ice-embedded particles . This cup-shaped structure, 15.0 nm in side length and 13.5 nm wide at its rim, extends obliquely upward on the back of the subunit . Other previously characterized features of the 40 S subunit can readily be identified: the head with its prominent beak structure, the body with its two back lobes expressed as relatively small-scale features, and the two widely separated feet that comprise the base of the subunit. FEMS Microbiol Lett, 1995 Feb 1, 126(1), 69 - 74 Cellular fatty acids and aldehydes of oral Eubacterium; Itoh U et al.; The cellular fatty acids and aldehydes of oral Eubacterium species were determined by gas chromatography-mass spectrometry . E . brachy and E . lentum contained mainly branched-chain fatty acids, whereas the others contained straight-chain acids . E . brachy, E . lentum, E . yurii ssp . yurii, E . yurii spp . margaretiae, E . limosum, E . plauti and E . aerofaciens also contained aldehydes with even carbon numbers . In addition to species-specific components, the compositional ratios of fatty acids and aldehydes characterized each individual species . The 10 species tested were divided into 5 groups by the principal component analysis . Cellular fatty acids and aldehydes would be chemical markers for interspecies differentiation of oral Eubacterium. Biol Chem Hoppe Seyler, 1995 Feb, 376(2), 119 - 26 Primary structure of the thermosome from Thermoplasma acidophilum; Waldmann T et al.; The thermosome, a chaperonin from the archaebacterium Thermoplasma acidophilum, consists of two subunits (M(r) 58,000 and 60,000) which assemble into a cylindrical complex of pseudo eight-fold rotational symmetry . The sequences of the two subunits are approximately 60% identical to each other and to TF55 from Sulfolobus shibatae, and are 30-40% identical to the subunits of the TCP1 containing ring complex (TRiC) from the eukaryotic cytosol . A dendrogram of this family of chaperonins contains eight eukaryotic branches of TRiC subunits and one archaebacterial branch of thermosome subunits . Alignment of thermosome/TRiC sequences with eubacterial and eukaryotic Hsp60 sequences reveals a statistically significant similarity in two large N- and C-terminal blocks of sequence . Based on this alignment and on the recently published crystal structure of GroEL, we propose that subunits of the thermosome/TRiC family of chaperonins have a similar equatorial domain and overall domain topology as GroEL but differ in the structure of the apical domain. Insect Mol Biol, 1995 Feb, 4(1), 47 - 59 Genetics of the tryptophan biosynthetic pathway of the prokaryotic endosymbiont (Buchnera) of the aphid Schlechtendalia chinensis; Lai CY et al.; Two DNA fragments (3941 and 7152 base pairs) from the procaryotic endosymbiont (Buchnera) of the aphid Schlechtendalia chinensis were cloned and sequenced . The smaller fragment contained trpEG and the larger fragment contained trpDC(F)BA, genes coding for enzymes of the tryptophan biosynthetic pathway which convert chorismate to tryptophan . Both of these gene clusters were present as one copy on the endosymbiont chromosome and probably constitute two transcription units . The deduced amino acid sequences of the proteins was 51-61% identical to the corresponding proteins were previously studied in Buchnera of the aphid Schizaphis graminum . In this endosymbiont, trpEG is amplified and located on a plasmid, whereas, in the endosymbiont of S . chinensis, as in other eubacteria, trpEG occurs as a single copy on the bacterial chromosome . Implications of these findings for the evolution of this mutualistic association are discussed. Microbiology, 1995 Feb, 141 ( Pt 2), 449 - 58 Characterization of the locus encoding the {Ni-Fe} sulfhydrogenase from the archaeon Pyrococcus furiosus: evidence for a relationship to bacterial sulfite reductases; Pedroni P et al.; The hydBGDA genes, which encode the four subunits beta, gamma, delta and alpha of the {Ni-Fe} hydrogenase from the archaeon Pyrococcus furiosus, have been isolated and sequenced using a PCR/IPCR-based strategy . From the sequence analysis it appears that the four structural genes are tightly linked and organized in a single transcription unit . The hydD and hydA gene products are related to the small and the large subunits of several archaeal and eubacterial {Ni-Fe} hydrogenases with an overall degree of sequence relatedness ranging from 35% to 50% (identity + similarity) . In particular, the amino acid sequence motifs involved in the accommodation of nickel and iron-sulfur clusters are conserved . In addition, the database search revealed that the hydB and hydG gene products are homologous to the asrA- and asrB-encoded subunits of the sulfite reductase enzyme from Salmonella typhimurium . This is particularly interesting in view of the recent finding that the P . furiosus hydrogenase appears to be a bifunctional enzyme endowed with both proton- and sulfur-reducing activities. FEMS Microbiol Lett, 1995 Jan 15, 125(2-3), 225 - 9 Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis; Park NY et al.; A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name 'Anguillina coli' . The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for 'A . coli' . The PCR was used to correctly identify DNA extracted from 43 isolates of 'A . coli' from humans and pigs, whilst no product was produced from Escherichia coli, or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi . The amplification provided a rapid and simple means of identifying DNA from isolates of 'A . coli', and could be used on boiled whole 'A . coli' cells, with a detection limit equivalent to 2.5 x 10(2) cells . The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs. Gene, 1995 Jan 11, 152(1), 93 - 8 Characterization of levJ, a sucrase/fructanase-encoding gene from Actinomyces naeslundii T14V, and comparison of its product with other sucrose-cleaving enzymes; Norman JM et al.; A library of Actinomyces naeslundii T14V DNA was constructed in plasmid pUC18 and from this several sucrose-positive clones were isolated . Evidence was obtained that all these clones contained the same gene . One clone, which carried a plasmid that was named pPNG102, was chosen for further study . It was found that the enzyme specified by this plasmid hydrolyzed sucrose, raffinose, inulin and levan, but not dextran, and did not synthesize fructan or glucan from sucrose . The sequence of the insert in pPNG102 was determined and was found to contain a large ORF that specifies a polypeptide of 99,319 Da with similarity to other sucrases . This gene was named levJ . The deduced amino acid (aa) sequence contained both a potential signal sequence and potential C-terminal cell envelope attachment domain . Alignments revealed an internal 331-aa domain not present in other levanases and sucrases . A neighbour-joining tree showed that sucrases of eukaryotic origin form a cluster with eubacterial sucrase/fructanases, and this cluster does not include other eubacterial sucrases . It is postulated that certain eukaryotic sucrase-encoding genes are of eubacterial origin. Gene, 1995 Jan 11, 152(1), 11 - 7 The ATP synthase (F1F0) of Streptomyces lividans: sequencing of the atp operon and phylogenetic considerations with subunit beta; Hensel M et al.; The DNA encoding the subunits of the ATP synthase (F1F0) of Streptomyces lividans 66 strain 1326 was identified using oligodeoxyribonucleotide probes derived from the N-terminal sequence of subunit gamma of the F1 complex . The complete nucleotide sequence of the operon was determined . The atp operon contains nine genes, atpIBEFHAGDC, encoding the eight structural components of the ATP synthase complex and the i protein, a polypeptide of unknown function . The gene order found is identical to that in other non-photosynthetic eubacteria . The determination of the N-terminal amino acid (aa) sequences of the F1 subunits alpha, beta, gamma, delta and epsilon allowed us to identify the translational start points and to define the primary structures of the proteins . The aa sequence deduced for subunit delta revealed an N-terminal extension of