Eubacterium

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, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples . The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals . This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates . The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1043 - 5
Cloning, overproduction, purification and crystallization of the DNA binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima; Christodoulou E et al.; The humar gene encoding for the histone-like DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima was efficiently overexpressed in Escherichia coli under the T7 promoter . The HU protein was purified using SP-Sepharose ion-exchange and heparin-affinity chromatography and was successfully crystallized in ammonium sulfate . The crystals were grown in the tetragonal form in space group P43 or P41 and have unit-cell dimensions a = b = 46.12, c = 77.56 A, alpha = beta = gamma = 90 degrees . The crystals diffract X-rays to 1.6 A resolution using synchrotron radiation and are suitable for determination of the HU structure at high resolution.

Trends Biochem Sci, 1998 Aug, 23(8), 286 - 90
A superfamily of proteins that contain the cold-shock domain; Graumann PL et al.; Members of a family of cold-shock proteins (CSPs) are found throughout the eubacterial domain and appear to function as RNA-chaperones . They have been implicated in various cellular processes, including adaptation to low temperatures, cellular growth, nutrient stress and stationary phase . The discovery of a domain--the cold-shock domain--that shows strikingly high homology and similar RNA-binding properties to CSPs in a growing number of eukaryotic nucleic-acid-binding proteins suggests that these proteins have an ancient origin.

EMBO J, 1998 Oct 1, 17(19), 5658 - 69
An essential protease involved in bacterial cell-cycle control; Jenal U et al.; Proteolytic inactivation of key regulatory proteins is essential in eukaryotic cell-cycle control . We have identified a protease in the eubacterium Caulobacter crescentus that is indispensable for viability and cell-cycle progression, indicating that proteolysis is also involved in controlling the bacterial cell cycle . Mutants of Caulobacter that lack the ATP-dependent serine protease ClpXP are arrested in the cell cycle before the initiation of chromosome replication and are blocked in the cell division process . ClpXP is composed of two types of polypeptides, the ClpX ATPase and the ClpP peptidase . Site-directed mutagenesis of the catalytically active serine residue of ClpP confirmed that the proteolytic activity of ClpXP is essential . Analysis of mutants lacking ClpX or ClpP revealed that both proteins are required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA . CtrA is a member of the response regulator family of two-component signal transduction systems and controls multiple cell-cycle processes in Caulobacter . In particular, CtrA negatively controls DNA replication and our findings suggest that specific degradation of the CtrA protein by the ClpXP protease contributes to G1-to-S transition in this organism.

Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11769 - 74
Amitochondriate amoebae and the evolution of DNA-dependent RNA polymerase II; Stiller JW et al.; Unlike parasitic protist groups that are defined by the absence of mitochondria, the Pelobiontida is composed mostly of free-living species . Because of the presence of ultrastructural and cellular features that set them apart from all other eukaryotic organisms, it has been suggested that pelobionts are primitively amitochondriate and may represent the earliest-evolved lineage of extant protists . Analyses of rRNA genes, however, have suggested that the group arose well after the diversification of the earliest-evolved protists . Here we report the sequence of the gene encoding the largest subunit of DNA-dependent RNA polymerase II (RPB1) from the pelobiont Mastigamoeba invertens . Sequences within RPB1 encompass several of the conserved catalytic domains that are common to eubacterial, archaeal, and eukaryotic nuclear-encoded RNA polymerases . In RNA polymerase II, these domains catalyze the transcription of all nuclear pre-mRNAs, as well as the majority of small nuclear RNAs . In contrast with rDNA-based trees, phylogenetic analyses of RPB1 sequences indicate that Mastigamoeba represents an early branch of eukaryotic evolution . Unlike sequences from parasitic amitochondriate protists that were included in our study, there is no indication that Mastigamoeba RPB1 is attracted to the base of the eukaryotic tree artifactually . In addition, the presence of introns and a heptapeptide C-terminal repeat in the Mastigamoeba RPB1 sequence, features that are typically associated with more recently derived eukaryotic groups, raise provocative questions regarding models of protist evolution that depend almost exclusively on rDNA sequence analyses.

J Exp Biol, 1998 Sep 22, 201(Pt 20), 2791 - 2799
Review: 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.

Comp Biochem Physiol B Biochem Mol Biol, 1998 Mar, 119(3), 493 - 503
Glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena pyriformis: enzyme purification and characterization of a gapC gene with primitive eukaryotic features; Hafid N et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC.1.2.1.12) was purified to electrophoretic homogeneity from an amicronucleated strain of the ciliate Tetrahymena pyriformis using a three-step procedure . The native enzyme is an homotetramer of 145 kDa exhibiting absolute specificity for NAD . In its catalytic properties it is similar to other glycolytic GAPDHs . Chromatofocusing analysis showed the presence of only one basic GAPDH isoform with an isoelectric point of 8.8 . Western blots using a monospecific polyclonal antibody raised against the T . pyriformis GAPDH showed a single 36-kDa band corresponding to the enzyme subunit in the cytosolic protein fraction of this strain and the closely related species, both from the class Oligohymenophorea, Paramecium tetraurelia . No bands were immunodetected in the ciliate Colpoda inflata (class Colpodea) and in the diverse eukaryotes and eubacteria tested . A 0.5-kb DNA fragment which corresponds to an internal region of a gapC gene was generated by polymerase chain reaction using cDNA of T . pyriformis as template . This gene codes for a basic GAPDH protein with eukaryotic-diplomonad signatures and exhibits a codon usage biased in the manner typical for T . pyriformis genes . Southern blots performed both under homologous and heterologous conditions using this amplified cDNA fragment as a probe, indicated that it should be the only gapC gene present in the macronuclear genome of this ciliate, its expression being confirmed by Northern blot analysis . These results are discussed in connection with the peculiar genomic organization of ciliates and in the context of protist evolution.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 965 - 72
Shewanella amazonensis sp . nov., a novel metal-reducing facultative anaerobe from Amazonian shelf muds; Venkateswaran K et al.; A new bacterial species belonging to the genus Shewanella is described on the basis of phenotypic characterization and sequence analysis of its 16S rRNA-encoding and gyrase B (gyrB) genes . This organism, isolated from shallow-water marine sediments derived from the Amazon River delta, is a Gram-negative, motile, polarly flagellated, facultatively anaerobic, rod-shaped eubacterium and has a G&C content of 51.7 mol% . Strain SB2BT is exceptionally active in the anaerobic reduction of iron, manganese and sulfur compounds . SB2BT grows optimally at 35 degrees C, with 1-3% NaCl and over a pH range of 7-8 . Analysis of the 16S rDNA sequence revealed a clear affiliation between strain SB2BT and members of the gamma subclass of the class Proteobacteria . High similarity values were found with certain members of the genus Shewanella, especially with Shewanella putrefaciens, and this was supported by cellular fatty acid profiles and phenotypic characterization . DNA-DNA hybridization between strain SB2BT and its phylogenetically closest relatives revealed low similarity values (24.6-42.7%) which indicated species status for strain SB2BT . That SB2BT represents a distinct bacterial species within the genus Shewanella is also supported by gyrB sequence analysis . Considering the source of the isolate, the name Shewanella amazonensis sp . nov . is proposed and strain SB2BT (= ATCC 700329T) is designated as the type strain.

Theor Popul Biol, 1998 Oct, 54(2), 91 - 104
Life's third domain (Archaea): an established fact or an endangered paradigm?
Gupta RS.
The three-domain proposal of Woese et al . (Proc . Natl . Acad . Sci . USA 87, 4576 (1990)) divides all living organisms into three primary groups or domains named Archaea (or archaebacteria), Bacteria (or eubacteria), and Eucarya (or eukaryotes), with Eucarya being relatives (or descendants) of Archaea . Although this proposal is currently widely accepted, sequence features and phylogenies derived from many highly conserved proteins are inconsistent with it and point to a close and specific relationship between archaebacteria and gram-positive bacteria, whereas gram-negative bacteria are indicated to be phylogenetically distinct . A closer relationship of archaebacteria to gram-positive bacteria in comparison to gram-negative bacteria is generally seen for the majority of the available gene/protein sequences . To account for these results, and the fact that both archaebacteria and gram-positive bacteria are prokaryotes surrounded by a single cell membrane, I propose that the primary division within prokaryotes is between Monoderm prokaryotes (surrounded by a single membrane) and Diderm prokaryotes (i.e., all true gram-negative bacteria containing both an inner cytoplasmic membrane and an outer membrane) . This proposal is consistent with both cell morphology and signature sequences in different proteins . Protein phylogenies and signature sequences also show that all eukaryotic cells have received significant gene contributions from both an archaebacterium and a gram- negative eubacterium . Thus, the hypothesis that archaebacteria and eukaryotes shared a common ancestor exclusive of eubacteria, or that the ancestral eukaryotic cell directly descended from an archaea, is erroneous . These results call into question the validity of the currently popular three-domain proposal and the assignment of a domain status to archaebacteria . A new classifica- tion of organisms consistent with phenotype and macromolecular sequence data is proposed .

J Mol Biol, 1998 Sep 11, 282(1), 167 - 79
The structure of a trimeric archaeal adenylate kinase; Vonrhein C et al.; The adenylate kinase from the hyperthermophilic archaean species Sulfolobus acidocaldarius has been cloned, expressed in Escherichia coli, purified and crystallized . The crystal structure was elucidated by multiple isomorphous replacement and non-crystallographic density averaging . The structure was refined at 2.6 A (1 A=0.1 nm) resolution . The enzyme is trimeric, in contrast to previous solution measurements that suggested a dimeric structure, and in contrast to the vast majority of adenylate kinases, which are monomeric . In large parts of each subunit the chain fold resembles the known enzyme structure from eubacteria and eukaryotes although the sequence homology is negligible . Since the asymmetric unit contains two trimers with and without bound AMP at the AMP sites and with an ADP at one of the six ATP sites, the analysis shows the enzyme in several states . The conformational differences between these states resemble those of other adenylate kinases . Because of sequence homology, the structure presented provides a good model for the methanococcal adenylate kinases .

Arch Microbiol, 1998 Oct, 170(4), 285 - 96
Characterization of the group 1 and group 2 sigma factors of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus; Gruber TM et al.; The group 1 and group 2 sigma70-type sigma factors of the green sulfur bacterium Chlorobium tepidum and of the green nonsulfur bacterium Chloroflexus aurantiacus were cloned and characterized . Cb . tepidum was found to contain one sigma70-type sigma factor; the expression of the gene was analyzed by Northern blot hybridization and primer-extension mapping . Cf . aurantiacus has genes encoding four sigma factors of groups 1 and 2 . The expression of these genes was examined in cells grown aerobically and anaerobically . The sigC gene was expressed at approximately equal levels under both conditions, resulting in its designation as the group 1 sigma factor of this organism . The only other detectable transcripts arose from the sigB gene, which was expressed at higher levels during aerobic growth . A phylogenetic tree was obtained using the group 1 sigma factors of Cb . tepidum, Cf . aurantiacus, and diverse eubacteria as the molecular marker . The resulting phylogenetic tree shows that Cb . tepidum and Cf . aurantiacus are related to each other and to the cyanobacteria . The relationship of the group 2 sigma factors of Cf . aurantiacus and the cyanobacteria was more specifically examined phylogenetically . The group 2 sigma factors of Cf . aurantiacus probably arose by gene duplication events after the split of the green nonsulfur bacteria from other photosynthetic eubacteria.

Microbiol Mol Biol Rev, 1998 Sep, 62(3), 725 - 74
Insertion sequences; Mahillon J et al.; Insertion sequences (ISs) constitute an important component of most bacterial genomes . Over 500 individual ISs have been described in the literature to date, and many more are being discovered in the ongoing prokaryotic and eukaryotic genome-sequencing projects . The last 10 years have also seen some striking advances in our understanding of the transposition process itself . Not least of these has been the development of various in vitro transposition systems for both prokaryotic and eukaryotic elements and, for several of these, a detailed understanding of the transposition process at the chemical level . This review presents a general overview of the organization and function of insertion sequences of eubacterial, archaebacterial, and eukaryotic origins with particular emphasis on bacterial elements and on different aspects of the transposition mechanism . It also attempts to provide a framework for classification of these elements by assigning them to various families or groups . A total of 443 members of the collection have been grouped in 17 families based on combinations of the following criteria: (i) similarities in genetic organization (arrangement of open reading frames); (ii) marked identities or similarities in the enzymes which mediate the transposition reactions, the recombinases/transposases (Tpases); (iii) similar features of their ends (terminal IRs); and (iv) fate of the nucleotide sequence of their target sites (generation of a direct target duplication of determined length) . A brief description of the mechanism(s) involved in the mobility of individual ISs in each family and of the structure-function relationships of the individual Tpases is included where available.

Gene, 1998 Jul 3, 214(1-2), 7 - 11
Cloning and expression of the gene coding for FtsH protease from Mycobacterium tuberculosis H37Rv; Anilkumar G et al.; This study was aimed at the molecular cloning and expression of the gene coding for FtsH protease of Mycobacterium tuberculosis H37Rv (virulent) . PCR on the genomic DNA of M . tuberculosis H37Ra (non-virulent) using the oligodeoxynucleotide primers, which were designed based on the codon usage pattern of M . tuberculosis and against the nucleotide (nt) sequence corresponding to two conserved domains of the FtsH protein of Escherichia coli, yielded a 363-bp product . The amino-acid sequence, deduced from the nt sequence of the PCR product, revealed the presence of two ATP-binding motifs and the AAA Signature motif (Second Region of Homology) that are characteristic features found conserved in the FtsH molecules from eubacteria, archaebacteria, and eukaryotes . Southern hybridisation of the NheI digest of the cosmid SCY6F7 containing part of the genomic DNA of M . tuberculosis H37Rv using the PCR fragment as the probe identified the full-length ftsH gene in the 7.2-kb fragment . The gene was subcloned into pBS (SK+) vector, and the FtsH product that was expressed in E . coli transformed with the vector was identified as an 85-kDa protein localised in the membrane.

Mycopathologia, 1998, 141(1), 15 - 9
Immunohistochemical detection for Actinomyces sp . in swine tonsillar abscess and granulomatous mastitis; Murakami S et al.; The tonsils of eleven pigs and the mammary glands of a sow were used to investigate actinomycotic lesions due to Actinomyces sp . infection . At necropsy, there was no abnormality on these tonsils, on the other hand, numerous abscesses containing sulfur granules were found in the mammary . Histopathologically, the Actinomyces sp . lesions were noted as crypt abscesses in the tonsils and as pus-forming granulomas in the mammary glands . The microorganisms in both lesions were composed of bead-like cocci, bacillary cells and short, branching filaments, those cells being positive by the Gram's and Grocott's methods . Clubs were formed around the microbial clumps in these lesions . Immunohistochemically, there were cross-reactions between antibodies of Actinomyces sp . Chiba 101 (101) and swine actinomycetes of 7 species: A . bovis, A . hyovaginalis, A . israeli, A . naeslundii, A . pyogenes, A . suis) formerly Eubacterium suits) and A . viscosus . However it was possible to differentiate Actinomyces sp . 101 from them by absorption and dilution of the antiserum, then the microorganisms in the tonsillar crypt abscesses and the granulomatous mastitis were labelled with an immunoperoxidase technique using the absorbed Actinomyces sp . 101 antiserum . Thus, these immunolabelling properties are suggestive of the presence of 'A . suis' (Grasser) Franke 1973.

Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10431 - 6
Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase; Felden B et al.; Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3'-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase . Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs . Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs . This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides . The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes . The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2-3 orders of magnitude . Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions.

EMBO J, 1998 Sep 1, 17(17), 5227 - 37
Crystal structure of aspartyl-tRNA synthetase from Pyrococcus kodakaraensis KOD: archaeon specificity and catalytic mechanism of adenylate formation; Schmitt E et al.; The crystal structure of aspartyl-tRNA synthetase (AspRS) from Pyrococcus kodakaraensis was solved at 1.9 A resolution . The sequence and three-dimensional structure of the catalytic domain are highly homologous to those of eukaryotic AspRSs . In contrast, the N-terminal domain, whose function is to bind the tRNA anticodon, is more similar to that of eubacterial enzymes . Its structure explains the unique property of archaeal AspRSs of accommodating both tRNAAsp and tRNAAsn . Soaking the apo-enzyme crystals with ATP and aspartic acid both separately and together allows the adenylate formation to be followed . Due to the asymmetry of the dimeric enzyme in the crystalline state, different steps of the reaction could be visualized within the same crystal . Four different states of the aspartic acid activation reaction could thus be characterized, revealing the functional correlation of the observed conformational changes . The binding of the amino acid substrate induces movement of two invariant loops which secure the position of the peptidyl moiety for adenylate formation . An unambiguous spatial and functional assignment of three magnesium ion cofactors can be made . This study shows the important role of residues present in both archaeal and eukaryotic AspRSs, but absent from the eubacterial enzymes.

Curr Genet, 1998 Aug, 34(2), 105 - 11
Cloning and characterization of a nuclear gene encoding a starch-branching enzyme from the marine red alga Gracilaria gracilis; Lluisma AO et al.; The biosynthesis of starch in red algae occurs in the cytosol, in contrast to green plants where it takes place in the plastid . We have cloned a nuclear gene from the red alga Gracilaria gracilis that encodes a homolog of starch-branching enzymes (SBEs); this gene, which is apparently intron-free, was designated as GgSBE1 . A potential TATA box, CAAT boxes, and other potential regulatory elements were observed in its 5' flanking region . The encoded 766-aa peptide shares significant sequence similarity with SBEs from green plants (at least 40%), and with glycogen-branching enzymes (GBEs) from human (46%) and Saccharomyces cerevisiae (45%) . Southern-hybridization analysis indicates that the gene is single-copy, although weaker signals suggest that related genes exist in the genome of G . gracilis . Phylogenetic analyses indicate that GgSBE1 groups within the eukaryote branching enzymes (BEs) and not with eubacterial GBEs, suggesting that its gene has not been derived directly from an endosymbiotic cyanobacterium, but instead is ancestrally eukaryotic.

Mol Microbiol, 1998 Aug, 29(3), 695 - 707
What are archaebacteria: life's third domain or monoderm prokaryotes related to gram-positive bacteria? A new proposal for the classification of prokaryotic organisms; Gupta RS; The evolutionary relationship within prokaryotes is examined based on signature sequences (defined as conserved inserts or deletions shared by specific taxa) and phylogenies derived from different proteins . Archaebacteria are indicated as being monophyletic by a number of proteins related to the information transfer processes . In contrast, for several other highly conserved proteins, common signature sequences are present in archaebacteria and Gram-positive bacteria, whereas Gram-negative bacteria are indicated as being distinct . For these proteins, archaebacteria do not form a phylogenetically distinct clade but show polyphyletic branching within Gram-positive bacteria . A closer relationship of archaebacteria to Gram-positive bacteria in comparison with Gram-negative bacteria is generally seen for the majority of the available gene/ protein sequences . To account for these results and the fact that both archaebacteria and Gram-positive bacteria are prokaryotes surrounded by a single cell membrane, I propose that the primary division within prokaryotes is between monoderm prokaryotes (surrounded by a single membrane) and diderm prokaryotes (i.e . all true Gram-negative bacteria containing both an inner cytoplasmic membrane and an outer membrane) . This proposal is consistent with both cell morphology and signature sequences in different proteins . The monophyletic nature of archaebacteria for some genes, and their polyphyletic branching within Gram-positive bacteria as suggested by others, is critically examined, and several explanations, including derivation of archaebacteria from Gram-positive bacteria in response to antibiotic selection pressure, are proposed . Signature sequences in proteins also indicate that the low-G+C Gram-positive bacteria are phylogenetically distinct from the high-G+C Gram-positive group and that the diderm prokaryotes (i.e . Gram-negative bacteria) appear to have evolved from the latter group . Protein phylogenies and signature sequences also show that all eukaryotic cells have received significant gene contributions from both an archaebacterium and a Gram-negative eubacterium . Thus, the hypothesis that archaebacteria and eukaryotes shared a common ancestor exclusive of eubacteria is not supported . These observations provide evidence for an alternate view of the evolutionary relationship among living organisms that is different from the currently popular three-domain proposal.

J Bacteriol, 1998 Sep, 180(17), 4739 - 41
Salmonella typhimurium nit is nadE: defective nitrogen utilization and ammonia-dependent NAD synthetase; Schneider BL et al.; S . typhimurium nit mutants are defective in nitrogen assimilation, despite having normal levels of assimilatory enzymes . Complementation, enzyme assays, and genetic mapping show that nit is nadE . We present evidence that ammonia, not glutamine, is the physiological substrate for eubacterial NAD synthetases and that low activity completely accounts for the mutant phenotype.

Mol Biol Evol, 1998 Aug, 15(8), 1055 - 61
Determining the evolutionary potential of a gene; Hall BG et al.; In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions . The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose . Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base-substitutions . Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote . A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta-galactosidase over 2 billion years ago . Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade . We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years . The evolutionary potential of Ebg is thus limited to those two replacements.

Clin Infect Dis, 1998 Aug, 27 Suppl 1, S54 - 63
Bacterial topoisomerases, anti-topoisomerases, and anti-topoisomerase resistance; Hooper DC; Topoisomerases are ubiquitous enzymes necessary for controlling the interlinking and twisting of DNA molecules . Among the four topoisomerases identified in eubacteria, two, DNA gyrase and topoisomerase IV have been exploited by nature and the pharmaceutical industry as antibacterial targets . Natural products that are inhibitors of one or both of these topoisomerases include the coumarin and cyclothialidine classes, which interfere with adenosine triphosphate hydrolysis, cinodine, flavones, and terpenoid derivatives . The plasmid-encoded bacterial peptides micron B17 and CcdB also inhibit DNA gyrase . The quinolones, a synthetic class of antibacterials that act on both DNA gyrase and topoisomerase IV have had the broadest clinical applications, however . Quinolone congeners differ in their relative potencies for DNA gyrase and topoisomerase IV Studies of an expanding set of resistant mutant enzymes and the crystal structure of the homologous enzyme in yeast have contributed to our understanding of interactions of these drugs with topoisomerase-DNA complexes and the ways in which mutations effect resistance.

FEBS Lett, 1998 Jul 17, 431(2), 138 - 42
RNase P RNA from Prochlorococcus marinus: contribution of substrate domains to recognition by a cyanobacterial ribozyme; Hess WR et al.; The molecular organisation of the Prochlorococcus marinus rnpB gene and the catalytic activity of the encoded RNA were characterised . Kinetic parameters for several pre-tRNA substrates were comparable to those from other eubacterial RNase P RNAs, although unusually high cation concentrations were required . The CCA-end of pre-tRNAs is essential for efficient turnover despite the lack of the canonical binding motif in P . marinus RNase P RNA . A trnR gene is located only 38 nt upstream the rnpB 5' end on the complementary strand . This arrangement resembles those in the plastids of Cyanophora and Porphyra but not in any other bacterium.

Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 9879 - 84
A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis; Takahashi S et al.; Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway . In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate . To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E . coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival . All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E . coli . The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp . PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis . The purified recombinant yaeM gene product was overexpressed in E . coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH . Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate . The enzyme required Mn2+, Co2+, or Mg2+ as well . These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E . coli.

Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 9761 - 6
Identification of an UP element consensus sequence for bacterial promoters; Estrem ST et al.; The UP element, a component of bacterial promoters located upstream of the -35 hexamer, increases transcription by interacting with the RNA polymerase alpha-subunit . By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter . A set of 31 of these upstream sequences increased transcription from 136- to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: -59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn -38 . The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the alpha C-terminal domain was similar to that of previously characterized UP elements . The identification of the UP element consensus should facilitate a detailed understanding of the alpha-DNA interaction . Based on the evolutionary conservation of the residues in alpha responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.

Eur J Clin Microbiol Infect Dis, 1998 Apr, 17(4), 272 - 4
Detection of staphylococcal genes directly from cerebrospinal and peritoneal fluid samples using a multiplex polymerase chain reaction; Schmitz FJ et al.; A multiplex polymerase chain reaction (PCR) that can simultaneously detect eubacterial isolates and the methicillin-susceptibility of staphylococcal isolates from cerebrospinal and peritoneal fluid samples was compared to conventional microbiological methods . Using conventional methods, bacteria were isolated from 8% (29/350) of the cerebrospinal fluid samples and from 5% (3/60) of the peritoneal fluid samples . All isolates except two Staphylococcus epidermidis isolates were also detected using the multiplex PCR . Coagulase-negative staphylococci and Staphylococcus aureus were correctly identified using both methods . The multiplex PCR can rapidly and simultaneously detect eubacteria, and the methicillin susceptibility of staphylococci from samples containing > or = 10(2) cfu/ml of bacteria.

