Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 83 - 7 A self-compartmentalizing protease in Rhodococcus: the 20S proteasome; De Mot R et al.; The 26S proteasome represents a major, energy-dependent and self-compartmentalizing protease system in eukaryotes . The proteolytic core of this complex, the 20S proteasome, is also ubiquitous in archaea . Although absent from most eubacteria, this multi-subunit protease was recently discovered in Rhodococcus and appears to be confined to actinomycetes . The eubacterial 20S proteasome represents an attractive complementary system to study proteasome assembly, quaternary structure, and catalytic mechanism . In addition, it is likely to contribute substantially to our understanding of the role of various self-compartmentalizing proteases in bacterial cells. Mol Cell Biol Res Commun, 1999 Apr, 1(1), 14 - 20 Tissue-specific and light-dependent expression within a family of nuclear-encoded sigma-like factors from Zea mays; Lahiri SD et al.; The principal transcription machinery functioning in chloroplasts of higher plants is encoded in two subcellular compartments . Subunits of the RNA polymerase catalytic core are plastid encoded, while sigma factors required for promoter recognition are encoded in the nucleus . We have isolated nuclear-encoded cDNAs, sig1, sig2, and sig3, specifying three sigma factors from maize (Zea mays) . The three deduced polypeptides have extensive sequence identity with the principal sigma factors of eubacteria . Two of the maize cDNAs, sig1 and sig3, encode NH2-terminal transit peptides which direct the uptake of a heterologous protein into chloroplasts in vitro . Transcripts for the sig3 gene were more abundant in green leaves than in roots and in light-treated seedlings than in dark-grown seedlings . In contrast, sig1 transcripts were readily detectable in all tissues examined . Thus, at least two promoter-selectivity factors function with the maize chloroplast RNA polymerase, one of which is constitutively expressed and the other is light activated. J Biol Chem, 1999 Mar 12, 274(11), 7182 - 9 Allosteric control of three B12-dependent (class II) ribonucleotide reductases . Implications for the evolution of ribonucleotide reduction; Eliasson R et al.; Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure . One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides . Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity . Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP . Binding of dATP to this site inhibits the activity of these enzymes . X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids . Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and Thermotoga maritima) . Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes . dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site . For the L . leichmannii and T . maritima enzymes, binding experiments also indicate the presence of only one allosteric site . Their primary sequences suggest that these enzymes lack the structural requirements for a second site . In contrast, the T . acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site . The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T . acidophilum enzyme . The other two B12 enzymes lost not only the function, but also the structural basis for the site . Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site . This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes. Science, 1999 Mar 5, 283(5407), 1476 - 81 Mitochondrial evolution; Gray MW et al.; The serial endosymbiosis theory is a favored model for explaining the origin of mitochondria, a defining event in the evolution of eukaryotic cells . As usually described, this theory posits that mitochondria are the direct descendants of a bacterial endosymbiont that became established at an early stage in a nucleus-containing (but amitochondriate) host cell . Gene sequence data strongly support a monophyletic origin of the mitochondrion from a eubacterial ancestor shared with a subgroup of the alpha-Proteobacteria . However, recent studies of unicellular eukaryotes (protists), some of them little known, have provided insights that challenge the traditional serial endosymbiosis-based view of how the eukaryotic cell and its mitochondrion came to be . These data indicate that the mitochondrion arose in a common ancestor of all extant eukaryotes and raise the possibility that this organelle originated at essentially the same time as the nuclear component of the eukaryotic cell rather than in a separate, subsequent event. Microbiol Res, 1999 Jan, 153(4), 309 - 17 Inhibitory metabolites production by the cyanobacterium Fischerella muscicola; Srivastava VC et al.; Broad-spectrum inhibitory metabolites were produced by a benthic cyanobacterium Fischerella muscicola (UTEX 1829) in batch culture . These metabolites inhibited the growth of eukaryotic algae, cyanobacteria and eubacteria . The effect of culture age on the production and leakage of these inhibitory metabolites from the cyanobacterium was studied . Confirmation of the presence of an allelochemical, possibly fischerellin was achieved using high performance liquid chromatography (HPLC) . The cyanobacterium produced inhibitory metabolites intracellularly at all stages of its growth cycle. J Biochem (Tokyo), 1999 Mar, 125(3), 460 - 8 An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor; Asayama M et al.; The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts . In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence . Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure . The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E . coli . In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry . These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription . The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium . This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria. J Struct Biol, 1998 Dec 15, 124(2-3), 244 - 56 Mollicutes-wall-less bacteria with internal cytoskeletons; Trachtenberg S; The structure and motility of the Mollicutes (Spiroplasma, Mycoplasma, and Acholeplasma) are briefly reviewed . The data are presented from the perspective of prokaryotic and eukaryotic motors, cytoskeletons, and cell motility . The Mollicutes are eubacteria derived from Clostridia by regressive evolution and genome reduction to produce the smallest and simplest free-living and self-replicating cells . Structurally, the Mollicutes are characterized by a complete lack of a cell wall and the presence of an internal cytoskeleton . Spiroplasma, which are helical cells with a flat, ribbon-like cytoskeleton, are amenable to structural and geometrical analysis . Motility and shape changes can be explained and modeled by the cytoskeleton acting as a linear motor . J Struct Biol, 1998 Dec 15, 124(2-3), 235 - 43 Crystal structure determination of FtsZ from Methanococcus jannaschii; Lowe J; FtsZ is the polymer-forming protein of bacterial cell division . It is part of a ring in the middle of the dividing cell that is required for constriction of cell membrane and cell envelope to yield two daughter cells . FtsZ is a GTPase and is the only bacterial protein showing significant sequence homology to the eukaryotic tubulins . FtsZ can polymerize into tubes, sheets, and rings in vitro and is ubiquitous in eubacteria and archaea . Full-length FtsZ1 from Methanococcus jannaschii has been over expressed in Escherichia coli, employing the hyperthermophilic properties of the protein . Crystals grown from PEG400 and ethanol belong to spacegroup I213 with a = b = c = 159.1 A . Isomorphous replacement using one Hg derivative yielded a interpretable electron density map at 4 A resolution . The structure for residues 23-356 and one GDP has been refined to an Rfree of 0.28 (Rf = 0.20) at 2.8 A resolution . FtsZ consists of two domains with a connecting core helix . The N-terminal domain and the core helix contain all residues involved in nucleotide binding and resemble the fold of dinucleotide-binding proteins . The structures of tubulin and FtsZ show striking similarity; together with the functional similarities, this provides a strong indication that FtsZ is a true homolog of tubulin . J Appl Microbiol, 1999 Jan, 86(1), 93 - 107 Amylase and 16S rRNA genes from a hyperthermophilic archaebacterium; Jones RA et al.; A hyperthermophilic and amylolytic prokaryote, designated Rt3, was isolated from a thermal spring near Rotorua, New Zealand . The 16S rRNA gene of Rt3 was cloned and sequenced with the aim of determining its phylogenetic affiliations . The phylogenetic analysis of this sequence, which included a selection of archaebacterial and eubacterial 16S rRNA sequences, indicates that Rt3 most likely belongs to the archaebacterial order Thermococcales . An amylase gene (amyA) from Rt3, encoding a highly thermostable amylase activity, was cloned and its DNA sequence determined . Transcriptional signals typical of archaebacteria were evident in this sequence . The sequence is homologous to a broad range of enzymes from the AMY superfamily and contains a typical N-terminal signal peptide . Phylogenetic analysis and comparison of structural features with other AMY superfamily enzymes reveals that, firstly, the closest homologues of the Rt3 amylase are members of the Bacillus and Plant alpha-amylase groups; and secondly, that the Rt3 amylase is closely related to only one other currently known archaebacterial enzyme, i.e . an (AMY superfamily) alpha-amylase from Natronococcus. Parasitology, 1999 Feb, 118 ( Pt 2), 125 - 34 Adonia variegata (Coleoptera: Coccinellidae) bears maternally inherited flavobacteria that kill males only; Hurst GD et al.; Inherited bacteria that parasitically distort the pattern of sex allocation of their host, biasing allocation towards female progeny, are found in many arthropods . One such manipulation is male-killing, where male progeny of infected females die during embryogenesis . We here provide evidence for a male-killing bacterium in the coccinellid beetle, Adonia variegata . We then address 3 questions . First, is this male-killing bacterium one that is found in other hosts, or does it represent a new transition to male-killing within the eubacteria? Using the sequence of the 16S rDNA of the bacterium, we found that the male-killing bacterium is a member of the Flavobacteria--Bacteroides group, most closely related to the male-killing bacterium in another ladybird beetle, Coleomegilla maculata . Secondly, is there any evidence that this bacterium affects female host physiology? In a paired test under nutritional stress, we found no evidence for a physiological benefit to infection, and weak evidence of a physiological cost, in terms of reduced fecundity . Thirdly, is there any evidence of host involvement in the transmission of the bacterium to the germ line? We found no evidence of host involvement . Rather, bacteria migrated to the ovariole independently of host cells . We conclude that the bacterium is a parasite, and discuss how 2 different species of ladybird come to be infected with 1 lineage of bacterium, and why case studies of male-killing bacteria have generally found little evidence of any symbiont contribution to host physiological functioning. Microb Comp Genomics, 1998, 3(4), 243 - 53 Relationship between codon usage and sequence-dependent curvature of genomes; Jauregui R et al.; Static DNA curvature distributions of full-sequenced genomes and large DNA contigs from different organisms were calculated . Very distinctive differences among histogram profiles coming from archaebacteria, eubacteria, and eukaryotes were observed . Eubacterial profiles were, on average, more curved than were archaeal and eukaryotic profiles . A comparative analysis between real and randomized DNA sequences revealed that eubacterial genomes presented, overall, higher curvature values than random sequences . An opposite portrait was exhibited by archaeal and eukaryotic genomes . They displayed a lower frequency of curved regions than their corresponding randomized sequences . The contributions of coding and intergenic regions to the curvature profile were also analyzed . Intergenic regions, on average, were found to be more curved than the overall genomic sequences, especially in prokaryotic organisms . Nevertheless, because of their small size with respect to coding regions, the contribution of intergenic sequences to the overall curvature profile tended to be minor . A clear relationship between codon usage and DNA curvature was demonstrated, and a proposal of the possible coevolution of both systems is discussed . Finally, we present a procedure to quantify the deviation of a curvature profile from randomness through a formal statistical analysis. RNA, 1999 Feb, 5(2), 281 - 9 In vivo and in vitro processing of the Bacillus subtilis transcript coding for glutamyl-tRNA synthetase, serine acetyltransferase, and cysteinyl-tRNA synthetase; Pelchat M et al.; In Bacillus subtilis, the adjacent genes gltX, cysE, and cysS encoding respectively glutamyl-tRNA synthetase, serine acetyl-transferase, and cysteinyl-tRNA synthetase, are transcribed as an operon but a gltX probe