about 90 aa, which is not present in any delta subunit or OSCP (oligomycin sensitivity-conferral protein) of other species studied so far . The phylogenetic relationship of eu- and archaebacteria was investigated using sequencing data of the highly conserved beta subunit of different ATP synthases including that of S . lividans . The calculations revealed that S . lividans beta does not form a phylogenetic group together with the Gram+ taxa of low G+C contents, but is more closely related to the beta subunit of Rhodobacteria. Gene, 1995 Jan 11, 152(1), 103 - 6 A gene from the hyperthermophile Pyrococcus furiosus whose deduced product is homologous to members of the prolyl oligopeptidase family of proteases; Robinson KA et al.; The mlr-2 gene from the hyperthermophilic archaeum Pyrococcus furiosus was identified from a family of clones whose expression was influenced by the presence of maltose in the medium . The sequence of 2100 bp of DNA containing mlr-2 and its flanking regions revealed a 616-amino-acid (71 kDa) open reading frame (ORF) . The ORF's initiation codon appeared 10 nt into the mlr-2 message and was not preceded by any apparent ribosome-binding site . The deduced product shared homology with prolyl endopeptidases from both eukaryotic and eubacterial sources (52-57% similarity, 30-37% identity) and signature domains containing the Ser-Asp-His triad, which is characteristic of this family of proteases, were present . Northern blot experiments revealed the presence of an approx . 2.0-kb transcript in P . furiosus extracts, corresponding in length to that expected from mlr-2 expression . Initiation of transcription occurred 23 bp downstream from a putative BoxA promoter element. Mol Biol Rep, 1995-96, 22(2-3), 135 - 8 The RNase P of Dictyostelium discoideum; Drainas D; Ribonuclease P (RNase P) is a key enzyme involved in tRNA biosynthesis . It catalyses the endonucleolytic cleavage of nearly all tRNA precursors to produce 5'-end matured tRNA . RNase P activity has been found in all organisms examined, from bacteria to mammals . Eubacterial RNase RNA is the only known RNA enzyme which functions in trans in nature . Similar behaviour has not been demonstrated in RNase P enzymes examined from archaebacteria or eukaryotes . Characterisation of RNase P enzymes from more diverse eukaryotic species, including the slime mold Dictyostelium discoideum, is useful for comparative analysis of the structure and function of eukaryotic RNase P. Mol Biol Rep, 1995-96, 22(2-3), 125 - 9 RNase P RNA of Mycoplasma capricolum; Ushida C et al.; There are at least six small stable RNAs in Mycoplasma capricolum cells besides tRNAs and rRNAs . One of them, MCS5 RNA, is a homolog of RNase P RNA . The predicted secondary structure of this RNA is essentially the same as that of other eubacterial RNase P RNAs . MCS5 RNA is more similar to the RNase P RNA of B . Subtilis than to that of E . coli . This is consistent with previous conclusions that mycoplasmas are phylogenetically related to the low G + C Gram-positive bacterial group . The major substrates for MCS5 RNA must be the precursors of tRNAs . The precursor of MCS6 RNA, which is a homolog of the E . coli 10Sa RNA, may also be a substrate for the MCS5 RNA because this RNA has a tRNA-like structure at its 5' and 3' ends. Virus Genes, 1995, 11(2-3), 271 - 84 Evolution of viral DNA-dependent RNA polymerases; Sonntag KC et al.; The DNA-dependent RNA polymerase (DdRP or RNAP) is an essential enzyme of transcription of replicating systems of prokaryotic and eukaryotic organisms as well as cytoplasmic DNA viruses . DdRPs are complex multisubunit enzymes consisting of 8-14 subunits, including two large subunits and several smaller polypeptides (small subunits) . An extensive search between the amino acid sequences of the known largest subunit of DNA-dependent RNA polymerases (RPO1) of different organisms indicates that all these polypeptides possess a universal heptapeptide NADFDGD in domain D . All RPO1 harbor a second well-conserved hexapeptide RQP(TS)LH upstream (26-31 amino acids) of the universal motif . The genes encoding the largest subunit of DdRP of insect iridescent virus type 6 (IIV6), fish lymphocystis disease virus (LCDV), and molluscum contagiosum virus (MCV-1), all members of the group of cytoplasmic DNA viruses, were identified by PCR technology . With the exception of IIV6, all other viral RPO1 possess the two C-terminal conserved regions G and H . The lack of C-terminal repetitive heptapeptide (YSPTSPS), which is a common feature of the largest subunit of eukaryotic RNAPII, is an additional characteristic of RPO1 proteins of LCDV and of MCV-1 . All viral RPO1 proteins were found to be lacking the amino acid N at a distinct position in domain F . This amino acid is known to be highly conserved in alpha-amanitin-sensitive eukaryotic RNA polymerases II . Comparison of the amino acid sequences of the RPO1 polypeptides of IIV6, LCDV, and MCV-1 with the corresponding prokaryotic, eukaryotic, and viral proteins revealed differences in amino acid similarity and phylogenetic relationships . IIV6 RPO1 possesses the closest similarity to the homologous subunit of eukaryotic RNAPII and lower but also significant similarity to that of eukaryotic RNAPI and RNAPIII, archaeal, eubacterial, and viral polymerases . The similarity between RPO1 of IIV6 and the cellular polymerase subunits is consistently higher than to the RPO1 of other cytoplasmic DNA viruses, for example, vaccinia and variola virus, African swine fever virus (ASFV), and MCV-1 . The RPO1 of LCDV shows the highest similarity to the RPO1 of IIV6 and significant lower similarity to the eukaryotic polymerases II and III as well as to the archaebacteral subunit . However, it is still considerably more similar to the cellular polymerase subunits than to the homologous viral proteins . The RPO1 of IIV6 possesses more similarity to cellular polymerases than the complete RPO1 of LCDV, indicating that there is a substantial difference in the organization of the RPO1 genes between these members of two genera of the Iridoviridae family . Analysis of the MCV-1 RPO1 revealed high amino acid homologies to the corresponding polypeptides of vaccinia and variola virus . The viral RPO1 proteins, including vaccinia and variola virus, MCV-1, ASFV, IIV6, and LCDV, share the common feature of showing the highest similarity to the largest subunit of eukaryotic RNAPII than to that of RNAPI, RNAPIII, and RPO1 of archaebacterias, eubacterias, ASFV, IIV6, and LCDV . Evolution of the individual largest subunit of DdRPs was tentatively investigated by generating phylogenetic trees using multiple amino acid alignments . These indicate that the RPO1 proteins of IIV6 and LCDV might have evolved from the largest subunit of eukaryotic RNAPII after divergence from the homologous subunits of RNAPI and RNAPIII . In contrast, evolutionary development of the RPO1 of vaccinia and variola virus, MCV-1, and ASFV seems to be quite different, with their common ancestor diverging from cellular homologues before the separation of the three types of eukaryotic ploymerases and having probably diverged earlier from their common lineage with cellular proteins. Biochimie, 1995, 77(9), 744 - 50 A family of homologous substrate-binding proteins with a broad range of substrate specificity and dissimilar biological functions; Wu LF et al.; The uptake of peptides is accomplished mainly by a family of homologous oligopeptide or dipeptide transporters in bacteria . Computer-aided sequence analyses expand members of the oligopeptide-binding protein family to nickel and heme permeases and other proteins, including an enzyme hyaluronate synthase . They are involved in human pathogenicity, bacterial virulence, substrate-sensing, bacterial conjugation and bacterial metabolic reactions distinct from nutrient uptake . These homologous proteins are found in both purple bacteria and Gram-positive bacteria, indicating the presence of a common ancestor before the appearance of the two eubacterial phyla . Nevertheless, the pheromone-binding proteins, involved in bacterial