Mol Microbiol, 1998 Jul, 29(1), 275 - 83
The ATP-dependent Clp protease is essential for acclimation to UV-B and low temperature in the cyanobacterium Synechococcus; Porankiewicz J et al.; ClpP is the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts . Cyanobacterial ClpP protein is encoded by a multigene family, producing up to four distinct isozymes . We have examined the importance of the first ClpP protein (ClpP1) isolated from the cyanobacterium Synechococcus sp . PCC 7942 for acclimation to ecologically relevant UV-B and low-temperature regimens . When the growth light of 50 mumol photons m-2 s-1 was supplemented with 0.5 W m-2 UV-B for 8 h, the constitutive level of ClpP1 rose eightfold after an initial lag of 1 h . Wild-type cells readily acclimated to this UV-B level, recovering after the initial stress to almost the same growth rate as that before UV-B exposure . Growth of a clpP1 null mutant (delta clpP1), however, was severely inhibited by UV-B, being eight times slower than the wild type after 8 h . In comparison, ClpP1 content increased 15-fold in wild-type cultures shifted from 37 degree C to 25 degree C for 24 h . Wild-type cultures readily acclimated to 25 degree C after 24 h, whereas the delta clpP1 strain did not and eventually lost viability with prolonged cold treatment . During acclimation to either UV-B or cold, photosynthesis in the wild type was initially inhibited upon the shift but then recovered . Photosynthesis in delta clpP1 cultures, however, was more severely inhibited by the stress treatment and failed to recover . Acclimation was also monitored by examining the exchange of photosystem II reaction centre D1 proteins that occurs in wild-type Synechococcus during conditions of excitation stress . During both cold and UV-B shifts, wild-type cultures replaced the acclimative form of D1 (D1:1) with the alternative D1 form 2 (D1:2) within the first hours . Once acclimated to either 25 degree C or 0.5 W m-2 UV-B, D1:2 was exchanged back for D1:1 . In delta clpP1 cultures, this second exchange between D1 forms did not occur, with D1:2 remaining the predominant D1 form . Our results demonstrate that the ATP-dependent Clp protease is an essential component of the cold and UV-B acclimation processes of Synechococcus.

RNA, 1998 Aug, 4(8), 984 - 97
Effects of polyvalent cations on the folding of an rRNA three-way junction and binding of ribosomal protein S15; Batey RT et al.; The Bacillus stearothermophilus ribosomal protein S15 binds to a phylogenetically conserved three-way junction formed by the intersection of helices 20, 21, and 22 of eubacterial 16S ribosomal RNA, inducing a large conformational change in the RNA . Like many RNA structures, this three-way junction can also be folded by the addition of polyvalent cations such as magnesium, as demonstrated by comparing the mobilities of the wild-type and mutant junctions in the absence and presence of polyvalent cations in nondenaturing polyacrylamide gels . Using a modification interference assay, critical nucleotides for folding have been identified as the phylogenetically conserved nucleotides in the three-way junction . NMR spectroscopy of the junction reveals that the conformations induced by the addition of magnesium or S15 are extremely similar . Thus, the folding of the junction is determined entirely by RNA elements within the phylogenetically conserved junction core, and the role of Mg2+ and S15 is to stabilize this intrinsically unstable structure . The organization of the junction by Mg2+ significantly enhances the bimolecular association rate (k(on)) of S15 binding, suggesting that S15 binds specifically to the folded form of the three-way junction via a tertiary structure capture mechanism.

J Bacteriol, 1998 Aug, 180(16), 4160 - 5
The Escherichia coli citrate carrier CitT: a member of a novel eubacterial transporter family related to the 2-oxoglutarate/malate translocator from spinach chloroplasts; Pos KM et al.; Under anoxic conditions in the presence of an oxidizable cosubstrate such as glucose or glycerol, Escherichia coli converts citrate to acetate and succinate . Two enzymes are specifically required for the fermentation of the tricarboxylic acid, i.e., a citrate uptake system and citrate lyase . Here we report that the open reading frame (designated citT) located at 13.90 min on the E . coli chromosome between rna and the citrate lyase genes encodes a citrate carrier . E . coli transformed with a plasmid expressing citT was capable of aerobic growth on citrate, which provides convincing evidence for a function of CitT as a citrate carrier . Transport studies with cell suspensions of the transformed strain indicated that CitT catalyzes a homologous exchange of citrate or a heterologous exchange against succinate, fumarate, or tartrate . Since succinate is the end product of citrate fermentation in E . coli, it is likely that CitT functions in vivo as a citrate/succinate antiporter . Analysis of the primary sequence showed that CitT (487 amino acids, 53.1 kDa) is a highly hydrophobic protein with 12 putative transmembrane helices . Sequence comparisons revealed that CitT is related to the 2-oxoglutarate/malate translocator (SODiT1 gene product) from spinach chloroplasts and five bacterial gene products, none of which has yet been functionally characterized . It is suggested that the E . coli CitT protein is a member of a novel family of eubacterial transporters involved in the transport of di- and tricarboxylic acids.

Genes Dev, 1998 Aug 1, 12(15), 2318 - 31
Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases; Tjalsma H et al.; Approximately 47% of the genes of the Gram-positive bacterium Bacillus subtilis belong to paralogous gene families . The present studies were aimed at the functional analysis of the sip gene family of B . subtilis, consisting of five chromosomal genes, denoted sipS, sipT, sipU, sipV, and sipW . All five sip genes specify type I signal peptidases (SPases), which are actively involved in the processing of secretory preproteins . Interestingly, strains lacking as many as four of these SPases could be obtained . As shown with a temperature-sensitive SipS variant, only cells lacking both SipS and SipT were not viable, which may be caused by jamming of the secretion machinery with secretory preproteins . Thus, SipS and SipT are of major importance for protein secretion . This conclusion is underscored by the observation that only the transcription of the sipS and sipT genes is temporally controlled via the DegS-DegU regulatory system, in concert with the transcription of most genes for secretory preproteins . Notably, the newly identified SPase SipW is highly similar to SPases from archaea and the ER membrane of eukaryotes, suggesting that these enzymes form a subfamily of the type I SPases, which is conserved in the three domains of life.

J Mol Evol, 1998 Aug, 47(2), 190 - 9
Phylogenetic relationships of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, from parabasalid flagellates; Viscogliosi E et al.; Over 90% of the open reading frame of gap genes for glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was obtained with PCR from five species of Parabasala . With gap1 from Trichomonas vaginalis obtained earlier, the data include two sequences each for three species . All sequences were colinear with T . vaginalis gap1 and shared with it as a synapomorphy a 10- to 11-residue insertion not found in any other gap and an S-loop with characteristic features of eubacterial GAPDH . All residues known to be highly conserved in this enzyme were present . The parabasalid sequences formed a robust monophyletic group in phylogenetic reconstructions with distance-based, maximum-parsimony, and maximum-likelihood methods . The two genes of the amphibian commensal, Trichomitus batrachorum, shared a common ancestor with the rest, which separate into two well-supported lineages . T . vaginalis and Tetratrichomonas gallinarum (both representatives of Trichomonadinae) formed one, while Monocercomonas sp . and Tritrichomonas foetus formed the other . These data agreed with and/or were close to published reconstructions based on other macromolecules . They did not support the ancestral position of Monocercomonas sp . proposed on the basis of morphological characteristics but confirmed an early emergence of Trichomitus batrachorum . The sequence pairs obtained from three species indicated either gene duplications subsequent to the divergence of the corresponding lineages or a strong gene conversion later in these lineages . The parabasalid clade was a robust part of the eubacterial radiation of GAPDH and showed no relationships to the clade that contained all other eukaryotic gap genes . The data clearly reveal that the members of this lineage use in their glycolytic pathway a GAPDH species with properties and an evolutionary history that are unique among all eukaryotes studied so far.

Eur J Biochem, 1998 Jun 15, 254(3), 620 - 5
Biosynthesis of vitamin B12 in anaerobic bacteria--experiments with Eubacterium limosum on the transformation of 5-hydroxy-6-methyl-benzimidazole, its nucleoside, its cobamide, and of 5-hydroxybenzimidazolylcobamide in vitamin B12; Schulze B et al.; In anaerobic bacteria 5-hydroxybenzimidazole and 5-hydroxy-6-methylbenzimidazole are precursors of the 5,6-dimethylbenzimidazole moiety of vitamin B12 . In order to elucidate the pathway from these bases to vitamin B12, experiments on the transformation of 5-hydroxy-6-methylbenzimidazole, of 5-hydroxy-6-methylbenzimidazole-alpha-D-ribofuranoside, of 5-hydroxybenzimidazolylcobamide and of 5-hydroxy-6-methylbenzimidazolylcobamide into vitamin B12 were carried out . The vitamin B12 synthesized by the anaerobe Eubacterium limosum in the presence of 5-hydroxy-6-methylbenzimidazole and L-{methyl-13C}methionine was subjected to NMR spectroscopy . It revealed that the methyl group at C5 of the 5,6-dimethylbenzimidazole moiety was 13C labeled, whereas the methyl group at C6 was unlabeled . This shows that the transformation of 5-hydroxy-6-methylbenzimidazole into the base moiety of vitamin B12 occurs regiospecifically . 5-Hydroxy-6-methylbenzimidazole-alpha-D-ribofuranoside as well as 5-hydroxybenzimidazolylcobamide and 5-hydroxy-6-methylbenzimidazolylcobamide were also transformed into vitamin B12 by E . limosum . When 5-hydroxy-6-methylbenzimidazolylcobamide 13C labeled at C2 of the base part and 14C labeled in the ribose was used for this experiment, the vitamin B12 obtained from this cobamide was 13C and 14C labeled in the same positions . This demonstrates that the alpha-glycosidic bond of the precursor cobamide is not split during the formation of vitamin B12 . It can be deduced from these results that the precursor bases are transformed regiospecifically into their alpha-nucleotides, and partially into their cobamides . The alpha-nucleotides are then transformed into alpha-ribazole-5'-phosphate and, subsequently, into vitamin B12 . Most likely the cobamides are degraded to the alpha-nucleotides before being used for the biosynthesis of vitamin B12 . A pathway for the latter process is suggested.

Nucleic Acids Res, 1998 Aug 15, 26(16), 3753 - 61
The tRNA(guanine-26,N2-N2) methyltransferase (Trm1) from the hyperthermophilic archaeon Pyrococcus furiosus: cloning, sequencing of the gene and its expression in Escherichia coli; Constantinesco F et al.; The structural gene pfTRM1 (GenBank accession no . AF051912), encoding tRNA(guanine-26, N 2- N 2) methyltransferase (EC 2.1.1.32) of the strictly anaerobic hyperthermophilic archaeon Pyrococcus furiosus, has been identified by sequence similarity to the TRM1 gene of Saccharomyces cerevisiae (YDR120c) . The pfTRM1 gene in a 3.0 kb restriction DNA fragment of P.furiosus genomic DNA has been cloned by library screening using a PCR probe to the 5'-part of the corresponding ORF . Sequence analysis revealed an entire ORF of 1143 bp encoding a polypeptide of 381 residues (calculated molecular mass 43.3 kDa) . The deduced amino acid sequence of this newly identified gene shares significant similarity with the TRM1- like genes of three other archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus), one eukaryon (Caenorhabditis elegans) and one hyperthermophilic eubacterium (Aquifex aeolicus) . Two short consensus motifs for S-adenosyl-l-methionine binding are detected in the sequence of pfTrm1p . Cloning of the P.furiosus TRM1 gene in an Escherichia coli expression vector allowed expression of the recombinant protein (pfTrm1p) with an apparent molecular mass of 42 kDa . A protein extract from the transformed E.coli cells shows enzymatic activity for the quantitative formation of N 2, N 2-dimethylguanosine at position 26 in a transcript of yeast tRNAPhe used as substrate . The recombinant enzyme was also shown to modify bulk E.coli tRNAs in vivo.

Genes Funct, 1997 Apr, 1(2), 119 - 29
Physical mapping of a collection of Mael-generating amber mutations in the beta gene of Escherichia coli RNA polymerase and the functional effect of internal deletions constructed through their manipulation; Buyukuslu N et al.; We report the fine mapping of 55 of our 95 amber mutations in the beta gene of Escherichia coli RNA polymerase by virtue of the unique MaeI restriction sites created by this subset of nonsense mutations (i.e . CTAG, where the amber codon is underlined) . {The full data are reported in Supplementary Publication SUP 50181 (12 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem . J . (1997) 321, 8-10.} The CTAG mutations, which have been positioned to within approx . 9-60 bp, are distributed almost along the entire length of the rpoB gene, the one exception being the interval 400-499 . The lack of amber fragments for mutations within the 5' approx . 265 codons suggests lability of the extreme N-terminal region; further potential destabilizing 'signals' may be present in the non-conserved 'spacer' regions . The locations of four of the eleven rpoB amber mutations that are strongly polar on expression of the downstream rpoC gene have been determined through a combination of MaeI mapping, PCR amplification and DNA sequencing . Surprisingly, one such mutant carries two tandem CTAG sites but is viable with three of the nonsense suppressors tested . These polar amber sites define three different amino acids (Gln-31, Gln-83 and Trp-183) that fall within three sequence-conservation blocks in the N-terminal region . Six of the MaeI/Am (where MaeI/Am is an amber mutation generating a MaeI restriction site) rpoB alleles (Gln-83, Gln-276, Gln-327, Gln-618, Gln-649 and Trp-183) have been used to generate small in-frame deletions (31-100 codons) within conserved and non-conserved regions of the beta gene, and the properties of these deletion variants were assessed in vivo . The smallest deletion reported in this study removes 31 amino acids from the middle of a region common to the eubacterial/chloroplast subgroup of beta homologues, and our results strongly suggest beta(deltaQ618-Q649) is assembled into a holoenzyme form capable of transcriptional initiation in vivo.

Plant Mol Biol, 1998 Jul, 37(5), 791 - 801
Inactivation of the clpP1 gene for the proteolytic subunit of the ATP-dependent Clp protease in the cyanobacterium Synechococcus limits growth and light acclimation; Clarke AK et al.; ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts . We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp . PCC 7942 . The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp . PCC 6803 . The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 micromol photons m(-2) s(-1) . A clpP1 inactivation strain (delta clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin . Many mutant cells (ca . 35%) were also severely elongated, up to 20 times longer than the wild type . The stress phenotype of delta clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2 . ClpP1 content also rose significantly during short-term photoinhibition, but its loss in delta clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.

Infect Immun, 1998 Aug, 66(8), 3727 - 35
Phospholipid composition of purified Chlamydia trachomatis mimics that of the eucaryotic host cell; Hatch GM et al.; Chlamydia trachomatis is an obligate intracellular eubacterial parasite capable of infecting a wide range of eucaryotic host cells . Purified chlamydiae contain several lipids typically found in eucaryotes, and it has been established that eucaryotic lipids are transported from the host cell to the parasite . In this report, we examine the phospholipid composition of C . trachomatis purified from host cells grown under a variety of conditions in which the cellular phospholipid composition was altered . A mutant CHO cell line, with a thermolabile CDP-choline synthetase, was used to show that decreased host cell phosphatidylcholine levels had no significant effect on C . trachomatis growth . However, less phosphatidylcholine was transported to the parasite and purified elementary bodies contained decreased levels of phosphatidylcholine . Brefeldin A, fumonisin B1, and exogenous sphingomyelinase were used to alter levels of host cell sphingomyelin . None of the agents had a significant effect on C . trachomatis replication . Treatment with fumonisin B1 and exogenous sphingomyelinase resulted in decreased levels of host cell sphingomyelin . This had no effect on glycerophospholipid trafficking to chlamydiae; however, sphingomyelin trafficking was reduced and elementary bodies purified from treated cells had reduced sphingomyelin content . Exposure to brefeldin A, which had no adverse effect on chlamydia growth, resulted in an increase in cellular levels of sphingomyelin and a concomitant increase in the amount of sphingomyelin in purified chlamydiae . Under the experimental conditions used, brefeldin A treatment had only a small effect on sphingomyelin trafficking to the host cell surface or to C . trachomatis . Thus, the final phospholipid composition of purified C . trachomatis mimics that of the host cell in which it is grown.

Gene, 1998 Jul 17, 215(1), 171 - 80
amlC, another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24; Yin XH et al.; The region located upstream of the alpha-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC . amlC Encodes a 993amino acid (aa) protein with a calculated molecular weight of 107.054kDa . On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1, 4-alpha-D-glucan glucanohydrolase family . amlC is transcribed as a unique 3kb leaderless monocistronic mRNA . Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences . amlC was successfully disrupted and was mapped at approx . 700kb from a chromosomal end of S . lividans TK24, 100kb on the right of the amplifiable unit AUD1 (Volff et al., 1996) . Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI-II fragment of the chromosome.

Gene, 1998 Jul 17, 215(1), 153 - 7
Localization and genomic structure of human deoxyhypusine synthase gene on chromosome 19p13.2-distal 19p13.1; Mantuano E et al.; The amino acid hypusine is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A, by two enzymes, namely deoxyhypusine synthase and deoxyhypusine hydroxylase . Hypusine is found in all eukaryotes and in some archaebacteria, but not in eubacteria . The deoxyhypusine synthase cDNA was cloned and mapped by fluorescence in situ hybridization on chromosome 19p13.11-p13.12 . Rare cDNAs containing internal deletions were also found . We localized the deoxyhypusine synthase gene on a high resolution cosmid/BAC contig map of chromosome 19 to a region in 19p13.2-distal 19p13.1 between MANB and JUNB . Analysis of the genomic exon/intron structure of the gene coding region showed that it consists of nine exons and spans a length of 6.6kb . From observation of the genomic structure, it seems likely that the internally deleted forms of mature RNA are the result of alternative splicing, rather than of artifacts.

Nat Struct Biol, 1998 Jul, 5(7), 546 - 50
Site-specific DNA binding using a variation of the double stranded RNA binding motif; Connolly KM et al.; The integrase family of site-specific recombinases catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria and yeast . The solution structure of the DNA binding domain of the integrase protein from the conjugative transposon Tn916 has been determined using NMR spectroscopy . The structure provides the first insights into distal site DNA binding by a site-specific integrase and reveals that the N-terminal domain is structurally similar to the double stranded RNA binding domain (dsRBD) . The results of chemical shift mapping experiments suggest that the integrase protein interacts with DNA using residues located on the face of its three stranded beta-sheet . This surface differs from the proposed RNA binding surface in dsRBDs, suggesting that different surfaces on the same protein fold can be used to bind DNA and RNA.

J Biol Chem, 1998 Jul 17, 273(29), 18099 - 108
Biosynthesis of the diterpene verrucosan-2beta-ol in the phototrophic eubacterium Chloroflexus aurantiacus . A retrobiosynthetic NMR study; Rieder C et al.; The biosynthesis of verrucosan-2beta-ol in the green phototrophic eubacterium Chloroflexus aurantiacus was investigated by in vivo incorporation of singly or doubly 13C-labeled acetate . The 13C labeling of the isolated diterpene was analyzed by one- and two-dimensional NMR spectroscopy . The 13C-labeling patterns of verrucosan-2beta-ol were compared with the labeling patterns of intermediary metabolites (acetyl-CoA, pyruvate, and glyceraldehyde 3-phosphate) which were deduced from amino acids and nucleosides by retrobiosynthetic analysis . The results show that verrucosan-2beta-ol is synthesized via mevalonate and not via the deoxyxylulose pathway, which was discovered recently in some eubacteria, algae, and plants . A scheme for the formation of the unusual tetracyclic ring system is offered . The cyclization process is initiated by the solvolysis of pyrophosphate from geranyllinaloyl pyrophosphate and the mechanism involves a Wagner-Meerwein rearrangement, a 1,5-hydride shift, and a cyclopropylcarbinyl to cyclopropylcarbinyl rearrangement.

Gene, 1998 Jul 3, 214(1-2), 205 - 13
Eubacterial origin of nuclear genes for chloroplast and cytosolic glucose-6-phosphate isomerase from spinach: sampling eubacterial gene diversity in eukaryotic chromosomes through symbiosis; Nowitzki U et al.; Higher plants possess two distinct nuclear-encoded glucose-6-phosphate isomerase (GPI) isoenzymes, a cytosolic enzmye of the Embden-Meyerhof pathway and a chloroplast enzyme essential to storage and mobilization of carbohydrate fixed by the Calvin cycle . We have purified spinach chloroplast GPI to homogeneity, determined amino acid sequences from the active enzyme, and cloned cDNAs for chloroplast and cytosolic GPI isoenzymes from spinach . Sequence comparisons reveal three distantly related families of GPI genes that are non-uniformly distributed among contemporary eubacteria and archaebacteria, suggesting that ancient gene diversity existed for this glycolytic enzyme . Spinach chloroplast GPI is much more similar to its homologue from the cyanobacterium Synechocystis PCC6803 than it is to the enzyme from any other source, providing strong evidence that the gene for chloroplast GPI was acquired by the nucleus via endosymbiotic gene transfer from the cyanobacterial antecedants of chloroplasts . Eukaryotic nuclear genes for cytosolic GPI are more similar to eubacterial than to archaebacterial homologues, suggesting that these too were acquired by eukaryotes from eubacteria, probably during the course of the endosymbiotic origin of mitochondria . Chloroplast and cytosolic GPI provide evidence for a eubacterial origin of yet another component of the eukaryotic glycolytic pathway.

EMBO J, 1998 Jun 1, 17(11), 3197 - 206
CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?
Shi PY, Maizels N, Weiner AM.
The CCA-adding enzyme repairs the 3'-terminal CCA sequence of all tRNAs . To determine how the enzyme recognizes tRNA, we probed critical contacts between tRNA substrates and the archaeal Sulfolobus shibatae class I and the eubacterial Escherichia coli class II CCA-adding enzymes . Both CTP addition to tRNA-C and ATP addition to tRNA-CC were dramatically inhibited by alkylation of the same tRNA phosphates in the acceptor stem and TPsiC stem-loop . Both enzymes also protected the same tRNA phosphates in tRNA-C and tRNA-CC . Thus the tRNA substrate must remain fixed on the enzyme surface during CA addition . Indeed, tRNA-C cross-linked to the S . shibatae enzyme remains fully active for addition of CTP and ATP . We propose that the growing 3'-terminus of the tRNA progressively refolds to allow the solitary active site to reuse a single CTP binding site . The ATP binding site would then be created collaboratively by the refolded CC terminus and the enzyme, and nucleotide addition would cease when the nucleotide binding pocket is full . The template for CCA addition would be a dynamic ribonucleoprotein structure.

Front Biosci, 1998 Jun 17, 3, D570 - 603
DNA strand exchange proteins: a biochemical and physical comparison; Bianco PR et al.; Homologous genetic recombination is an essential biological process that involves the pairing and exchange of DNA between two homologous chromosomes or DNA molecules . It is of fundamental importance to the preservation of genomic integrity, the production of genetic diversity, and the proper segregation of chromosomes . In Escherichia coli, the RecA protein is essential to recombination, and biochemical analysis demonstrates that it is responsible for the crucial steps of homologous pairing and DNA strand exchange . The presence of RecA-like proteins, or their functional equivalents, in bacteriophage, other eubacteria, archaea, and eukaryotes, confirms that the mechanism of homologous pairing and DNA strand exchange is conserved throughout all forms of life . This review focuses on the biochemical and physical characteristics of DNA strand exchange proteins from three diverse organisms: RecA protein from E . coli, UvsX protein from Bacteriophage T4, and RAD51 protein from Saccharomyces cerevisiae.

FEMS Microbiol Lett, 1998 Jun 1, 163(1), 11 - 7
Constitutive expression of a heterologous Eubacterium ruminantium xylanase gene (xynA) in Butyrivibrio fibrisolvens; Kobayashi Y et al.; An Eubacterium ruminantium xylanase gene (xynA) was inserted into pYK4, a shuttle vector replicable in both Escherichia coli and Butyrivibrio fibrisolvens, and the resultant chimeric plasmid (pYK4XT) was electroporated into B . fibrisolvens OB156C in an attempt to obtain a more xylanolytic B . fibrisolvens . Electrotransformants were screened by the development of erythromycin resistance, followed by an activity staining and Southern hybridization . The presence of mRNA from xynA in the transformant, B . fibrisolvens NO4, was confirmed by Northern hybridization . Xylanase activity of the transformant NO4 was apparently enhanced regardless of carbon sources in the medium . When grown on glucose or cellobiose . NO4 had approximately 5-6 times higher intracellular activity than the parent OB156C on a culture volume basis as well as protein basis . The transformant showed extracellular xylanase activity much higher (between 7- and 10(4)-fold) than the parent . Transformant NO4 recorded the highest activity when grown on xylan . Most (> 90%) of the activity was extracellular . The extracellular activity was 2-fold greater in NO4 . These findings indicate that the introduced xynA was expressed constitutively and the xylanase protein was exported into the culture supernatant . Growth of NO4 on glucose was similar to that of OB156C, which suggests little extra load for plasmid maintenance and foreign xylanase production in the transformant . The plasmid pYK4XT was maintained stably in the transformant for more than 100 generations.

J Mol Evol, 1998 Jun, 46(6), 716 - 20
Signature sequences in diverse proteins provide evidence of a close evolutionary relationship between the Deinococcus-thermus group and cyanobacteria; Gupta RS et al.; A number of proteins have been identified that contain prominent sequence signatures that are uniquely shared by the members of the Deinococcus-Thermus genera and the cyanobacterial species but which are not found in any of the other eubacterial or archaebacterial homologs . The proteins containing such sequence signatures include (1) the DnaJ/Hsp40 family of proteins, (2) DNA polymerase I, (3) the protein synthesis elongation factor EF-Tu, and (4) the elongation factor EF-Ts . A strong affinity of the Deinococcus-Thermus species to cyanobacteria is also seen in the phylogenetic trees based on Hsp70 and DnaJ sequences . These results provide strong evidence of a close and specific evolutionary relationship between species belonging to these two eubacterial divisions.