reveals only the presence of a monocistronic gltX mRNA (Gagnon et al., 1994, J Biol Chem 269:7473-7482) . The transcript of the gltX-cysE intergenic region contains putative alternative secondary structures forming a p-independent terminator or an antiterminator, and a conserved sequence (T-box) found in the leader of most aminoacyl-tRNA synthetase and many amino acid biosynthesis genes in B . subtilis and in other Gram-positive eubacteria . The transcription of these genes is initiated 45 nt upstream from the first codon of gltX and is under the control of a sigmaA-type promoter . Analysis of the in vivo transcript of this operon revealed a cleavage site immediately downstream from the p-independent terminator structure . In vitro transcription analysis, using RNA polymerases from Escherichia coli, B . subtilis, and that encoded by the T7 phage, in the presence of various RNase inhibitors, shows the same cleavage . This processing generates mRNAs whose 5'-end half-lives differ by a factor of 2 in rich medium, and leaves putative secondary structures at the 3' end of the gltX transcript and at the 5' end of the cysE/S mRNA, which may be involved in the stabilization of these mRNAs . By its mechanism and its position, this cleavage differs from that of the other known transcripts encoding aminoacyl-tRNA synthetases in B . subtilis. Mol Cell Biol, 1999 Mar, 19(3), 2389 - 99 NMD3 encodes an essential cytoplasmic protein required for stable 60S ribosomal subunits in Saccharomyces cerevisiae; Ho JH et al.; A mutation in NMD3 was found to be lethal in the absence of XRN1, which encodes the major cytoplasmic exoribonuclease responsible for mRNA turnover . Molecular genetic analysis of NMD3 revealed that it is an essential gene required for stable 60S ribosomal subunits . Cells bearing a temperature-sensitive allele of NMD3 had decreased levels of 60S subunits at the nonpermissive temperature which resulted in the formation of half-mer polysomes . Pulse-chase analysis of rRNA biogenesis indicated that 25S rRNA was made and processed with kinetics similar to wild-type kinetics . However, the mature RNA was rapidly degraded, with a half-life of 4 min . Nmd3p fractionated as a cytoplasmic protein and sedimented in the position of free 60S subunits in sucrose gradients . These results suggest that Nmd3p is a cytoplasmic factor required for a late cytoplasmic assembly step of the 60S subunit but is not a ribosomal protein . Putative orthologs of Nmd3p exist in Drosophila, in nematodes, and in archaebacteria but not in eubacteria . The Nmd3 protein sequence does not contain readily recognizable motifs of known function . However, these proteins all have an amino-terminal domain containing four repeats of Cx2C, reminiscent of zinc-binding proteins, implicated in nucleic acid binding or protein oligomerization. Biol Pharm Bull, 1999 Jan, 22(1), 80 - 2 Purification and characterization of glycyrrhetic acid mono-glucuronide beta-D-glucuronidase in Eubacterium sp . GLH; Akao T; Glycyrrhetic acid mono-glucuronide (GAMG), 1-(18beta-glycyrrhet-3-yl)-beta-D-glucopyranuroic acid, was hydrolyzed to glycyrrhetic acid (GA) by GAMG beta-D-glucuronidase in Eubacterium sp . GLH from human intestinal bacteria . The enzyme had an optimum pH of 5.0 and was purified from a crude extract by Butyl Toyopearl 650 S, Toyopearl HW-55 S, Hydroxyapatite and DEAE-Toyopearl 650 M column chromatography . The purified enzyme showed a specific activity of 495 nmol/min/mg protein and a single band on Coomassie brilliant blue staining and a molecular weight of about 43 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . The apparent molecular weight was 49.5 kDa, as estimated by Toyopearl HW-55 S column chromatography . Also, the enzyme seemed to have a sulfhydryl group(s) in its active site with a Km value of 77 x 10(-3) M. Mol Microbiol, 1999 Jan, 31(1), 1 - 8 Negative regulation of bacterial heat shock genes; Narberhaus F; The expression of eubacterial heat shock genes is efficiently controlled at the transcriptional level by both positive and negative mechanisms . Positive control operates by the use of alternative sigma factors that target RNA polymerase to heat shock gene promoters . Alternatively, bacteria apply repressor-dependent mechanisms, in which transcription of heat shock genes is initiated from a classical housekeeping promoter and cis-acting DNA elements are used in concert with a cognate repressor protein to limit transcription under physiological conditions . Eight examples of negative regulation will be presented, among them the widespread CIRCE/HrcA system and the control by HspR in Streptomyces . Both mechanisms are designed to permit simple feedback control at the level of gene expression . Many bacteria have established sophisticated regulatory networks, often combining positive and negative mechanisms, in order to allow fine-tuned heat shock gene expression in an environmentally responsive way. J Bacteriol, 1999 Feb, 181(4), 1256 - 63 Purification, characterization, and cloning of a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in biosynthesis of terpenoids; Takahashi S et al.; The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) was purified 3,000-fold from Streptomyces sp . strain CL190 to apparent homogeneity with an overall yield of 2.1% . The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies . The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer . The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 microM for NADPH and 7.7 microM for HMG-CoA . A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme . The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes . These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme. Prog Nucleic Acid Res Mol Biol, 1999, 62, 329 - 67 Regulation of the Bacillus subtilis pyrimidine biosynthetic operon by transcriptional attenuation: control of gene expression by an mRNA-binding protein; Switzer RL et al.; The pyrimidine nucleotide biosynthetic (pyr) operon of Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which termination of transcription at points upstream of the genes being regulated is promoted by the binding of a regulatory protein, PyrR, to specific sequences in the pyr mRNA . Binding of PyrR to pyr mRNA is stimulated by uridine nucleotides and causes changes in the mRNA secondary structure . This model is supported by extensive molecular genetic analysis . PyrR, which is encoded by the first gene of the pyr operon, is also a uracil phosphoribosyltransferase, although it has little amino acid sequence resemblance to other bacterial uracil phosphoribosyltransferases . Purified B . subtilis pyrR promotes attenuation of pyr transcription in vitro and binds specifically to pyr RNA sequences . The crystallographic structure of PyrR demonstrates the similarity of its tertiary structure to other phosphoribosyltransferases and suggests the surface to which RNA binds . PyrR is widely distributed among eubacteria and appears to regulate pyr genes not only by the attenuation mechanism found in B . subtilis, but also by a coupled transcription-translation attenuation mechanism and by acting as a translational repressor . PyrR illustrates the concept that transcriptional attenuation is a much more widespread and mechanistically versatile mechanism for the regulation of gene expression in bacteria than is generally recognized. J Mol Biol, 1999 Feb 12, 286(1), 279 - 90 Domain dislocation: a change of core structure in periplasmic binding proteins in their evolutionary history; Fukami-Kobayashi K et al.; Periplasmic binding proteins (PBPs) serve as receptors for various water-soluble ligands in ATP-binding cassette (ABC) transport systems, and form one of the largest protein families in eubacterial and archaebacterial genomes . They are considered to be derived from a common ancestor, judging from their similarities of three-dimensional structure, their mechanism of ligand binding and the operon structure of their genes . Nevertheless, there are two types of topological arrangements of the central beta-sheets in their core structures . It follows that there must have been differentiation in the core structure, which we call "domain dislocation", in the course of evolution of the PBP family . To find a clue as to when the domain dislocation occurred, we constructed phylogenetic trees for PBPs based on their amino acid sequences and three-dimensional structures, respectively . The trees show that the proteins of each type clearly cluster together, strongly indicating that the change in the core structure occurred only once in the evolution of PBPs . We also constructed a phylogenetic tree for the ABC proteins that are encoded by the same operon of their partner PBP, and obtained the same result . Based on the phylogenetic relationship and comparison of the topological arrangements of PBPs, we obtained a reasonable genealogical chart of structural changes in the PBP family . The present analysis shows that the unidirectional change of protein evolution is clearly deduced at the level of protein three-dimensional structure rather than the level of amino acid sequence . Microb Ecol, 1999 Feb, 37(2), 140 - 151 Degradation of Soil Humic Extract by Wood- and Soil-Associated Fungi, Bacteria, and Commercial Enzymes; Gramss G et al.; > Abstract An alkaline humic extract (HE) of a black calcareous forest mull was exposed to 36 fungal and 9 eubacterial isolates in liquid standing culture . At 21 d in fungi, and 4 d in bacteria, the groups of wood-degrading basidiomycetes, terricolous basidiomycetes, ectomycorrhizal fungi, soil-borne microfungi, and eubacteria had reduced the absorbance (A340) of HE media by 57, 28, 19, 26 and 5%, respectively . Gel permeation chromatography revealed that the large humic acid molecules were more readily degraded than the smaller fulvic acid molecules and served as a sole source of carbon and energy . The more active HE degraders reduced the overall molecular weight of humic and fulvic acids by 0.25 to 0.47 kDa . They also reduced the chemical reactivity of HE to tetrazotized o-dianisidine, indicating the degradation of hydroxylated aromatic molecules (which are responsible for this reaction) . Decreases in absorbance, molecular weight, and reactivity were caused by fungal manganese peroxidase, horseradish peroxidase, beta-glucosidase, and abiotic oxidants such as H2O2 and Mn(III) acetate . It is concluded that fungi, some of which are propagated in contaminated soils to control xenobiotics, metabolize HE compounds enzymatically . They use enzymes which are also involved in the degradation of soil xenobiotics . Because of reductions in the molecular weight of HE, which is a potential carrier of heavy metal ions and xenobiotics, solubility and motility of humic substances in soil and surface waters are increased. Nucleic Acids Res, 1999 Feb 15, 27(4), 1039 - 46 DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli; Connelly JC et al.; The SbcCD protein is a member of a group of nucleases found in bacteriophage T4 and T5, eubacteria, archaebacteria, yeast, Drosophila, mouse and man . Evidence from electron microscopy has revealed a distinctive structure consisting of two globular domains linked by a long region of coiled coil, similar to that predicted for the members of the SMC family . That a nuclease should have such an unusual structure suggests that its mode of action may be complex . Here we show that the protein degrades duplex DNA in a 3'-->5' direction . This degradation releases products half the length of the original duplex suggesting simultaneous degradation from two duplex ends . This may provide a link to the unusual structure of the protein since our data are consistent with recognition and cleavage of DNA ends followed by 3'-->5' nicking by two nucleolytic centres within a single nuclease molecule that releases a half length limit product . We also show that cleavage is not simply at the point of a single-strand/double-stand transition and that despite the dominant 3'-->5' polarity of degradation, a 5' single-strand can be cleaved when attached to duplex DNA . The implications of this mechanism for the processing of hairpins formed during DNA replication are discussed. Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 875 - 80 Induced fit of a peptide loop of methionyl-tRNA formyltransferase triggered by the initiator tRNA substrate; Ramesh V et al.; A 16-aa insertion loop present in eubacterial methionyl-tRNA formyltransferases (MTF) is critical for specific recognition of the initiator tRNA in Escherichia coli . We have studied the interactions between this region of the E . coli enzyme and initiator methionyl-tRNA (Met-tRNA) by using two complementary protection experiments: protection of MTF against proteolytic cleavage by tRNA and protection of tRNA against nucleolytic cleavage by MTF . The insertion loop in MTF is uniquely sensitive to cleavage by trypsin . We show that the substrate initiator Met-tRNA protects MTF against trypsin cleavage, whereas a formylation-defective mutant initiator Met-tRNA, which binds to MTF with approximately the same affinity, does not . Also, mutants of MTF within the insertion loop (which are defective in formylation) are not protected by the initiator Met-tRNA . Thus, a functional enzyme-substrate complex is necessary for protection of MTF against trypsin cleavage . Along with other data, these results strongly suggest that a segment of the insertion loop, which is exposed and unstructured in MTF, undergoes an induced fit in the functional MTF.Met-tRNA complex but not in the nonfunctional one . Footprinting experiments show that MTF specifically protects the acceptor stem and the 3'-end region of the initiator Met-tRNA against cleavage by double and single strand-specific nucleases . This protection also depends on formation of a functional MTF.Met-tRNA complex . Thus, the insertion loop interacts mostly with the acceptor stem of the initiator Met-tRNA, which contains the critical determinants for formylation. EMBO J, 1999 Feb 1, 18(3), 709 - 16 A mutation in region 1.1 of sigma70 affects promoter DNA binding by Escherichia coli RNA polymerase holoenzyme; Bowers CW et al.; The sigma subunit of eubacterial RNA polymerase is essential for initiation of transcription at promoter sites . It directs recognition of DNA sequences by holoenzyme (alpha2betabeta'sigma) and facilitates subsequent steps in the initiation pathway . The primary sigma factor from Escherichia coli, sigma70, has four regions that are conserved among members of the sigma70 family . Previous work has shown that region 1.1 modulates DNA binding by regions 2 and 4 when sigma is separated from the core subunits, and is required for efficient progression through the later steps of initiation in the context of holoenzyme . In this report, we show that an amino acid substitution at position 53 in region 1.1, which converts isoleucine to alanine (I53A), creates a sigma factor that associates with the core subunits to form holoenzyme, but the holoenzyme is severely deficient for promoter binding . The I53A phenotype can be suppressed by truncation of five amino acids from the C-terminus of sigma70 . We propose that the behavior of sigma70-I53A is a consequence of impaired ability to undergo a critical conformational change upon binding to the core subunits, which is needed to expose the DNA-binding domains and confer promoter recognition capability upon holoenzyme. Insect Mol Biol, 1999 Feb, 8(1), 133 - 9 Invasion of one insect species, Adalia bipunctata, by two different male-killing bacteria; Hurst GD et al.; Male-killing bacteria, which are inherited through the female line and kill male progeny only, are known from five different orders of insect . Our knowledge of the incidence of these elements has stemmed from discovery of their phenotype in different species . Our estimate of the frequency with which insects have been invaded by these elements therefore depends on each observation of the male-killing phenotype within a species being associated with a single microorganism . We here record an example of a single insect species being infected with two taxonomically distinct male-killing bacteria . Western European populations of the two-spot ladybird, Adalia bipunctata, have previously been shown to bear a male-killing Rickettsia . However, we here show that the majority of the male-killing lines tested from Central and Eastern Europe do not bear this bacterium . Rather, 16S rDNA sequence analysis suggests male-killing is associated with a member of the genus Spiroplasma . We discuss this conclusion in relation to the evolutionary genetics of male-killing bacteria, and the evolution of male-killing behaviour in the eubacteria. Hum Pathol, 1999 Jan, 30(1), 59 - 65 Chronic recurrent multifocal osteomyelitis in children: diagnostic value of histopathology and microbial testing; Girschick HJ et al.; Chronic recurrent, unifocal or multifocal osteomyelitis (CRMO), an inflammatory disorder of unknown origin, involves different osseous sites and may be associated with palmoplantar pustulosis . Bacterial cultures of affected tissue were reported negative in nearly all cases . Radiological and magnetic resonance imaging features of CRMO have been described, but differential diagnosis remains difficult, including rheumatic diseases, bacterial osteomyelitis, and malignancy . Although definite diagnosis relies on histopathologic confirmation by biopsy, histopathologic criteria have not been defined . Because CRMO may be treated with nonsteroidal antiinflammatory drugs, but not antibiotics, distinguishing CRMO from bacterial osteomyelitis is of major importance . Histopathologic analysis of 12 patients with CRMO indicated a wide variation of reparative changes of bone, but chronic inflammation could not be found at all sites in the same biopsy . The inflammatory infiltrate was mostly scattered, consisting mainly of lymphocytes, plasma cells, histiocytes, and also few neutrophil granulocytes . Immunohistochemistry showed a predominance of CD3(+), CD45RO(+) T-cells, which were mainly CD8(+) . In addition, CD20(+) B cells and CD68(+) macrophages were abundant in each biopsy specimen . Mild lymphocytic and granulocytic infiltrates were also detected in three synovial biopsy specimens obtained from adjacent joints . All bacterial and fungal cultures from native biopsy tissues were negative . Amplification of partial-length 16S ribosomal DNA by polymerase chain reaction (PCR) using broad-range eubacterial primers was below the detection limit in all patients . Because histopathologic features alone may not provide conclusive evidence, CRMO should be included in the differential diagnosis of chronic inflammatory bone lesions in children, and the definite diagnosis should be made by the clinical picture, x-ray studies, bone scan, bacterial culture, and histopathologic analysis in a multidisciplinary approach. Biochemistry, 1999 Jan 19, 38(3), 1009 - 17 Global conformational changes upon receptor stimulation in photoactive yellow protein; Hoff WD et al.; Biological signal transduction starts with the activation of a receptor protein . Two central questions in signaling are the mechanism of activation by a stimulus and the nature and extent of the protein conformational changes involved . We report extensive evidence for the occurrence of large structural changes upon the light activation of photoactive yellow protein (PYP), a eubacterial photosensor . Absorption of a blue photon by the p-coumaric acid (pCA) chromophore in pG, the initial state of PYP, results in the formation of pB, a putative signaling state . In the presence of an adequate hydration shell, large structural changes in the protein backbone, involving both solvent accessible and core regions, were detected using Fourier transform infrared (FTIR) difference spectroscopy . A significant part (23%) of the amide groups which are buried in pG become exposed to the solvent in pB, as measured through light-induced H/D exchange, using both electrospray ionization mass spectrometry and FTIR spectroscopy . Exposure of previously buried hydrophobic sites would lead to an increase in heat capacity during pB formation and a decrease in heat capacity during pB decay . Thermodynamic studies indeed show that the heat capacity change of pB activation is -2.35 +/- 0.08 kJ/(mol/K), independent of pH from pH 2.4-7.5 . A model for photoactivation of PYP is proposed, which provides a framework for a deeper understanding of receptor activation in general. Br J Ophthalmol, 1998 Sep, 82(9), 1078 - 82 Polymerase chain reaction in the diagnosis of bacterial endophthalmitis; Therese KL et al.; BACKGROUND: Microbiological investigations of vitreous fluid (VF) and aqueous humour (AH) specimens have often failed to detect the infecting agent in infectious endophthalmitis, resulting in a clinical dilemma regarding therapy . In this study, the polymerase chain reaction (PCR) was evaluated in the diagnosis of bacterial and Propionibacterium acnes endophthalmitis . METHODS: 58 intraocular specimens (30 VF and 28 AH) from 55 cases of endophthalmitis and 20 specimens (14 VF and 6 AH) as controls from non-infective disorders were processed for microbiological investigations . Nested PCR directed at the 16S rDNA using universal primers for eubacterial genome was done . PCR for P acnes was performed on specimens microbiologically negative by conventional techniques but eubacterial genome positive . RESULTS: Of the 20 controls from non-infective cases, one (5%) was positive using eubacterial primers and none with P acnes primers . PCR for eubacterial genome showed 100% correlation with 20 (34.5%) bacteriologically positive specimens . Eubacterial genome, was detected in 17 (44.7%) of 38 bacteriologically negative specimens and nine (52.9%) out of the 17 were positive for P acnes genome . Among the 21 eubacterial PCR negative specimens, seven were fungus positive . By inclusion of PCR, microbiologically positive specimens increased from 46.5% to 75.8% . PCR on AH was as sensitive as that on VF for the detection of both eubacterial and the P acnes genome . CONCLUSION: PCR performed on AH and VF is a reliable tool for the diagnosis of bacterial and P acnes endophthalmitis particularly in smear and culture negative specimens. Annu Rev Microbiol, 1998, 52, 231 - 86 The anti-sigma factors; Hughes KT et al.; A mechanism for regulating gene expression at the level of transcription utilizes an antagonist of the sigma transcription factor known as the anti-sigma (anti-sigma) factor . The cytoplasmic class of anti-sigma factors has been well characterized . The class includes AsiA form bacteriophage T4, which inhibits Escherichia coli sigma 70; FlgM, present in both gram-positive and gram-negative bacteria, which inhibits the flagella sigma factor sigma 28; SpoIIAB, which inhibits the sporulation-specific sigma factor, sigma F and sigma G, of Bacillus subtilis; RbsW of B . subtilis, which inhibits stress response sigma factor sigma B; and DnaK, a general regulator of the heat shock response, which in bacteria inhibits the heat shock sigma factor sigma 32 . In addition to this class of well-characterized cytoplasmic anti-sigma factors, a new class of homologous, inner-membrane-bound anti-sigma factors has recently been discovered in a variety of eubacteria . This new class of anti-sigma factors regulates the expression of so-called extracytoplasmic functions, and hence is known as the ECF subfamily of anti-sigma factors . The range of cell processes regulated by anti-sigma factors is highly varied and includes bacteriophage phage growth, sporulation, stress response, flagellar biosynthesis, pigment production, ion transport, and virulence. Nucleic Acids Res, 1999 Feb 1, 27(3), 721 - 9 tRNA recognition and evolution of determinants in seryl-tRNA synthesis; Lenhard B et al.; We have analyzed the evolution of recognition of tRNAsSerby seryl-tRNA synthetases, and compared it to other type 2 tRNAs, which contain a long extra arm . In Eubacteria and chloroplasts this type of tRNA is restricted to three families: tRNALeu, tRNASer and tRNATyr . tRNALeuand tRNASer also carry a long extra arm in Archaea, Eukarya and all organelles with the exception of animal mitochondria . In contrast, the long extra arm of tRNATyr is far less conserved: it was drastically shortened after the separation of Archaea and Eukarya from Eubacteria, and it is also truncated in animal mitochondria . The high degree of phylo-genetic divergence in the length of tRNA variable arms, which are recognized by both class I and class II aminoacyl-tRNA synthetases, makes type 2 tRNA recognition an ideal system with which to study how tRNA discrimination may have evolved in tandem with the evolution of other components of the translation machinery. Arch Environ Contam Toxicol, 1999 Feb, 36(2), 124 - 31 Copper speciation and microbial activity in long-term contaminated soils; Dumestre A et al.; Most soil quality guidelines do not distinguish among the various forms of metals in soils; insoluble, nonreactive, and nonbioavailable forms are deemed as hazardous as highly soluble, reactive, and toxic forms . The objective of this study was to better understand the long-term effects of copper on microorganisms in relation to its chemical speciation in the soil environment . Carbon mineralization processes and the global structure of different microbial communities (fungi, eubacteria, actinomycetes) are still affected after more than 50 years of copper contamination in 20 soils sampled from two different agricultural sites . The microbial respiration lag period (LP) preceding the beginning of mineralization process increases with the level of soil copper contamination and is not significantly affected by other environmental factors such as soil pH and soil organic matter (SOM) content . The total copper concentration showed the best correlation with the LP when each site is considered separately . However, when considering the whole set of data, soil solution free Cu2+ activity (pCu2+) is the best predictor of Cu toxicity determined by LP (quite likely because pCu2+ integrates the soil physicochemical variability) . The maximum mineralization rate (MMR), even if well correlated with the pCu2+, appears not to be a good