conjugation, and the hyaluronate synthase are present only in the low G-C Gram-positive eubacteria subdivision, which suggests that these proteins diverged from the common ancestor after the appearance of this subdivision. Postepy Hig Med Dosw, 1995, 49(1), 149 - 60 {Initiation of DNA replication in Streptomyces; organizational comparison of the oriC region in prokaryotic organisms}; Zakrzewska-Czerwinska J; DnaA protein and its binding sequence (DnaA-box) are two elements essential for the initiation of chromosomal replication in Escherichia coli and other bacteria . Recently these two elements have been found to be conserved in four Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus, Mycoplasma capricolum and Streptomyces lividans) . The structures of eubacterial DnaA-box regions and their locations on the chromosome and than functional aspects od DnaA protein have been compared. Nucleic Acids Symp Ser, 1995, (33), 89 - 91 Analysis of conserved positions in nuclear RNase P RNA; Pagan-Ramos E et al.; Secondary structure models of eubacterial and eukaryotic nuclear RNase P RNA subunits show extensive structural similarities, allowing the identification of highly conserved nucleotide positions and molecular modeling of the enzyme-substrate complex in three dimensions . Based on this information, we present a preliminary tertiary structure model of the yeast nuclear RNase P RNA . In addition, the most conserved positions in the structure have been subjected to sequence randomization, with viable sequence variations identified by selection in vivo and characterized for phenotypic consequences. Annu Rev Microbiol, 1995, 49, 55 - 94 Genetics, physiology, and evolutionary relationships of the genus Buchnera: intracellular symbionts of aphids; Baumann P et al.; Evolutionary studies suggest that 200-250 million years ago an aphid ancestor was infected with a free-living eubacterium . The latter became established within aphid cells . Host and endosymbiont (genus Buchnera) became interdependent and unable to survive without each other . The growth of Buchnera became integrated with that of the aphids, which acquired the endosymbionts from their mothers before birth . Speciation of host lineages was paralleled by divergence of associated endosymbiont lineages, resulting in parallel evolution of Buchnera and aphids . Present day Buchnera retains many of the properties of its free-living ancestor, containing genes for proteins involved in DNA replication, transcription, and translation, as well as chaperonins and proteins involved in secretion, energy-yielding metabolism, and amino acid biosynthesis . Some of these processes are also observed in isolated endosymbiont cells . Genetic and physiological studies indicate that Buchnera can synthesize methionine, cysteine, and tryptophan and supply these amino acids to the aphid host . In the case of some fast-growing species of aphids, the overproduction of tryptophan by Buchnera involves plasmid-amplification of the gene coding for anthranilate synthase, the first enzyme of the tryptophan biosynthetic pathway . These recent studies provide a beginning in our understanding of Buchnera and its role in the endosymbiosis with aphids. Mol Biol Evol, 1995 Jan, 12(1), 16 - 27 Assessing horizontal transfer of nifHDK genes in eubacteria: nucleotide sequence of nifK from Frankia strain HFPCcI3; Hirsch AM et al.; The structural genes for nitrogenase, nifK, nifD, and nifH, are crucial for nitrogen fixation . Previous phylogenetic analysis of the amino acid sequence of nifH suggested that this gene had been horizontally transferred from a proteobacterium to the gram-positive/cyanobacterial clade, although the confounding effects of paralogous comparisons made interpretation of the data difficult . An additional test of nif gene horizontal transfer using nifD was made, but the NifD phylogeny lacked resolution . Here nif gene phylogeny is addressed with a phylogenetic analysis of a third and longer nif gene, nifK . As part of the study, the nifK gene of the key taxon Frankia was sequenced . Parsimony and some distance analyses of the nifK amino acid sequences provide support for vertical descent of nifK, but other distance trees provide support for the lateral transfer of the gene . Bootstrap support was found for both hypotheses in all trees; the nifK data do not definitively favor one or the other hypothesis . A parsimony analysis of NifH provides support for horizontal transfer in accord with previous reports, although bootstrap analysis also shows some support for vertical descent of the orthologous nifH genes . A wider sampling of taxa and more sophisticated methods of phylogenetic inference are needed to understand the evolution of nif genes . The nif genes may also be powerful phylogenetic tools . If nifK evolved by vertical descent, it provides strong evidence that the cyanobacteria and proteobacteria are sister groups to the exclusion of the firmicutes, whereas 16S rRNA sequences are unable to resolve the relationships of these three major eubacterial lineages. Mol Biol Evol, 1995 Jan, 12(1), 1 - 6 Protein-based phylogenies support a chimeric origin for the eukaryotic genome; Golding GB et al.; The phylogenetic position of the archaebacteria and the place of eukaryotes in the history of life remain a question of debate . Recent studies based on some protein-sequence data have obtained unusual phylogenies for these organisms . We therefore collected the protein sequences that were available with representatives from each of the major forms of life: the gram-negative bacteria, gram-positive bacteria, archaebacteria, and eukaryotes . Monophyletic, unrooted phylogenies based on these sequence data show that seven of 24 proteins yield a significant gram-positive-archaebacteria clade/gram-negative-eukaryotic clade . The phylogenies for these seven proteins cannot be explained by the traditional three-way split of the eukaryotes, archaebacteria, and eubacteria . Nine of the 24 proteins yield the traditional gram-positive-gram-negative clade/archaebacteria-eukaryotic clade . The remaining eight proteins give phylogenies that cannot be statistically distinguished . These results support the hypothesis of a chimeric origin for the eukaryotic cell nucleus formed from the fusion of an archaebacteria and a gram-negative bacteria. J Bacteriol, 1995 Jan, 177(1), 82 - 5 Lack of production of (p)ppGpp in Halobacterium volcanii under conditions that are effective in the eubacteria; Scoarughi GL et al.; The stringent halobacterial strain Haloferax volcanii was subjected to a set of physiological conditions different from amino acid starvation that are known to cause production of guanosine polyphosphates {(p)pp Gpp} in eubacteria via the relA-independent (spoT) pathway . The conditions used were temperature upshift, treatment with cyanide, and total starvation . Under none of these conditions were detectable levels of (p)ppGpp observed . This result, in conjunction with our previous finding that (p)ppGpp synthesis does not occur under amino acid starvation, leads to the conclusion that in halobacteria both growth rate control and stringency are probably governed by mechanisms that operate in the absence of ppGpp . During exponential growth, a low level of phosphorylated compounds with electrophoretic mobilities similar, but not identical, to that of (p)ppGpp were observed . The intracellular concentration of these compounds increased considerably during the stationary phase of growth and with all of the treatments used . The compounds were identified as short-chain polyphosphates identical to those found under similar conditions in Saccharomyces cerevisiae. Dtsch Tierarztl Wochenschr, 1995 Jan, 102(1), 21 - 7 {The therapy of urinary tract infections in sows}; Wendt M et al.; Fifteen sows spontaneously infected with Eubacterium suis were treated with enrofloxacin and, if necessary, with ampicillin . Before and after treatment different blood and urine parameters were determined with regard to renal function and the condition of the urinary bladder was examined by cystoscopy . Besides results