Genes Dev, 1998 Jun 1, 12(11), 1678 - 90
Purification and characterization of the nuclear RNase P holoenzyme complex reveals extensive subunit overlap with RNase MRP; Chamberlain JR et al.; Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that cleaves precursor tRNA transcripts to give mature 5' ends . RNase P in eubacteria has a large, catalytic RNA subunit and a small protein subunit that are required for precursor tRNA cleavage in vivo . Although the eukaryotic holoenzymes have similar, large RNA subunits, previous work in a number of systems has suggested that the eukaryotic enzymes require a greater protein content . We have purified the Saccharomyces cerevisiae nuclear RNase P to apparent homogeneity, allowing the first comprehensive analysis of an unexpectedly complex subunit composition . Peptide sequencing by ion trap mass spectrometry identifies nine proteins that copurify with the nuclear RNase P RNA subunit, totaling 20-fold more protein than in the bacterial enzyme . All of these proteins are encoded by genes essential for RNase P activity and for cell viability . Previous genetic studies suggested that four proteins might be subunits of both RNase P and RNase MRP, the related rRNA processing enzyme . We demonstrate that all four of these proteins, Pop1p, Pop3p, Pop4p, and Rpp1p, are integral subunits of RNase P . In addition, four of the five newly identified protein subunits, Pop5p, Pop6p, Pop7p, and Pop8p, also appear to be shared between RNase P and RNase MRP . Only one polypeptide, Rpr2p, is unique to the RNase P holoenzyme by genetic depletion and immunoprecipitation studies . The large increase in the number of protein subunits over eubacterial RNase P is consistent with an increase in functional complexity in eukaryotes . The degree of structural similarity between nuclear RNase P and RNase MRP suggests that some aspects of their functions in pre-tRNA and pre-rRNA processing pathways might overlap or be coordinated.

J Biol Chem, 1998 May 8, 273(19), 11413 - 6
Structure of peptide deformylase and identification of the substrate binding site; Becker A et al.; Peptide deformylase is an essential metalloenzyme required for the removal of the formyl group at the N terminus of nascent polypeptide chains in eubacteria . The Escherichia coli enzyme uses Fe2+ and nearly retains its activity on substitution of the metal ion by Ni2+ . We have solved the structure of the Ni2+ enzyme at 1.9-A resolution by x-ray crystallography . Each of the three monomers in the asymmetric unit contains one Ni2+ ion and, in close proximity, one molecule of polyethylene glycol . Polyethylene glycol is shown to be a competitive inhibitor with a KI value of 6 mM with respect to formylmethionine under conditions similar to those used for crystallization . We have also solved the structure of the inhibitor-free enzyme at 2.5-A resolution . The two structures are identical within the estimated errors of the models . The hydrogen bond network stabilizing the active site involves nearly all conserved amino acid residues and well defined water molecules, one of which ligates to the tetrahedrally coordinated Ni2+ ion.

Mol Microbiol, 1998 Apr, 28(2), 235 - 47
Control of cell shape and elongation by the rodA gene in Bacillus subtilis; Henriques AO et al.; The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively . While rodA and ftsW are essential genes in E . coli, the B . subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan . In this work, we report on the characterization of a B . subtilis gene, designated rodA, encoding a homologue of E . coli RodA . We found that the growth of a B . subtilis strain carrying a fusion of rodA to the IPTG-inducible Pspac promoter is inducer dependent . Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed . An increase in the level of IPTG induced a sphere-to-short rod transition that re-established viability . Higher levels of inducer restored normal cell length . Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions . Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability . In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell . Electron microscopy observations support these conclusions . The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cell's inability to construct a division septum capable of spanning the enlarged cell . RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation) . Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.

Genetics, 1998 Jun, 149(2), 1051 - 61
quemao, a Drosophila bristle locus, encodes geranylgeranyl pyrophosphate synthase; Lai C et al.; The quemao (qm) locus of Drosophila melanogaster is characterized by a P-element-associated mutant lacking most of the large bristles on the thorax and by several EMS-induced recessive lethals . quemao was cloned using a transposon tagging strategy . P-element-mediated transformation demonstrated that the cloned qm DNA sequence (from the 65F cytological region) rescues the mutant phenotype . A 2.3-kb qm transcript was identified by Northern blot analysis by sequencing of the isolated qm cDNA clones and by 5' rapid amplification cDNA end (RACE) . The predicted amino acid sequence (338 residues) of the coding region of the qm transcript shares 42, 31, 13, 20, and 12% identical amino acid sequences with the geranylgeranyl pyrophosphate synthase (GGPPS) of fungi, yeast, plants, archaebacteria, and eubacteria, respectively . It also contains five highly conserved domains common among all known isoprenyl pyrophosphate synthases . The P element associated with the original qm mutant is inserted in the 5' untranslated region of the transcript . An EMS-induced qm nonsense mutation at the 12th codon leads to recessive lethality at the first larval instar, indicating the essential role of qm in the isoprenoid biosynthesis of insects.

J Bacteriol, 1998 Jun, 180(11), 2854 - 61
Molecular characterization of the gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae; Balas D et al.; The gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae was cloned and sequenced . The gyrA gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria . Translation of the gene in an Escherichia coli expression system revealed a 92-kDa polypeptide . A sequence-directed DNA curvature was identified in the promoter region of gyrA . The bend center was mapped and located between the -35 and -10 regions of the promoter . Primer extension analysis showed that gyrA transcription initiates 6 bp downstream of an extended -10 promoter . The possible implications of the bent DNA region as a regulatory element in the transcription of gyrA are discussed.

J Mol Evol, 1998 May, 46(5), 499 - 507
A new aspect to the origin and evolution of eukaryotes; Vellai T et al.; One of the most important omissions in recent evolutionary theory concerns how eukaryotes could emerge and evolve . According to the currently accepted views, the first eukaryotic cell possessed a nucleus, an endomembrane system, and a cytoskeleton but had an inefficient prokaryotic-like metabolism . In contrast, one of the most ancient eukaryotes, the metamonada Giardia lamblia, was found to have formerly possessed mitochondria . In sharp contrast with the traditional views, this paper suggests, based on the energetic aspect of genome organization, that the emergence of eukaryotes was promoted by the establishment of an efficient energy-converting organelle, such as the mitochondrion . Mitochondria were acquired by the endosymbiosis of ancient alpha-purple photosynthetic Gram-negative eubacteria that reorganized the prokaryotic metabolism of the archaebacterial-like ancestral host cells . The presence of an ATP pool in the cytoplasm provided by this cell organelle allowed a major increase in genome size . This evolutionary change, the remarkable increase both in genome size and complexity, explains the origin of the eukaryotic cell itself . The loss of cell wall and the appearance of multicellularity can also be explained by the acquisition of mitochondria . All bacteria use chemiosmotic mechanisms to harness energy; therefore the periplasm bounded by the cell wall is an essential part of prokaryotic cells . Following the establishment of mitochondria, the original plasma membrane-bound metabolism of prokaryotes, as well as the funcion of the periplasm providing a compartment for the formation of different ion gradients, has been transferred into the inner mitochondrial membrane and intermembrane space . After the loss of the essential function of periplasm, the bacterial cell wall could also be lost, which enabled the naked cells to establish direct connections among themselves . The relatively late emergence of mitochondria may be the reason why multicellularity evolved so slowly.

Mol Gen Genet, 1998 Apr, 258(1-2), 166 - 73
Mutagenesis of the genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone-oxidoreductase in tobacco by polyethylene glycol-mediated plastome transformation; Kofer W et al.; Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the metabolic function of the enzyme is unknown . The enzyme consists of at least eleven subunits (A-K), which are all encoded on the plastid chromosome . We have mutagenized ndhC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertion, of a cassette which carried a spectinomycin resistance gene as a marker . The transformation was carried out by the polyethylene glycol-mediated plastid transformation method . Southern analysis revealed that even after repeated regeneration cycles each of the four different types of transformants had retained 1-5% of wild-type gene copies . This suggests that complete deletion of ndh genes is not compatible with viability . The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark after illumination with actinic light for 5 min; in the wild-type this dark-rise reflects a transient reduction of the plastoquinone pool by reduction equivalents generated in the stroma; and (ii) transformants with defects in the ndhC-K-J operon accumulate starch, indicating inefficient oxidation of glucose via glycolysis and the oxidative pentose phosphate pathway . Both observations support the theory of chlororespiration, which postulates that the NDH complex acts as a valve to remove excess reduction equivalents in the chloroplast.

Biochemistry (Mosc), 1998 Feb, 63(2), 139 - 48
A new alternative non-mevalonate pathway for isoprenoid biosynthesis in eubacteria and plants; Paseshnichenko VA; Data concerning the discovery of an alternative non-mevalonate pathway for isoprenoid biosynthesis leading to isopentenyl diphosphate formation are reviewed . This pathway has been discovered in experiments with several eubacteria producing triterpenoids of the hopane series . 13C-labeled acetate, glucose, and triose phosphates were used as precursors . The 13C-labeling patterns in isoprenoids were studied by 13C-NMR spectrometry . In eubacteria the universal C5 precursor--isopentenyl diphosphate--did not appear to form via the classical acetate/mevalonate pathway, but via a novel glyceraldehyde 3-phosphate/pyruvate pathway . It is postulated that the condensation of the C2 unit formed as a result of pyruvate decarboxylation with the C3 unit (glyceraldehyde 3-phosphate) and the next transposition leads to the formation of the branched C5 precursor--isopentenyl diphosphate . In Scenedesmus obliquus not only all plastid isoprenoids (carotenoids and prenyl side chains of chlorophylls and plastoquinone-9) were formed via this novel pathway, but also the non-plastid cytoplasmic sterols . In higher plants the plastid isoprenoids were formed via the glyceraldehyde 3-phosphate/pyruvate pathway, while the cytoplasmic sterols were formed via the acetate/mevalonate pathway.

J Mol Biol, 1998 Apr 3, 277(3), 573 - 92
Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities; Paulsen IT et al.; We have conducted genome sequence analyses of seven prokaryotic microorganisms for which completely sequenced genomes are available (Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Mycoplasma genitalium, Synechocystis PCC6803 and Methanococcus jannaschii) . We report the distribution of encoded known and putative polytopic cytoplasmic membrane transport proteins within these genomes . Transport systems for each organism were classified according to (1) putative membrane topology, (2) protein family, (3) bioenergetics, and (4) substrate specificities . The overall transport capabilities of each organism were thereby estimated . Probable function was assigned to greater than 90% of the putative transport proteins identified . The results show the following: (1) Numbers of transport systems in eubacteria are approximately proportional to genome size and correspond to 9.7 to 10.8% of the total encoded genes except for H . pylori (5.4%), Synechocystis (4.7%) and M . jannaschii (3.5%) which exhibit substantially lower proportions . (2) The distribution of topological types is similar in all seven organisms . (3) Transport systems belonging to 67 families were identified within the genomes of these organisms, and about half of these families are also found in eukaryotes . (4) 12% of these families are found exclusively in Gram-negative bacteria, but none is found exclusively in Gram-positive bacteria, cyanobacteria or archaea . (5) Two superfamilies, the ATP-binding cassette (ABC) and major facilitator (MF) superfamilies account for nearly 50% of all transporters in each organism, but the relative representation of these two transporter types varies over a tenfold range, depending on the organism . (6) Secondary, pmf-dependent carriers are 1.5 to threefold more prevalent than primary ATP-dependent carriers in E . coli, H . influenzae, H . pylori and B . subtilis while primary carriers are about twofold more prevalent in M . genitalium and Synechocystis . M . jannaschii exhibits a slight preference for secondary carriers . (7) Bioenergetics of transport generally correlate with the primary forms of energy generated via available metabolic pathways but ecological niche and substrate availability may also be determining factors . (8) All organisms display a similar range of transport specificities with quantitative differences presumably reflective of disparate ecological niches . (9) M . jannaschii and Synechocystis have a two to threefold increased proportion of transporters for inorganic ions with a concomitant decrease in transporters for organic compounds . (10) 6 to 18% of all transporters in these bacteria probably function as drug export systems showing that these systems are prevalent in non-pathogenic as well as pathogenic organisms . (11) All seven prokaryotes examined encode proteins homologous to known channel proteins, but none of the channel types identified occurs in all of these organisms . (12) The phosphoenolpyruvate:sugar phosphotransferase system is prevalent in the large genome organisms, E . coli and B . subtilis, and is present in the small genome organisms, H . influenzae and M . genitalium, but is totally lacking in H . pylori, Synechocystis and M . jannaschii . Details of the information summarized in this article are available on our web sites, and this information will be periodically updated and corrected as new sequence and biochemical data become available .

J Cell Biol, 1998 Apr 20, 141(2), 385 - 95
A SecY homologue is required for the elaboration of the chloroplast thylakoid membrane and for normal chloroplast gene expression; Roy LM et al.; Results of in vitro and genetic studies have provided evidence for four pathways by which proteins are targeted to the chloroplast thylakoid membrane . Although these pathways are initially engaged by distinct substrates and involve some distinct components, an unresolved issue has been whether multiple pathways converge on a common translocation pore in the membrane . A homologue of eubacterial SecY called cpSecY is localized to the thylakoid membrane . Since SecY is a component of a protein-translocating pore in bacteria, cpSecY likely plays an analogous role . To explore the role of cpSecY, we obtained maize mutants with transposon insertions in the corresponding gene . Null cpSecY mutants exhibit a severe loss of thylakoid membrane, differing in this regard from mutants lacking cpSecA . Therefore, cpSecY function is not limited to a translocation step downstream of cpSecA . The phenotype of cpSecY mutants is also much more pleiotropic than that of double mutants in which both the cpSecA- and DeltapH-dependent thylakoid-targeting pathways are disrupted . Therefore, cpSecY function is likely to extend beyond any role it might play in these targeting pathways . CpSecY mutants also exhibit a defect in chloroplast translation, revealing a link between chloroplast membrane biogenesis and chloroplast gene expression.

FEMS Microbiol Lett, 1998 May 1, 162(1), 53 - 60
The phylogenetic relationships of Chlorobium tepidum and Chloroflexus aurantiacus based upon their RecA sequences; Gruber TM et al.; Using RecA as the phylogenetic marker, the relationships of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus to other eubacteria were investigated . The recA genes of the two organisms were cloned, and the resulting protein sequences aligned with 86 other eubacterial RecA sequences . Cb . tepidum was placed as the nearest relative to the Cytophaga/Flexibacter/Bacteriodes group, a relationship supported by results obtained with several phylogenetic markers . Cf . aurantiacus was placed near Chlamydia trachomatis and the high-GC Gram-positives; however, this branching pattern was not strongly supported statistically by bootstrap analyses . Possible reasons for this ambiguity are discussed.

Mol Microbiol, 1998 Apr, 28(1), 179 - 92
The redox- and fixed nitrogen-responsive regulatory protein NIFL from Azotobacter vinelandii comprises discrete flavin and nucleotide-binding domains; Soderback E et al.; Azotobacter vinelandii NIFL is a nitrogen fixation-specific regulatory flavoprotein that modulates the activity of the transcriptional activator NIFA in response to oxygen and fixed nitrogen in vivo . NIFL is also responsive to ADP in vitro . Limited proteolysis of NIFL indicates that it comprises a relatively stable N-terminal domain and a C-terminal domain that is protected from trypsin digestion in the presence of adenosine nucleotides . ATP protects the protein from cleavage in the vicinity of potential nucleotide-binding sites in the C-terminus, whereas ADP protects the entire C-terminal domain . NIFL has an apparent Kd of 130 microM for ATP and 16 microM for ADP . The purified N-terminal domain has an identical UV/visible absorption spectrum to the wild-type protein and is reduced by sodium dithionite, demonstrating that it is a flavin-binding domain . The isolated N-terminal domain does not inhibit NIFA activity . A subdomain fragment containing 160 residues of the C-terminal region, including the nucleotide-binding sites, is also not competent to inhibit NIFA . Removal of the first 146 residues of NIFL, which includes a conserved S-motif (PAS-like domain), found in a large family of sensory proteins from eubacteria, archea and eukarya eliminates the redox response . However, this truncated protein remains competent to inhibit NIFA activity in response to ADP in vitro and to the level of fixed nitrogen in vivo . The redox and nitrogen-sensing functions of A . vinelandii NIFL are therefore separable and are discrete functions of the protein.

Mol Microbiol, 1998 Apr, 28(1), 55 - 67
In vivo studies on the positive control function of NifA: a conserved hydrophobic amino acid patch at the central domain involved in transcriptional activation; Gonzalez V et al.; The eubacterial enhancer-binding proteins activate transcription by binding to distant sites and, simultaneously, contacting the RNA polymerase r54 promoter complex (Esigma54) . The positive control function is located at the central domain of these proteins, but it is not know which specific region has the determinants for the interaction with Esigma54 . Here, we present genetic evidence that a small region of hydrophobic amino acids, previously denominated C3, at the central domain of Bradyrhizobium japonicum NifA is involved in positive control . We obtained 26 missense mutants along this conserved region . Among these, only strains expressing the NifA(F307-->Y) and NifA(A310-->S) mutant proteins retained some of the transcriptional activity (<20%), whereas those carrying NifA(E298-->D) and NifA(T308-->S) had very low but detectable activity (< 1.0%) . The rest of the NifA mutants did not induce any measurable transcriptional activity . When expressed in the presence of wild-type NifA, the great majority of the mutants displayed a dominant phenotype, suggesting that their oligomerization determinants were not altered . In vivo dimethyl-sulphate footprinting experiments for a subset of the NifA mutants showed that they were still able to bind specifically to DNA . Analysis of intragenic supressors highlight the functional role of a hydroxyl group at position 308 to activate transcription.

Zhonghua Kou Qiang Yi Xue Za Zhi, 1996 Sep, 31(5), 303 - 6
{The effect of tinidazole in the treatment of adult periodontitis}; Wang R et al.; Fifty-two adult periodontitis patients were treated by the tablets of tinidazole (TNZ), and 23 patients treated by metronidazole (MNZ) served as control group . The effective rate of TNZ in adult periodontitis patients was 73.1%, which was significantly higher than that of the control group (43.5%) . Antimicrobial studies showed that the capability of TNZ to kill the periodontal dominant anaerobic bacteria, especially B . gingivalis and B . melaninogenicus, was better than that of MNZ . The minimal inhibitory concentration (MIC) of TNZ to Bacteroides, Fusobacterium, Veillonella, Eubacterium, Antinomyces, Peotostreptococus and Anaerobic Streptococus was lower than that of MNZ and actylspiramycine.

Nat Struct Biol, 1998 May, 5(5), 393 - 9
The NMR structure of the RNA binding domain of E . coli rho factor suggests possible RNA-protein interactions; Briercheck DM et al.; Rho protein is an essential hexameric RNA-DNA helicase that binds nascent mRNA transcripts and terminates transcription in a wide variety of eubacterial species . The NMR solution structure of the RNA binding domain of rho, rho130, is presented . This structure consists of two sub-domains, an N-terminal three-helix bundle and a C-terminal beta-barrel that is structurally similar to the oligosaccharide/oligonucleotide binding (OB) fold . Chemical shift changes of rho130 upon RNA binding and previous mutagenetic analyses of intact rho suggest that residues Asp 60, Phe 62, Phe 64, and Arg 66 are critical for binding and support the hypothesis that ssRNA/ssDNA binding is localized in the beta-barrel sub-domain . On the basis of these studies and the tertiary structure of rho130, we propose that residues Asp 60, Phe 62, Phe 64, Arg 66, Tyr 80, Lys 105, and Arg 109 participate in RNA-protein interactions.

Nat Struct Biol, 1998 May, 5(5), 352 - 6
Crystal structure of the RNA-binding domain from transcription termination factor rho; Allison TJ et al.; Transcription termination factor rho is an ATP-dependent hexameric helicase found in most eubacterial species . The Escherichia coli rho monomer consists of two domains, an RNA-binding domain (residues 1-130) and an ATPase domain (residues 131-419) . The ATPase domain is homologous to the beta subunit of F1-ATPase . Here, we report that the crystal structure of the RNA-binding domain of rho (rho130) at 1.55 A confirms that rho130 contains the oligosaccharide/oligonucleotide-binding (OB) fold, a five stranded beta-barrel . The beta-barrel of rho130 is also surprisingly similar to the N-terminal beta-barrel of F1 ATPase, extending the applicability of F1 ATPase as a structural model for hexameric rho.

Biochem Mol Biol Int, 1998 Apr, 44(4), 665 - 72
The effect of ribosome-inactivating proteins on the ribosome from the hyperthermophilic archaeon Sulfolobus solfataricus; Raimo G et al.; Protein synthesis in the thermoacidophilic archaeon Sulfolobus solfataricus (Ss) was inhibited by polynucleotide:adenosine glycosylase activity of some type 1 ribosome-inactivating proteins (RIP) . The target of RIP was S . solfataricus rRNA that was depurinated thus producing inactive ribosomes . The amount of RIP required to half-inactivated Ss-ribosomes was comparable to that needed for eubacterial ribosomes, but two orders of magnitude higher than that required for mammalian ribosomes . In addition, RIP treated Ss-ribosomes were also less efficient in stimulating the ribosome dependent GTPase activity of the S . solfataricus elongation factor 2 (SsEF-2) thus suggesting that the inhibition of protein synthesis was probably due to the lack of the interaction between depurinated Ss-ribosomes and SsEF-2 . Since SsEF-2 protects Ss-ribosomes against RIP activity it can be hypothesised that also on Ss-ribosomes the sites of interaction for the translocation factor 2 and the RIP are topographically close.

Mol Biol Evol, 1998 May, 15(5), 528 - 37
The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis . Structural and phylogenetic relationships within family-16 glycoside hydrolases; Barbeyron T et al.; We report here cloning from the marine gliding bacterium Cytophaga drobachiensis of kappa-carrageenase, a glycoside hydrolase involved in the degradation of kappa-carrageenan . Structural features in the nucleotide sequence are pointed out, including the presence of an octameric omega sequence similar to the ribosome-binding sites of various eukaryotes and prokaryotes . The cgkA gene codes for a protein of 545 aa, with a signal peptide of 35 aa and a 229-aa-long posttranslationaly processed C-terminal domain . The enzyme displays the overall folding and catalytic domain characteristics of family 16 of glycoside hydrolases, which comprises other beta-1,4-alpha-1,3-D/L-galactan hydrolases, beta-1,3-D-glucan hydrolases (laminarinases), beta-1,4-1,3-D-glucan hydrolases (lichenases), and beta-1,4-D-xyloglucan endotransglycosylases . In order to address the origin and evolution of CgkA, a comprehensive phylogenetic tree of family 16 was built using parsimony analysis . Family-16 glycoside hydrolases cluster according to their substrate specificity, regardless of their phylogenetic distribution over eubacteria and eukaryotes . Such a topology suggests that the general homology between laminarinases, agarases, kappa-carrageenases, lichenases, and xyloglucan endotransglycosylases has arisen through gene duplication, likely from an ancestral protein with laminarinase activity.

Cell Mol Life Sci, 1998 Mar, 54(3), 253 - 62
Structure and assembly of the 20S proteasome; Gerards WL et al.; The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex . The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances . This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly . Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions . While the outer rings contain alpha-type subunits, the inner ones comprise beta-type subunits . The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly . The available data suggest a cooperative assembly of the alpha-type and beta-type subunits into half proteasome-like complexes followed by dimerization into proteasomes . During or after dimerization of half proteasomes, the beta-type subunits are processed . The prosequence of the beta-type subunits is essential for the assembly proves and prevents protease activity of immature proteasomes.

J Clin Microbiol, 1998 May, 36(5), 1399 - 403
Molecular epidemiology of oral treponemes associated with periodontal disease; Moter A et al.; Periodontitis, a disease responsible for tooth loss worldwide, is characterized by chronic inflammation of the periodontium, eventually leading to destruction of periodontal ligaments and supporting alveolar bone . Spirochetes, identified by dark-field microscopy as being the most predominant bacteria in advanced lesions, are thought to play a causative role . Various spirochetal morphotypes were observed, but most of these morphotypes are as yet uncultivable . To assess the role of these organisms we designed oligonucleotide probes for the identification of both cultivable and so far uncultivable spirochetes in periodontitis patients . Subgingival plaque specimens taken from diseased sites (n = 200) and healthy control sites (n = 44) from 53 patients with rapidly progressive periodontitis (RPP) were submitted to direct in situ hybridization or dot blot hybridization after prior amplification with eubacterial primers . Spirochetes were found in all patients, but their distributions varied considerably . Parallel use of oligonucleotide probes specific for cultivable or so far uncultivable treponemes suggested the presence of novel yet unknown organisms at a high frequency . These uncultivable treponemes were visualized by fluorescence in situ hybridization, and their morphologies, sizes, and numbers could be estimated . All RPP patients included in this study harbored oral treponemes that represent either novel species, e.g., Treponema maltophilum, or uncultivable phylotypes . Therefore, it is necessary to include these organisms in etiologic considerations and to strengthen efforts to cultivate these as yet uncultivable treponemes.