biomonitor of copper contamination in soils since it is highly sensitive to soil characteristics such as SOM content . This study emphasizes the importance of the physicochemical properties of the environment on soil heavy metal toxicity and on soil toxicological measurements . These properties must be characterized in soil toxicological studies with respect to (1) their interactions with heavy metals, and (2) their direct impact on the selected biological test . The measurement of pCu2+ to characterize the level of soil contamination and of lag period as a bioindicator of metal effects in the soil are recognized as useful tools for the evaluation of the biological quality of soils. Biochemistry, 1999 Jan 12, 38(2), 643 - 50 Determination of substrate specificity for peptide deformylase through the screening of a combinatorial peptide library; Hu YJ et al.; Peptide deformylase is an essential Fe2+ metalloenzyme that catalyzes the removal of the N-terminal formyl group from nascent polypeptides in eubacteria . In vivo, the deformylase is capable of deformylating most of the polypeptides in a bacterial cell, which contain diverse N-terminal sequences . In this work, we have developed a combinatorial method to systematically examine the sequence specificity of peptide deformylase . A peptide library that contains all possible N-terminally formylated tetrapeptides was constructed on TentaGel resin, with a unique peptide sequence on each resin bead . Limited treatment with the Escherichia coli deformylase resulted in the deformylation of those peptides that are the most potent substrates of the enzyme . By using an enzyme-linked assay, the beads containing the deformylated peptides were identified and isolated . Peptide sequence analysis using matrix-assisted laser desorption ionization mass spectrometry revealed a consensus sequence, formyl-Met-X-Z-Tyr (X = any amino acid except for aspartate and glutamate; Z = lysine, arginine, tyrosine, or phenylalanine), for the E . coli enzyme . The deformylase is also capable of efficient deformylation of formyl-Phe-Tyr-(Phe/Tyr) peptides . These results demonstrate that, despite being a broad-specificity enzyme, the peptide deformylase deformylates different peptides at drastically different rates . In addition, the selectivity of peptide deformylase for the N-formyl over the N-acetyl group has been studied with N-alpha-fluoroacetyl peptides, and the results suggest that both electronic and steric factors are responsible for the observed specificity . The deformylase was also shown to exhibit esterase activity . These results will facilitate the design of specific deformylase inhibitors as potential antibacterial agents . This combinatorial method should be generally applicable to the study of the substrate specificity of other acylases and peptidases. J Bacteriol, 1999 Jan, 181(2), 552 - 5 Helicobacter pylori: a eubacterium lacking the stringent response; Scoarughi GL et al.; Accumulation of 16S rRNA and production of guanosine polyphosphates (pppGpp and ppGpp) were studied during amino acid starvation in three wild-type strains of Helicobacter pylori . All strains exhibit a relaxed phenotype with respect to accumulation of 16S rRNA . This constitutes the first example of a wild-type eubacterium showing a relaxed phenotype . The guanosine polyphosphate levels do not rise as a result of amino acid starvation, as expected for relaxed organisms . However, in both growing and starved cells, basal levels of the two polyphosphates appeared to be present, demonstrating that the enzymatic machinery for guanosine polyphosphate production is present in this organism . These findings are discussed within the framework of the hypothesis that stringent control is a physiological control mechanism more important for the fitness of prokaryotes growing in the general environment than for those that inhabit protected niches. J Biol Chem, 1999 Jan 15, 274(3), 1698 - 707 The role of lipoprotein processing by signal peptidase II in the Gram-positive eubacterium bacillus subtilis . Signal peptidase II is required for the efficient secretion of alpha-amylase, a non-lipoprotein; Tjalsma H et al.; Computer-assisted analyses indicate that Bacillus subtilis contains approximately 300 genes for exported proteins with an amino-terminal signal peptide . About 114 of these are lipoproteins, which are retained in the cytoplasmic membrane . We have investigated the importance of lipoprotein processing by signal peptidase II (SPase II) for cellular homeostasis, using cells lacking SPase II . The results show that lipoprotein processing is important for cell viability at low and high temperatures, suggesting that lipoproteins are essential for growth under these conditions . Although certain lipoproteins are required for the development of genetic competence, sporulation, and germination, these developmental processes were not affected in the absence of SPase II . Cells lacking SPase II accumulated lipid-modified precursor and mature-like forms of PrsA, a folding catalyst for secreted proteins . These forms of PrsA seem to have a reduced activity, as the secretion of alpha-amylase was strongly impaired . Unexpectedly, type I signal peptidases, which process secretory preproteins, were not involved in alternative amino-terminal processing of pre-PrsA in the absence of SPase II . In conclusion, processing of lipoproteins by SPase II in B . subtilis is not strictly required for lipoprotein function, which is surprising as lipoproteins and type II SPases seem to be conserved in all eubacteria. Eur J Biochem, 1998 Dec 1, 258(2), 813 - 9 Glutamate dehydrogenase, the marker protein of Plasmodium falciparum--cloning, expression and characterization of the malarial enzyme; Wagner JT et al.; The gene of an NADP+-specific glutamate dehydrogenase was cloned from Plasmodium falciparum, the causative agent of tropical malaria . Southern-blot analysis indicates a single-copy gene . The gene encodes a protein with 470 residues which has 50% of all residues identical with those of the glutamate dehydrogenases from other low eukaryotes and eubacteria . In contrast, the sequence identity with the human enzyme is marginal, which underlines the long evolutionary distance between parasite and host . The gene was overexpressed in Escherichia coli . The kinetic properties of the recombinant enzyme are in good agreement with those of the authentic enzyme . The parasite enzyme is inhibited by D-glutamate and glutarate, but not by chloroquine . Like other coenzyme-specific glutamate dehydrogenases, but in contrast to the dual-specific mammalian enzymes, the P . falciparum enzyme is not affected by GTP and ADP . The physical and chemical properties of the protein are in accordance with the cytosol being the major localization . The gene does not encode a cleavable mitochondrial presequence and the Mr of the recombinant protein and the protein isolated from the parasite are indistinguishable on SDS/PAGE . Western-blot analysis of stage-specific parasites shows that glutamate dehydrogenase is present in all intraerythrocytic stages . The signal increased continuously from rings, early trophozoites to late trophozoites and decreased slightly in the segmenter stage . Glutamate dehydrogenase, suggested to be the major source of NADPH in the parasite, is an attractive target molecule for the rational development of new antimalarial drugs. J Lipid Res, 1999 Jan, 40(1), 17 - 23 The bile acid-inducible baiF gene from Eubacterium sp . strain VPI 12708 encodes a bile acid-coenzyme A hydrolase; Ye HQ et al.; The human intestinal Eubacterium sp . strain VPI 12708 has been shown to have a multistep biochemical pathway for bile acid 7alpha-dehydroxylation . A bile acid-inducible operon encoding 9 open reading frames has been cloned and sequenced from this organism . Several of the genes in this operon have been shown to catalyze specific reactions in the 7alpha-dehydroxylation pathway . The baiF gene from this operon was cloned, expressed in Escherichia coli, and found to encode a novel bile acid-coenzyme A (CoA) hydrolase . The subunit molecular mass of the purified bile acid-CoA hydrolase was calculated to be 47,466 daltons and the native enzyme had a relative molecular weight of 72,000 . The K m and Vmax for cholyl-coenzyme A (CoA) hydrolysis was approximately 175 microm and 374 micromol/min per mg protein, respectively . The enzyme used cholyl-CoA, 3-dehydrocholyl-CoA, and chenodeoxycholyl-CoA as substrates . No hydrolytic activity was detected using acetyl-CoA, isovaleryl-CoA, palmitoyl-CoA, or phenylacetyl-CoA as substrates . Amino acid sequence database searches showed no significant similarity of bile acid-CoA hydrolase to other thioesterases, but significant amino acid sequence identity was found with Escherichia coli carnitine dehydratase . The characteristic thioesterase active site Gly-X-Ser-X-Gly motif was not found in the amino acid sequence of this enzyme.Bile acid-CoA hydrolase from Eubacterium sp . strain VPI 12708 may represent a new family of thioesterases. J Exp Biol, 1998 Oct, 201 ( Pt 20), 2791 - 9 Evolutionary link between prokaryotic and eukaryotic K+ channels; Derst C et al.; Considering the importance of K+ channels in controlling the crucial K+ gradient across the plasma membranes of all living cells, it comes as no surprise that, besides being present in every eukaryotic cell, these integral membrane proteins have recently also been identified in prokaryotes . Today, approximately a dozen successfully completed and many more ongoing sequencing projects permit a search for genes related to K+ channels in the genomes of both eubacteria and archaea . The coding regions of homologues show a remarkable variety in primary structure . They predict membrane proteins with one, two, three and six hydrophobic segments surrounding a putative K(+)-selective pore (H5) and the presence or absence of a cytosolic putative NAD(+)-binding domain (PNBD) that probably senses the reducing power of the cell . The analysis of kinships on the basis of phylogenetic algorithms identifies sequences closely related to eukaryotic voltage-dependent Kv channels, but also defines members of a primordial class of prokaryotic K+ channel (containing the 2TMS/PNBD motif) . Considering the unique mechanisms that may account for the assembly of modern proteins from different ancestral genes, and with more primary sequence data soon to appear, a scheme for the evolutionary origin of K+ channels comes within reach. Microb Ecol, 1998 Nov, 36(3), 221 - 230 Microbial Phototrophic, Heterotrophic, and Diazotrophic Activities Associated with Aggregates in the Permanent Ice Cover of Lake Bonney, Antarctica; Paerl HW et al.; Abstract The McMurdo Dry Valley lakes, Antarctica, one of the Earth's southernmost ecosystems containing liquid water, harbor some of the most environmentally extreme (cold, nutrient-deprived) conditions on the planet . Lake Bonney has a permanent ice cover that supports a unique microbial habitat, provided by soil particles blown onto the lake surface from the surrounding, ice-free valley floor . During continuous sunlight summers (Nov.-Feb.), the dark soil particles are heated by solar radiation and melt their way into the ice matrix . Layers and patches of aggregates and liquid water are formed . Aggregates contain a complex cyanobacterial-bacterial community, concurrently conducting photosynthesis (CO2 fixation), nitrogen (N2) fixation, decomposition, and biogeochemical zonation needed to complete essential nutrient cycles . Aggregate-associated CO2- and N2-fixation rates were low and confined to liquid water (i.e., no detectable activities in the ice phase) . CO2 fixation was mediated by cyanobacteria; both cyanobacteria and eubacteria appeared responsible for N2 fixation . CO2 fixation was stimulated primarily by nitrogen (NO3-), but also by phosphorus (PO43-) . PO43- and iron (FeCl3 + EDTA) enrichment stimulated of N2 fixation . Microautoradiographic and physiological studies indicate a morphologically and metabolically diverse microbial community, exhibiting different cell-specific photosynthetic and heterotrophic activities . The microbial community is involved in physical (particle aggregation) and chemical (establishing redox gradients) modification of a nutrient- and organic matter-enriched microbial "oasis," embedded in the desertlike (i.e., nutrient depleted) lake ice cover . Aggregate-associated production and nutrient cycling represent microbial self-sustenance in a microenvironment supporting "life at the edge," as it is known on Earth. Mol Biochem Parasitol, 1998 Oct 30, 96(1-2), 69 - 81 Gene structure, activity and localization of a catalase from intracellular bacteria in Onchocerca volvulus; Henkle-Duhrsen K et al.; Within the context of studies on the antioxidant enzymes in Onchocerca volvulus, DNA clones encoding catalase (CAT) were isolated from an O . volvulus adult lambda zapII cDNA library . Analysis of their nucleotide and encoded amino acid sequences revealed that they derive from intracellular bacteria, rather than the O . volvulus nuclear genome . The endobacterial CAT gene was found