of a controlled treatment in a breeding herd with a high frequency of urinary tract infections are referred . Duration of treatment with appropriate dosage should be at least 10 days . Even when a haemorrhagic inflammation was present therapy of infections with Eubacterium suis showed no problems, if the alterations were restricted to the bladder . In sows with renal insufficiency in case of ascending infection treatment is also possible on principle, but intensive infusion therapy is necessary in addition to antibiosis . Reliable estimation of the renal function can be obtained by measurement of creatinine and urea blood levels . Enrofloxacin treatment of urinary tract infections was successful in a breeding herd . An insufficient water supply and a high incidence of crystalluria were important predisposing factors. Mol Microbiol, 1995 Jan, 15(1), 1 - 11 Evolution of the chaperonin families (Hsp60, Hsp10 and Tcp-1) of proteins and the origin of eukaryotic cells; Gupta RS; The members of the 10 kDa and 60 kDa heat-shock chaperonin proteins (Hsp10 and Hsp60 or Cpn10 and Cpn60), which form an operon in bacteria, are present in all eubacteria and eukaryotic cell organelles such as mitochondria and chloroplasts . In archaebacteria and eukaryotic cell cytosol, no close homologues of Hsp10 or Hsp60 have been identified . However, these species (or cell compartments) contain the Tcp-1 family of proteins (distant homologues of Hsp60) . Phylogenetic analysis based on global alignments of Hsp60 and Hsp10 sequences presented here provide some evidence regarding the evolution of mitochondria from a member of the alpha-subdivision of Gram-negative bacteria and chloroplasts from cyanobacterial species, respectively . This interference is strengthened by the presence of sequence signatures that are uniquely shared between Hsp60 homologues from alpha-purple bacteria and mitochondria on one hand, and the chloroplasts and cyanobacterial hsp60s on the other . Within the alpha-purple subdivision, species such as Rickettsia and Ehrlichia, which live intracellularly within eukaryotic cells, are indicated to be the closest relatives of mitochondrial homologues . In the Hsp60 evolutionary tree, rooted using the Tcp-1 homologue, the order of branching of the major groups was as follows: Gram-positive bacteria--cyanobacteria and chloroplasts--chlamydiae and spirochaetes--beta- and gamma-Gram-negative purple bacteria--alpha-purple bacteria--mitochondria . A similar branching order was observed independently in the Hsp10 tree . Multiple Hsp60 homologues, when present in a group of species, were found to be clustered together in the trees, indicating that they evolved by independent gene-duplication events . This review also considers in detail the evolutionary relationship between Hsp60 and Tcp-1 families of proteins based on two different models (viz . archaebacterial and chimeric) for the origin of eukaryotic cell nucleus . Some predictions of the chimeric model are also discussed. Schweiz Arch Tierheilkd, 1995, 137(4), 129 - 36 {Serologic examinations for the detection of antibodies against Eubacterium suis in swine}; Wendt M et al.; Determination of antibodies against Eubacterium suis (E . suis) in the serum of pigs was carried out by an indirect immunofluorescence test . Therefore 35 sows with urinary tract infections as well as 43 healthy control sows were investigated . E . suis could be detected in the urine of 19 sows . Serological results were compared with the bacteriological examinations . To reach a specificity of 100% for detection of animals actually infected with E . suis, titres from 1:16++ can be accepted as positive . Therefore sensitivity gave a level of 78.9% . Seroconversion and development of antibody titers were studied on 15 sows, which were experimentally infected with E . suis . Antibodies could be shown 3 weeks after infection at the earliest, they were preferably demonstrated in sows with a haemorrhagic cystitis . No correlation existed between the affection of the kidneys and the development and the level of titres respectively . Serological investigations can turn out negative in spite of E . suis infection, if immunological response still has not taken place or development of antibodies has failed to appear after short-time infection of the bladder. Biochimie, 1995, 77(1-2), 7 - 15 Pseudouridine and O2'-methylated nucleosides . Significance of their selective occurrence in rRNA domains that function in ribosome-catalyzed synthesis of the peptide bonds in proteins; Lane BG et al.; Pseudouridine (5-ribosyluracil, psi) was the first of a host of modified nucleoside constituents detected in cellular RNA and it remains the most abundant, being broadly distributed in the RNA of archaebacteria, eubacteria and eukaryotes . Like some other modifications, psi is particularly abundant in more complex organisms, reaching 2-3% of the total nucleoside constituents in tRNA, snRNA and rRNA of multicellular plants and animals . Like all other modified nucleosides, psi arises by site-specific, enzymically catalyzed modification of a nucleoside residue in an RNA molecule . Unlike all other modified nucleosides, psi arises by isomerisation (not substitution) of a classical nucleoside, uridine (1-ribosyluracil) . There have been suggestions that key processes such as ribosome assembly and peptidyl transfer may rely, more than is generally appreciated, on RNA modifications such as O2'-methylation and pseudouridylation, respectively . However, a persuasive case for the view that secondary modifications are of primary importance in ribosome function has not been convincingly made . Accordingly, we think it is timely to broaden what is generally meant by the 'catalytic properties of rRNA', and to ask, to what extent do modifications contribute to in vivo rates of ribosome assembly and ribosomal peptide-bond synthesis? The first part of this article sets forth the evidence that there is a conspicuous association between modified nucleosides and cellular RNAs that participate in group-transfer reactions . The second part reviews evidence in support of the view that the functions of psi and other modified nucleosides are likely of central importance for understanding the dynamics and stereostructural modeling at functionally significant sites in the ribosome. Annu Rev Biochem, 1995, 64, 435 - 61 Structure and activities of group II introns; Michel F et al.; Group II introns are found in eubacteria and eubacterial-derived, organellar genomes . They have ribozymic activities, by which they direct and catalyze the splicing of the exons flanking them . This chapter reviews the secondary structure and known tertiary interactions of the ribozymic component of group II introns in relation to the problems of specifying splice sites and building a catalytic core . We pay special attention to the relationship between the transesterification and hydrolytic modes of initiating splicing and the stereospecificities of these reactions . A number of group II introns encode proteins of the reverse transcriptase family; the activity of these proteins enables the host introns to change genomic locations by mechanisms that are only beginning to be deciphered . Finally, we briefly discuss multipartite and post-transcriptionally edited group II introns, together with the intron microcosm of Euglena gracilis chloroplasts and the possible relationships between group II and spliceosome-catalyzed splicing processes. Gene, 1994 Dec 30, 151(1-2), 173 - 6 Isolation, sequence and characterization of the maltose-regulated mlrA gene from the hyperthermophilic archaeum Pyrococcus furiosus; Robinson KA et al.; The mlrA (maltose regulated) gene from the hyperthermophilic archaeum Pyrococcus furiosus was identified from a family of clones whose expression was influenced by the presence of maltose in the medium . Sequencing of the 2276 bp of DNA containing mlrA and flanking regions revealed a 753-amino-acid (aa) (88 kDa) open reading frame (ORF) . The ORF is preceded by a bacterial-like ribosome-binding site . The deduced product shared extensive homology with pyruvate dikinases (PDK) from both eukaryal and eubacterial sources (35-61% similarity) and the signature domains characteristic of this class of proteins were present . Northern blot experiments demonstrated the presence of an approx . 