Oral Microbiol Immunol, 1998 Feb, 13(1), 23 - 9
Restriction fragment-length polymorphism analysis of 16S rDNA from oral asaccharolytic Eubacterium species amplified by polymerase chain reaction; Sato T et al.; Restriction fragment-length polymorphism (RFLP) analysis of 16S rDNA amplified by polymerase chain reaction was used to generate restriction profiles of the type strains of oral asaccharolytic Eubacterium species, that is, Eubacterium brachy, Eubacterium exiguum, Eubacterium lentum, Eubacterium minutum, Eubacterium nodatum, Eubacterium saphenum, Eubacterium timidum and 33 asaccharolytic Eubacterium strains isolated from oral sites . The 16S rRNA gene sequences from isolated genomic DNA samples were amplified by polymerase chain reaction (PCR) . PCR products were purified and characterized by single digestions with 7 restriction endonucleases . Among the 7 endonucleases, HpaII was found to discriminate the respective reference strains . Twenty-three isolates, out of 33, were assigned to one of the reference species, on the basis of their restriction profiles by digestion with HpaII . The remaining 10 isolates could not be assigned to any of the established species and constituted 4 distinct groups, each of which may be a new species.

Gene, 1998 Apr 14, 210(2), 277 - 85
Two types of differentially photo-regulated nuclear genes that encode sigma factors for chloroplast RNA polymerase in the red alga Cyanidium caldarium strain RK-1; Oikawa K et al.; A nuclear gene, sigA, that encodes a sigma factor for chloroplast RNA polymerase has previously been identified and characterized in the primitive red alga Cyanidium caldarium strain RK-1 . Southern hybridization analysis indicated the presence of two additional sigma factor genes, which have now been cloned and shown to encode virtually identical proteins that are homologous to eubacterial sigma factors . These genes, which are also present in the nuclear genome, have therefore been named sigB and sigC . The substantial sequence similarity of sigB and sigC to sigA of the same strain as well as to cyanobacterial principal sigma factors and other chloroplast sigma factors strongly suggests that the nuclear genome of C . caldarium contains three genes that encode two types of chloroplast sigma factors . Each of the three recombinant Sig proteins showed sigma factor activity in vitro when combined with the Escherichia coli RNA polymerase core enzyme . Northern blot analysis revealed that, whereas the overall abundance of sigA transcripts was not affected by light, the amount of sigB and sigC mRNAs was greater in the light than in the dark . Thus, multiple sigma factors appear to contribute to light-regulated gene expression in the chloroplast.

J Med Microbiol, 1998 Apr, 47(4), 335 - 40
Development of a multiplex-PCR for direct detection of the genes for enterotoxin B and C, and toxic shock syndrome toxin-1 in Staphylococcus aureus isolates; Schmitz FJ et al.; As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production . These toxins might be a cause of the toxic shock syndrome (TSS) . For that reason an easy and quick test system for determining the toxin production pattern of S . aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier . In the present investigation, a new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation . This procedure generated information concerning the presence of seb, sec-1 and tst genes within 4 h in a single test . To analyse the sensitivity and the specificity of this procedure, 100 methicillin-resistant S . aureus (MRSA), 50 coagulase-negative staphylococci and 50 other eubacterial isolates were tested initially with sets of single primer pairs followed by a combined multiplex-PCR . Results of this amplification technique were compared to a conventional and widely used method for toxin detection, reversed passive latex agglutination (RPLA) . With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and specificity of the sec-1 primer set were 100% and 82%, respectively . With the sec-1 primer set, two isolates were identified as carrying the corresponding toxin gene although the RPLA test did not show any detectable toxin . The multiplex-PCR rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains and could be easily integrated into a multiplex procedure described previously . The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and mecA genes.

Protein Sci, 1998 Apr, 7(4), 1029 - 38
Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms; Wallin E et al.; We have carried out detailed statistical analyses of integral membrane proteins of the helix-bundle class from eubacterial, archaean, and eukaryotic organisms for which genome-wide sequence data are available . Twenty to 30% of all ORFs are predicted to encode membrane proteins, with the larger genomes containing a higher fraction than the smaller ones . Although there is a general tendency that proteins with a smaller number of transmembrane segments are more prevalent than those with many, uni-cellular organisms appear to prefer proteins with 6 and 12 transmembrane segments, whereas Caenorhabditis elegans and Homo sapiens have a slight preference for proteins with seven transmembrane segments . In all organisms, there is a tendency that membrane proteins either have many transmembrane segments with short connecting loops or few transmembrane segments with large extra-membraneous domains . Membrane proteins from all organisms studied, except possibly the archaeon Methanococcus jannaschii, follow the so-called "positive-inside" rule; i.e., they tend to have a higher frequency of positively charged residues in cytoplasmic than in extra-cytoplasmic segments.

J Bioenerg Biomembr, 1997 Dec, 29(6), 533 - 40
The intriguing evolution of the "b" and "G" subunits in F-type and V-type ATPases: isolation of the vma-10 gene from Neurospora crassa; Hunt IE et al.; We have characterized the vma-10 gene which encodes the G subunit of the vacuolar ATPase in Neurospora crassa . The gene is somewhat unusual in filamentous fungi because it contains five introns, comprising 71% of the region between the translation start and stop codons . The 5' untranslated region of the gene contains several elements that have been identified in other genes that encode subunits of the vacuolar ATPase in N . crassa . A comparison of G subunits from N . crassa, S . cerevisiae, and animal cells showed that the N-terminal half of the polypeptide shows the highest degree of sequence conservation . Most striking is the observation that this region could form an alpha helix in which all of the conserved residues are clustered on one face . Subunit G appears to be homologous to the b subunit found in F-type ATPases . The major difference between the b and G subunits is the lack of a membrane-spanning region in the G subunit . We have also identified homologous subunits in the operons which encode V-type ATPases in a eubacterium, Enterrococcus hirae, and an archaebacterium, Methanococcus jannaschii . As in eukaryotic vacuolar ATPases the G subunit homologs lack a membrane-spanning region . Although the b and G subunits appear to be derived from a common ancestor, significant changes have evolved . In F-type and V-type ATPases these subunits can have zero, one, or two membrane-spanning regions and can also differ significantly in the number of copies per enzyme.

J Mol Evol, 1998 Apr, 46(4), 401 - 8
A unique fungal lysine biosynthesis enzyme shares a common ancestor with tricarboxylic acid cycle and leucine biosynthetic enzymes found in diverse organisms; Irvin SD et al.; Fungi have evolved a unique alpha-amino-adipate pathway for lysine biosynthesis . The fungal-specific enzyme homoaconitate hydratase from this pathway is moderately similar to the aconitase-family proteins from a diverse array of taxonomic groups, which have varying modes of obtaining lysine . We have used the similarity of homoaconitate hydratase to isopropylmalate isomerase (serving in leucine biosynthesis), aconitase (from the tricarboxylic acid cycle), and iron-responsive element binding proteins (cytosolic aconitase) from fungi and other eukaryotes, eubacteria, and archaea to evaluate possible evolutionary scenarios for the origin of this pathway . Refined sequence alignments show that aconitase active site residues are highly conserved in each of the enzymes, and intervening sequence sites are quite dissimilar . This pattern suggests strong purifying selection has acted to preserve the aconitase active site residues for a common catalytic mechanism; numerous other substitutions occur due to adaptive evolution or simply lack of functional constraint . We hypothesize that the similarities are the remnants of an ancestral gene duplication, which may not have occurred within the fungal lineage . Maximum likelihood, neighbor joining, and maximum parsimony phylogenetic comparisons show that the alpha-aminoadipate pathway enzyme is an outgroup to all aconitase family proteins for which sequence is currently available.

Chem Biol, 1998 Mar, 5(3), 135 - 46
Heterologously expressed acyl carrier protein domain of rat fatty acid synthase functions in Escherichia coli fatty acid synthase and Streptomyces coelicolor polyketide synthase systems; Tropf S et al.; INTRODUCTION: Fatty acid synthases (FASs) catalyze the de novo biosynthesis of long-chain saturated fatty acids by a process common to eubacteria and eukaryotes, using either a set of monofunctional proteins (Type II FAS) or a polypeptide containing several catalytic functions (Type I FAS) . To compare the features of a Type I domain with its Type II counterpart we expressed and characterized an acyl carrier protein (ACP) domain of the Type I rat FAS . RESULTS: An ACP domain of rat FAS was defined that allows expression of a small percentage of active holo-ACP both in Escherichia coli, increasing fivefold upon co-expression with an E . coli holo-ACP synthase, and in Streptomyces coelicolor . The rat ACP domain functions with some components of the E . coli FAS, and can replace the actinorhodin polyketide synthase (PKS) ACP in S . coelicolorA3(2) . Purification of the rat ACP domain from E . coli resulted in loss of its functionality . Purified apo-ACP could be converted to its holo-form upon incubation with purified E . coli holo-ACP synthase in vitro, however, suggesting that the loss of functionality was not due to a conformational change . CONCLUSIONS: Functionality of the recombinant rat ACP was shown in distantly related and diverse enzyme systems, suggesting that Type I and Type II ACPs have a similar conformation . A procedure was described that might permit the production of rat FAS holo-ACP for structural and further biochemical characterization.

Biochim Biophys Acta, 1998 Mar 9, 1396(2), 228 - 36
Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family; Fleury Y et al.; Dermaseptins are a family of cationic (Lys-rich) antimicrobial peptides that are abundant in the skin secretions of the arboreal frogs Phyllomedusa bicolor and P . sauvagii . In vitro, these peptides are microbicidal against a wide variety of microorganisms including Gram-positive and Gram-negative bacteria, yeasts, protozoa and fungi . To date, 6 dermaseptin B mature peptides, 24-34 residues long, 2 dermaseptin B cDNAs and 2 gene sequences have been identified in P . bicolor . To assess dermaseptin related genes further, we screened a P . bicolor genomic library with 32P-labeled cDNAs coding either for prepro-dermaseptins B1 or B2 (adenoregulin) . A gene sequence was identified that coded a novel dermaseptin B, termed Drg3, which exhibits 23-42% amino acids identities with other members of the family . Analysis of the cDNAs coding precursors for several opioid and antimicrobial peptides originating from the skin of various amphibian species revealed that the 25-residue preproregion of these preproforms are all encoded by conserved nucleotides encompassed by the first coding exon of the Drg3 gene . Synthetic dermaseptin Drg3 exhibited a bactericidal activity towards several species of mollicutes (wall-less eubacteria), firmicutes (Gram-positive eubacteria), and gracilicutes (Gram-negative eubacteria), with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 microM . Experiments performed on Acholeplasma laidlawii cells revealed that this peptide is membranotropic and that if efficiently depolarizes the plasma membrane.

J Bacteriol, 1998 Apr, 180(7), 1642 - 6
An archaeal aerotaxis transducer combines subunit I core structures of eukaryotic cytochrome c oxidase and eubacterial methyl-accepting chemotaxis proteins; Brooun A et al.; Signal transduction in the archaeon Halobacterium salinarum is mediated by three distinct subfamilies of transducer proteins . Here we report the complete htrVIII gene sequence and present analysis of the encoded primary structure and its functional features . HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B . At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome c oxidase . The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein . The HtrVIII protein mediates aerotaxis: a strain with a deletion of the htrVIII gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis . We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response.

Science, 1998 Mar 20, 279(5358), 1946 - 50
Energy transduction on the nanosecond time scale: early structural events in a xanthopsin photocycle; Perman B et al.; Photoactive yellow protein (PYP) is a member of the xanthopsin family of eubacterial blue-light photoreceptors . On absorption of light, PYP enters a photocycle that ultimately transduces the energy contained in a light signal into an altered biological response . Nanosecond time-resolved x-ray crystallography was used to determine the structure of the short-lived, red-shifted, intermediate state denoted {pR}, which develops within 1 nanosecond after photoelectronic excitation of the chromophore of PYP by absorption of light . The resulting structural model demonstrates that the {pR} state possesses the cis conformation of the 4-hydroxyl cinnamic thioester chromophore, and that the process of trans to cis isomerization is accompanied by the specific formation of new hydrogen bonds that replace those broken upon excitation of the chromophore . Regions of flexibility that compose the chromophore-binding pocket serve to lower the activation energy barrier between the dark state, denoted pG, and {pR}, and help initiate entrance into the photocycle . Direct structural evidence is provided for the initial processes of transduction of light energy, which ultimately translate into a physiological signal.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2100 - 4
A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway; Lange BM et al.; Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms . However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors . Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway . This synthase gene contains an ORF of 2,172 base pairs . When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa . The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants.

FEMS Microbiol Lett, 1998 Mar 15, 160(2), 237 - 46
The deconvolution of pyrolysis mass spectra using genetic programming: application to the identification of some Eubacterium species; Taylor J et al.; Pyrolysis mass spectrometry was used to produce complex biochemical fingerprints of Eubacterium exiguum, E . infirmum, E . tardum and E . timidum . To examine the relationship between these organisms the spectra were clustered by canonical variates analysis, and four clusters, one for each species, were observed . In an earlier study we trained artificial neural networks to identify these clinical isolates successfully; however, the information used by the neural network was not accessible from this so-called 'black box' technique . To allow the deconvolution of such complex spectra (in terms of which masses were important for discrimination) it was necessary to develop a system that itself produces 'rules' that are readily comprehensible . We here exploit the evolutionary computational technique of genetic programming; this rapidly and automatically produced simple mathematical functions that were also able to classify organisms to each of the four bacterial groups correctly and unambiguously . Since the rules used only a very limited set of masses, from a search space some 50 orders of magnitude greater than the dimensionality actually necessary, visual discrimination of the organisms on the basis of these spectral masses alone was also then possible.

Genet Anal, 1998 Jan, 14(3), 75 - 83
Cloning, sequence analysis and expression in E . coli of the DNA polymerase I gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium; Tvermyr M et al.; We have cloned and sequenced the polA gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium, and expressed the recombinant protein in Escherichia coli . One open reading frame encodes a protein with 942 amino acids showing 38% identity with DNA polymerase I from E . coli . Sequence alignments with other members of DNA polymerase family A and analysis of the separate domains show that the central 3'-5' exonuclease domain is 30% identical to the corresponding E . coli domain and that three sequence motifs associated with 3'-5' exonuclease activity are conserved . Also, a protein fraction from E . coli expressing the Chloroflexus polymerase contains a thermostable 3'-5' exonucleolytic activity, indicating that this activity is present in the enzyme, in agreement with the sequence analysis . The N-terminal 5'-3' exonuclease domain and the C-terminal polymerase domain show 31 and 46% identity, respectively, with the corresponding E . coli domains and all sequence motifs associated with these two enzymatic activities also are conserved . Since several DNA polymerase I enzymes lack the proofreading activity associated with the central domain it has been suggested that the ancestral polA gene contained only the two more conserved N- and C-terminal domains and that the proofreading 3'-5' exonuclease domain was introduced later in those eubacterial branches that have this activity . Our data indicate a different scenario where the ancestral polA gene contained both the exonucleolytic activities in addition to the polymerase activity and where several eubacterial branches lost the polymerase-associated proofreading activity during evolution.

Mol Gen Genet, 1998 Feb, 257(3), 271 - 82
Shine-Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli; Fargo DC et al.; Initiation of translation in Escherichia coli and related eubacteria involves well-defined interactions between a conserved Shine-Dalgarno (SD) sequence immediately upstream of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3' end of the 16S rRNA . SD-like sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in location, size, and base composition compared to those in E . coli, while anti-SD sequences in the respective 16S rRNAs remain highly conserved . We have examined the function of the SD-like sequences found in the leaders of four chloroplast genes of the green alga Chlamydomonas reinhardtii using replacement mutagenesis to eliminate complementarity with the anti-SD sequences and insertion of canonical SD sequences (GGAGG) at positions -9 to -5 relative to the initiation codon . Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diversity of chloroplast SD-like sequences were fused to aadA and uidA reporter genes encoding spectinomycin resistance and GUS activity respectively . Analysis of chloroplast transformants of C . reinhardtii and transformants of E . coli carrying the wild-type and mutant reporter constructs revealed that mutagenic replacement of the putative SD sequences had no effect on the expression of either the aadA or uidA reporter genes . Chloroplast transformants with the canonical SD sequence also showed no differences in reporter gene expression, whereas expression of the reporter genes was increased by 10 to 30% in the E . coli transformants . Collectively our results suggest that even though SD-dependent initiation predominates in E . coli, this bacterium also has the capacity to initiate translation by an SD-independent mechanism . In contrast, plant chloroplasts, and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation of most mRNAs.

EMBO J, 1998 Feb 16, 17(4), 868 - 76
Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes; Burrows PA et al.; The plastid genomes of several plants contain homologues, termed ndh genes, of genes encoding subunits of the NADH:ubiquinone oxidoreductase or complex I of mitochondria and eubacteria . The functional significance of the Ndh proteins in higher plants is uncertain . We show here that tobacco chloroplasts contain a protein complex of 550 kDa consisting of at least three of the ndh gene products: NdhI, NdhJ and NdhK . We have constructed mutant tobacco plants with disrupted ndhC, ndhK and ndhJ plastid genes, indicating that the Ndh complex is dispensible for plant growth under optimal growth conditions . Chlorophyll fluorescence analysis shows that in vivo the Ndh complex catalyses the post-illumination reduction of the plastoquinone pool and in the light optimizes the induction of photosynthesis under conditions of water stress . We conclude that the Ndh complex catalyses the reduction of the plastoquinone pool using stromal reductant and so acts as a respiratory complex . Overall, our data are compatible with the participation of the Ndh complex in cyclic electron flow around the photosystem I complex in the light and possibly in a chloroplast respiratory chain in the dark.

J Mol Biol, 1998 Feb 13, 276(1), 249 - 64
A symmetric-iterated multiple alignment of protein sequences; Brocchieri L et al.; A new symmetric-iterative method for multiple alignment of protein sequences is presented . The method can be described as a combination of motif finding and dynamic programming procedures . It uses each sequence as a standard to which all sequences are aligned based on the significant segment pair alignment (SSPA) protocol . Sequences are further matched using a reduced scoring threshold to provide fillers and extensions between highly significant segment pair matches . The method produces alignment blocks that accommodate indels and are separated by variable-length unaligned segments . Construction of consensus sequences is iterative, assigning greater weights to more distantly related sequences . A consensus sequence and various measures of conservation at each aligned position can be used for comparisons between protein families, for data base searches, and for analysis of functional and evolutionary features . The method is illustrated on the extended family of prokaryotic and eukaryotic RecA-like sequences . The RecA-like sequences reveal extended alignments among eubacterial RecA and separately among eukaryotic/archaebacterial Rad51/RadA . Eleven conserved blocks are common to both groups, two of them encompassing the ATP-binding A and B-sites . Among the most conserved positions are glycine residues . For example, they occur twice as doublets putatively serving as hinge connections that provide opportunity for alternative structural conformations . Also several charged/polar residues are highly conserved, probably consequent upon the extensive intermonomer interactions in RecA/Rad51 filament formation and possibly relevant protein-protein and protein-nucleic acid interactions.

Gene, 1998 Jan 19, 207(1), 97 - 103
Determination of Coxiella burnetii rpoB sequence and its use for phylogenetic analysis; Mollet C et al.; The nucleotide sequence of the rpoB, encoding the beta-subunit of RNA polymerase of the obligate intracellular bacterium Coxiella burnetii, was determined using a polymerase chain reaction amplification and direct sequencing methodology . Comparison between C . burnetii and other eubacterial rpoB sequences indicated sequence similarity ranging from 53.6% to 67.6% . Coxiella burnetii rpoB consists of 4128 base pairs with a 45.3% GC content encoding 1375 amino acids with a calculated molecular mass of 153.67 kDa . Comparison of 512 bases of the rpoB variable region I, from eight C . burnetii strains isolated from various sources, revealed fewer than four base differences, although the distribution of these did not correlate with previously determined genotypic groupings with the species . Phylogenetic analysis of C . burnetii based on comparison of its rpoB sequence with sequences available for other bacteria is consistent with those previously derived from 16S rRNA gene sequence, and indicate that C . burnetii belongs to the gamma-group of Proteobacteria . Furthermore, phylogeny inferred from comparison of RpoB, or homologous sequences including Archae, Bacteria and Eukarya, concurred with these results.

Nature, 1998 Mar 5, 392(6671), 37 - 41
The hydrogen hypothesis for the first eukaryote; Martin W et al.; A new hypothesis for the origin of eukaryotic cells is proposed, based on the comparative biochemistry of energy metabolism . Eukaryotes are suggested to have arisen through symbiotic association of an anaerobic, strictly hydrogen-dependent, strictly autotrophic archaebacterium (the host) with a eubacterium (the symbiont) that was able to respire, but generated molecular hydrogen as a waste product of anaerobic heterotrophic metabolism . The host's dependence upon molecular hydrogen produced by the symbiont is put forward as the selective principle that forged the common ancestor of eukaryotic cells.

RNA, 1998 Mar, 4(3), 276 - 84
A top-half tDNA minihelix is a good substrate for the eubacterial CCA-adding enzyme; Shi PY et al.; The CCA-adding enzyme {ATP(CTP):tRNA nucleotidyltransferase} catalyzes the addition and regeneration of the 3'-terminal CCA sequence of tRNAs . We show that the CCA-adding enzyme will specifically add a CCA terminus to synthetic full-length tDNA and to DNA oligonucleotides corresponding to the "top half" of tRNA-the acceptor stem and TpsiC stem-loop of tRNA . CCA addition to the top half tDNA minihelices requires a 2' as well as a 3' OH at the 3' terminus of the tDNA . Addition also depends on the length of the base paired stem, and is facilitated by, but is not dependent upon, the presence of a TpsiC loop . These results provide further evidence for independent functions of the top and bottom halves of tRNA, and support the hypothesis that these two structurally distinct and functionally independent domains evolved independently.

RNA, 1998 Mar, 4(3), 257 - 67
Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation; Nitta I et al.; Experimental results are presented suggesting that 23S rRNA is directly involved in the peptide bond formation usually performed on the ribosome . Although several reports have indicated that the eubacterial peptidyltransferase reaction does not necessarily require all the ribosomal proteins, the reconstitution of peptidyltransferase activity by a naked 23S rRNA without the help of any of the ribosomal proteins has not been reported previously . It is demonstrated that an E . coli 23S rRNA transcript synthesized by T7 RNA polymerase in vitro was able to promote peptide bond formation in the presence of 0.5% SDS . The reaction was inhibited by the peptidyltransferase-specific antibiotics chloramphenicol and carbomycin, and by digestion with RNases A and T1 . Site-directed mutageneses at two highly conserved regions close to the peptidyltransferase center ring, G2252 to U2252 and C2507G2581 to U2507A2581, also suppressed peptide bond formation . These findings strongly suggest that 23S rRNA is the peptidyltransferase itself.

J Clin Microbiol, 1998 Mar, 36(3), 752 - 5
Cellular fatty acid composition, soluble-protein profile, and antimicrobial resistance pattern of Eubacterium lentum; Mosca A et al.; Phenotypic heterogeneity among isolates of Eubacterium lentum has been recognized for many years . To better delineate their taxonomic relatedness, 29 clinical isolates of E . lentum were examined for soluble-protein content, cellular fatty acid profile, and antimicrobial resistance pattern in order to ascertain whether differences in these characteristics could be correlated with differences in biochemical activities . Among 29 isolates we could identify 6 that were different from all the others . These strains were coccobacilli with translucent colonies; they were catalase and H2S negative, not fluorescent under UV light, and susceptible to beta-lactam drugs; growth was not stimulated by arginine; and fatty acid analysis revealed the presence of straight-chain fatty acids . The remainder of the strains, including the type species, were pleomorphic bacilli with speckled colonies and were catalase and H2S positive; all but two were fluorescent under UV light; they were resistant to beta-lactam antibiotics; growth was greatly stimulated by arginine; and they demonstrated saturated branched-chain fatty acids . Our data suggest that E . lentum can be further differentiated into different types.