to lie in a gene cluster, followed by a ferritin gene and an excinuclease gene . The endobacterial CAT gene encodes a functional enzyme capable of detoxifying H2O2, demonstrated by producing an active recombinant protein in an E . coli expression system . The purified 54 kDa protein has CAT activity over a broad pH range, with a specific activity of 103,000 +/- 3000 U mg(-1) . The optical spectrum of the endobacterial CAT shows that it is a ferric haem-containing protein with a Soret band at 405 nm . To investigate the phylogeny of the intracellular bacterium in O . volvulus, a segment of the 16S rRNA gene was amplified from total genomic DNA by a polymerase chain reaction using universal eubacterial primers . A phylogenetic analysis of the O . volvulus-derived 16S rRNA sequence revealed that the endobacterium belongs to a distinct Wolbachia clade of the order Rickettsiales . Onchocercomata and biopsies containing different onchocercal species were immunohistochemically stained using polyclonal antibodies raised against the recombinant endobacterial CAT . CAT was detected in the endobacteria in the hypodermis of adult male and female O . volvulus, O . ochengi, O . gibsoni and O . fasciata . The endobacterial enzyme was also detected in onchocercal oocytes and all embryonic stages including intrauterine microfilariae as well as skin microfilariae . O . volvulus thus harbours Wolbachia-like endosymbionts which are transovarially transmitted and show particular affinity for the hypodermal tissues of the lateral chords. J Mol Evol, 1998 Dec, 47(6), 739 - 50 The pyrophosphate-dependent phosphofructokinase of the protist, Trichomonas vaginalis, and the evolutionary relationships of protist phosphofructokinases; Mertens E et al.; The pyrophosphate-dependent phosphofructokinase (PPi-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified . The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation . The protein was fragmented and a number of peptides were sequenced . Based on this information a PCR product was obtained from T . vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones . Southern analysis indicated the presence of five genes . One open reading frame (ORF) was completely sequenced and for two others the 5' half of the gene was determined . The sequences were highly similar . The complete ORF corresponded to a polypeptide of about 46 kDa . All the peptide sequences obtained were present in the derived sequences . The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half . The T . vaginalis enzyme was most similar to PPi-PFK of the mitochondriate heterolobosean, Naegleria fowleri . Most of the residues shown or assumed to be involved in substrate binding in other PPi-PFKs were conserved in the T . vaginalis enzyme . Direct comparison and phylogenetic reconstruction revealed a significant divergence among PPi-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists . The separation of these groups is supported with a high percentage of bootstrap proportions . The short T . vaginalis PFK shares a most recent common ancestor with the enzyme from N . fowleri . This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the alpha- and beta-subunits of plant PPi-PFKs . The third group ("X") containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E . histolytica pfk1, and a second sequence from B . burgdorferi . The fourth group ("Y") comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences . The well-studied PPi-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups . These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier . These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. J Mol Evol, 1998 Dec, 47(6), 691 - 6 Base composition skews, replication orientation, and gene orientation in 12 prokaryote genomes; McLean MJ et al.; Variation in GC content, GC skew and AT skew along genomic regions was examined at third codon positions in completely sequenced prokaryotes . Eight out of nine eubacteria studied show GC and AT skews that change sign at the origin of replication . The leading strand in DNA replication is G-T rich at codon position 3 in six eubacteria, but C-T rich in two Mycoplasma species . In M . genitalium the AT and GC skews are symmetrical around the origin and terminus of replication, whereas its GC content variation has been shown to have a centre of symmetry elsewhere in the genome . Borrelia burgdorferi and Treponema pallidum show extraordinary extents of base composition skew correlated with direction of DNA replication . Base composition skews measured at third codon positions probably reflect mutational biases, whereas those measured over all bases in a sequence (or at codon positions 1 and 2) can be strongly affected by protein considerations due to the tendency in some bacteria for genes to be transcribed in the same direction that they are replicated . Consequently in some species the direction of skew for total genomic DNA is opposite to that for codon position 3. J Mol Evol, 1998 Dec, 47(6), 649 - 55 Evolutionary relationship between translation initiation factor eIF-2gamma and selenocysteine-specific elongation factor SELB: change of function in translation factors; Keeling PJ et al.; Eubacterial and eukaryotic translation initiation systems have very little in common, and therefore the evolutionary events that gave rise to these two disparate systems are difficult to ascertain . One common feature is the presence of initiation, elongation, and release factors belonging to a large GTPase superfamily . One of these initiation factors, the gamma subunit of initiation factor 2 (eIF-2gamma), is found only in eukaryotes and archaebacteria . We have sequenced eIF-2gamma gene fragments from representative diplomonads, parabasalia, and microsporidia and used these new sequences together with new archaebacterial homologues to examine the phylogenetic position of eIF-2gamma within the GTPase superfamily . The archaebacterial and eukaryotic eIF-2gamma proteins are found to be very closely related, and are in turn related to SELB, the selenocysteine-specific elongation factor from eubacteria . The overall topology of the GTPase tree further suggests that the eIF-2gamma/SELB group may represent an ancient subfamily of GTPases that diverged prior to the last common ancestor of extant life. Nat Struct Biol, 1998 Dec, 5(12), 1053 - 8 Iron center, substrate recognition and mechanism of peptide deformylase; Becker A et al.; Eubacterial proteins are synthesized with a formyl group at the N-terminus which is hydrolytically removed from the nascent chain by the mononuclear iron enzyme peptide deformylase . Catalytic efficiency strongly depends on the identity of the bound metal . We have determined by X-ray crystallography the Fe2+, Ni2+ and Zn2+ forms of the Escherichia coli enzyme and a structure in complex with the reaction product Met-Ala-Ser . The structure of the complex, with the tripeptide bound at the active site, suggests detailed models for the mechanism of substrate recognition and catalysis . Differences of the protein structures due to the identity of the bound metal are extremely small and account only for the observation that Zn2+ binds more tightly than Fe2+ or Ni2+ . The striking loss of catalytic activity of the Zn2+ form could be caused by its reluctance to change between tetrahedral and five-fold metal coordination believed to occur during catalysis . N-terminal formylation and subsequent deformylation Nucleic Acids Res, 1999 Jan 1, 27(1), 158 - 60 5S Ribosomal RNA Data Bank; Szymanski M et al.; This paper presents the updated version of the data base of ribosomal 5S ribonucleic acids (5S rRNA) and their genes (5S rDNA) . This edition of the data bank contains 1889 primary structures of 5S rRNA and 5S rDNA . These include 60 archaebacterial, 439 eubacterial, 63 plastid, 9 mitochondrial and 1318 eukaryotic sequences . The nucleotide sequences of 5S rRNAs or 5S rDNAs are divided according to the taxonomic position of organisms . The sequences stored in the database can be viewed and retrieved using the taxonomic browser at the URL: + FEBS Lett, 1998 Nov 20, 439(3), 235 - 40 C-terminal truncation of yeast SerRS is toxic for Saccharomyces cerevisiae due to altered mechanism of substrate recognition; Lenhard B et al.; Like all other eukaryal cytosolic seryl-tRNA synthetase (SerRS) enzymes, Saccharomyces cerevisiae SerRS contains a C-terminal extension not found in the enzymes of eubacterial and archaeal origin . Overexpression of C-terminally truncated SerRS lacking the 20-amino acid appended domain (SerRSC20) is toxic to S . cerevisiae possibly because of altered substrate recognition . Compared to wild-type SerRS the truncated enzyme displays impaired tRNA-dependent serine recognition and is less stable . This suggests that the C-terminal peptide is important for the formation or maintenance of the enzyme structure optimal for substrate binding and catalysis. Eur J Immunol, 1998 Nov, 28(11), 3857 - 66 Correlation between chlamydial infection and autoimmune response: molecular mimicry between RNA polymerase major sigma subunit from Chlamydia trachomatis and human L7; Hemmerich P et al.; L7 is one of the ribosomal proteins frequently targeted by autoantibodies in rheumatic autoimmune diseases . A computer search revealed a region within the immunodominant epitope of L7 (peptide II) that is highly homologous to amino acid sequence 264-286 of the RNA polymerase major sigma factor of the eubacterium Chlamydia trachomatis . Anti-L7 autoantibodies affinity purified from the immunodominant epitope were able to recognize this sequence as they reacted with purified recombinant sigma factor . Immunofluorescence labeling experiments on C . trachomatis lysates revealed a punctate staining pattern of numerous spots when incubated with the affinity-purified anti-peptide II autoantibodies . Binding of autoantibodies to peptide II was inhibited by the homologous sigma peptide . This is the first demonstration of epitope mimicry between a human and a chlamydial protein on the level of B cells . Antibody screening revealed a significant correlation between the presence of anti-L7 autoantibodies and C . trachomatis infection in patients with systemic lupus erythematosus and mixed connective tissue disease . Our results suggest that molecular mimicry is involved in the initiation of anti-L7 autoantibody response and may represent a first glance into the immunopathology of Chlamydia with respect to systemic rheumatic diseases. EMBO J, 1998 Dec 1, 17(23), 6819 - 26 Crystal structure of methionyl-tRNAfMet transformylase complexed with the initiator formyl-methionyl-tRNAfMet; Schmitt E et al.; The crystal structure of Escherichia coli methionyl-tRNAfMet transformylase complexed with formyl-methionyl-tRNAfMet was solved at 2.8 A resolution . The formylation reaction catalyzed by this enzyme irreversibly commits methionyl-tRNAfMet to initiation of translation in eubacteria . In the three-dimensional model, the methionyl-tRNAfMet formyltransferase fills in the inside of the L-shaped tRNA molecule on the D-stem side . The anticodon stem and loop are away from the protein . An enzyme loop is wedged in the major groove of the acceptor helix . As a result, the C1-A72 mismatch characteristic of the initiator tRNA is split and the 3' arm bends inside the active centre . This recognition mechanism is markedly distinct from that of elongation factor Tu, which binds the acceptor arm of aminoacylated elongator tRNAs on the T-stem side. Biochemistry, 1998 Nov 10, 37(45), 15925 - 32 Functional interaction of an arginine conserved in the sixteen amino acid insertion module of Escherichia coli methionyl-tRNA formyltransferase with determinants for formylation in the initiator tRNA; Ramesh V et al.; Formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in eubacteria . The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA . Previous studies suggested that a 16 amino acid insertion loop, present in all eubacterial MTF's (residues 34-49 in the E . coli enzyme), plays an important role in specific recognition of the initiator tRNA . Here, we have analyzed the effect of site-specific mutations of amino acids within this region . We show that an invariant arginine at position 42 within the loop plays a very important role both in the steps of substrate binding and in catalysis . The kinetic parameters of the R42K and R42L mutant enzymes using acceptor stem mutant initiator tRNAs as substrates suggest that arginine 42 makes functional contacts with the determinants at the 3:70 and possibly also the 2:71 base pairs in the acceptor stem of the initiator tRNA . The kinetic parameters of the G41R/R42L double mutant enzyme are essentially the same as those of R42L mutant, suggesting that the requirement for arginine at position 42 cannot be fulfilled by an arginine at position 41 . Along with other data, this result suggests that the insertion loop, which is normally unstructured and flexible, adopts a defined conformation upon binding to the tRNA. Microbiol Mol Biol Rev, 1998 Dec, 62(4), 1435 - 91 Protein phylogenies and signature sequences: A reappraisal of evolutionary