2.4-kb transcript in P . furiosus extracts, corresponding in length to that expected from expression of mlrA . P . furiosus cultures grown in the presence of maltose were found to contain approx . 5-10-fold greater mlrA mRNA than those grown without maltose . Initiation of transcription under both cultural conditions occurred at the same transcription start point (tsp), 23 bp downstream from a putative BoxA promoter element. Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12842 - 6 Which bacterium is the ancestor of the animal mitochondrial genome? Karlin S, Campbell AM. We present considerable data supporting the hypothesis that a Sulfolobus- or Mycoplasma-like endosymbiont, rather than an alpha-proteobacterium, is the ancestor of animal mitochondrial genomes . This hypothesis is based on pronounced similarities in oligonucleotide relative abundance extremes common to animal mtDNA, Sulfolobus, and Mycoplasma capricolum and pronounced discrepancies of these relative abundance values with respect to alpha-proteobacteria . In addition, genomic dinucleotide relative abundance measures place Sulfolobus and M . capricolum among the closest to animal mitochondrial genomes, whereas the classical eubacteria, especially the alpha-proteobacteria, are at excessive distances . There are also considerable molecular and cellular phenotypic analogies among mtDNA, Sulfolobus, and M . capricolum. FEBS Lett, 1994 Dec 19, 356(2-3), 345 - 50 Primary structure of a multimeric protein, homologous to the PEP-utilizing enzyme family and isolated from a hyperthermophilic archaebacterium; Cicicopol C et al.; A large protein complex (approx . 2000 kDa) was found in the cytosol of the hyperthermophilic archaebacterium Staphylothermus marinus . The purified protein was shown to be a homomultimer of 93 kDa subunits, the primary structure of which was determined by nucleotide sequence analysis . The protein belongs to the family of phosphoenolpyruvate-utilizing enzymes and represents the first member characterized in archaebacteria . Its homomultimeric organisation differs from the typically dimeric structure of its eubacterial and eukaryotic counterparts. Eur J Biochem, 1994 Dec 15, 226(3), 981 - 91 Purification, properties and structural aspects of a thermoacidophilic alpha-amylase from Alicyclobacillus acidocaldarius atcc 27009 . Insight into acidostability of proteins; Schwermann B et al.; The alpha-amylase from the thermoacidophilic eubacterium Alicyclobacillus (Bacillus) acidocaldarius strain ATCC 27009 was studied as an example of an acidophilic protein . The enzyme was purified from the culture fluid . On an SDS/polyacrylamide gel, the protein an apparent molecular mass of 160 kDa, which is approximately 15% higher than that predicted from the nucleotide sequence . The difference is due to the enzyme being a glycoprotein . Deglycosylation or synthesis of the enzyme in Escherichia coli gave a product with the mass expected for the mature protein . The amylase hydrolyzed starch at random and from the inside, and its main hydrolysis products were maltotriose and maltose . It also formed glucose from starch (by hydrolysing the intermediate product maltotetraose to glucose and maltotriose) and exhibited some pullulanase activity . the pH and temperature optima were pH3 and 75 degrees C, respectively, characterizing the enzyme as being thermoacidophilic . Alignment of the sequence of the enzyme with that of its closest neutrophilic relatives and with that of alpha-1,4 or alpha-1,6 glycosidic-bond hydrolyzing enzymes of known three-dimensional structure showed that the acidophilic alpha-amylase contains approximately 30% less charged residues than do its closest relatives, that these residues are replaced by neutral polar residues, and that hot spots for these exchanges are likely to be located at the surface of the protein . Literature data show that similar effects are observed in three other acidophilic proteins . It is proposed that these proteins have adapted to the acidic environment by reducing the density of both positive and negative charges at their surface, that this effect circumvents electrostatic repulsion of charged groups at low pH, and thereby contributes to the acidostability of these proteins. Gene, 1994 Dec 2, 150(1), 9 - 15 Escherichia coli contains a set of genes homologous to those involved in protein secretion, DNA uptake and the assembly of type-4 fimbriae in other bacteria; Whitchurch CB et al.; A specialised system involved in a diverse array of functions, including the biogenesis of fimbriae, protein secretion and DNA uptake, has recently been found to be widespread in the eubacteria . These systems have in common several sets of related genes, including those encoding proteins containing leader sequences homologous to that of the type-4 fimbrial subunit (prepilin), a prepilin-type leader peptidase, a cytoplasmic nucleotide-binding protein, and other proteins located in the inner and outer membranes {Hobbs, M . and Mattick, J.S., Mol Microbiol . 10 (1993) 233-243} . Here, we show that Escherichia coli contains at least nine homologs of this system, and present complete sequence data for five of the genes involved (ppdD . hopB, hopC, hopD and pshM), as well as for an adjacent gene (nadC), which encodes quinolic acid phosphoribosyltransferase . Insertional mutagenesis of hopB and hopD failed to reveal any obvious effects on cell viability, morphogenesis of M13 phage, conjugative transfer of the F plasmid, or protein secretion. Gene, 1994 Dec 2, 150(1), 81 - 5 Cloning, analysis and expression of an rpoS homologue gene from Pseudomonas aeruginosa PAO1; Tanaka K et al.; A homologue of the rpoS gene of Escherichia coli was cloned from Pseudomonas aeruginosa PAO1 by hybridization with an oligodeoxyribonucleotide probe designed from an amino-acid stretch conserved among the principal sigma factors of eubacteria . Two open reading frames, the pcm gene and the orf-297 of unknown function, were found in the upstream region of rpoS, and in the same order as in E . coli . The rpoS gene of P . aeruginosa was expressed in E . coli and complemented the catalase deficiency of the rpoS mutant of E . coli . The RpoS protein of P . aeruginosa was identified by Western blot analysis in both P . aeruginosa (Pa) and the transformed E . coli . Levels of RpoS of Pa increased drastically at the onset of the stationary growth phase. J Bacteriol, 1994 Dec, 176(24), 7711 - 8 Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium; Hackett NR et al.; Phenotypic variants of Halobacterium salinarium NRC-1 arise at a frequency of 10(-2) . These result from transpositions of halobacterial insertion sequences and rearrangements mediated by halobacterial insertion sequences . We have tested the hypothesis that such mutations are confined to only a portion of the genome by comparing the chromosomal restriction map of H . salinarium NRC-1 and that of the derivative S9, which was made in 1969 . The two chromosomes were mapped by using two-dimensional pulsed-field gel electrophoresis and the restriction enzymes AflII, AseI, and DraI . A comparison of the two deduced maps showed a domain of about 210 kbp to be subject to many rearrangements, including an inversion in S9 relative to NRC-1 . However, the rest of the chromosome was conserved among NRC-1, S9, and an independent Halobacterium isolate, GRB, previously mapped by St . Jean et al . (A . St . Jean, B . A . Trieselmann, and R . L . Charlebois, Nucleic Acids Res . 