Int J Parasitol, 1998 Jan, 28(1), 149 - 64
Anaerobic bacterial metabolism in the ancient eukaryote Giardia duodenalis; Brown DM et al.; The protozoan parasite, Giardia duodenalis, shares many metabolic and genetic attributes of the bacteria, including fermentative energy metabolism which relies heavily on pyrophosphate rather than adenosine triphosphate and as a result contains two typically bacterial glycolytic enzymes which are pyrophosphate dependent . Pyruvate decarboxylation and subsequent electron transport to as yet unidentified anaerobic electron acceptors relies on a eubacterial-like pyruvate:ferredoxin oxidoreductase and an archaebacterial/eubacterial-like ferredoxin . The presence of another 2-ketoacid oxidoreductase (with a preference for alpha-ketobutyrate) and multiple ferredoxins in Giardia is also a trait shared with the anaerobic bacteria . Giardia pyruvate:ferredoxin oxidoreductase is distinct from the pyruvate dehydrogenase multienzyme complex invariably found in mitochondria . This is consistent with a lack of mitochondria, citric acid cycle, oxidative phosphorylation and glutathione in Giardia . Giardia duodenalis actively consumes oxygen and yet lacks the conventional mechanisms of oxidative stress management, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes . In their place Giardia contains a prokaryotic H2O-producing NADH oxidase, a membrane-associated NADH peroxidase, a broad-range prokaryotic thioredoxin reductase-like disulphide reductase and the low molecular weight thiols, cysteine, thioglycolate, sulphite and coenzyme A . NADH oxidase is a major component of the electron transport pathway of Giardia which, in conjunction with disulphide reductase, protects oxygen-labile proteins such as ferredoxin and pyruvate:ferredoxin oxidoreductase against oxidative stress by maintaining a reduced intracellular environment . As the terminal oxidase, NADH oxidase provides a means of removing excess H+, thereby enabling continued pyruvate decarboxylation and the resultant production of acetate and adenosine triphosphate . A further example of the bacterial-like metabolism of Giardia is the utilisation of the amino acid arginine as an energy source . Giardia contain the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, but not in other eukaryotes apart from trichomonads and Chlamydomonas reinhardtii . The pathway includes substrate level phosphorylation and is sufficiently active to make a major contribution to adenosine triphosphate production . Two enzymes of the pathway, arginine deiminase and carbamate kinase, are rare in eukaryotes and do not occur in higher animals . Arginine is transported into the trophozoite via a bacterial-like arginine:ornithine antiport . Together these metabolic pathways in Giardia provide a wide range of potential drug targets for future consideration.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 1319 - 24
The plastid ndh genes code for an NADH-specific dehydrogenase: isolation of a complex I analogue from pea thylakoid membranes; Sazanov LA et al.; The plastid genomes of several plants contain ndh genes-homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria . From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established . Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum . The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step . The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting . The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors . This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase . Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 1236 - 41
The Arthromitus stage of Bacillus cereus: intestinal symbionts of animals; Margulis L et al.; In the guts of more than 25 species of arthropods we observed filaments containing refractile inclusions previously discovered and named "Arthromitus" in 1849 by Joseph Leidy {Leidy, J . (1849) Proc . Acad . Nat . Sci . Philadelphia 4, 225-233} . We cultivated these microbes from boiled intestines of 10 different species of surface-cleaned soil insects and isopod crustaceans . Literature review and these observations lead us to conclude that Arthromitus are spore-forming, variably motile, cultivable bacilli . As long rod-shaped bacteria, they lose their flagella, attach by fibers or fuzz to the intestinal epithelium, grow filamentously, and sporulate from their distal ends . When these organisms are incubated in culture, their life history stages are accelerated by light and inhibited by anoxia . Characterization of new Arthromitus isolates from digestive tracts of common sow bugs (Porcellio scaber), roaches (Gromphodorhina portentosa, Blaberus giganteus) and termites (Cryptotermes brevis, Kalotermes flavicollis) identifies these flagellated, spore-forming symbionts as a Bacillus sp . Complete sequencing of the 16S rRNA gene from four isolates (two sow bug, one hissing roach, one death's head roach) confirms these as the low-G+C Gram-positive eubacterium Bacillus cereus . We suggest that B . cereus and its close relatives, easily isolated from soil and grown on nutrient agar, enjoy filamentous growth in moist nutrient-rich intestines of healthy arthropods and similar habitats.

J Bacteriol, 1998 Mar, 180(5), 1248 - 55
Isolation of Rhodospirillum centenum mutants defective in phototactic colony motility by transposon mutagenesis; Jiang ZY et al.; The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation . To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity . A majority of the ca . 250 mutants identified lost the ability to form motile swarm cells on an agar surface . These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella . Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus . Several photosensory mutants also were obtained with defects in the perception and transmission of light signals . Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception . It was shown that screening for photosensory defective R . centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.

Eur J Biochem, 1998 Feb 1, 251(3), 549 - 57
Human aldehyde dehydrogenase gene family; Yoshida A et al.; Twelve aldehyde dehydrogenase (ALDH) genes have been identified in humans . These genes, located on different chromosomes, encode a group of enzymes which oxidizes varieties of aliphatic and aromatic aldehydes . Metabolic disorders and clinical problems associated with mutations of ALDH1, ALDH2, ALDH4, ALDH10 and succinic semialdehyde (SSDH) genes have been emerged . Comparison of the human ALDHs indicates a wide range of divergency (> 80 - < 15% identity at the protein sequence level) among them . However, several protein regions, some of which are implicated in functional activities, are conserved in the family members . The phylogenic tree constructed of 56 ALDH sequences of humans, animals, fungi, protozoa and eubacteria, suggests that the present-day human ALDH genes were derived from four ancestral genes that existed prior to the divergence of Eubacteria and Eukaryotes . The neighbor-joining tree derived from 12 human ALDHs and antiquitin indicates that diversification within the ALDH1/2/5/6 gene cluster occurred during the Neoproterozoic period (about 800 million years ago) . Duplication in the ALDH 3/10/7/8 gene cluster occurred in Phanerozoic period (about 300 million years ago) . Separations of ALDH3/ALDH10 and that of ALDH7/ALDH8 had occurred during the period of appearance and radiation of mammalian species.

Plant Mol Biol, 1998 Feb, 36(3), 493 - 6
Characterisation of transcript initiation sites in ribosome-deficient barley plastids; Hubschmann T et al.; Transcription of plastid genes in higher plants is driven by two RNA polymerases . One is encoded in the chloroplast, the other is encoded in the nucleus . RNA synthesis in ribosome-deficient plastids is performed exclusively by the nuclear-encoded enzyme . In vitro capping was used to identify the transcriptional start sites of the genes clpP and rpl23 in ribosome-free plastids of the barley mutant albostrians . No transcript initiation was found at sequences similar to eubacterial promoters . Instead, transcription started near the motif 5'-YRTA-3', which is also conserved in mitochondrial promoters of higher plants . Our data suggest that the nuclear encoded RNA polymerase is active in mature chloroplasts and is the sole polymerase involved in transcription of rpl123.

J Mol Evol, 1997 Dec, 45(6), 688 - 95
Analysis of the cluster of ribosomal protein genes in the plastid genome of a unicellular red alga Cyanidioschyzon merolae: translocation of the str cluster as an early event in the rhodophyte-chromophyte lineage of plastid evolution; Ohta N et al.; The nucleotide sequence of a cluster of ribosomal protein genes in the plastid genome of a unicellular red alga, Cyanidioschyzon merolae, which has been supposed to be the most primitive alga, was determined . The phylogenetic tree inferred from the amino acid sequence of ribosomal proteins of two rhodophytes, a chromophyte, a glaucophyte, two chlorophytes (land plants), a cyanobacterium, and three eubacteria suggested a close relationship between the cyanobacterium Synechocystis PCC6803 and the plastids of various species in the kingdom Plantae, which is consistent with the hypothesis of the endosymbiotic origin of plastids . In this tree, the two species of rhodophytes were grouped with the chromophyte, and the glaucophyte was grouped with the chlorophytes . Analysis of the organization of the genes encoding the ribosomal proteins suggested that the translocation of the str cluster occurred early in the lineage of rhodophytes and chromophytes after these groups had been separated from chlorophytes and glaucophytes.

J Mol Evol, 1997 Dec, 45(6), 671 - 81
On the evolution of the single-subunit RNA polymerases; Cermakian N et al.; Many eukaryotic nuclear genomes as well as mitochondrial plasmids contain genes displaying evident sequence similarity to those encoding the single-subunit RNA polymerase (ssRNAP) of bacteriophage T7 and its relatives . We have collected and aligned these ssRNAP sequences and have constructed unrooted phylogenetic trees that demonstrate the separation of ssRNAPs into three well-defined and nonoverlapping clusters (phage-encoded, nucleus-encoded, and plasmid-encoded) . Our analyses indicate that these three subfamiles of T7-like RNAPs shared a common ancestor; however, the order in which the groups diverged cannot be inferred from available data . On the basis of structural similarities and mutational data, we suggest that the ancestral ssRNAP gene may have arisen via duplication and divergence of a DNA polymerase or reverse transcriptase gene . Considering the current phylogenetic distribution of ssRNAP sequences, we further suggest that the origin of the ancestral ssRNAP gene closely paralleled in time the introduction of mitochondria into eukaryotic cells through a eubacterial endosymbiosis.

J Mol Evol, 1997 Dec, 45(6), 661 - 70
On the origin of protein synthesis factors: a gene duplication/fusion model; Cousineau B et al.; Sequence similarity has given rise to the proposal that IF-2, EF-G, and EF-Tu are related through a common ancestor . We evaluate this proposition and whether the relationship can be extended to other factors of protein synthesis . Analysis of amino acid sequence similarity gives statistical support for an evolutionary affiliation among IF-1, IF-2, IF-3, EF-Tu, EF-Ts, and EF-G and suggests further that this association is a result of gene duplication/fusion events . In support of this mechanism, the three-dimensional structures of IF-3, EF-Tu, and EF-G display a predictable domain structure and overall conformational similarity . The model that we propose consists of three consecutives duplication/fusion events which would have taken place before the divergence of the three superkingdoms: eubacteria, archaea, and eukaryotes . The root of this protein superfamily tree would be an ancestor of the modern IF-1 gene sequence . The repeated fundamental motif of this protein superfamily is a small RNA binding domain composed of two alpha-helices packed along side of an antiparallel beta-sheet.

Nucleic Acids Res, 1998 Jan 1, 26(1), 156 - 9
5S rRNA Data Bank; Szymanski M et al.; In this paper we present the updated version of the compilation of 5S rRNA and 5S rDNA nucleotide sequences . It contains 1622 primary structures of 5S rRNAs and 5S rRNA genes from 888 species . These include 58 archaeal, 427 eubacterial, 34 plastid, nine mitochondrial and 1094 eukaryotic DNA or RNA nucleotide sequences . The sequence entries are divided according to the taxonomic position of the organisms . All individual sequences deposited in the 5S rRNA Database can be retrieved using the WWW-based, taxonomic browser at + or fu-berlin.de/fb_chemie/agerdmann/5S_rRNA.html . The files with complete sets of data as well as sequence alignments are available via anonymous ftp.

Biochim Biophys Acta, 1998 Jan 8, 1379(1), 76 - 82
Occurrence of peptidyl D-amino acids in soluble fractions of several eubacteria, archaea and eukaryotes; Nagata Y et al.; The occurrence of peptidyl D-amino acids in the aqueous soluble fractions was investigated in various eubacteria, some archaea and some eukaryotes . The contents of the D-enantiomers of serine, alanine, proline, glutamate (glutamine), aspartate (asparagine) and phenylalanine were determined with cell- and tissue-extracts, by means of acid hydrolysis and high-performance liquid chromatography . The rate of D-enantiomer (%, the ratio in molar concentration of a D-amino acid to the total of the D-amino acid and the corresponding L-amino acid) of alanine and glutamate were high in some Gram-positive eubacteria: 11.7% in Staphylococcus epidermidis and 10.3% in Streptococcus pyogenes for alanine, and 22.3% for glutamate in Bacillus YN-1 . The D-glutamate content was also high (8.0%) in the Gram-negative eubacterium, Thiobacillus ferrooxidans . D-Aspartate was common, as was D-glutamate: the highest D-aspartate content was detected in an archaeum, Pyrobaculum islandicum (4.0%) . However, the content of D-aspartate was low, 0.2-1.8% in most other bacteria . The presence of D-serine was shown in some organisms, but that of D-proline was scarce . The D-enantiomer of phenylalanine was not detected in any of the organisms examined . These results indicate that of the bacteria examined herein most Gram-negative and some Gram-positive eubacteria, as well as archaea contain only low levels of D-amino acids in the soluble peptidyl fraction, and the levels were comparable to those in eukaryotes examined . To our knowledge, the general presence of peptidyl D-amino acids in these organisms, especially archaea and eukaryotic cells including those from rat liver tissues, has been shown here for the first time.

Oral Microbiol Immunol, 1997 Oct, 12(5), 318 - 22
Evaluation of root canal bacteria and their antimicrobial susceptibility in teeth with necrotic pulp; Le Goff A et al.; This study aimed to evaluate the microbiota of necrotic pulp in teeth without carious lesions where the crown and root were intact and to test the sensitivity of this microbiota to antibiotics in order to improve treatment . The necrotic pulp was sampled from 26 single-rooted teeth in intact pulp chambers . A total of 84 strains were isolated . The number of species isolated per tooth varied from 2 to 8, with a strong component (81%) of anaerobic bacteria . The most commonly represented species were Bacteroides gracilis, Propionibacterium acnes, Fusobacterium nucleatum, Prevotella buccae and Eubacterium lentum . The sensitivity of these organisms to amoxicillin, amoxicillin combined with clavulanate and tetracycline was evaluated by Etest on 38 isolates . For all strains tested, the minimum inhibitory concentration values obtained were low and substantially below effective serum concentrations for these antibiotics . These data enable us to devise suitable treatments for acute development of apical lesions and to prevent dissemination of this source of infection to the rest of the host.

J Clin Microbiol, 1998 Feb, 36(2), 462 - 6
Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H . felis from related bacteria by restriction fragment length polymorphism analysis; Messick JB et al.; The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced . Based on this sequence data, we designed a set of H . felis-specific primers . These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H . felis from each of four experimentally infected cats at peak parasitemia . No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis . Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment . The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats . By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data . In this PCR assay, the minimum detectable number of H . felis organisms was determined to be between 50 and 704 . The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H . felis from closely related bacteria was examined . This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H . felis infection in cats.

Int Immunol, 1997 Dec, 9(12), 1867 - 74
Bet v 1, the major birch pollen allergen, conjugated to crystalline bacterial cell surface proteins, expands allergen-specific T cells of the Th1/Th0 phenotype in vitro by induction of IL-12; Jahn-Schmid B et al.; Modulation of allergic immune responses by using adequate adjuvants is a promising concept for future immunotherapy of type I hypersensitivity . In the present study, recombinant Bet v 1 (rBet v 1, the major birch pollen allergen) was conjugated to cross-linked crystalline surface layer proteins (SL) derived from Gram-positive eubacteria . T cell lines (TCL) and clones (TCC) were established from peripheral blood of birch pollen-allergic patients . TCL and TCC were induced either using rBet v 1 alone or rBet v 1/SL conjugates (rBet v 1/SL) as initial antigen stimulus . Cytokine production after re-stimulation with rBet v 1 was investigated . TCL initiated with rBet v 1/SL showed significantly increased IFN-gamma production as compared to rBet v 1 -selected TCL . TCC were established from TCL of five patients . As expected, the majority of CD4+ TCC induced by rBet v 1 (55%) displayed a Th2-like pattern of cytokine production . However, only 21% of Bet v 1-specific TCC isolated from TCL established with the Bet v 1/SL revealed this phenotype . The majority of SL-specific TCC (80%) belonged to the Th1 phenotype . In cultures of peripheral blood mononuclear cells, both, SL and Bet v 1/SL (but not rBet v 1) stimulated the production of high levels of IL-12, a pivotal mediator of Th1 responses . Moreover, stimulation of rBet v 1-induced TCC with rBet v 1/SL led to an increased IFN-gamma production . This effect could be reversed by neutralizing anti-IL-12 mAb . Together these results indicate an adjuvant effect of SL mediated by IL-12 . Our results indicate that bacterial components, such as SL, displaying adjuvant effects may be suitable for immunotherapeutical vaccines for type I allergy.

Z Naturforsch {C}, 1997 Nov-Dec, 52(11-12), 789 - 98
Subunit II (b') and not subunit I (b) of photosynthetic ATP synthases is equivalent to subunit b of the ATP synthases from nonphotosynthetic eubacteria . Evidence for a new assignment of b-type F0 subunits; Tiburzy HJ et al.; Subunit I of chloroplast ATP synthase is reviewed until now to be equivalent to subunit b of Escherichia coli ATP synthase, whereas subunit II is suggested to be an additional subunit in photosynthetic ATP synthases lacking a counterpart in E . coli . After publication of some sequences of subunits II a revision of this assignment is necessary . Based on the analysis of 51 amino acid sequences of b-type subunits concerning similarities in primary structure, isoelectric point and a discovered discontinuous structural feature, our data provide evidence that chloroplast subunit II (subunit b' of photosynthetic eubacteria) and not chloroplast subunit I (subunit b of photosynthetic eubacteria) is the equivalent of subunit b of nonphotosynthetic eubacteria, and therefore does have a counterpart in e.g . E . coli . In consequence, structural features essential for function should be looked for on subunit II (b').

J Mol Evol, 1997, 44 Suppl 1, S57 - 64
Genome plasticity as a paradigm of eubacteria evolution; Watanabe H et al.; To test the hypotheses that eubacterial genomes leave evolutionarily stable structures and that the variety of genome size is brought about through genome doubling during evolution, the genome structures of Haemophilus influenzae, Mycoplasma genitalium, Escherichia coli, and Bacillus subtilis were compared using the DNA sequences of the entire genome or substantial portions of genome . In these comparisons, the locations of orthologous genes were examined among different genomes . Using orthologous genes for the comparisons guaranteed that differences revealed in physical location would reflect changes in genome structure after speciation . We found that dynamic rearrangements have so frequently occurred in eubacterial genomes as to break operon structures during evolution, even after the relatively recent divergence between E . coli and H . influenzae . Interestingly, in such eubacterial genomes of high plasticity, we could find several highly conservative regions with the longest conserved region comprising the S10, spc, and alpha operons . This suggests that such exceptional conservative regions have undergone strong structural constraints during evolution.

Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13524 - 9
Suppressor mutations in Escherichia coli methionyl-tRNA formyltransferase: role of a 16-amino acid insertion module in initiator tRNA recognition; Ramesh V et al.; The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF; EC 2.1.2.9) is important for the initiation of protein synthesis in eubacteria and in eukaryotic organelles . The determinants for formylation in the tRNA are clustered mostly in the acceptor stem . As part of studies on the molecular mechanism of recognition of the initiator tRNA by MTF, we report here on the isolation and characterization of suppressor mutations in Escherichia coli MTF, which compensate for the formylation defect of a mutant initiator tRNA, lacking a critical determinant in the acceptor stem . We show that the suppressor mutant in MTF has a glycine-41 to arginine change within a 16-amino acid insertion found in MTF from many sources . A mutant with glycine-41 changed to lysine also acts as a suppressor, whereas mutants with changes to aspartic acid, glutamine, and leucine do not . The kinetic parameters of the purified wild-type and mutant Arg-41 and Lys-41 enzymes, determined by using the wild-type and mutant tRNAs as substrates, show that the Arg-41 and Lys-41 mutant enzymes compensate specifically for the strong negative effect of the acceptor stem mutation on formylation . These and other considerations suggest that the 16-amino acid insertion in MTF plays an important role in the specific recognition of the determinants for formylation in the acceptor stem of the initiator tRNA.

Biol Chem, 1997 Oct, 378(10), 1103 - 17
Glutaminyl-tRNA synthetase; Freist W et al.; Among the twenty aminoacyl-tRNA synthetases glutaminyl-tRNA synthetase occupies a special position: it is one of only two enzymes of this family which is not found in all organisms, being mainly absent from gram positive eubacteria, archaebacteria and organelles . The E . coli GlnRS is relatively small with 553 amino acids and a molecular mass of 64.4 kDa and functions as a monomer . The mammalian enzymes are somewhat larger and can be parts of multienzyme complexes . Crystal structures were solved of E . coli GlnRS complexed with tRNA(Gln) and ATP, of this complex containing tRNA(Gln) replaced by unmodified tRNA(Gln), and of three complexes with mutated GlnRS enzymes . The GlnRS molecule consists of four domains, the catalytic site is located in the Rossman fold, typical for class I synthetases, and the reaction mechanism follows the normal adenylate pathway . The enzyme shows many similarities with glutamyl-tRNA synthetase; a common ancestor of both molecules is well established . In the E . coli system recognition of the cognate tRNA has been studied in many details using both natural and artificial mutants of tRNA(Gln) and of the enzyme: GlnRS recognizes mainly conventional parts of the tRNA molecule, namely some bases of the anticodon loop and parts of the acceptor stem.

FEMS Microbiol Lett, 1998 Jan 1, 158(1), 39 - 43
Studies on the cyanobacterial family C DNA polymerase; Ito J et al.; Our studies showed that family C DNA polymerase (pol III) of the cyanobacterium Synechocystis sp . strain PCC 6803 is phylogenetically close to the Gram-negative dna E group, rather than to the Gram-positive group . However, in contrast to the dna E genes of most of the eubacteria, the cyanobacterial dna E gene has split into two genes, dna E1 and dna E2 . The evolutionary origin of the split dna E gene is discussed.

Biochemistry, 1998 Jan 6, 37(1), 377 - 86
The lon protease from Mycobacterium smegmatis: molecular cloning, sequence analysis, functional expression, and enzymatic characterization; Roudiak SG et al.; We have charterized a Mycobacterium smegmatis gene encoding a homolog of the ATP-dependent protease Lon (La) . Our identification of a Lon homolog, in conjunction with our previous work, identifies M . smegmatis as the first known example of a eubacterium containing both Lon and a complete 20S proteasome (containing both alpha- and beta-subunits) . Despite the significant primary sequence divergence between M . smegmatis Lon (Ms-Lon) and E . coli Lon (Ec-Lon), expression of Ms-Lon was only moderately toxic to E . coli cells . The ability of E . coli cells to tolerate expression of Ms-Lon reveals that Ms-Lon does not recognize and degrade essential E . coli proteins . We conclude that discrimination against nonsubstrate proteins is broadly conserved between Ec-Lon and Ms-Lon . Additional conservation of substrate recognition was demonstrated by the ability of Ms-Lon to degrade efficiently RcsA, a natural substrate of Ec-Lon . Purified Ms-Lon displays chymotrypsin-like specificity in peptidase assays that are stimulated by unfolded protein and supported by nonhydrolyzed nucleotide analogs . Maximal peptidase activity requires ATP or dATP . Replacement of Ms-Lon's catalytic Ser with Ala (S675A), Thr (S675T), or Cys (S675C) reduced to background levels Ms-Lon's in vitro peptidase activity . However, by employing a sensitive in vivo assay, based on the degradation of RcsA, we demonstrated that the S675C variant retained specific protease activity . Finally, variants of Ms-Lon, with substututions at or near S675, reduce the enzyme's basal ATPase activity, suggesting a structural interaction between the peptidase and ATPase active sites of Ms-Lon.

Zentralbl Veterinarmed B, 1997 Nov, 44(9), 547 - 50
The first isolation of Eubacterium suis in Hungary; Biksi I et al.; Eubacterium suis was isolated from the preputium of seven out of 16 mature boars on two farms and from the urinary bladder of one out of five sows originating from a third herd . The morphological and biochemical characteristics of the isolated strains were identical to that of the reference strain of E . suis ATTC 33144 . Three out of four strains isolated from Farm A were successfully subcultured aerobically, and then anaerobically again . E . suis together with Proteus mirabilis was isolated from cystitis of a sow 4 days after artificial insemination . These are the first strains of E . suis isolated in Hungary.

FEBS Lett, 1997 Dec 22, 420(1), 54 - 6
Functional expression of pharaonis phoborhodopsin in Escherichia coli; Shimono K et al.; Pharaonis phoborhodopsin, the photoreceptor of the negative phototaxis of archaebacterial Natronobacterium pharaonis, was functionally expressed in the heterologous system of Escherichia coli . Flash-photolysis on a millisecond time scale indicated that the photochemical properties of ppR expressed in E . coli were the same as those of the native ppR in N . pharaonis . We concluded that the integral membrane protein ppR is correctly folded in vivo in the eubacterial E . coli membrane.

Biol Pharm Bull, 1997 Dec, 20(12), 1274 - 8
Enzymes responsible for the metabolism of saikosaponins from Eubacterium sp . A-44, a human intestinal anaerobe; Kida H et al.; From a human intestinal bacterium, Eubacterium sp . A-44, which is capable of hydrolyzing saikosaponins to saikogenins, two glycosidases, beta-D-glucosidase and a novel type of beta-D-fucosidase, were isolated and characterized as saikosaponin-hydrolyzing beta-D-glucosidase and prosaikogenin-hydrolyzing beta-D-fucosidase . Relative to the hydrolyzing activities toward saikosaponins a, b1 and b2, the beta-D-glucosidase showed lower ability to hydrolyze saikosaponin d, but no ability to hydrolyze saikosaponin c or prosaikogenins . By Sephacryl S-300 column chromatography, the molecular weight of prosaikogenin-hydrolyzing beta-D-fucosidase was estimated to be about 130 kDa . The beta-D-fucosidase could hydrolyze prosaikogenins A and F, but not prosaikogenins D and G or saikosaponins . Relative to p-nitrophenyl beta-D-fucoside-hydrolyzing activity, this enzyme had 32.0% and 22.2% of its hydrolyzing ability toward p-nitrophenyl beta-D-glucoside and p-nitrophenyl beta-D-galactoside, respectively . p-Nitrophenyl beta-D-fucoside-hydrolyzing activity was inhibited by D-fucose, and was weakly inhibited by D-glucose, D-glucono delta-lactone, D-galactose and D-galactono delta-lactone . By combining these two glycosidases, saikosaponins a and b1 were converted to their saikogenins via the corresponding prosaikogenins.