relationships among archaebacteria, eubacteria, and eukaryotes; Gupta RS; The presence of shared conserved insertion or deletions (indels) in protein sequences is a special type of signature sequence that shows considerable promise for phylogenetic inference . An alternative model of microbial evolution based on the use of indels of conserved proteins and the morphological features of prokaryotic organisms is proposed . In this model, extant archaebacteria and gram-positive bacteria, which have a simple, single-layered cell wall structure, are termed monoderm prokaryotes . They are believed to be descended from the most primitive organisms . Evidence from indels supports the view that the archaebacteria probably evolved from gram-positive bacteria, and I suggest that this evolution occurred in response to antibiotic selection pressures . Evidence is presented that diderm prokaryotes (i.e., gram-negative bacteria), which have a bilayered cell wall, are derived from monoderm prokaryotes . Signature sequences in different proteins provide a means to define a number of different taxa within prokaryotes (namely, low G+C and high G+C gram-positive, Deinococcus-Thermus, cyanobacteria, chlamydia-cytophaga related, and two different groups of Proteobacteria) and to indicate how they evolved from a common ancestor . Based on phylogenetic information from indels in different protein sequences, it is hypothesized that all eukaryotes, including amitochondriate and aplastidic organisms, received major gene contributions from both an archaebacterium and a gram-negative eubacterium . In this model, the ancestral eukaryotic cell is a chimera that resulted from a unique fusion event between the two separate groups of prokaryotes followed by integration of their genomes. Hepatogastroenterology, 1998 Sep-Oct, 45(23), 1643 - 50 Deconjugation ability of bacteria isolated from the jejunal fluid of patients with progressive systemic sclerosis and its gastric pH; Shindo K et al.; BACKGROUND/AIMS: Our goal was to demonstrate the role of bacteria in altered bile acid metabolism, which overgrow in the upper small intestine of patients with progressive systemic sclerosis . We identified the bacterial species, isolated from the jejunal fluid obtained from patients with progressive systemic sclerosis, who had previously shown an increase in 14CO2, specific activity on breath test, and normal controls . After which, we investigated the deconjugation ability of the isolated bacteria and the relationship between 14CO2, specific activity and gastric pH . METHODOLOGY: Bile acid breath tests were performed on 12 patients, and 19 normal controls using 5 microCi of oral glycine-1-(14)C-labeled glycocholate . Jejunal fluid was aspirated through a double lumen-tube with a rubber cover on the tip . Deconjugation ability was examined by thin-layer chromatography using conjugated bile acids in ox gall . RESULTS: The following species were identified in jejunal fluid samples obtained from patients: Bacteroides vulgatus, Eubacterium lentum, enterococcus, Lactobacillus bifidus, Escherichia (E) coli, Aerobacter (A) aerogenes . Except for E . coli and A . aerogenes, these species were capable of hydrolyzing conjugated bile acids in ox gall . The administration of chloramphenicol (1 g orally per day for 14 days in divided doses) significantly reduced the 14CO2, specific activity (p<0.05) in the patients with progressive systemic sclerosis . On the other hand, nineteen healthy control subjects demonstrated no increase in CO2 excretion, and 16 of the 19 had no bacteria isolated from jejunal fluid . The remaining healthy man showed an overgrowth of E . coli and Pseudomonas (P) aeruginosa, but the E . coli and P . aeruginosa did not have the ability of deconjugation . CO2 specific activity of expired breath samples in the patients with progressive systemic sclerosis was correlated with gastric pH (n=12, r=0.588, p<0.05) . CONCLUSIONS: Our results demonstrated that some of the bacterial species that overgrow in the upper small intestine of patients with progressive systemic sclerosis can deconjugate bile acids, and that a shift to neutral pH in gastric juice, may promote the bacterial overgrowth related to their impaired peristaltic activity. Appl Environ Microbiol, 1998 Dec, 64(12), 4939 - 43 Carbon monoxide oxidation by bacteria associated with the roots of freshwater macrophytes Rich JJ, King GM. The potential rates and control of aerobic root-associated carbon monoxide (CO) consumption were assessed by using excised plant roots from five common freshwater macrophytes . Kinetic analyses indicated that the maximum potential uptake velocities for CO consumption ranged from 0.4 to 2.7 &mgr;mol of CO g (dry weight)-1 h-1 for the five species . The observed rates were comparable to previously reported rates of root-associated methane uptake . The apparent half-saturation constants for CO consumption ranged from 50 to 370 nM CO; these values are considerably lower than the values obtained for methane uptake . The CO consumption rates reached maximum values at temperatures between 27 and 32 degreesC, and there was a transition to CO production at >/=44 degreesC, most likely as a result of thermochemical organic matter decomposition . Incubation of roots with organic substrates (e.g., 5 mM syringic acid, glucose, alanine, and acetate) dramatically reduced the rate of CO consumption, perhaps reflecting a shift in metabolism by facultative CO oxidizers . Based on responses to a suite of antibiotics, most of the CO consumption (about 90%) was due to eubacteria rather than fungi or other eucaryotes . Based on the results of acetylene inhibition experiments, methanotrophs and ammonia oxidizers were not active CO consumers. FEMS Microbiol Lett, 1998 Nov 15, 168(2), 269 - 76 Cloning and expression of the gene encoding RNA polymerase alpha subunit from alkaliphilic Bacillus sp . strain C-125; Nakasone K et al.; The rpoA gene, encoding the alpha subunit of RNA polymerase, was isolated from alkaliphilic Bacillus sp . strain C-125 by the PCR method . A 3-kb HindIII fragment containing the complete rpoA gene was cloned and sequenced . The alpha subunit gene was found to encode a protein consisting of 314 amino acid residues with a molecular mass of 34,805 Da . Compared with the amino acid sequences of other known eubacterial RNA polymerase alpha subunits, the gene has 84% identity to that of B . subtilis, while showing 48% and 47% identity to that of Streptomyces coelicolor and Escherichia coli, respectively . Six conserved regions, which are observed in the case of other eubacteria, were found in the RNA polymerase alpha subunit of this strain . Five of them are located in the N-terminal domain involved in assembly of the core enzyme, while one is located in the C-terminal domain, which interacts with several transcriptional factors and a specific DNA element . By means of recombinant plasmids, a hexahistidine-tagged derivative of the RNA polymerase alpha subunit of strain C-125 and two deletion derivatives (C- and N-terminal domains) of this protein were overexpressed in E . coli cells and purified to near homogeneity. Gen Physiol Biophys, 1998 Sep, 17(3), 193 - 210 Malate dehydrogenase: distribution, function and properties; Musrati RA et al.; Malate dehydrogenase (MDH) (EC 1.1.1.37) catalyzes the conversion of oxaloacetate and malate . This reaction is important in cellular metabolism, and it is coupled with easily detectable cofactor oxidation/reduction . It is a rather ubiquitous enzyme, for which several isoforms have been identified, differing in their subcellular localization and their specificity for the cofactor NAD or NADP . The nucleotide binding characteristics can be altered by a single amino acid change . Multiple amino acid sequence alignments of MDH show that there is a low degree of primary structural similarity, apart from several positions crucial for catalysis, cofactor binding and the subunit interface . Despite the low amino acids sequence identity their 3-dimensional structures are very similar . MDH is a group of multimeric enzymes consisting of identical subunits usually organized as either dimer or tetramers with subunit molecular weights of 30-35 kDa . MDH has been isolated from different sources including archaea, eubacteria, fungi, plant and mammals. Lett Appl Microbiol, 1998 Nov, 27(5), 302 - 6 Enumeration of Carnobacterium divergens V41, Carnobacterium piscicola V1 and Lactobacillus brevis LB62 by in situ hybridization-flow cytometry; Connil N et al.; The specific detection and enumeration of Lactobacillus brevis LB62, Carnobacterium divergens V14 and Carnobacterium piscicola VI were studied by in situ hybridization-flow cytometry . The method was performed on the exponential growth phase with three probes targeting 16S rRNA labelled with fluorescein isothicyanate (FITC): EUB338 probe universal for Eubacteria, Lb probe specific for Lact . brevis and Cb probe specific for the genus Carnobacterium . EUB338 was used to determine the permeabilization and hybridization conditions for the cells . The Lb probe gave no hybridization signal whereas the Cb probe allowed the detection and quantification by flow cytometry at 520 nm of the two Carnobacterium strains in pure culture or in mixtures with Listeria innocua F. J Bacteriol, 1998 Dec, 180(23), 6389 - 91 Aspartate transcarbamylase from the hyperthermophilic eubacterium Thermotoga maritima: fused catalytic and regulatory polypeptides form an allosteric enzyme; Chen P et al.; In the allosteric aspartate transcarbamylase (ATCase) from the hyperthermophilic eubacterium Thermotoga maritima, the catalytic and regulatory functions, which in class B ATCases are carried out by specialized polypeptides, are combined on a single type of polypeptide assembled in trimers . The ATCases from T . maritima and Treponema denticola present intriguing similarities, suggesting horizontal gene transfer. J Bacteriol, 1998 Dec, 180(23), 6276 - 82 The Bacillus subtilis nucleotidyltransferase is a tRNA CCA-adding enzyme; Raynal LC et al.; There has been increased interest in bacterial polyadenylation with the recent demonstration that 3' poly(A) tails are involved in RNA degradation . Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes . Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences . Gram-negative organisms from the beta and gamma subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E . coli homologues . Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable . The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity . We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase . We suggest that the papS gene should be renamed cca, following the notation for its E . coli counterpart . The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B . subtilis has a PAP similar to E . coli PAP I . Thus, the activity involved in RNA 3' polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria. Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14136 - 41 A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli; Spinelli SL et al.; tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2'-phosphotransferase to transfer the splice junction 2'-phosphate from ligated tRNA to NAD, producing ADP ribose 1"-2" cyclic phosphate (Appr>p) . We show here that functional 2'-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules . Surprisingly, functional 2'-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2'-phosphate . Analysis of the database shows that likely members of the 2'-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii) . Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2'-phosphotransferase into Eubacteria, suggesting that the 2'-phosphotransferase has been present there since close to the time that the three kingdoms diverged . Although 2'-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E . coli, the continued presence of 2'-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein. J Pharm Pharmacol, 1998 Oct, 50(10), 1155 - 60 Intestinal bacterial hydrolysis is required for the appearance of compound K in rat plasma after oral administration of ginsenoside Rb1 from Panax ginseng; Akao T et al.; Ginsenoside Rb1 from Panax ginseng root is transformed into compound K via ginsenosides Rd and F2 by intestinal bacterial flora . Among 31 defined intestinal strains from man, only Eubacterium sp . A-44 transformed ginsenoside Rb1 into compound K via ginsenoside Rd . The ginsenoside Rb1-hydrolysing enzyme isolated from Eubacterium sp . A-44 was identical to a previously purified geniposide-hydrolysing beta-D-glucosidase . When ginsenoside Rb1 (200 mg kg-1) was administered orally to germ-free rats, neither compound K nor any other metabolite was detected in the plasma, intestinal tract or cumulative faeces 7 or 15 h after administration . Most of the ginsenoside Rb1 administered was recovered from the intestinal tract, especially the caeca, and cumulative faeces indicating poor absorption of ginsenoside Rb1 . When ginsenoside Rb1 was administered orally to gnotobiote rats mono-associated with Eubacterium sp . A-44, a significant amount of compound K was detected in the plasma and considerable amounts were found in the caecal contents and cumulative faeces 7 and 15 h after administration . A small amount of ginsenoside Rb1 was detected in the caecal contents only 7 h after administration . These results indicate that orally administered ginsenoside Rb1 is poorly absorbed from the gut but that its metabolite compound K, produced by ginsenoside Rb1-hydrolysing bacteria such as Eubacterium sp . A-44 in the lower part of intestine, is absorbed. FEMS Microbiol Rev, 1998 Sep, 22(3), 127 - 50 Eubacterial sigma-factors; Wosten MM; The initiation of transcription is the most important step for gene regulation in eubacteria . To initiate transcription, RNA polymerase has to associate with a small protein, known as a sigma-factor . The sigma-factor directs RNA polymerase to a specific class of promoter sequences . Most bacterial species synthesize several different sigma-factors that recognize different consensus sequences . This variety in sigma-factors provides bacteria with the opportunity to maintain basal gene expression as well as for regulation of gene expression in response to altered environmental or developmental signals . This review focuses on the function, regulation and distribution of the 14 different classes of sigma-factors that are presently known. Zhonghua Liu Xing Bing Xue Za Zhi, 1997 Feb, 18(1), 22 - 5 {Serological investigation on Yang Xiao-xia's "mysterious disease"}; Yang HM et al.; The famous "mysterious disease" of Yang Xiao-xia, a girl from Shangdong Province, was diagnosed as multi-bacterial synergistic gangrene based on bacteriological, serological and antibiotics sensitivity findings . In order to uncover the initial pathogenic bacteria and to investigate their relationship with environment, a total number of 18 serum samples were collected from volunteers living around patient's home and from patient's relatives . Serological study on 4 bacterial strains isolated from patient, named as Eubacterim lentum, Stereptococcus acidominimus, Y6 and Yp was conducted with Western blot, using whole cell protein preparation was carried out, and data obtained was statistically analysed . Our findings indicated that YP and Y6 strains were interrelated with environment of patient's home and Y6 from the pool where patient visited often and several hours before she intially experienced the disease . The strains of Y6 and YP are virulent to animials, indicating that they serve as potential pathogens . Eubacteriumlentum and Streptococcus acidominimus are irrelevant to environment. FEMS Microbiol Lett, 1998 Oct 15, 167(2), 229 - 37 Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria; Eremeeva ME et al.; Heat shock proteins (Hsp) of four Rickettsia species, three Bartonella species, two Ehrlichia species, Orientia tsutsugamushi and seventeen other eubacterial species were characterized by the enhanced chemiluminescence Western blotting (WB) technique with antibodies raised against recombinant Hsp from Escherichia coli and purified GroES from R . typhi . Although E . coli DnaK and GroEL have epitopes that are highly conserved among the homologous proteins found in Rickettsia, Ehrlichia, O . tsutsugamushi, Bartonella and other Proteobacteria, anti-E . coli DnaK and GroEL monoclonal antibodies (Dasch et al . (1990) Ann . N.Y . Acad . Sci . 590, 352-369) recognize less conserved epitopes . In contrast, epitopes on E . coli DnaJ, GrpE and GroES are much less conserved since anti-E . coli DnaJ, GrpE and GroES polyclonal antibodies did not recognize DnaJ, GrpE or GroES homologues in Rickettsia, Bartonella, Orientia, Ehrlichia and Legionella . Polyclonal antiserum prepared against GroES from R . typhi reacted strongly with purified 10 kDa GroES peptide from Rickettsia and Bartonella, and strongly bound to proteins of varying electrophoretic mobility from Wolbachia, Legionella, Proteus and Shigella flexneri and more weakly to other GroES homologues including that found in E . coli . Consequently, commercially available anti-DnaJ, anti-GrpE and anti-GroES polyclonal antibodies and anti-DnaK monoclonal antibody raised against their respective recombinant E . coli Hsp are not suitable for detection and identification of homologues of these proteins in a wide range of eubacteria. Microbiology, 1998 Oct, 144 ( Pt 10), 2783 - 90 In situ detection of bacteria in continuous-flow cultures of seawater sediment suspensions with fluorescently labelled rRNA-directed oligonucleotide probes; Bruns A et al.; rRNA-targeted and fluorescently labelled oligonucleotide probes were used to study the composition of natural bacterial populations in continuous-flow cultures of seawater sediment suspensions . The cultures were run as enrichment cultures with increasing dilution rates, and hexadecane as the sole carbon source . Total cell numbers were analysed by counting DAPI (4',6-diamidino-2-phenylindole)-stained cells . To differentiate the population composition, oligonucleotide probes for eubacteria, for Cytophaga/Flavobacteria, and for four subclasses of the Proteobacteria (alpha, beta, gamma and delta) were used . About 40-80% of the DAPI-stained cells could be detected with the EUB338 probe . Moreover, it was possible to detect a shift in the composition of the natural bacterial population with increasing dilution rate of the continuous culture, from large amounts of Cytophaga/Flavobacteria to large numbers of members of the gamma-Proteobacteria . The cell recovery rate for bacteria labelled with specific oligonucleotide probes was analysed with defined cell numbers of Rhodospirillum rubrum, Comamonas testosteroni and Desulfovibrio vulgaris subsp . vulgaris introduced into the seawater sediment suspension, and was determined to be 13.9-33.5% . The standard deviation determined for this method applied to sediment suspensions was +/- 8.3% . The results suggest that the application of the in situ hybridization technique allows a good insight into the structure of populations growing in sediment suspensions. Nucleic Acids Res, 1998 Nov 15, 26(22), 5045 - 51 Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family; Shiba K et al.; We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS) . The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes . The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme . The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes . The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase. Biochemistry, 1998 Nov 3, 37(44), 15254 - 60 DNA polymerase III of Gram-positive eubacteria is a zinc metalloprotein conserving an essential finger-like domain; Barnes MH et al.; DNA polymerase III (pol III) of Gram-positive eubacteria is a catalytically bifunctional DNA polymerase:3'-5' exonuclease {Low, R . L., Rashbaum, S . A., and Cozzarelli, N . R . (1976) J . Biol.Chem . 251, 1311-1325} . The pol III protein conserves, between its exonuclease and dNTP binding sites, a 35-residue segment of primary structure with the potential to form a zinc finger-like structure {Berg, J . M . (1990) Ann . Rev . Biochem . 19, 405-421} . This paper describes results of experiments which probe the capacity of this segment to bind zinc and the role of this segment in enzyme function . The results of metal and mutational analysis of a model pol III derived from Bacillus subtilis indicate that (i) the Gram-positive pol III is a metalloprotein containing tightly bound zinc in a stoichiometry of 1, (ii) the zinc atom is bound within the 35-residue segment, likely in one of two probable finger-like structures, and (iii) the integrity of the zinc-bound structure is specifically critical to the formation and/or function of the enzyme's polymerase site. EMBO J, 1998 Nov 2, 17(21), 6377 - 84 The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA synthetases; Stoldt M et al.; The structure of the Escherichia coli ribosomal protein L25 has been determined to an r.m.s . displacement of backbone heavy atoms of 0.62 +/- 0.14 A by multi-dimensional heteronuclear NMR spectroscopy on protein samples uniformly labeled with 15N or 15N/13C . L25 shows a new topology for RNA-binding proteins consisting of a six-stranded beta-barrel and two alpha-helices . A putative RNA-binding surface for L25 has been obtained by comparison of backbone 15N chemical shifts for L25 with and without a bound cognate RNA containing the eubacterial E-loop that is the site for binding of L25 to 5S ribosomal RNA . Sequence comparisons with related proteins, including the general stress protein, CTC, show that the residues involved in RNA binding are highly conserved, thereby providing further confirmation of the binding surface . Tertiary structure comparisons indicate that the six-stranded beta-barrels of L25 and of the tRNA anticodon-binding domain of glutaminyl-tRNA synthetase are similar. Gene, 1998 Sep 14, 217(1-2), 83 - 90 Gene organization in the trxA/B-oriC region of the Streptomyces coelicolor chromosome and comparison with other eubacteria; Gal-Mor O et al.; The gene organization was determined in the trxA/B-rnpA region of the Streptomyces coelocolor chromosome, near to the origin of replication, oriC . Previously, we showed that the trxA and trxB genes, coding for thioredoxin and thioredoxin reductase, respectively, occur in S . coelicolor as a gene cluster and are contained on a cosmid H24 that carries oriC and several genes involved in DNA replication . Here we show that the trxA/B locus is positioned approx . 9.4kb from oriC, present the nucleotide sequence of the trxA/B-rnpA region and use sequence analysis to identify the nature of the intervening genes . Seven open reading frames were found, all oriented in the same direction, five of which were identified as the S . coelicolor homologs of SpoIIIJ, Jag, GidB, Soj and SpoOJ in Bacillus subtilis and which have been ascribed different functions in this and other bacteria for either DNA replication, chromosomal partitioning or morphological development . The arrangement of the genes coding for the above five proteins in the trxA/B-rnpA region in S . coelicolor resembles that in Mycobacterium leprae, Mycobacterium tuberculosis, B . subtilis and Pseudomonas putida, and supports the view that many of the genes necessary for development and cell division in bacteria are organized in a similar fashion . In B . subtilis and P . putida, however, the trxA/B genes are not present in the above gene arrangement. J Biol Chem, 1998 Nov 6, 273(45), 29693 - 700 The CCA-adding enzyme has a single active site; Yue D et al.; The CCA-adding enzyme (tRNA nucleotidyltransferase) synthesizes and repairs the 3'-terminal CCA sequence of tRNA . The eubacterial, eukaryotic, and archaeal CCA-adding enzymes all share a single active-site signature motif, which identifies these enzymes as belonging to the nucleotidyltransferase superfamily . Here we show that mutations at Asp-53 or Asp-55 of the Sulfolobus shibatae signature sequence abolish addition of both C and A, demonstrating that a single active site is responsible for addition of both nucleotides . Mutations at Asp-106 (and to a lesser extent, at Glu-173 and Asp-215) selectively impaired addition of A, but not C . We have previously demonstrated that the tRNA acceptor stem remains fixed on the surface of the CCA-adding enzyme during C and A addition (Shi, P.-Y., Maizels, N., and Weiner, A . M . (1998) EMBO J . 17, 3197-3206) . Taken together with this new evidence that there is a single active site for catalysis, our data suggest that specificity of nucleotide addition is determined by a process of collaborative templating: as the single active site catalyzes addition of each nucleotide, the growing 3'-end of the tRNA would progressively refold to create a binding pocket for addition of the next nucleotide. J Bacteriol, 1998 Nov, 180(21), 5601 - 11 Gigantism in a bacterium, Epulopiscium fishelsoni, correlates with complex patterns in arrangement, quantity, and segregation of DNA; Bresler V et al.; Epulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2, 000-fold in volume), and undergoes a complex daily life cycle . In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm . Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell . Fluorescence measurements of DNA, RNA, and other cell components indicate the following . DNA quantity is proportional to cell volume over cell lengths of approximately 30 micrometers to >500 micrometers . For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA . And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size . The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and "pinching" of DNA near the middle of the cell in the absence of new wall formation . Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines. Biochem Biophys Res Commun, 1998 Oct 9, 251(1), 173 - 81 Structure prediction in a post-genomic environment: a secondary and tertiary structural model for the initiation factor 5A family; Gerloff DL et al.; Two predictions have been prepared for the fold of initiation factor 5A (IF5A) starting from a set of homologous sequences . In the first, a secondary structural model was predicted for the protein in 1994, when only eleven homologs (and no eubacterial homologs) had been sequenced . The second was made recently, after genome projects had generated a total of 33 sequences for the protein family from species of all three kingdoms of life . With the second set of sequences, but not with the first, it was possible to predict that the N-terminal domain of the protein folds in a possibly open beta-barrel/sandwich core structure, with a short helix capping one side of the barrel . We place the pair of predictions in the public domain before an experimental structure is known . This example illustrates the impact of genome sequencing projects on structure prediction from sequence alignments . Orv Hetil, 1998 Sep 27, 139(39), 2313 - 8 {The role of Lactobacillus acidophilus in the prevention and adjuvant therapy of certain infectious diseases}; Halmy C et al.; Authors call attention to the role of lactic acid bacteria in the prevention and adjuvant therapy of certain infective diseases . It has special importance in the prevention and adjuvant therapy of new-born and childhood enteritis, different urogenital inflammations and antibiotic associated diarrhoea . Administration of lactic acid bacteria create eubiosis between the human organism and the world of bacteria, that is, eubacteriosis is developed instead of a pathogen flora, assuring normal physiologic functions for the well-being of the organism. Proc Natl Acad Sci U S A, 1998 Oct 27, 95(22), 12872 - 7 Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII; Frank P et al.; Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription . RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell . By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides . This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI . The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE . Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI . Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E . coli, an enzyme of unknown function and previously judged as a minor activity . This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII . The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced. FEMS Microbiol Lett, 1998 Oct 1, 167(1), 19 - 25 Mutations in the Escherichia coli surE gene increase isoaspartyl accumulation in a strain lacking the pcm repair methyltransferase but suppress stress-survival phenotypes; Visick JE et al.; The Escherichia coli surE gene is co-transcribed with pcm, encoding the L-isoaspartyl protein repair methyltransferase, and is highly conserved among both the Eubacteria and the Archaea; however, no biochemical function has yet been identified for this gene . Isoaspartyl accumulation during stationary phase was much higher in a pcm surE double mutant than in either single mutant, suggesting that the two genes may represent two parallel pathways by which E . coli can respond to protein damage . A null mutation in surE also suppressed stress-survival defects previously observed in a pcm mutant strain, providing further evidence for an interaction between the two gene products. Biopolymers, 1997, 44(4), 405 - 21 DNA recognition by structure-selective nucleases; Suck D; The nucleases discussed in this review show little sequence specificity but instead recognize certain structural features of their respective DNA substrates . The level of their structural selectivity ranges from simple discrimination between single- and double-stranded DNA (nucleases P1 and S1), the recognition of helical parameters like groove width and flexibility (DNase I), the recognition of helical distortions caused by abasic sites (exonuclease III, HAP1), to the recognition of specialized structures like flap DNA (5'-nucleases of eukaryotes, phages, and eubacterial DNA polymerases) and four-way junctions (T4 endonuclease VII, RuvC) . The discussion is focused on the structural basis of the recognition process . In most cases the available x-ray structures of the nucleases and/or their DNA complexes have revealed the presence of structural motifs explaining the observed structural selectivity. RNA, 1998 Oct, 4(10), 1282 - 94 Metal ion probing of rRNAs: evidence for evolutionarily conserved divalent cation binding pockets; Polacek N et al.; Ribosomes are multifunctional RNP complexes whose catalytic activities absolutely depend on divalent metal ions . It is assumed that structurally and functionally important metal ions are coordinated to highly ordered RNA structures that form metal ion binding pockets . One potent tool to identify the structural surroundings of high-affinity metal ion binding pockets is metal ion-induced cleavage of RNA . Exposure of ribosomes to divalent metal ions, such as Pb2+, Mg2+, Mn2+, and Ca2+, resulted in site-specific cleavage of rRNAs . Sites of strand scission catalyzed by different cations accumulate at distinct positions, indicating the existence of general metal ion binding centers in the highly folded rRNAs in close proximity to the cleavage sites . Two of the most efficient cleavage sites are located in the 5' domain of both 23S and 16S rRNA, regions that are known to self-fold even in the absence of ribosomal proteins . Some of the efficient cleavage sites were mapped to the peptidyl transferase center located in the large ribosomal subunit . Furthermore, one of these cleavages was clearly diminished upon AcPhe-tRNA binding to the P site, but was not affected by uncharged tRNA . This provides evidence for a close physical proximity of a metal ion to the amino acid moiety of charged tRNAs . Interestingly, comparison of the metal ion cleavage pattern of eubacterial 70S with that of human 80S ribosomes showed that certain cleavage sites are evolutionarily highly conserved, thus demonstrating an identical location of a nearby metal ion . This suggests that cations, bound to evolutionarily constrained binding sites, are reasonable candidates for being of structural or functional importance. Mol Microbiol, 1998 Aug, 29(4), 945 - 54 A glycyl radical solution: oxygen-dependent interconversion of pyruvate formate-lyase; Sawers G et al.; Pyruvate formate-lyase (PFL) catalyses the non-oxidative dissimilation of pyruvate to formate and acetyl-CoA using a radical-chemical mechanism . The enzyme is enzymically interconverted between inactive and active forms, the active form contains an organic free radical located on a glycyl residue in the C-terminal portion of the polypeptide chain . Introduction of the radical into PFL only occurs anaerobically, and the activating enzyme responsible is an iron-sulphur protein that uses S-adenosyl methionine as cofactor and reduced flavodoxin as reductant . As the radical form of PFL is inactivated by molecular oxygen it is safeguarded during the transition to aerobiosis by conversion back to the radical-free, oxygen-stable form . This reaction is catalysed by the anaerobically induced multimeric enzyme alcohol dehydrogenase . The genes encoding PFL and its activating enzyme are adjacent on the chromosome but form discrete transcriptional units . This genetic organization is highly conserved in many, but not all, organisms that have PFL . Recent studies have shown that proteins exhibiting significant similarity to PFL and its activating enzyme are relatively widespread in facultative and obligate anaerobic eubacteria, as well as archaea . The physiological function of many of these PFL-like enzymes remains to be established . It is becoming increasingly apparent that glycyl radical enzymes are more prevalent than previously surmised . They represent a class of enzymes with unusual biochemistry and probably predate the appearance of molecular oxygen. Res Microbiol, 1997 Nov, 148(8), 649 - 59 Universal ribotyping method using a chemically labelled oligonucleotide probe mixture; Regnault B et al.; Some of the present problems in ribotyping are associated with a lack of uniform reactivity of probes when bacterial DNAs are of phylogenetically diverse origins . To overcome these problems, a set of five oligonucleotides (referred to as OligoMix5) was selected to react with conserved sequences located near both extremities of rrs (16S rRNA gene) and near both extremities and the middle of rrl (23S rRNA gene) . DNA samples from 13 bacterial species selected to represent various phylogenetic branches within the Eubacteria were cleaved by a restriction endonuclease and electrophoresed in 0.8% agarose, and the fragments were vacuum-transferred to nylon membranes and hybridized with digoxigenin-labelled OligoMix5, plasmid DNA from pKK3535 (cloned rrn operon from Escherichia coli) or pBA2 (cloned rrs from Bacillus subtilis), or acetylamino-fluorene-labelled E . coli 16 + 23S rRNA . The results showed OligoMix5 to visualize patterns in DNA from phylogenetically diverse bacteria with comparable intensity . Banding patterns (not band intensity) obtained with OligoMix5 were identical with those obtained with 16 + 23S rRNA or plasmid pKK3535 for each strain studied and represented complete ribotypes . For DNA from Gram-positive bacteria, complete ribotypes were observed after prolonged enzymatic detection of bands when probes were either E . coli 16 + 23S rRNA or pKK3535 . Patterns given by plasmid pBA2 were subsets of the complete ribotypes for 9/13 strains . Each oligonucleotide of the OligoMix5 set was used as a probe to determine its contribution to the complete ribotype . The five oligonucleotide probes, used individually, visualized one to four patterns per DNA sample . Use of DNA from Xenorhabdus sp . CIP 105189 cleaved by EcoRI is suggested to control the quality of the oligonucleotide probes composing OligoMix5 . Probe OligoMix5 was found to be an essential tool for ribotyping phylogenetically diverse eubacteria. Res Microbiol, 1997 Mar-Apr, 148(3), 191 - 200 A cheA cheW operon in Borrelia burgdorferi, the agent of Lyme disease; Trueba GA et al.; Borrelia burgdorferi sensu stricto homologues of cheA and cheW were cloned and characterized . A combination of strategies such as polymerase chain reaction (PCR) using degenerate primers, random-primed gene walking PCR and construction of a lamda library were used to identify the putative cheA gene . Sequence analysis of the DNA fragments obtained from the CT strain identified a 2,592-bp open reading frame (ORF) encoding an 864-amino-acid protein with significant similarity (53-64.6% identical residues) to the CheA of several genera of eubacteria . In particular, hallmarks of a histidine kinase family were found such as the location of the histidine autophosphorylation domain very close to the NH2 terminus and the nucleotide-binding site . A second ORF located immediately downstream from the putative borrelial cheA gene encoded a 195-amino-acid protein which displayed a high level of similarity to bacterial CheW . Using reverse transcription PCR, we demonstrated that cheA and cheW form an operon with an upstream, unidentified ORF . The cheA and cheW homologues were located at 722-737 kbp, 738-768 kbp and 743-824 kbp on the linear chromosomes of B . burgdorferi sensu stricto, B . garinii and B . afzelii, respectively . Identification of cheA and cheW is the first step toward elucidation of a possible role of chemotaxis in virulence of the Lyme disease borreliae. Plant Cell, 1998 Oct, 10(10), 1713 - 22 rbcL Transcript levels in tobacco plastids are independent of light: reduced dark transcription rate is compensated by increased mRNA stability; Shiina T et al.; The plastid rbcL gene, encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase, in higher plants is transcribed from a sigma70 promoter by the eubacterial-type RNA polymerase . To identify regulatory elements outside of the rbcL -10/-35 promoter core, we constructed transplastomic tobacco plants with uidA reporter genes expressed from rbcL promoter derivatives . Promoter activity was characterized by measuring steady state levels of uidA mRNA on RNA gel blots and by measuring promoter strength in run-on transcription assays . We report here that the rbcL core promoter is sufficient to obtain wild-type rates of transcription . Furthermore, the rates of transcription were up to 10-fold higher in light-grown leaves than in dark-adapted plants . Although the rates of transcription were lower in the dark, rbcL mRNA accumulated to similar levels in light-grown and dark-adapted leaves . Accumulation of uidA mRNA from most rbcL promoter deletion derivatives directly reflected the relative rates of transcription: high in the light-grown and low in the dark-adapted leaves . However, uidA mRNA accumulated to high levels in a light-independent fashion as long as a segment encoding a stem-loop structure in the 5' untranslated region was included in the promoter construct . This finding indicates that lower rates of rbcL transcription in the dark are compensated by increased mRNA stability. Appl Environ Microbiol, 1998 Oct, 64(10), 3683 - 9 Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences; Wood J et al.; We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes . The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA . Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow . Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, toget