22:1476-1483, 1994) . This concurs with data from eubacteria suggesting strong selective forces maintaining gene order even in the face of rearrangement events occurring at a high frequency. J Bacteriol, 1994 Dec, 176(24), 7506 - 15 Interplasmidic recombination following irradiation of the radioresistant bacterium Deinococcus radiodurans; Daly MJ et al.; Deinococcus radiodurans R1 and other members of the eubacterial family Deinococcaceae are extremely resistant to ionizing radiation and many other agents that damage DNA . For example, after irradiation, D . radiodurans can repair > 100 DNA double-strand breaks per chromosome without lethality or mutagenesis, while most other organisms can survive no more than 2 or 3 double-strand breaks . The unusual resistance of D . radiodurans is recA dependent, but the repair pathway(s) is not understood . Recently, we described how a plasmid present in D . radiodurans (plasmid copy number, approximately 6 per cell; chromosome copy number, approximately 4 per cell) during high-dose irradiation undergoes extreme damage like the chromosome and is retained by the cell without selection and fully repaired with the same efficiency as the chromosome . In the current work, we have investigated the repair of two similar plasmids within the same cell . These two plasmids were designed to provide both restriction fragment polymorphisms and a drug selection indicator of recombination . This study presents a novel system of analysis of in vivo damage and recombinational repair, exploiting the unique ability of D . radiodurans to survive extraordinarily high levels of DNA damage . We report that homologous recombination among plasmids following irradiation is extensive . For example, 2% of Tcs plasmids become Tcr as a result of productive recombination within a 929-bp region of the plasmids after repair . Our results suggest that each plasmid may participate in as many as 6.7 recombinational events during repair, a value that extrapolates to > 700 events per chromosome undergoing repair simultaneously . These results indicate that the study of plasmid recombination within D . radiodurans may serve as an accurate model system for simultaneously occurring repair in the chromosome. Curr Opin Genet Dev, 1994 Dec, 4(6), 816 - 22 Archaebacterial genomes: eubacterial form and eukaryotic content; Keeling PJ et al.; Since the recognition of the uniqueness and coherence of the archaebacteria (sometimes called Archaea), our perception of their role in early evolution has been modified repeatedly . The deluge of sequence data and rapidly improving molecular systematic methods have combined with a better understanding of archaebacterial molecular biology to describe a group that in some ways appears to be very similar to the eubacteria, though in others is more like the eukaryotes . The structure and contents of archaebacterial genomes are examined here, with an eye to their meaning in terms of the evolution of cell structure and function. Plant Mol Biol, 1994 Dec, 26(6), 1961 - 73 Chloroplast and cytosolic triosephosphate isomerases from spinach: purification, microsequencing and cDNA cloning of the chloroplast enzyme; Henze K et al.; Chloroplast and cytosolic triosephosphate isomerases from spinach were separated and purified to homogeneity . Both enzymes were partially sequenced by Edman degradation . Using degenerate primers designed against the amino acid sequences, a homologous probe for the chloroplast enzyme was amplified and used to isolate several full-size cDNA clones . Chloroplast triosephosphate isomerase is encoded by a single gene in spinach . Analysis of the chloroplast cDNA sequence in the context of its homologues from eukaryotes and eubacteria reveals that the gene arose through duplication of its preexisting nuclear counterpart for the cytosolic enzyme during plant evolution. Microbiol Rev, 1994 Dec, 58(4), 700 - 54 Chloroplast ribosomes and protein synthesis; Harris EH et al.; Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria . Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions . Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis . This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression . It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins . A description of the synthesis and assembly of chloroplast ribosomes follows . The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. Anal Chem, 1994 Dec 1, 66(23), 4171 - 6 Electrospray tandem mass spectrometry for analysis of native muramic acid in whole bacterial cell hydrolysates; Black GE et al.; Muramic acid is an amino sugar found in eubacterial cell walls and not elsewhere in nature . This study explored the use of electrospray tandem mass spectrometry (ESI MS/MS) in analysis of underivatized muramic acid in bacterial hydrolysates . Fungal hydrolysates were used as negative controls . The only processing used was hydrolysis in sulfuric acid followed by extraction with an organic base (N,N-dioctylmethylamine) to remove the acid prior to ESI MS/MS analysis . Compared with pure muramic acid, bacterial hydrolysates produced more complex ESI mass spectra, such that the protonated molecular ion at m/z 252 was barely detectable . In contrast, product ion spectra of m/z 252 were identical among pure muramic acid, Gram positive bacteria, and Gram negative bacteria . However, no characteristic product ion spectrum was manifested from m/z 252 in fungal samples . This allowed ready, visual differentiation of bacteria and fungi . Multiple reaction monitoring (MRM) following muramic acid fragmentations (m/z 252-->144 and m/z 252-->126) increased sensitivity and allowed quantitative differentiation when compared with the MRM of the internal standard N-methyl-D-glucamine (m/z 196-->44) . ESI MS/MS required minimal sample preparation and allowed rapid sample throughput for analysis of muramic acid in whole bacterial cell hydrolysates. Biol Pharm Bull, 1994 Dec, 17(12), 1573 - 6 Enzymic studies on the animal and intestinal bacterial metabolism of geniposide; Akao T et al.; Geniposide, a main iridoid glucoside of Gardenia fruit, is transformed to genipin, a genuine choleretic, in vivo in rats (Aburada et al., J . Pharmacobio-Dyn., 1, 81 (1978)) . As geniposide was not hydrolyzed to any metabolite by rat liver homogenate, which has beta-D-glucosidase and esterase activities, beta-D-glucosidases in intestinal bacteria seem to be required for an exhibition of its choleretic action . The crude extract of Eubacterium sp . A-44, a human intestinal anaerobe, hydrolyzed geniposide, but that of Ruminococcus sp . PO1-3, another human anaerobe, did not, though both extracts had beta-D-glucosidase activities for p-nitrophenyl beta-D-glucopyranoside . Only one of three beta-D-glucosidases from E . sp . A-44 and none of two from R . sp . PO1-3 hydrolyzed geniposide to genipin . However, carboxylesterases from E . sp . A-44 and pig liver were unable to hydrolyze geniposide to geniposidic acid, but hydrolyzed genipin to an aglycone of geniposidic acid, indicating that geniposide is first hydrolyzed to genipin by beta-D-glucosidases and subsequently to the aglycone of geniposidic acid by esterases . Thus, when geniposide is orally administered, genipin seems to be effectively produced in the intestine and then absorbed to act as a genuine choleretic. Curr Biol, 1994 Dec 1, 4(12), 1104 - 14 Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus; Gupta RS et al.; BACKGROUND: The evolutionary relationships between archaebacteria, eubacteria and eukaryotic cells are of central importance in biology . The current view is that each of these three groups of organisms constitutes a monophyletic domain, and that eukaryotic cells have evolved fom an archaebacterial ancestor . Recent studies on a number of highly conserved protein sequences do not, however, support this view and raise important questions concerning the evolutionary relationships between all extant organisms, particularly regarding the origin of eukaryotic cells . RESULTS: RESULTS: We have used sequences of 70 kD heat shock protein (hsp70)--the most conserved protein found to date in all species--to examine the evolutionary relationship between various species . We have obtained two new archaebacterial hsp70 sequences from the species, Thermoplasma acidophilum and Halobacterium cutirubrum . A global comparison of hsp70 sequences, including our two new sequences, shows that all known archaebacterial homologs share a number of sequence signatures with the