Biol Pharm Bull, 1997 Dec, 20(12), 1245 - 9
Hydrolysis of glycyrrhetyl mono-glucuronide to glycyrrhetic acid by glycyrrhetyl mono-glucuronide beta-D-glucuronidase of Eubacterium sp . GLH; Akao T; Glycyrrhetyl mono-glucuronide (GAMG) is an intermediate in the hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid (GA) . An enzyme responsible for its hydrolysis, characterized as a GAMG beta-D-glucuronidase of Eubacterium sp . (species) GLH, has been isolated from human intestinal bacteria . The pattern of GAMG beta-D-glucuronidase activity was different from that of GL beta-D-glucuronidase activity by Butyl-Toyopearl 650 S column chromatography . Thus, these enzymes showed differences in the purification ratio and substrate specificity . After this step, GAMG beta-D-glucuronidase was completely separated from GL beta-D-glucuronidase by gel filtration through Toyopearl HW-55 S, indicating that the GAMG beta-D-glucuronidase is a novel type of beta-D-glucuronidase which hydrolyzes one glucuronic acid linkage of GA.

J Biol Chem, 1997 Dec 26, 272(52), 33009 - 14
Substrate recognition by the leucyl/phenylalanyl-tRNA-protein transferase . Conservation within the enzyme family and localization to the trypsin-resistant domain; Ichetovkin IE et al.; The leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) from Escherichia coli catalyzes a peptidyltransferase reaction that results in the N-terminal aminoacylation of acceptor proteins using Leu-, Phe-, and Met-tRNAs as amino acid donors . We demonstrated that L/F-transferase homologs are widely distributed throughout the eubacteria, supporting our proposal that the enzyme family is ancient and catalyzes early peptide bond synthesis . However, here we present data suggesting that the L/F-transferase is not a homolog of the peptidyltransferase enzymes involved in cell wall peptidoglycan biosynthesis in Gram-positive species, such as Staphylococcus aureus . A sequence comparison of the known L/F-transferase homologs began to identify the essential residues required to catalyze a peptidyltransferase reaction and revealed that <20% of the residues were invariant within the L/F-transferase family . Despite this sequence variation, substrate specificity was broadly conserved, and L/F-transferase homologs from Providencia stuartii, Vibrio cholerae, Neisseria gonorrhoeae, and the cyanobacterium Synechocystis sp . all complemented an E . coli aat mutant (lacking L/F-transferase activity) for the degradation of N-end rule substrates . In vitro comparison of the most divergent L/F-transferase homologs, from E . coli and the cyanobacterium Synechocystis sp., revealed near-complete conservation of both substrate specificity and secondary structure . Finally, we demonstrated that variants of the E . coli L/F-transferase, lacking either 33 or 78 N-terminal residues, retained measurable peptidyltransferase activity and wild type substrate specificity . Overall, our results identified an essential core of an L/F-transferase and revealed that a peptidyltransferase catalyst may be constructed from approximately 120 amino acids.

Annu Rev Genet, 1997, 31, 1 - 31
Antisense RNA-regulated programmed cell death; Gerdes K et al.; Eubacterial plasmids and chromosomes encode multiple killer genes belonging to the hok gene family . The plasmid-encoded killer genes mediate plasmid stabilization by killing plasmid-free cells . This review describes the genetics, molecular biology, and evolution of the hok gene family . The complicated antisense RNA-regulated control-loop that regulates posttranscriptional and postsegregational activation of killer mRNA translation in plasmid-free cells is described in detail . Nucleotide covariations in the mRNAs reveal metastable stem-loop structures that are formed at the mRNA 5' ends in the nascent transcripts . The metastable structures prevent translation and antisense RNA binding during transcription . Coupled nucleotide covariations provide evidence for a phylogenetically conserved mRNA folding pathway that involves sequential dynamic RNA rearrangements . Our analyses have elucidated an intricate mechanism by which translation of an antisense RNA-regulated mRNA can be conditionally activated . The complex phylogenetic relationships of the plasmid- and chromosome-encoded systems are also presented and discussed.

Annu Rev Cell Dev Biol, 1997, 13, 395 - 424
Bacterial cell division; Bramhill D; Bacteria usually divide by building a central septum across the middle of the cell . This review focuses on recent results indicating that the tubulin-like FtsZ protein plays a central role in cytokinesis as a major component of a contractile cytoskeleton . Assembly of this cytoskeletal element abutting the membrane is a key point for regulation . The characterization of FtsZ homologues in Mycoplasmas, Archaea, and chloroplasts implies that the constriction mechanism is conserved and that FtsZ can constrict in the absence of peptidoglycan synthesis . In most Eubacteria, the internal cytoskeleton must also regulate synthesis of septal peptidoglycan . The Escherichia coli septum-specific penicillin-binding protein 3 (PBP3) forms a complex with other enzymes involved in murein metabolism, suggesting a centrally located transmembrane complex capable of splicing multiple new strands of peptidoglycan into the cell wall . Important questions remain about the spatial and temporal control of bacterial division.

Gene, 1997 Dec 19, 204(1-2), 55 - 62
Metazoan nuclear genes for mitoribosomal protein S12; Shah ZH et al.; We have characterized nuclear genes for mitoribosomal protein S12 (mt-rps12) a major component of the ribosomal accuracy centre, in human, mouse and Drosophila melanogaster . In human and Drosophila, and probably also in mouse, there is a single intron within the coding region, located in the mitochondrial targeting pre-sequence . In humans, the mRNA structure is highly suggestive of translational regulation . In all three species, there is an amino-acid substitution with respect to eubacterial homologues in a residue implicated in aminoglycoside resistance . The only viable mutant allele of the Drosophila gene, associated with a bang-sensitive phenotype (paralysis upon mechanical vibration, arising from a mechanoreceptor cell defect) also has a novel substitution in a conserved region implicated in translational fidelity . Given the involvement of the mitoribosomal accuracy centre in human sensorineural deafness by virtue of rRNA mutations, our results indicate that this fly mutant may be a useful animal model of this disorder, and earmark the gene for mt-rps12 as a candidate in human hearing impairment.

Nature, 1998 Jan 8, 391(6663), 203 - 6
Crystal structure of the bacterial cell-division protein FtsZ; Lowe J et al.; Bacterial cell division ends with septation, the constriction of the cell wall and cell membranes that leads to the formation of two daughter cells . During septation, FtsZ, a protein of relative molecular mass 40,000 which is ubiquitous in eubacteria and is also found in archaea and chloroplasts, localizes early at the division site to form a ring-shaped septum . This septum is required for the mechanochemical process of membrane constriction . FtsZ is a GTPase with weak sequence homology to tubulins . The nature of FtsZ polymers in vivo is unknown, but FtsZ can form tubules, sheets and minirings in vitro . Here we report the crystal structure at 2.8 A resolution of recombinant FtsZ from the hyperthermophilic methanogen Methanococcus jannaschii . FtsZ has two domains, one of which is a GTPase domain with a fold related to one found in the proteins p21ras and elongation factor EF-Tu . The carboxy-terminal domain, whose function is unknown, is a four-stranded beta-sheet tilted by 90 degrees against the beta-sheet of the GTPase domain . The two domains are arranged around a central helix . GDP binding is different from that typically found in GTPases and involves four phosphate-binding loops and a sugar-binding loop in the first domain, with guanine being recognized by residues in the central connecting helix . The three-dimensional structure of FtsZ is similar to the structure of alpha- and beta-tubulin.

J Bacteriol, 1998 Jan, 180(1), 65 - 72
Characterization of the dnaG locus in Mycobacterium smegmatis reveals linkage of DNA replication and cell division; Klann AG et al.; We have isolated a UV-induced temperature-sensitive mutant of Mycobacterium smegmatis that fails to grow at 42 degrees C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division . Complementation of this mutant with an M . smegmatis genomic library and subsequent subcloning reveal that the defect lies within the M . smegmatis dnaG gene encoding DNA primase . Sequence analysis of the mutant dnaG allele reveals a substitution of proline for alanine at position 496 . Thus, dnaG is an essential gene in M . smegmatis, and DNA replication and cell division are coupled processes in this species . Characterization of the sequences flanking the M . smegmatis dnaG gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a dgt gene encoding dGTPase . The organization of this operon is conserved in Mycobacterium tuberculosis and Mycobacterium leprae, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.

J Bacteriol, 1998 Jan, 180(1), 10 - 9
Metabolic roles of a Rhodobacter sphaeroides member of the sigma32 family; Karls RK et al.; We report the role of a gene (rpoH) from the facultative phototroph Rhodobacter sphaeroides that encodes a protein (sigma37) similar to Escherichia coli sigma32 and other members of the heat shock family of eubacterial sigma factors . R . sphaeroides sigma37 controls genes that function during environmental stress, since an R . sphaeroides deltaRpoH mutant is approximately 30-fold more sensitive to the toxic oxyanion tellurite than wild-type cells . However, the deltaRpoH mutant lacks several phenotypes characteristic of E . coli cells lacking sigma32 . For example, an R . sphaeroides deltaRpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1, as well as its wild-type parent . In characterizing the response of R . sphaeroides to heat, we found that its growth temperature profile is different when cells generate energy by aerobic respiration, anaerobic respiration, or photosynthesis . However, growth of the deltaRpoH mutant is comparable to that of a wild-type strain under each of these conditions . The deltaRpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42 degrees C, but it exhibited altered induction kinetics of approximately 120-, 85-, 75-, and 65-kDa proteins . There was also reduced accumulation of several presumed heat shock transcripts (rpoD P(HS), groESL1, etc.) when aerobically grown deltaRpoH cells were placed at 42 degrees C . Under aerobic conditions, it appears that another sigma factor enables the deltaRpoH mutant to mount a heat shock response, since either RNA polymerase preparations from an deltaRpoH mutant, reconstituted Esigma37, or a holoenzyme containing a 38-kDa protein (sigma38) each transcribed E . coli Esigma32-dependent promoters . The lower growth temperature profile of photosynthetic cells is correlated with a difference in heat-inducible gene expression, since neither wild-type cells or the deltaRpoH mutant mount a typical heat shock response after such cultures were shifted from 30 to 37 degrees C.

Microbiology, 1997 Dec, 143 ( Pt 12), 3913 - 9
Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA; Zhou J et al.; Genomic DNA was isolated from the active layer of tundra soil collected from the Kolyma lowland, Northeast Eurasia, near the Arctic Ocean coast . The SSU (small subunit) rRNA genes were amplified with eubacterial primers from the bulk genomic community DNA and cloned into plasmid vectors . Forty-three SSU rDNA clones were obtained, and all of them had different RFLP patterns . Phylogenetic analysis based on partial sequences (about 300 bp) established with the maximum likelihood method revealed the presence of three major and several minor groups that fell into 11 of the established lines of bacteria, and one sequence that could not be assigned to any of the described groups . Most of the clones belonged to the alpha (20.9%) and delta (25.6%) subdivisions of the Proteobacteria, with lesser proportions in the beta (9.3%) and gamma (4.7%) subdivisions, groups typically isolated from soil by culture methods . Fewer than 12% of the clones belonged to Gram-positive bacteria, and 16% of the clones were related to Fibrobacter . The majority of the clones (70%) had sequences that were 5-15% different from those in the current databases, and 7% of the clones had sequences that differed by more than 20% from those in the database . The results suggest that these tundra-derived clones are very diverse in phylogeny, and that many probably reflect new genera or families . Hence, most of the tundra soil bacterial community has never been isolated and thus the physiology and function of its dominant members appears to be unknown.

Yi Chuan Xue Bao, 1997 Aug, 24(4), 380 - 4
{Plasmid DNA fragments from Halobacterium halobium active as eubacteria promoters in Escherichia coli}; Sun G et al.; In this paper; a promoter-probe plasmid pKK232-8 was used as a vector which functioned in E . coli . The plasmid pHH205 of Halobacterium halobium J7 was digested by two groups of restriction endonucleases BamHI-SalI and HindIII -SalI, recombined in vitro and transformed into E . coli . The transformants were selected on resistance plates to contain ampicillin (Am) and chloramphenicol (Cm) . From random-selected 20 strains of transformants we obtained transformants T1 and T2, whose level of Cm resistance got to 110 micrograms/ml . The recombinant plasmids from T1 and T2 were named pJH and pJB, respectively . The resultes of analysis by restriction endonucleases digestion and molecular hybridization showed that recombinant plasmid pJH carried a inserted fragment with size of 800bp from plasmid pHH205 . Experimental results of retransfomation proved further that the DNA fragment had promoter function in E . coli . Thus, this indicated that DNA fragments from plasmid of archaebaeteria (H halobium) may function as eubacteria (E . coil) promoters.

Microbiol Mol Biol Rev, 1997 Dec, 61(4), 456 - 502
Archaea and the prokaryote-to-eukaryote transition; Brown JR et al.; Since the late 1970s, determining the phylogenetic relationships among the contemporary domains of life, the Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes), has been central to the study of early cellular evolution . The two salient issues surrounding the universal tree of life are whether all three domains are monophyletic (i.e., all equivalent in taxanomic rank) and where the root of the universal tree lies . Evaluation of the status of the Archaea has become key to answering these questions . This review considers our cumulative knowledge about the Archaea in relationship to the Bacteria and Eucarya . Particular attention is paid to the recent use of molecular phylogenetic approaches to reconstructing the tree of life . In this regard, the phylogenetic analyses of more than 60 proteins are reviewed and presented in the context of their participation in major biochemical pathways . Although many gene trees are incongruent, the majority do suggest a sisterhood between Archaea and Eucarya . Altering this general pattern of gene evolution are two kinds of potential interdomain gene transferrals . One horizontal gene exchange might have involved the gram-positive Bacteria and the Archaea, while the other might have occurred between proteobacteria and eukaryotes and might have been mediated by endosymbiosis.

Biochemistry, 1997 Nov 11, 36(45), 13910 - 8
Purification, characterization, and inhibition of peptide deformylase from Escherichia coli; Rajagopalan PT et al.; Peptide deformylase (EC 3.5.1.31) catalyzes the removal of a formyl group from the N-termini of nascent ribosome-synthesized polypeptides, an obligatory step during protein maturation in eubacteria . Since its discovery in crude Escherichia coli extracts 3 decades ago, the deformylase has resisted all attempts of purification or characterization due to its extraordinary lability . By placing the coding sequence (def gene) of Escherichia coli deformylase behind a bacteriophage T7 promoter, we have, however, been able to overexpress this deformylase in Escherichia coli . Overproduction has allowed the purification of > 50 mg of deformylase enzyme from each liter of cell culture . Purified deformylase is highly active toward N-formylated peptide substrates . A new, sensitive assay for the deformylase has been developed by measuring the amount of released formate using a formate dehydrogenase . This has allowed for the assessment of the catalytic properties of peptide deformylase using a series of synthetic N-formylated peptides as substrates . The deformylase exhibits strong preference for an L-methionine or the isosteric norleucine at the N-terminus of a substrate and has broad specificity for the rest of the residues . Small divalent metal chelators strongly inhibit the E . coli deformylase . In particular, certain 1,2- and 1,3-dithiol compounds act as potent, time-dependent inhibitors of the peptide deformylase.

Nature, 1997 Dec 11, 390(6660), 580 - 6
Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi; Fraser CM et al.; The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs . The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions . Because B . burgdorferi and M . genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors . Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families . The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.

Mol Microbiol, 1997 Nov, 26(3), 469 - 80
The role of ribosomal RNAs in macrolide resistance; Sander P et al.; Macrolides are bacteriostatic antibiotics which interfere with the peptidyltransfer function of the ribosome . We have investigated the molecular mechanisms underlying macrolide resistance in Mycobacterium smegmatis, an eubacterium carrying two rRNA operons . Surprisingly, drug resistance was associated not with alterations in ribosomal proteins, but with a single point mutation in the peptidyltransferase region of one of the two 23S RNA genes, i.e . A2058-->G or A2059-->G . This mutation resulted in a heterozygous organism with a mutated and a wild-type rRNA operon respectively . Reverse transcriptase sequencing indicated the expression of both wild-type and mutated rRNAs . The mutated operon was introduced into genetically engineered rrn- strains of M . smegmatis carrying a single functional rRNA operon and into parental M . smegmatis with two chromosomal rRNA operons, using gene transfer as well as gene replacement techniques . The results obtained demonstrate the dominant nature of resistance . As exemplified in our results on macrolide resistance, a complete set of genetic tools is now available, which allows questions of dominance vs . recessivity and gene dosage effects in eubacterial ribosomal nucleic acids to be addressed experimentally in vivo.

Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 13116 - 21
Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction; Ni X et al.; The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source . Analysis of 5' and 3' deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation . The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation . Single base changes at -10, -12, -32, -33, -35, and -37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction . The sequences centered around -10 (TATTCT) and -35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters . Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence . Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation . Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression . The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion . The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression.

Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 13028 - 33
Determining divergence times with a protein clock: update and reevaluation; Feng DF et al.; A recent study of the divergence times of the major groups of organisms as gauged by amino acid sequence comparison has been expanded and the data have been reanalyzed with a distance measure that corrects for both constraints on amino acid interchange and variation in substitution rate at different sites . Beyond that, the availability of complete genome sequences for several eubacteria and an archaebacterium has had a great impact on the interpretation of certain aspects of the data . Thus, the majority of the archaebacterial sequences are not consistent with currently accepted views of the Tree of Life which cluster the archaebacteria with eukaryotes . Instead, they are either outliers or mixed in with eubacterial orthologs . The simplest resolution of the problem is to postulate that many of these sequences were carried into eukaryotes by early eubacterial endosymbionts about 2 billion years ago, only very shortly after or even coincident with the divergence of eukaryotes and archaebacteria . The strong resemblances of these same enzymes among the major eubacterial groups suggest that the cyanobacteria and Gram-positive and Gram-negative eubacteria also diverged at about this same time, whereas the much greater differences between archaebacterial and eubacterial sequences indicate these two groups may have diverged between 3 and 4 billion years ago.

FEMS Immunol Med Microbiol, 1997 Oct, 19(2), 151 - 7
Simultaneous detection of Streptococcus pneumoniae and Haemophilus influenzae by nested PCR amplification from cerebrospinal fluid samples; Saruta K et al.; Haemophilus influenzae and Streptococcus pneumoniae are often the cause of serious diseases such as meningitis . We designed a nested PCR assay to identify these pathogens from cerebrospinal fluid samples . The first-step PCR was able to detect eubacterial rRNA genes with a unified set of universal primers . In the second-step PCR, the identification primers, HI I and II and SP I and II, could detect H . influenzae and S . pneumoniae respectively through amplification of the rRNA spacer between the 16S and 23S rRNA genes . We suggest that the two-step PCR assay can be used as a novel method for the immediate and retrospective diagnosis of bacterial meningitis caused by H . influenzae and S . pneumoniae.

Microbiology, 1997 Nov, 143 ( Pt 11), 3591 - 8
Characterization of the initiator tRNA gene locus and identification of a strong promoter from Mycobacterium tuberculosis; Vasanthakrishna M et al.; An initiator tRNA gene, metA, and a closely linked fragment of a second initiator-tRNA-like sequence, metB, from Mycobacterium tuberculosis H37Ra have been cloned and characterized . The promoter region of metA shows the presence of conserved sequence elements, TAGCCT and TTGGCG, with resemblance to -10 and -35 promoter regions . The deduced sequence of the mature tRNA contains the three unique features of the eubacterial initiator tRNAs represented by (i) a C:U mismatch at position 1:72, (ii) three consecutive base pairs, 29-31G:C39-41 in the anticodon stem, and (iii) a purine:pyrimidine (A:U) base pair at position 11:24 in the dihydrouridine stem . A putative hairpin structure consisting of an 11 bp stem and a three-base loop found in the 3' flanking region is followed by a stretch of T residues and may serve as a transcription terminator . Analysis of the expression of metA and of its promoter using chloramphenicol acetyltransferase fusion constructs in Mycobacterium smegmatis shows that metA is a functional gene driven by a strong promoter . Furthermore, the overexpressed transcripts are fully processed and formylated in vivo . The metB clone shows the presence of sequences corresponding to those downstream of position 30 of the tRNA . However, the CCA sequence at the 3' end has been mutated to CCG . Interestingly, the 3' flanking sequences of both the genes are rich in GCT repeats . The metB locus also harbours a repeat element, IS6110 . A method to prepare total RNA from mycobacteria (under acidic conditions) to analyse in vivo status of tRNAs is described.

Immunotechnology, 1996 Jun, 2(2), 103 - 13
Immunoreactivity of allergen (Bet v 1) conjugated to crystalline bacterial cell surface layers (S-layers); Jahn-Schmid B et al.; BACKGROUND: Crystalline cell surface layers (S-layers) from Gram-positive eubacteria had been demonstrated as carrier/adjuvants for chemically synthesized tumor-associated oligosaccharide haptens and capsular polysaccharide antigens of Streptococcus pneumoniae strains . OBJECTIVES: The applicability of S-layers as vaccine carrier for treatment of Type I allergy was investigated . STUDY DESIGN: Native or cross-linked S-layer self-assembly products and cell wall preparations from Bacillus sphaericus CCM 2177 and Thermoanaerobacter thermohydrosulfuricus L111-69 and L110-69 were used for immobilization of recombinant major birch pollen allergen Bet v 1 . RESULTS AND CONCLUSIONS: Depending on the carrier used, amounts of approximately 20-40 micrograms allergen per mg conjugate could be immobilized . By application of L-glutamic acid dimethyl ester as a spacer, this value could be increased approximately 10-fold . The functionality of the rBet v 1-conjugates was assessed in immunological systems . (i) The presence of intact B-cell epitopes was demonstrated in inhibition experiments using human Bet v 1-specific IgE . (ii) The rBet v 1-S-layer conjugates were immunogenic in mice . (iii) The proliferation of rBet v 1-specific T-cell clones suggested that the peptides created by processing of immobilized Bet v 1 were similar to those derived from natural allergen . (iv) Stimulation of human allergen-specific TH2 lymphocytes with S-layer-conjugated Bet v 1 led to a modulation of the cytokine production pattern from TH2 to TH0/TH1 . This study indicates that S-layers may be suitable carriers for few immunotherapeutical vaccines for Type 1 hypersensitivity.

Gene, 1997 Oct 1, 198(1-2), 275 - 80
Xenopus HDm, a maternally expressed histone deacetylase, belongs to an ancient family of acetyl-metabolizing enzymes; Ladomery M et al.; Modification of core histones can alter chromatin structure, facilitating the activation and repression of genes . A key example is the acetylation of N-terminal lysines of the core histones . Recently, the mammalian histone deacetylase HD1 was cloned from Jurkat T cells, and shown to be 60% identical to the yeast global gene regulator Rpd3 (Taunton et al., 1996) . Here we report the cloning of HDm, a maternally expressed putative deposition histone deacetylase from Xenopus laevis . Comparison of the amino acid sequences of histone deacetylases from diverse eukaryotes shows high levels of identity within a putative enzyme core region . Further alignment with other types of protein: acetoin-utilizing enzymes from eubacteria; acetylpolyamine hydrolases from mycoplasma and cyanobacteria; and a protein of unknown function from an archaebacterium, reveals an apparently conserved core, and suggests that histone deacetylases belong to an ancient family of enzymes with related functions.

J Urol, 1997 Dec, 158(6), 2291 - 5
Detection of eubacteria in interstitial cystitis by 16S rDNA amplification; Heritz DM et al.; OBJECTIVE: To determine what role non-culturable microorganisms play in the etiology of interstitial cystitis (IC) . MATERIALS AND METHODS: Thirty patients fulfilling NIH criteria for the diagnosis of interstitial cystitis and sixteen control patients with culture negative urine gave written informed consent and underwent bladder biopsy . Polymerase chain reaction (PCR) using two sets of universal primers for bacterial 16S rDNA was performed on urine from the cystoscope and on a cold cup bladder biopsy specimen . Of the PCR positive bladder biopsies, three patients with interstitial cystitis and three controls were randomly selected and cloned . Ten clones from each were sequenced and putative taxonomic assignments made . RESULTS: 12/26 (46%) IC and 5/12 (42%) control urine specimens and 16/30 (53%) and 9/15 (60%) bladder biopsies were PCR positive, respectively . The bacterial populations in the two patient groups tested appeared to be different based upon analysis of the 16S rRNA sequences . CONCLUSIONS: Both IC and control patients had non-culturable bacteria in their bladders . A random sampling of the two populations revealed that the bacterial populations are different, suggesting a possible link between one or more bacterial species and IC.