Gram-positive group of bacteria that are not found in any other prokaryotic or eukaryotic species . In contrast, the eukaryotic homologs are shown to share a number of unique sequence features with the Gram-negative bacteria that are not present in any archaebacteria . Detailed phylogenetic analyses of hsp70 sequences strongly support a specific evolutionary relationship between archaebacteria and Gram-positive bacteria on the one hand, and Gram-negative bacteria and eukaryotes on the other . The phylogenetic analyses also indicate a polyphyletic branching of archaebacteria within the Gram-positive species . The possibility that the observed relationships are due to horizontal gene transfers can be excluded on the basis of sequence characteristics of different groups of homologs . CONCLUSIONS: Our results do not support the view that archaebacteria constitute a monophyletic domain, but instead suggest a close evolutionary linkage between archaebacteria and Gram-positive bacteria . Furthermore, in contrast to the presently accepted view, eukaryotic hsp70s show a close and specific relationship to those from Gram-negative species . To explain the phylogenies based on different gene sequences, a chimeric model for the origin of the eukaryotic cell nucleus involving fusion between an archaebacterium and a Gram-negative eubacterium is proposed . Several predictions from the chimeric model are discussed. Microbiology, 1994 Dec, 140 ( Pt 12), 3357 - 65 Transcription analysis of the Streptomyces coelicolor A3(2) rrnA operon; van Wezel GP et al.; Transcription start sites and processing sites of the Streptomyces coelicolor A3(2) rrnA operon have been investigated by a combination of in vivo and in vitro transcription analyses . The data from these approaches are consistent with the existence of four in vivo transcription sites, corresponding to the promoters P1-P4 . The transcription start sites are located at -597, -416, -334 and -254 relative to the start of the 16S rRNA gene . Two putative processing sites were identified, one of which is similar to a sequence reported earlier in S . coelicolor and other eubacteria . The P1 promoter is likely to be recognized by the RNA polymerase holoenzyme containing sigma hrdB, the principal sigma factor in S . coelicolor . P2 also shares homology with the consensus for vegetative promoters, but has a sequence overlapping the consensus -35 region that is also present in the -35 regions of P3 and P4 . The -35 sequence common to P2, P3 and P4 is not similar to any other known consensus promoter sequence . In fast-growing mycelium, P2 appears to be the most frequently used promoter . Transcription from all of the rrnA promoters decreased during the transition from exponential to stationary phase, although transcription from P1 and P2 ceased several hours before that from P3 and P4. J Bacteriol, 1994 Dec, 176(24), 7748 - 53 Cloning of the hsp70 (dnaK) genes from Rhizobium meliloti and Pseudomonas cepacia: phylogenetic analyses of mitochondrial origin based on a highly conserved protein sequence; Falah M et al.; The genes for hsp70 (or dnaK) have been cloned and sequenced from Rhizobium meliloti and Pseudomonas cepacia, two bacterial species belonging to the alpha- and beta-subdivisions of gram-negative proteobacteria, respectively . On the basis of global alignment of HSP70 proteins, several sequence signatures have been identified that are distinctive of mitochondrial homologs and gram-negative proteobacteria on the one hand and the chloroplasts and cyanobacteria on the other . Detailed phylogenetic analyses of HSP70 sequences from various eubacteria and eukaryotic organellar and cytosolic homologs support the inference regarding the origin of mitochondria from a member of the alpha-proteobacteria and of chloroplasts from cyanobacteria . The analysis presented here also suggests a monophyletic origin of the mitochondrial homologs. Biochemistry, 1994 Nov 29, 33(47), 13959 - 62 Thiol ester-linked p-coumaric acid as a new photoactive prosthetic group in a protein with rhodopsin-like photochemistry; Hoff WD et al.; A number of Eubacteria contain a photoactive yellow protein which has a photosensory function in negative phototaxis . It has been proposed that the cofactor responsible for the intense yellow color of this protein is retinal {McRee, D . E., et al . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 6533-6537} . This would make it the first eubacterial rhodopsin . Here we report the chemical structure of this chromophoric group to be p-coumaric acid, which is covalently bound to a unique cysteine in the apoprotein via a thiol ester bond, and thus not retinal . This makes PYP the first example of a protein containing p-coumaric acid, a metabolite previously found only in plants, as a prosthetic group and establishes the photoactive yellow proteins as a new type of photochemically active receptor molecule. J Mol Biol, 1994 Nov 18, 244(1), 125 - 32 Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of hydrolases with diverse specificity . Application of an iterative approach to database search; Koonin EV et al.; Using an iterative approach to sequence database search that combines scanning with individual amino acid sequences and with alignment blocks, we show that bacterial haloacid dehalogenases (HADs) belong to a large superfamily of hydrolases with diverse substrate specificity . The superfamily also includes epoxide hydrolases, different types of phosphatases, and numerous uncharacterized proteins from eubacteria, eukaryotes, and Archaea . Nine putative proteins of the HAD superfamily with functions unknown, in addition to two known enzymes, were found in Escherichia coli alone, making it one of the largest groups of enzymes and indicating that a variety of hydrolytic enzyme activities remain to be described . Many of the proteins with known enzymatic activities in the HAD superfamily are involved in detoxification of xenobiotics or metabolic by-products . All the proteins in the superfamily contain three conserved sequence motifs . Along with the conservation of the predicted secondary structure, motifs I, II, and III include a conserved aspartic acid residue, a lysine, and a nucleophile, namely aspartic acid or serine, respectively . A specific role in the catalysis of the hydrolysis of carbon-halogen and other bonds is assigned to each of these residues. Nucleic Acids Res, 1994 Nov 11, 22(22), 4756 - 67 Intrinsic and extrinsic approaches for detecting genes in a bacterial genome; Borodovsky M et al.; The unannotated regions of the Escherichia coli genome DNA sequence from the EcoSeq6 database, totaling 1,278 'intergenic' sequences of the combined length of 359,279 basepairs, were analyzed using computer-assisted methods with the aim of identifying putative unknown genes . The proposed strategy for finding new genes includes two key elements: i) prediction of expressed open reading frames (ORFs) using the GeneMark method based on Markov chain models for coding and non-coding regions of Escherichia coli DNA, and ii) search for protein sequence similarities using programs based on the BLAST algorithm and programs for motif identification . A total of 354 putative expressed ORFs were predicted by GeneMark . Using the BLASTX and TBLASTN programs, it was shown that 208 ORFs located in the unannotated regions of the E . coli chromosome are significantly similar to other protein sequences . Identification of 182 ORFs as probable genes was supported by GeneMark and BLAST, comprising 51.4% of the GeneMark 'hits' and 87.5% of the BLAST 'hits' . 