Science, 1997 Nov 7, 278(5340), 1119 - 22
A euryarchaeal lysyl-tRNA synthetase: resemblance to class I synthetases; Ibba M et al.; The sequencing of euryarchaeal genomes has suggested that the essential protein lysyl-transfer RNA (tRNA) synthetase (LysRS) is absent from such organisms . However, a single 62-kilodalton protein with canonical LysRS activity was purified from Methanococcus maripaludis, and the gene that encodes this protein was cloned . The predicted amino acid sequence of M . maripaludis LysRS is similar to open reading frames of unassigned function in both Methanobacterium thermoautotrophicum and Methanococcus jannaschii but is unrelated to canonical LysRS proteins reported in eubacteria, eukaryotes, and the crenarchaeote Sulfolobus solfataricus . The presence of amino acid motifs characteristic of the Rossmann dinucleotide-binding domain identifies M . maripaludis LysRS as a class I aminoacyl-tRNA synthetase, in contrast to the known examples of this enzyme, which are class II synthetases . These data question the concept that the classification of aminoacyl-tRNA synthetases does not vary throughout living systems.

Infect Immun, 1997 Nov, 65(11), 4515 - 24
Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry; Sonnenberg MG et al.; A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity . However, a complete analysis of the protein composition of this fraction has been lacking . By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M . tuberculosis H37Rv were generated . In total, 205 protein spots were observed . The coupling of this electrophoretic technique with Western blot analysis allowed the identification and mapping of 32 proteins . Further molecular characterization of abundant proteins within this fraction was achieved by N-terminal amino acid sequencing and liquid chromatography-mass spectrometry . Eighteen proteins were subjected to N-group analysis; of these, only 10 could be sequenced by Edman degradation . Among the most interesting were a novel 52-kDa protein demonstrating significant homology to an alpha-hydroxysteroid dehydrogenase of Eubacterium sp . strain VPI 12708, a 25-kDa protein corresponding to open reading frame 28 of the M . tuberculosis cosmid MTCY1A11, and a 31-kDa protein exhibiting an amino acid sequence identical to that of antigen 85A and 85B . This latter product migrated with an isoelectric point between those of antigen 85A and 85C but did not react with the antibody specific for this complex, suggesting that there is a fourth member of the antigen 85 complex . Novel N-terminal amino acid sequences were obtained for three additional culture filtrate proteins; however, these did not yield significant homology to known protein sequences . A protein cluster of 85 to 88 kDa, recognized by the monoclonal antibodies IT-57 and IT-42 and known to react with sera from a large proportion of tuberculosis patients, was refractory to N-group analysis . Nevertheless, mass spectrometry of peptides obtained from one member of this complex identified it as the M . tuberculosis KatG catalase/peroxidase . Thus, the detailed mapping of M . tuberculosis proteins, combined with state-of-the-art analytical techniques such as mass spectrometry, provides a basis for further analysis and rapid identification of biologically relevant molecules.

Appl Environ Microbiol, 1997 Oct, 63(10), 3933 - 40
Characterization of an endosymbiont infecting wood ticks, Dermacentor andersoni, as a member of the genus Francisella; Niebylski ML et al.; A microorganism (Dermacantor andersoni symbiont {DAS}) infecting Rocky Mountain wood ticks (D . andersoni) collected in the Bitterroot Mountains of western Montana was characterized as an endosymbiont belonging to the genus Francisella . Previously described as Wolbachia like, the organism's DNA was amplified from both naturally infected tick ovarial tissues and Vero cell cultures by PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA) and Francisella membrane protein genes . The 16S rDNA gene sequence of the DAS was most similar (95.4%) to that of Francisella tularensis subsp . tularensis . Through a combination of Gimenez staining, PCR assay, and restriction fragment length polymorphism analysis, 102 of 108 female ticks collected from 1992 to 1996 were infected . Transovarial transmission to female progeny was 95.6%, but we found no evidence of horizontal transmission.

Appl Environ Microbiol, 1997 Oct, 63(10), 3926 - 32
Endosymbionts of ticks and their relationship to Wolbachia spp . and tick-borne pathogens of humans and animals; Noda H et al.; The presence, internal distribution, and phylogenetic position of endosymbiotic bacteria from four species of specific-pathogen-free ticks were studied . These included the hard ticks Ixodes scapularis (the black-legged tick), Rhipicephalus sanguineus (the brown dog tick), and Haemaphysalis longicornis and the African soft tick Ornithodoros moubata . PCR assays for bacteria, using two sets of general primers for eubacterial 16S and 23S rRNA genes (rDNAs) and seven sets of specific primers for wolbachial, rickettsial, or Francisella genes, indicated that I . scapularis possessed symbiotic rickettsiae in the ovaries and that the other species harbored eubacteria in both the ovaries and Malpighian tubules . Phylogenetic analysis based on the sequence of 16S rDNA indicated that the symbiont of I . scapularis belonged to the alpha subgroup of proteobacteria and was closely related to the members of the genus Rickettsia . The other species had similar microorganisms in the ovaries and Malpighian tubules, which belonged to the gamma subgroup of proteobacteria, and formed a monophyletic group with the Q-fever pathogen, Coxiella burnetii . O . moubata harbored another symbiont, which formed a monophyletic group with Francisella tularensis and Wolbachia persica, the latter a symbiont previously isolated from Malpighian tubules of the soft tick Argas (Persicargas) arboreus . Thus, the symbionts of these four tick species were not related to the Wolbachia species found in insects . The two symbionts that live in the Malpighian tubules, one closely related to C . burnetii and the other closely related to F . tularensis, appear to be of ancient origin and be widely distributed in ticks.

Appl Environ Microbiol, 1997 Oct, 63(10), 3895 - 901
Seasonal changes in the relative abundance of uncultivated sulfate-reducing bacteria in a salt marsh sediment and in the rhizosphere of Spartina alterniflora; Rooney-Varga JN et al.; Phylogenetic diversity and community composition of sulfate-reducing bacteria in a salt marsh sediment and in the rhizosphere of Spartina alterniflora were investigated . Uncultivated Desulfobacteriaceae family-related phylotypes were studied by selectively amplifying 16S rRNA gene fragments from DNA extracted from salt marsh rhizosphere samples . Two novel phylotypes were retrieved from rhizosphere samples, with A01 having 89.1% sequence similarity with Desulfococcus multivorans and 4D19 having 96.3% sequence similarity with Desulfosarcina variabilis . Additionally, six sequences that were extremely closely related to Desulfococcus multivorans (99% sequence similarity) were found . Reference RNAs containing sequences identical to corresponding cloned regions of A01 or 4D19 16S rRNA were synthesized via in vitro transcription and were used in subsequent quantitative membrane hybridization experiments . Oligonucleotide probes A01-183 and 4D19-189 were designed to specifically target these two novel phylotypes and were tested for target specificity against synthesized RNA and reference RNAs extracted from pure cultures . The newly designed probes were then used, together with eubacterial probes, to determine the relative abundances of the novel phylotypes in the salt marsh sediment and the rhizosphere . Mean relative abundances of A01-183 and 4D19-189 targets were 7.5 and 3.4%, respectively, suggesting that the target organisms of A01-183 and, to a lesser extent, of 4D19-189 play an important role in the salt marsh sediment and the Spartina rhizosphere . A seasonal trend of increased A01 relative abundance during the period of vegetative plant growth was evident, suggesting a close interaction between A01 and S . alterniflora.

Mol Cell Biol, 1997 Nov, 17(11), 6465 - 71
Characterization of a Holliday junction-resolving enzyme from Schizosaccharomyces pombe; White MF et al.; The rearrangement and repair of DNA by homologous recombination involves the creation of Holliday junctions, which are cleaved by a class of junction-specific endonucleases to generate recombinant duplex DNA products . Only two cellular junction-resolving enzymes have been identified to date: RuvC in eubacteria and CCE1 from Saccharomyces cerevisiae mitochondria . We have identified a protein from Schizosaccharomyces pombe which has 28% sequence identity to CCE1 . The YDC2 protein has been cloned and overexpressed in Escherichia coli, and the purified recombinant protein has been shown to be a Holliday junction-resolving enzyme . YDC2 has a high degree of specificity for the structure of the four-way junction, to which it binds as a dimer . The enzyme exhibits a sequence specificity for junction cleavage that differs from both CCE1 and RuvC, and it cleaves fixed junctions at the point of strand exchange . The conservation of the mechanism of Holliday junction cleavage between two organisms as diverse as S . cerevisiae and S . pombe suggests that there may be a common pathway for mitochondrial homologous recombination in fungi, plants, protists, and possibly higher eukaryotes.

Annu Rev Microbiol, 1997, 51, 629 - 59
Genetics of eubacterial carotenoid biosynthesis: a colorful tale; Armstrong GA; Carotenoids represent one of the most widely distributed and structurally diverse classes of natural pigments, with important functions in photosynthesis, nutrition, and protection against photooxidative damage . In the eubacterial community, yellow, orange, and red carotenoids are produced by anoxygenic photosynthetic bacteria, cyanobacteria, and certain species of nonphotosynthetic bacteria . Many eukaryotes, including all algae and plants, as well as some fungi, also synthesize these pigments . In noncarotenogenic organisms, such as mammals, birds, amphibians, fish, crustaceans, and insects, dietary carotenoids and their metabolites also serve important biological roles . Within the last decade, major advances have been made in the elucidation of the molecular genetics, the biochemistry, and the regulation of eubacterial carotenoid biosynthesis . These developments have important implications for eukaryotes, and they make increasingly attractive the genetic manipulation of carotenoid content for biotechnological purposes.

Plant Physiol, 1997 Oct, 115(2), 501 - 10
Sequence analysis of the cloned glossy8 gene of maize suggests that it may code for a beta-ketoacyl reductase required for the biosynthesis of cuticular waxes; Xu X et al.; The gl8 locus of maize (Zea mays L.) was previously defined by a mutation that reduces the amount and alters the composition of seedling cuticular waxes . Sixty independently derived gl8 mutant alleles were isolated from stocks that carried the Mutator transposon system . A DNA fragment that contains a Mu8 transposon and that co-segregates with one of these alleles, gl8-Mu3142, was identified and cloned . DNA flanking the Mu8 transposon was shown via allelic cross-referencing experiments to represent the gl8 locus . The gl8 probe revealed a 1.4-kb transcript present in wild-type seedling leaves and, in lesser amounts, in other organs and at other developmental stages . The amino acid sequence deduced from an apparently full-length gl8 cDNA exhibits highly significant sequence similarity to a group of enzymes from plants, eubacteria, and mammals that catalyzes the reduction of ketones . This finding suggests that the GL8 protein probably functions as a reductase during fatty acid elongation in the cuticular wax biosynthetic pathway.

J Mol Evol, 1997 Nov, 45(5), 549 - 63
The evolution of the conserved ATPase domain (CAD): reconstructing the history of an ancient protein module; Swaffield JC et al.; The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes . The AAA proteins are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD . Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete groups that are related not only in structure but in cellular function . Evolutionary analyses also indicate that the CAD was present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes . The eubacterial CADs are found in metalloproteases, while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory, NSF/Sec, and Pas proteins . The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change that characterize the AAA proteins.

J Mol Evol, 1997 Nov, 45(5), 535 - 48
Molecular evolution of the photolyase-blue-light photoreceptor family; Kanai S et al.; The photolyase-blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors . CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively . CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity . Blue-light photoreceptors are essential light detectors for the early development of plants . The amino acid sequence of the receptor is similar to those of the photolyases, although the receptor does not show the activity of photoreactivation . To investigate the functional divergence of the family, the amino acid sequences of the proteins were aligned . The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases (class I photolyases) are different from those of class II CPD photolyases (class II photolyases) . We reconstructed the phylogenetic trees based on the alignment by the NJ method and the ML method . The phylogenetic analysis suggested that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11819 - 26
Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation; Curnow AW et al.; The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned . Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity . The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles . Disruption of this operon is lethal . This demonstrates that transamidation is the only pathway to Gln-tRNAGln in B . subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential component of the translational apparatus.

Gene, 1997 Sep 15, 197(1-2), 37 - 45
Cloning and characterization of a new alpha-amylase gene from Streptomyces lividans TK24; Yin XH et al.; Streptomyces lividans TK24 possesses a very weak amylolytic activity, nevertheless Southern blot analysis carried out at high stringency revealed that this strain does contain a gene strongly related to the well expressed alpha-amylase gene (amlSL) of Streptomyces limosus . To clone this related gene, three genomic banks of S . lividans TK24 were constructed into the multicopy plasmid vector pIJ699 and transformed into the same strain . Two different genes were isolated . One (amlA) has been previously described, whereas the other (amlB) has never been described . Sub-cloning experiments localized amlB to a 3 kb BamHI-NotI fragment that was sequenced . Frame analysis on sequence data revealed the presence of a 1719 bp long open reading frame encoding a 573 amino acid protein of 61214 kDa . Northern blot analysis identified a unique 1.8 kb monocistronic transcript . Primer extension allowed the localization of the transcription start point 108 bp upstream of the translational start codon and demonstrated that the gene was transcribed from a unique typical eubacterial-like promoter . AmlB shares 74.7% amino acid identity with the alpha-amylase of S . limosus and only 27.2% with the amylolytic enzyme encoded by amlA.

Infect Immun, 1997 Oct, 65(10), 4322 - 9
Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines; Brenner C et al.; Mycoplasmas are bacteria which can cause respiratory, arthritic, and urogenital diseases . During the early phase of infection, mycoplasmas usually induce an inflammatory response and a humoral response preferentially directed against their membrane-bound, surface-exposed lipoproteins . In this report, we describe the effects on immune cells of spiralin, a well-characterized mycoplasmal lipoprotein . Purified spiralin stimulated the in vitro proliferation of human peripheral blood mononuclear cells and murine splenocytes . The stimulation pathway was probably different from that followed by Escherichia coli lipopolysaccharide because the effect of spiralin was not abolished by polymyxin B . Comparison of the effects of whole, native spiralin with those induced by proteinase K-digested spiralin or by the C-terminal half of spiralin (peptide p{13.5}T) revealed that the first half of the protein, which contains the lipoylated N terminus, is responsible for the mitogenic activity . In contrast to whole spiralin, proteinase K-digested spiralin did not trigger murine B-cell differentiation and immunoglobulin G and M secretion . Stimulation of human or murine immune cells led to early secretion of proinflammatory cytokines (human tumor necrosis factor alpha and murine interleukin 1 or 6) . Spiralin induced the T-cell-independent blastogenesis of murine B cells but did not stimulate T cells . Altogether, our data demonstrate that spiralin possesses potent immunostimulating activity, similar to that reported for lipoproteins of pathogenic gracilicutes (gram-negative eubacteria; e.g., Borrelia burgdorferi OspA and E . coli Braun lipoprotein), and are consistent with the fact that lipoproteins are major antigens during mycoplasma infections.

FEMS Microbiol Lett, 1997 Sep 15, 154(2), 173 - 80
Identification and sequence analysis of Sulfolobus solfataricus purE and purK genes; Sorensen IS et al.; From a genomic library of Sulfolobus solfataricus DSM1617 we have isolated and identified the purEK locus . Two open reading frames are identified as homologs of the purE and purK purine biosynthetic genes in Escherichia coli . The C-terminus of purE overlaps with the N-terminus of purK . When either of the genes is expressed from an E . coli promoter they can complement the corresponding purE and purK mutations in E . coli . PurE seems to be more closely related to eubacteria than to other archaea and to eukaryotes . Also the purK gene, which has not yet been found in other archaea, is more closely related to eubacteria than to eukaryotes.

J Membr Biol, 1997 Sep 1, 159(1), 1 - 20
Membrane topology motifs in the SGLT cotransporter family; Turk E et al.; Homologues of the Na+/glucose cotransporter, the SGLT family, include sequences of mammalian, eubacterial, yeast, insect and nematode origin . The cotransported substrates are sugars, inositol, proline, pantothenate, iodide, urea and undetermined solutes . It is reasonable to expect that the SGLT family members share a similar or identical topology of membrane spanning elements, by virtue of their common ancestry and similar coupling of solute transport to downhill sodium flux . Here we examine their membrane topologies as deduced from diverse analyses of their primary sequences, and from their sequence correlations with the experimentally determined topology of the human Na+/ glucose contransporter SGLT1 . Our analyses indicate that all family members share a common core of 13 transmembrane helices, but that some, like SGLT1 itself, have one additional span appended to the C-terminus, and still others, two . One bacterial member incorporates an additional span at the N-terminus . Sequence comparisons indicative of common ancestry of the SGLT and the {Na+ + Cl-} transporter families are introduced, and evaluated in light of their topologies . New evidence concerning the previously asserted common ancestry of SGLT1 and an N-acetylglucosamine permease of the bacterial phosphotransferase system is considered . Finally, we analyze observations which lead us to conjecture that the experimental strategy most commonly employed to reveal the topology of bacterial transporters (i.e., the fusion of reporter enzymes such as phoA alkaline phosphatase, beta-lactamase or beta-galactosidase, to progressively C-truncated fragments of the transporter) has often instead so perturbed local topology as to have entirely missed pairs of adjacent membrane spans.

Curr Genet, 1997 Jul, 32(1), 1 - 18
The evolution of the Calvin cycle from prokaryotic to eukaryotic chromosomes: a case study of functional redundancy in ancient pathways through endosymbiosis; Martin W et al.; The evolutionary histories of the 12 enzymes that catalyze the reactions of the Calvin cycle in higher-plant chloroplasts are summarized . They are shown to be encoded by a mixture of nuclear genes of cyanobacterial and proteobacterial origin . Moreover, where cytosolic isoforms of these enzymes are found they are almost invariably encoded by genes of clearly endosymbiont origin . We infer that endosymbiosis resulted in functional redundancy that was eliminated through differential gene loss, with intruding eubacterial genes repeatedly replacing pre-existing nuclear counterparts to which they were either functionally or structurally homologous . Our findings fail to support the 'product-specificity corollary', which predicts re-targeting of nuclear-encoded gene products to the organelle from whose genome they originated . Rather it would appear that the enzymes of central carbohydrate metabolism have evolved novel targeting possibilities regardless of their origins . Our findings suggest a new hypothesis to explain organelle genome persistence, based on the testable idea that some organelle-encoded gene products might be toxic when present in the cytosol or other inappropriate cellular compartments.

J Biol Chem, 1997 Sep 26, 272(39), 24137 - 40
Tethering of the large subunits of Escherichia coli RNA polymerase; Severinov K et al.; The rpoB and rpoC genes of eubacteria and archaea, coding, respectively, for the beta and beta'-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC . Here, we show that in Escherichia coli the two genes can be fused and that the resulting 2751-amino acid beta::beta' fusion polypeptide assembles into functional RNA polymerase in vivo and in vitro . The results establish that the C terminus of the beta subunit and the N terminus of the beta' subunit are in close proximity to each other on the surface of the assembled RNA polymerase during all phases of the transcription cycle and also suggest that RNA polymerase assembly in vivo may occur co-translationally.

J Mol Evol, 1997 Sep, 45(3), 271 - 7
Taxonomy of 5 S ribosomal RNA by the linguistic technique: probing with mitochondrial and mammalian sequences; Guimaraes RC et al.; Linguistic similarities and dissimilarities between 5 S rRNA sequences allowed taxonomical separation of species and classes . Comparisons with the molecule from mammals distinguished fungi and plants from protists and animals . Similarities to mammalians progressively increased from protists to invertebrates and to somatic-type molecules of the vertebrates lineage . In this, deviations were detected in avian, oocyte type, and pseudogene sequences . Among bacteria, actinobacteria were most similar to the mammalians, which could be related to the high frequency of associations among members of these groups . Some archaebacterial species most similar to the mammalians belonged to the Thermoproteales and Halobacteria groups . Comparisons with the soybean mitochondrial molecule revealed high internal homogeneity among plant mitochondria . The eubacterial groups most similar to it were Thermus and Rhodobacteria gamma-1 and alpha-2 . Other procedures have already indicated similarities of Rhodobacteria alpha to mitochondria but the linguistic similarities were on the average higher with the first two groups.

Arch Dis Child, 1997 Aug, 77(2), 148 - 9
Eubacterial approach to the diagnosis of bacterial infection; Ley BE et al.; Computed tomography revealed a frontal empyema in an 8 month old boy who presented with a prolonged convulsion . Before admission he had received oral antibiotics and all specimens were negative on culture . The presence of bacteria in cerebrospinal and empyema fluid was demonstrated using the polymerase chain reaction (PCR) with 16S rRNA gene primers . A presumptive identification of Streptococcus pneumoniae was made by comparison of PCR products with those derived from known bacteria using denaturing gradient gel electrophoresis.

Gene, 1997 Aug 11, 195(1), 73 - 9
Organization of a large gene cluster encoding ribosomal proteins in the cyanobacterium Synechococcus sp . strain PCC 6301: comparison of gene clusters among cyanobacteria, eubacteria and chloroplast genomes; Sugita M et al.; The structure of a large gene cluster containing 22 ribosomal protein (r-protein) genes of the cyanobacterium Synechococcus sp . strain PCC6301 is presented . Based on DNA and protein sequence analyses, genes encoding r-proteins L3, L4, L23, L2, S19, L22, S3, L16, L29, S17, L14, L24, L5, S8, L6, L18, S5, L15, L36, S13, S11, L17, SecY, adenylate kinase (AK) and the alpha subunit of RNA polymerase were identified . The gene order is similar to that of the E . coli S10, spc and alpha operons . Unlike the corresponding E . coli operons, the genes for r-proteins S4, S10, S14 and L30 are not present in this cluster . The organization of Synechococcus r-protein genes also resembles that of chloroplast (cp) r-protein genes of red and brown algal species . This strongly supports the endosymbiotic theory that the cp genome evolved from an ancient photosynthetic bacterium.

Biochem Biophys Res Commun, 1997 Sep 18, 238(2), 370 - 6
Protein kinase associated with ribosomes phosphorylates ribosomal proteins of Streptomyces collinus; Mikulik K et al.; Protein kinase activity associated with ribosomes of a kirromycin-producing strain of Streptomyces collinus was detected . The enzyme utilizes {gamma-32P}ATP to phosphorylate proteins, yielding acid-stable phosphoamino acids . Two-dimensional electrophoresis of proteins from a crude ribosomal fraction revealed 17 phosphoproteins . Eleven of the phosphoproteins exhibited electrophoretic mobility identical to that of S . collinus ribosomal proteins S3, S4, S12, S13, S14, S18, L2, L7, L16, L17, and L23 . Protein L2 was identified by microsequencing of internal peptide fragments . Immunodetection with monoclonal antibodies indicated that the ribosomal proteins are phosphorylated on serine and threonine residues . Phosphorylation of ribosomal proteins led to the reduction of activity of ribosomes in the translation of poly(U) . These results provide the first evidence of phosphorylation of ribosomal proteins in bacteriophage-uninfected cells of eubacteria .

Electrophoresis, 1997 Aug, 18(8), 1207 - 16
Strategies for whole microbial genome sequencing and analysis; Fraser CM et al.; The introduction of methods for automated DNA sequence analysis nearly a decade ago, together with more recent advances in the field of bioinformatics, have revolutionized biology and medicine and have ushered in a new era of genomic science, the study of genes and genomes . These new technologies have had an impact on many areas of research, including the association between genes and disease, in DNA-based diagnostics, and in the sequencing of genomes from human and other model organisms . The demonstration in 1995, that automated DNA sequencing methods could be used to decipher the entire genome sequence of a free-living organism, Haemophilus influenzae, was a milestone in both the genomics and microbial fields {1} . Since the first report of the complete sequence of H . influenzae, these methodologies have been adopted by laboratories around the world . The complete genomic sequence of five eubacterial species {1-5}, one archaea {6}, and the eukaryote, Saccharomyces cerevisiae {7}, have been reported in the last 18 months . At the beginning of 1997 more than a dozen microbial genome projects are at or near completion, with many others in progress . It is likely that in the next few years we will see the complete sequence of perhaps as many as 30-40 microbial genomes . In this article, we will review methods for whole genome sequencing and analysis and examine how this information can be exploited to better understand microbial physiology and evolution.

Ann Pharmacother, 1997 Sep, 31(9), 999 - 1002
Clarithromycin-induced digoxin intoxication; Laberge P et al.; OBJECTIVE: To report a case of clarithromycin-induced digoxin intoxication . CASE SUMMARY: A 78-year-old white man with ischemic cardiomyopathy and chronic renal insufficiency was admitted 4 days after being prescribed clarithromycin for a suspected episode of bronchitis . He reported weakness, asthenia, and gastrointestinal symptoms; the digoxin serum concentration was measured at 3.89 ng/mL . The patient recovered uneventfully after digoxin and clarithromycin were discontinued . DISCUSSION: Erythromycin frequently interacts with other drugs that are also metabolized by the CYP3A4 isoenzyme . However, erythromycin is hypothesized to interact with digoxin by inhibiting Eubacterium lentum, which is a normal inhabitant of the human gut and is responsible for intestinal metabolism of digoxin in 10% of patients . Since clarithromycin shares a comparable antibacterial spectrum with erythromycin, the possibility of a drug interaction with digoxin remains . Only four cases of clarithromycin interacting with digoxin have been reported to date . Clinically, this interaction may have been more obvious because of our patient's moderate renal dysfunction and serum digoxin concentrations in the upper therapeutic range prior to clarithromycin initiation . Other causes for digoxin intoxication could not be identified . CONCLUSIONS: Clarithromycin may inhibit the growth of E . lentum, which can lead to an increase in digoxin bioavailability and blood concentrations in patients in whom this intestinal metabolic pathway is present . Patients at risk include those with renal dysfunction, with serum concentrations in the upper therapeutic range, or with measured digoxin concentrations that are much lower than predicted by pharmacokinetic calculations . For these patients, appropriate therapy includes the selection of an alternative, noninteracting antibiotic or, if this is not possible, a temporary reduction of digoxin dosage.