73 putative new genes, comprising 20.6% of the GeneMark predictions, belong to ancient conserved protein families that include both eubacterial and eukaryotic members . This value is close to the overall proportion of highly conserved sequences among eubacterial proteins, indicating that the majority of the putative expressed ORFs that are predicted by GeneMark, but have no significant BLAST hits, nevertheless are likely to be real genes . The majority of the putative genes identified by BLAST search have been described since the release of the EcoSeq6 database, but about 70 genes have not been detected so far . Among these new identifications are genes encoding proteins with a variety of predicted functions including dehydrogenases, kinases, several other metabolic enzymes, ATPases, rRNA methyltransferases, membrane proteins, and different types of regulatory proteins. Nature, 1994 Nov 10, 372(6502), 193 - 6 mRNAs can be stabilized by DEAD-box proteins; Iost I et al.; Eubacterial messenger RNAs are synthesized and translated simultaneously; moreover the speed of ribosomes usually matches that of RNA polymerase . We report here that when in Escherichia coli the host RNA polymerase is replaced by the eightfold faster bacteriophage T7 enzyme for the transcription of the lacZ gene, the beta-galactosidase yield per transcript is depressed 100-fold . But the overexpression of DEAD-box proteins greatly improves this low yield by stabilizing the corresponding transcripts . More generally, it stabilizes inefficiently translated E . coli mRNAs . Ribosome-free mRNA regions, such as those lying behind the fast T7 enzyme or between successive ribosomes on inefficiently translated transcripts, are often unstable and we propose that DEAD-box proteins protect them from endonucleases . These results pinpoint the importance of transcription-translation synchronization for mRNA stability, and reveal an undocumented property of DEAD-box RNA helicases . These proteins have been implicated in a variety of processes involving RNA but not mRNA stability. Arch Biochem Biophys, 1994 Nov 1, 314(2), 399 - 404 Isolation and characterization of idsA: the gene for the short chain isoprenyl diphosphate synthase from Methanobacterium thermoautotrophicum; Chen A et al.; The gene that encodes the bifunctional short chain isoprenyl diphosphate synthase (idsA) for synthesis of farnesyl diphosphate and geranylgeranyl diphosphate in Methanobacterium thermoautotrophicum, a strict archaebacterial anaerobe, was isolated from a genomic DNA library by colony-lift hybridization and sequenced . Amino acid sequences were obtained for the N-terminus of the enzyme and for internal peptide fragments generated by proteolysis and treatment with cyanogen bromide . Degenerate primers based on the amino acid sequences were used in PCR to synthesize a 220-bp probe from genomic DNA . The probe was radiolabeled and used to isolate idsA . DNA sequencing revealed a 975-bp open reading frame located within an operon . The encoded 325-amino-acid protein contained five conserved regions found in eubacterial and eukaryotic farnesyl diphosphate and geranylgeranyl diphosphate synthases, including aspartate-rich motifs commonly found in prenyltransferases. J Exp Biol, 1994 Nov, 196, 109 - 21 y(+)-type cationic amino acid transport: expression and regulation of the mCAT genes; MacLeod CL et al.; The transport of cationic amino acids across animal cell membranes is largely mediated by a small group of well-described transport system (y+, bo,+, Bo,+) . Only recently have genes encoding transport proteins in some of these systems been isolated . Two genes, mCAT-1 and mCAT-2, encode related multiple membrane-spanning proteins that share substantial amino acid sequence identity and virtually superimposable hydrophilicity profiles . mCAT-1 and mCAT-2 proteins expressed in Xenopus oocytes are functionally indistinguishable and similar to transport system y+, but have distinct tissue distribution patterns . mCAT-1 expression is nearly ubiquitous and produces a single protein, while mCAT-2 is highly tissue-specific, has two distinct protein isoforms encoded by a single gene and is expressed in different tissues using at least two widely separated promoters . All three proteins facilitate the ion-independent transport of arginine, lysine and ornithine . Both mCAT-1 and mCAT-2 proteins have low amino acid sequence similarity but strikingly similar hydrophilicity profiles with amino acid antiporters, uniporters and symporters of yeast, fungi and eubacteria . Current work will elucidate whether any of the mCAT proteins interact with members of a newly identified family of single membrane-spanning proteins, such as rBAT, 4F2 and NAA-Tr, which are thought to modulate or activate y+L and/or bo,+ transport systems. Biol Chem Hoppe Seyler, 1994 Nov, 375(11), 785 - 8 Mode of transformation of {1-15N}5-hydroxybenzimidazole and {1-15N}5-hydroxy-6-methylbenzimidazole into the 5,6-dimethylbenzimidazole moiety of vitamin B12; Schulze B et al.; The transformation of {1-15N}5-hydroxybenzimidazole and {1-15N}5-hydroxy-6-methylbenzimidazole into the 5,6-dimethylbenzimidazole moiety of vitamin B12 by Eubacterium limosum-cultures was studied . The vitamin B12 obtained was exclusively 15N-labeled in N-1 of the base part, as revealed by NMR-measurements . This indicates that either the unsubstituted 5,6-dimethylbenzimidazole presumably formed is not released from the enzyme until the ribose-5'-phosphate substituent is introduced, or that the precursors are first transformed into their alpha-nucleotide-5'-phosphates which then react to form 5,6-dimethylbenzimidazole-alpha-D-ribofuranoside- 5'-phosphate (alpha-ribazole-5'-phosphate). Biol Chem Hoppe Seyler, 1994 Nov, 375(11), 747 - 57 Genes for DNA cytosine methyltransferases and structural proteins, expressed during lytic growth by the phage phi H of the archaebacterium Halobacterium salinarium; Stolt P et al.; Lytic genes and transcription from the Halobacterium salinarium phage phi H were studied . Genes for three structural proteins were located to the left arm of the linear phage genome . The right arm was shown to encode three DNA cytosine methyltransferases, the first such sequences reported from an archaebacterium . One cytosine methyltransferase is of the N(4)-methyltransferase type . The other two open reading frames (ORFs) seem to be parts of the same gene, which has been split by a recombination event . This gene product is of the C5-methyltransferase type . The methyltransferase genes are the first phi H genes detected showing high homology to eubacterial proteins . Five of the six described gene products have a higher proportion acidic over basic amino acid residues, a common characteristic of halobacterial proteins . Lytic phi H transcription was shown to produce three RNA species, two shorter species encoding the methyltransferase genes and one large species transcribed from both the right and the left phage arm and subsequently being processed upstream of the region encoding the structural proteins. Arch Oral Biol, 1994 Nov, 39(11), 967 - 72 The metabolism of phenylalanine and leucine by a cell suspension of Eubacterium brachy and the effects of metronidazole on metabolism; Hamid MA et al.; Degradation of phenylalanine and leucine by resting cells of Eubacterium brachy ATCC 33089 were studied under strict anaerobic conditions . The effects of metronidazole and air on the metabolism were also studied . The organism principally produced phenylpropionate and isocaproate from phenylalanine and leucine, respectively . Other products were cinnamate and phenylacetate from phenylalanine, and alpha-ketoisocaproate and isovalerate from leucine . The organism also produced hydroxylated end-products, i.e . phenyllactate from phenylalanine and hydroxyisocaproate from leucine . When metronidazole was added to the reaction mixture, the production of phenylpropionate, cinnamate, phenylacetate, isocaproate, alpha-ketoisocaproate and isovalerate was inhibited, while that of hydroxylated products was not, suggesting that the organism has metronidazole-sensitive and -tolerant pathways of metabolism . A similar inhibitory effect was also found when the reaction was done aerobically, suggesting that the inhibitory mode of metronidazole is similar to that of oxygen. FEBS Lett, 1994 Oct 24, 353(3), 301 - 4 A cytoplasmic domain is required for the functional interaction of SRI and HtrI in archaeal signal transduction; Krah M et al.; Phototaxis in the archaeon Halobacterium salinarium is mediated by a stable complex of the photoreceptor sensory rhodopsin I and its transducer HtrI, whic