J Bacteriol, 1997 Sep, 179(18), 5720 - 7
Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components; Jiang ZY et al.; The chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum contains five open reading frames (ORFs) that have significant sequence homology to chemotaxis genes from other bacteria . To elucidate the functions of each ORF, we have made various mutations in the gene cluster and analyzed their phenotypic defects . Deletion of the entire che operon (delta che), as well as nonpolar disruptions of cheAY, cheW, and cheR, resulted in a smooth-swimming phenotype, whereas disruption of cheB resulted in a locked tumbly phenotype . Each of these mutants was defective in chemotactic response . Interestingly, disruption of cheY resulted in a slight increase in the frequency of tumbling/reversal with no obvious defects in chemotactic response . In contrast to observations with Escherichia coli and several other bacteria, we found that all of the che mutant cells were capable of differentiating into hyperflagellated swarmer cells when plated on a solid agar surface . When viewed microscopically, the smooth-swimming che mutants exhibited active surface motility but were unable to respond to a step-down in light intensity . Both positive and negative phototactic responses were abolished in all che mutants, including the cheY mutant . These results indicate that eubacterial photosensory perception is mediated by light-generated signals that are transmitted through the chemotaxis signal transduction cascade.

J Med Microbiol, 1997 Sep, 46(9), 773 - 8
Specific information concerning taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by a multiplex PCR; Schmitz FJ et al.; The use of DNA amplification techniques such as the polymerase chain reaction (PCR) in modern diagnostic microbiology not only allows the sensitive and specific identification of micro-organisms but also the detection of specific antibiotic resistance genes . This study describes a multiplex PCR on bacterial colonies picked directly from agar plates without preceding DNA preparation . Eubacteria and staphylococci were identified by, 16S rRNA specific PCR products . In parallel, specific primers were used for the detection of staphylococcal coa and mecA genes . This 4-h multiplex PCR, consisting of four sets of primers, was evaluated for rapid and specific differential diagnosis of methicillin-resistant and methicillin-susceptible strains of Staphylococcus aureus and coagulase-negative staphylococci . To analyse specificity of the amplification products, 100 non-staphylococcal, eubacterial isolates and 20 Candida albicans strains were tested . In a first step, specificity of all four single sets of primers was evaluated before the co-amplification within the multiplex PCR procedure was performed . The results were compared with those of conventional susceptibility and typing methods . The specific 16S rRNA PCR product for eubacterial isolates (n = 786) and staphylococci (686) was found in all strains tested . The coa gene was detected only in S . aureus (488) strains with a specificity of 100%, and was not detected in any of the coagulase-negative staphylococci (198) . The mecA gene was detected in 98% of methicillin-resistant staphylococci (393) and in 2% of all methicillin-susceptible staphylococci (293) . The multiplex PCR with co-amplification of different determinants provides rapid reliable information on staphylococcal identification and methicillin susceptibility supporting the diagnosis, treatment and control of staphylococcal infections.

Biochem J, 1997 Sep 1, 326 ( Pt 2), 449 - 54
Biosynthesis of the labdane diterpene marrubiin in Marrubium vulgare via a non-mevalonate pathway; Knoss W et al.; The biosynthesis of the furanic labdane diterpene marrubiin has been studied in plantlets and shoot cultures of Marrubium vulgare (Lamiaceae) . The use of {2-14C}acetate, {2-14C}pyruvate, {2-14C}mevalonic acid and {U-14C}glucose incorporation experiments showed that the labelling of sterols in etiolated shoot cultures of M . vulgare was in accordance with their biosynthesis via the acetate-mevalonate pathway . In contrast, the incorporation rates of these precursors into the diterpene marrubiin could not be explained by biosynthesis of this compound via the acetate-mevalonate pathway . Cultivation of etiolated shoot cultures of M . vulgare on medium containing {1-13C}glucose and subsequent 13C-NMR spectroscopy of marrubiin led to the conclusion that the biosynthesis of marrubiin follows a non-mevalonate pathway . All isoprenic units of 13C-labelled marrubiin were enriched in those carbons that correspond to positions 1 and 5 of a putative precursor isopentenyl diphosphate . This labelling pattern from {1-13C}glucose is consistent with an alternative pathway via trioses, which has already been shown to occur in Eubacteria and Gymnospermae . The labdane skeleton is a precursor of many other skeletal types of diterpenes . Therefore it becomes obvious that in connection with the few known examples of a non-mevalonate pathway to isoprenoids the formation of some isoprenoids in plants via a non-mevalonate pathway might be quite common.

Nucleic Acids Res, 1997 Sep 15, 25(18), 3693 - 7
Histone deacetylases, acetoin utilization proteins and acetylpolyamine amidohydrolases are members of an ancient protein superfamily; Leipe DD et al.; Searches of several sequence databases reveal that human HD1, yeast HDA1, yeast RPD3 and other eukaryotic histone deacetylases share nine motifs with archaeal and eubacterial enzymes, including acetoin utilization protein (acuC) and acetylpolyamine amidohydrolase . Histone deacetylase and acetylpolyamine amidohydrolase also share profound functional similarities in that both: (i) recognize an acetylated aminoalkyl group; (ii) catalyze the removal of the acetyl group by cleaving an amide bond; (iii) increase the positive charge of the substrate . Stabilization of nucleosomal DNA-histone interaction brought about by the change in charge has been implicated as the underlying cause for histone deacetylase-mediated transcriptional repression . We speculate that the eukaryotic histone deacetylases originated from a prokaryotic enzyme similar to the acetylpolyamine amidohydrolases that relied on reversible acetylation and deacetylation of the aminoalkyl group of a DNA binding molecule to achieve a gene regulatory effect.

Mol Mar Biol Biotechnol, 1997 Sep, 6(3), 180 - 8
Sulfur-oxidizing symbionts have not co-evolved with their hydrothermal vent tube worm hosts: an RFLP analysis; Laue BE et al.; A fine-scale phylogenetic comparison was made among the symbionts of different genera of hydrothermal vent tube worms . These included Riftia pachyptila and Tevnia jerichonona, which inhabit sites along the east Pacific Rise, and Ridgeia piscesae from the Juan de Fuca Ridge . An analysis of restriction fragment length polymorphism (RFLP) was employed using three symbiont-specific gene probes: eubacterial 16S rRNA, RuBPC/O Form II, and ATP sulfurylase (recently cloned from the Riftia symbiont) . Results indicated that all of the symbionts from the three different hosts were conspecific and the Riftia and Tevnia symbionts were indistinguishable over and 1800-km range . Significantly, this indicates that the symbionts have not co-evolved with their respective hosts, which are known to belong to separate families . This study strongly supports the conclusion that the symbionts are acquired de novo by each generation of juvenile tube worms from a common source in the surrounding sea water.

Nucleic Acids Res, 1997 Sep 1, 25(17), 3478 - 85
Nucleoprotein complex formation by the enhancer binding protein nifA; Wang XY et al.; The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme . Binding sites for NifA, upstream activator sequences (UASs), are remotely located . Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF) . We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS in the presence and absence of holoenzyme and IHF . Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates . Results establish that the consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA . Reactivity suggests that each UAS thymidine is not structurally equivalent . Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter . Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact . Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion . Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation . Since T-9 is melted in open complexes the closed complex appears poised for melting . Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 8928 - 35
Considerations of transcriptional control mechanisms: do TFIID-core promoter complexes recapitulate nucleosome-like functions?
Hoffmann A, Oelgeschlager T, Roeder RG.
The general transcription initiation factor TFIID was originally identified, purified, and characterized with a biochemical assay in which accurate transcription initiation is reconstituted with multiple, chromatographically separable activities . Biochemical analyses have demonstrated that TFIID is a multiprotein complex that directs preinitiation complex assembly on both TATA box-containing and TATA-less promoters, and some TFIID subunits have been shown to be molecular targets for activation domains in DNA-binding regulatory proteins . These findings have most commonly been interpreted to support the view that transcriptional activation by upstream factors is the result of enhanced TFIID recruitment to the core promoter . Recent insights into the architecture and cell-cycle regulation of the multiprotein TFIID complex prompt both a reassessment of the functional role of TFIID in gene activation and a review of some of the less well-appreciated literature on TFIID . We present a speculative model for diverse functional roles of TFIID in the cell, explore the merits of the model in the context of published data, and suggest experimental approaches to resolve unanswered questions . Finally, we point out how the proposed functional roles of TFIID in eukaryotic class II transcription fit into a model for promoter recognition and activation that applies to both eubacteria and eukaryotes.

FEBS Lett, 1997 Aug 18, 413(2), 309 - 13
Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana: evidence for the sigma factor heterogeneity in higher plant plastids; Tanaka K et al.; By database search analysis, we identified three Arabidopsis EST (Expression Sequence Tag) entries having similarity to eubacterial RNA polymerase sigma factors . cDNA clones corresponding to these partial sequences were isolated, and the complete nucleotide sequences were determined . All three sequences encode proteins highly homologous to cyanobacterial and plastid sigma factors, and the gene products have N-terminal extensions which are assumed to function as plastid-targeting transit peptides . Thus we have concluded that the gene products are RNA polymerase sigma factors of plastids, and named sigA, sigB and sigC, respectively . Expression of these genes was analyzed by RNA gel-blot analysis and shown to be induced by illumination after a short-term dark adaptation . This strongly suggests that light regulation of the nuclear encoded sigma factor genes is involved in light-dependent activation of plastid promoters.

J Biol Chem, 1997 Aug 8, 272(32), 19731 - 7
Tryptophan fluorescence reports nucleotide-induced conformational changes in a domain of the ArsA ATPase; Zhou T et al.; The ars operon of plasmid R773 encodes an ATP-dependent extrusion pump for arsenite and antimonite in Escherichia coli . The ArsA ATPase is the catalytic subunit of the pump protein, with two nucleotide binding consensus sequences, one in the NH2-terminal half and one in the COOH-terminal half of the protein . A 12-residue consensus sequence (DTAPTGHTIRLL) has been identified in ArsA homologs from eubacteria, archebacteria, fungi, plants, and animals . ArsA enzymes were constructed containing single tryptophan residues at either end of this conserved sequence . The emission spectrum of the fluorescence of the tryptophan on the COOH-terminal end (Trp-159) indicated a relatively hydrophilic environment for this residue . An increase in intrinsic tryptophan fluorescence and a blue shift of the maximum emission wavelength were observed upon addition of MgATP, indicating movement of Trp-159 into a relatively less polar environment . No fluorescence response was observed with MgADP, with nonhydrolyzable ATP analogs, or with MgATP by catalytically inactive enyzmes . This suggests that the location Trp-159 is shifted only during hydrolysis of ATP . In contrast, the emission spectrum of Trp-141, located on the NH2-terminal side of the consensus sequence, indicated a relatively nonpolar environment . The maximum emission wavelength red shifted upon addition of MgADP . MgATP slowly produced a response that correlated with product formation, suggesting that the environment of Trp-141 is sensitive only to MgADP binding . Thus, during ATP hydrolysis the COOH-terminal end of the conserved domain moves into a less polar environment, whereas the NH2-terminal end moves into a more hydrophilic environment as product is formed . A hypothesis is presented in which the conserved domain of ArsA and homologs is an energy transduction domain involved in transmission of the energy of ATP hydrolysis to biological functions such as transport.

Science, 1997 Aug 8, 277(5327), 809 - 11
Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis; Hedtke B et al.; In addition to the RNA polymerases (RNAPs) transcribing the nuclear genes, eukaryotic cells also require RNAPs to transcribe the genes of the mitochondrial genome and, in plants, of the chloroplast genome . The plant Arabidopsis thaliana was found to contain two nuclear genes similar to genes encoding the mitochondrial RNAP from yeast and RNAPs of bacteriophages T7, T3, and SP6 . The putative transit peptides of the two polymerases were capable of targeting fusion proteins to mitochondria and chloroplasts, respectively, in vitro . The results indicate that the mitochondrial RNAP in plants is a bacteriophage-type enzyme . A gene duplication event may have generated the second RNAP, which along with the plastid-encoded eubacteria-like RNAP could transcribe the chloroplast genome.

Comput Appl Biosci, 1997 Aug, 13(4), 431 - 8
Micado--a network-oriented database for microbial genomes; Biaudet V et al.; MOTIVATION: We created Micado, a database for managing genomic information, as part of the Bacillus subtilis genome programs . Its content will be progressively extended to the whole microbial world . RESULTS: A relational schema is defined for selective queries . It links eubacterial and archaeal sequences, genetic maps for Bacillus subtilis and Escherichia coli, and information on mutants . The latter comes from a new functional analysis project of unknown genes in B subtilis, and the database allows the community to curate information . To help queries from users, a graphical interface is built on SQL access to the database and provided through the WWW . We have automated imports of microbial sequences, and E . coli genetic map, by programming parsers of flat file distributions . These ensure smooth updates from molecular biology repositories on the Internet . Hyperlinks are created as a complement, to reference other general and specialized related information resources.

RNA, 1997 Aug, 3(8), 815 - 20
Interaction of thiostrepton with an RNA fragment derived from the plastid-encoded ribosomal RNA of the malaria parasite; Rogers MJ et al.; Although eukaryotes are not generally sensitive to thiostrepton, growth of the human malaria parasite Plasmodium falciparum is severely inhibited by the drug . The proposed target in P . falciparum is the ribosome of the plastid-like organelle (35 kb circular genome) of unknown function . Positive identification of the drug target would confirm that the organelle is essential for blood-stage development of Plasmodium and help clarify the plastid's biological role . The action of thiostrepton as an antibiotic relates to its affinity for a conserved domain of eubacterial rRNA . Its effect on organelles is unknown . Because a number of different point mutations within the Escherichia coli domain abrogates thiostrepton binding, extensive sequence differences between eubacterial and plastid domains brings into question the site of drug action . We have examined temperature-dependent hyperchromicity profiles of synthetic RNAs corresponding to domains in the plastid and cytoplasmic RNAs of P . falciparum . Thiostrepton induces a tertiary structure in the plastid-like fragment similar to that seen in eubacterial rRNA, even though the two share only about 60% sequence identity . A single point mutation in the plastid-like fragment removes thiostrepton-dependent tertiary structure formation . Thus, the plastid and eubacterial RNAs share a stabilized tertiary structure induced by the drug . This direct indicator of drug sensitivity in eubacteria suggests that the plastid-encoded ribosome is similarly sensitive to thiostrepton and that the plastid is the site of drug action . Correlation of thiostrepton-sensitive and -resistant phenotypes with physical parameters suggests thiostrepton resistance as a selectable marker for plastid transformation.

J Mol Evol, 1997 Aug, 45(2), 193 - 205
The sequences of heat shock protein 40 (DnaJ) homologs provide evidence for a close evolutionary relationship between the Deinococcus-thermus group and cyanobacteria; Bustard K et al.; The genes encoding for heat shock protein 40 (Hsp40 or DnaJ) homologs were cloned and sequenced from the archaebacterium Halobacterium cutirubrum and the eubacterium Deinococcus proteolyticus to add to sequences from the gene banks . These genes were identified downstream of the Hsp70 (or DnaK) genes in genomic fragments spanning this region and, as in other prokaryotic species, Hsp70-Hsp40 genes are likely part of the same operon . The Hsp40 homolog from D . proteolyticus was found to be lacking a central 204 base pair region present in H . cutirubrum that encodes for the four cysteine-rich domains of the repeat consensus sequence CxxCxGxG (where x is any amino acid), present in most Hsp40 homologs . The available sequences from various archaebacteria, eubacteria, and eukaryotes show that the same deletion is also present in the homologs from Thermus aquaticus and two cyanobacteria, but in no other species tested . This unique deletion and the clustering of homologs from the Deinococcus-Thermus group and cyanobacterial species in the Hsp40 phylogenetic trees suggest a close evolutionary relationship between these groups as was also shown recently for Hsp70 sequences (R.S . Gupta et al., J Bacteriol 179:345-357, 1997) . Sequence comparisons indicate that the Hsp40 homologs are not as conserved as the Hsp70 sequences . Phylogenetic analysis provides no reliable information concerning evolutionary relationship between prokaryotes and eukaryotes and their usefulness in this regard is limited . However, in phylogenetic trees based on Hsp40 sequences, the two archaebacterial homologs showed a polyphyletic branching within Gram-positive bacteria, similar to that seen with Hsp70 sequences.

J Clin Microbiol, 1997 Aug, 35(8), 2087 - 92
PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species; Sumner JW et al.; Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii . The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria . A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences . Nucleotide sequences obtained from clinical samples containing E . equi, E . phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical . Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E . equi and the HGE agent) and sequences from the European species, E . phagocytophila . A single pair of PCR primers derived from these sequences was used to detect E . chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.

Biochim Biophys Acta, 1997 Jul 17, 1353(1), 7 - 12
cDNA sequence of a translational elongation factor Ts homologue from Caenorhabditis elegans: mitochondrial factor-specific features found in the nematode homologue peptide; Watanabe Y et al.; The cDNA for a homologue of elongation factor Ts which probably functions in mitochondria has been sequenced from a nematode Caenorhabditis elegans . The deduced amino acid sequence (316 amino acids long) has a possible transit peptide sequence at the amino terminus and several common specific features for mammalian mitochondrial EF-Ts . The amino acid identities in the protein from C . elegans compared with those of bovine mitochondria and Escherichia coli are 29.5% and 24.0%, respectively . The C . elegans sequence was classified as a long EF-Ts (ca . 280 amino acids long) similar to peptides from mammalian mitochondria and eubacteria other than Thermus and cyanobacteria (except Spirulina platensis), rather than short EF-Ts (ca . 200 amino acids long) as those of Thermus, cyanobacteria and plastids.

Mol Microbiol, 1997 Jul, 25(2), 375 - 83
Inactivation of the 20S proteasome in Mycobacterium smegmatis; Knipfer N et al.; The 20S proteasome is an essential component of the cytosolic protein turnover apparatus of eukaryotic cells . In higher eukaryotes, the 20S proteasome is responsible for most cytosolic protein turnover and also generates peptides for subsequent presentation by the MHC class I pathway . Structurally, the eukaryotic 20S proteasome is extremely complex, being composed of 14 different subunits . Proteasomes with simplified subunit composition have been identified in certain eubacteria and archaebacteria but, in each case, the proteasome-containing organism is recalcitrant to further molecular genetic analyses . As a result, no in vivo characterization of a simplified eubacterial or archaebacterial proteasome has been reported . We have shown that the genetically tractable eubacterium Mycobacterium smegmatis contains a 20S proteasome, allowing the first in vivo characterization of a simplified 20S proteasome . We use a positive/negative selection scheme to inactivate the genes encoding 20S proteasome subunits and demonstrate that, in contrast to eukaryotic cells, M . smegmatis cells lacking intact proteasome genes are viable and phenotypically indistinguishable from congenic strains containing proteasomes . Implications for the evolution of the protein turnover apparatus are discussed.

Lett Appl Microbiol, 1997 Jul, 25(1), 30 - 3
Direct extraction of microbial community DNA from humified upland soils; Clegg CD et al.; This paper describes a protocol effective at extracting high yields of high-purity microbial community DNA from humified soils . DNA was extracted from soil by lysozyme, SDS and freeze-thaw lysis, precipitated and then subjected to a double caesium chloride density gradient centrifugation stage before concentrating and washing . Evaluation using three soils yielded up to 30 micrograms DNA g-1 dry soil, with absorbance ratios at 260:230 nm and 260:280 nm of 1.6-2.0 . The DNA extracted from the three soils was digested by four restriction enzymes and a 16S rDNA eubacterial product was amplified by PCR . These tests indicated that the DNA obtained by the protocol was sufficiently pure for molecular biological analysis.

J Mol Evol, 1997 Jul, 45(1), 9 - 16
Evidence for the early divergence of tryptophanyl- and tyrosyl-tRNA synthetases; Brown JR et al.; Each amino acid is attached to its cognate tRNA by a distinct aminoacyl-tRNA synthetase (aaRS) . The conventional evolutionary view is that the modern complement of synthetases existed prior to the divergence of eubacteria and eukaryotes . Thus comparisons of prokaryotic and eukaryotic aminoacyl-tRNA synthetases of the same type (charging specificity) should show greater sequence similarities than comparisons between synthetases of different types-and this is almost always so . However, a recent study {Ribas de Pouplana L, Furgier M, Quinn CL, Schimmel P (1996) Proc Natl Acad Sci USA 93:166-170} suggested that tryptophanyl- (TrpRS) and tyrosyl-tRNA (TyrRS) synthetases of the Eucarya (eukaryotes) are more similar to each other than either is to counterparts in the Bacteria (eubacteria) . Here, we reexamine the evolutionary relationships of TyrRS and TrpRS using a broader range of taxa, including new sequence data from the Archaea (archaebacteria) as well as species of Eucarya and Bacteria . Our results differ from those of Ribas de Pouplana et al.: All phylogenetic methods support the separate monophyly of TrpRS and TyrRS . We attribute this result to the inclusion of the archaeal data which might serve to reduce long branch effects possibly associated with eukaryotic TrpRS and TyrRS sequences . Furthermore, reciprocally rooted phylogenies of TrpRS and TyrRS sequences confirm the closer evolutionary relationship of Archaea to eukaryotes by placing the root of the universal tree in the Bacteria.

J Bacteriol, 1997 Jul, 179(13), 4195 - 205
Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis; Pettersson B et al.; Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C . pecorum, C . pneumoniae, C . psittaci, and C . trachomatis were investigated . In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C . pneumoniae and C . pecorum were subjected to solid-phase DNA sequencing of both strands . The human isolates of C . pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains . Interestingly, horse isolate N16 of C . pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences . The type strain and koala isolates of C . pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference . Furthermore, the C . pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species . This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria . The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences . All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C . pneumoniae cluster and the C . psittaci cluster . The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis . The former cluster contained the human isolates of C . pneumoniae and equine strain N16 . The latter cluster consisted of C . psittaci, C . pecorum, and C . trachomatis . The members of the C . pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C . pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences . C . psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine . Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content . Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C . pneumoniae and C . psittaci forming early branches of their respective cluster and with C . trachomatis being the more recently evolved species within the genus Chlamydia.

J Mol Biol, 1997 Jun 27, 269(5), 647 - 64
Recognition and manipulation of branched DNA structure by junction-resolving enzymes; White MF et al.; The junction-resolving enzymes are a class of nucleases that introduce paired cleavages into four-way DNA junctions . They are important in DNA recombination and repair, and are found throughout nature, from eubacteria and their bacteriophages through to higher eukaryotes and their viruses . These enzymes exhibit structure-selective binding to DNA junctions; although cleavage may be more or less sequence-dependent, binding affinity is purely related to the branched structure of the DNA . Binding and cleavage events can be separated for a number of the enzymes by mutagenesis, and mutant proteins that are defective in cleavage while retaining normal junction-selective binding have been isolated . Critical acidic residues have been identified in several resolving enzymes, suggesting a role in the coordination of metal ions that probably deliver the hydrolytic water molecule . The resolving enzymes all bind to junctions in dimeric form, and the subunits introduce independent cleavages within the lifetime of the enzyme-junction complex to ensure resolution of the four-way junction . In addition to recognising the structure of the junction, recent data from four different junction-resolving enzymes indicate that they also manipulate the global structure . In some cases this results in severe distortion of the folded structure of the junction . Understanding the recognition and manipulation of DNA structure by these enzymes is a fascinating challenge in molecular recognition.

Gene, 1997 Jun 19, 192(2), 241 - 3
A ribosomal RNA operon from Pseudomonas stutzeri Zobell; Kerkhof L; A ribosomal RNA operon from the marine bacterium, Pseudomonas stutzeri Zobell, was cloned and characterized by Southern hybridization and sequence analysis . The 16S rRNA, 23S rRNA, 5S rRNA and 2 tRNA genes (alanine and isoleucine) were identified by homology with sequences in GenBank . The rRNA gene exhibited typical eubacterial organization (16S-tRNAs-23S-5S) . A putative ribosomal promoter and anti-terminator regions were also identified and described . Significant differences in spacing of the anti-terminator regulatory elements were observed between P . stutzeri Zobell and Escherichia coli.

What Is Molecular Microbiology?, What Is Yeast?, What Is Biofilter?, What Is Fermentation?, What Is Genetics?, e, Bacteria, c, Microbes, c, Bacterium, n, Bacteriology, a, Microorganism, c, Biofilms, n, Cryptococci, s, Bacillus subtilis, a, Prokaryotes, i, Salmonella typhimurium, o, Eubacterium, a, Escherichia coli, r, Microorganism, i, Streptococcal, c, Biodegradation, n, Cell suspensions, s, Microorganisms, n, Fermentations, c, Escherichia coli, r, Bacteriological, c, Bacillus subtilis, c, Lactobacillus, n, Microorganisms, c, Enterobacteriacea, n, Haemophilus, e, Bacteriophages

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005