Curr Med Chem, 2002 Feb, 9(3), 385 - 409 Methionine in and out of proteins: targets for drug design; Vaughan MD et al.; The increasing need for new antibiotics to overcome rapidly developing resistance mechanisms observed in clinical isolates of Gram-positive and Gram-negative eubacteria has placed critical emphasis on the search for new antibacterial enzyme targets and the structural and mechanistic investigation of such targets . Among these potential targets, the enzymes responsible for integrating the amino acid methionine into proteins, along with its subsequent post-translational modification and repair, have emerged as promising candidates for the development of novel antibiotics . As well, there is increasing evidence for the importance of several of these enzymes in the development of anti-cancer, anti-parasitic, and anti-atherosclerotic drugs . Within the last three years, the crystal structures of all of these enzymes have been determined, which offers an unprecedented source of structural information for inhibitor design . The development of combinatorial chemistry and high throughput screening procedures has quickly provided several potent, specific inhibitors for a number of these enzymes, particularly the peptide deformylase, methionine aminopeptidase, and methionyl-tRNA synthetase enzymes . This review critically analyzes the future potential for inhibition of enzymes in this pathway, allowing for a pragmatic view of the success of inhibitor developments and highlighting areas in which further investigations are warranted. Amino Acids, 2001 Dec, 21(4), 393 - 9 Conservation of the basic pattern of cellular amino acid composition of archaeobacteria during biological evolution and the putative amino acid composition of primitive life forms; Sorimachi K et al.; Previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs such as eubacteria, protozoa, fungi and mammalian cells . These results suggest that the difference in the cellular amino acid composition reflects biological changes as the result of evolution . However, the basic pattern of cellular amino acid composition was relatively constant in all organisms examined . In the present study, we examined archaeobacteria, because they are considered important in understanding the relationship between biological evolution and cellular amino acid composition . The cellular amino acid compositions of Archaeoglobus fulgidus, Pyrococcus horikoshii, Methanobacterium thermoautotrophicum and Methanococcus jannaschii differed slightly from each other, but were similar to those determined from codon usage data, based on the complete genomes . Thus, the cellular amino acid composition reflects biological evolution . We suggest that primitive forms of life appearing on earth at the end of prebiotic evolution had a similar-cellular amino acid composition. Clin Microbiol Infect, 1999 Feb, 5(2), 92 - 96 Polymerase chain reaction, with sequencing, as a diagnostic tool in culture---negative bacterial meningitis; Lorino G et al.; OBJECTIVE: To evaluate the feasibility of using 16S rDNA universal primer PCR (followed by sequencing) and 65-kDa heat shock Mycobacterium tuberculosis protein gene PCR as a method to determine a bacterial etiology in culture---negative cerebrospinal fluid (CSF) samples . METHODS: One hundred and forty-nine CSF samples from 128 patients were processed . DNA was extracted from the CSF samples and amplified with the eubacterial 16S rDNA primers P11E and P13B, and with the 65-kDa heat shock protein gene mycobacterial primers . The amplicons were identified by sequencing and specific oligoprobe hybridization . RESULTS: Overall, a microbiological diagnosis was made in 11 of 125 ultimately culture-negative cases . The use of 65-kDa heat shock protein gene PCR was needed to improve the diagnosis of tuberculous meningitis; in four patients, prospectively studied, the outcome of antituberculous therapy could also be followed . CONCLUSIONS: In culture-negative bacterial meningitis it is possible to improve the microbiological diagnosis by use of 16S rDNA amplification and sequencing, together with amplification of a more specific gene in mycobacteria. Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3701 - 5 Epub 2002 Feb 19. A spliceosomal intron in Giardia lamblia; Nixon JE et al.; Short introns occur in numerous protist lineages, but there are no reports of intervening sequences in the protists Giardia lamblia and Trichomonas vaginalis, which may represent the deepest known branches in the eukaryotic line of descent . We have discovered a 35-bp spliceosomal intron in a gene encoding a putative {2Fe-2S} ferredoxin of G . lamblia . The Giardia intron contains a canonical splice site at its 3' end (AG), a noncanonical splice site at its 5' end (CT), and a branch point sequence that fits the yeast consensus sequence of TACTAAC except for the first nucleotide (AACTAAC) . We have also identified several G . lamblia genes with spliceosomal peptides, including homologues of eukaryote-specific spliceosomal peptides (Prp8 and Prp11), several DExH-box RNA-helicases that have homologues in eubacteria, but serve essential functions in the splicing of introns in eukaryotes, and 11 predicted archaebacteria-like Sm and like-Sm core peptides, which coat small nuclear RNAs . Phylogenetic analyses show the Giardia Sm core peptides are the products of multiple, ancestral gene duplications followed by divergence, but they retain strong similarity to Sm and like-Sm peptides of other eukaryotes . Although we have documented only a single intron in Giardia, it likely has other introns and fully functional, spliceosomal machinery . If introns were added during eukaryotic evolution (the introns-late hypothesis), then these results push back the date of this event before the branching of G . lamblia. Eur J Biochem, 2002 Feb, 269(3), 868 - 83 Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants . A case study of endosymbiotic gene transfer; Schnarrenberger C et al.; The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes . The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose . Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution . Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants . The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues . A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria . Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor. Environ Microbiol, 2001 Nov, 3(11), 710 - 9 Comparative analysis of polychlorinated biphenyl-dechlorinating communities in enrichment cultures using three different molecular screening techniques; Watts JE et al.; The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques . Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species . To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community . These methods were applied to enrichment cultures that selectively dechlorinated double-flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl . The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture . However, each method was effectively qualitative and identified the same organisms: a low G + C Gram-positive eubacterium, an organism most similar to the green non-sulphur bacteria, an Aminobacterium sp . and a Desulfovibrio sp . Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture . TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library . ARDRA provided an in-depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA . These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture . In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units). Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 115 - 22 Taxonomic characterization of Mogibacterium diversum sp . nov . and Mogibacterium neglectum sp . nov., isolated from human oral cavities; Nakazawa F et al.; Novel isolates, strains HM-7, HM-6, HH-31, P9a-hT and UJB13-d, which were isolated from tongue plaque and necrotic dental pulp, were studied taxonomically and phylogenetically . These organisms were anaerobic, non-spore-forming, gram-positive, rod-shaped bacteria that were inert in most of the conventional biochemical tests and phenotypically resemble Mogibacterium species or asaccharolytic Eubacterium species . The G+C contents of the DNAs from the novel isolates ranged from 41 to 42 mol % . DNA-DNA hybridization studies demonstrated that these strains might be assigned to the genus Mogibacterium but not to the previously described species . It was also apparent that strain HM-7 belonged to the same species as strains HM-6 and HH-31, and that strains P9a-hT and UJB13-d belonged to a second species . The levels of DNA-DNA relatedness to asaccharolytic Eubacterium species, including Eubacterium brachy, Eubacterium nodatum, Eubacterium saphenum and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2% . The results of 16S rDNA sequence comparisons revealed that these organisms represent novel lineages distinct from all previously described species of gram-positive, rod-shaped bacteria . On the basis of phenotypic characteristics, DNA-DNA hybridization data and phylogenetic analysis with 16S rRNA gene sequence data, new species are proposed, namely Mogibacterium diversum (for strains HM-7, HM-6 and HH-31) and Mogibacterium neglectum (for strains P9a-hT and UJB13-d) . HM-7T (= ATCC 700923T = JCM 11205T) is the type strain of the former and P9a-hT (= ATCC 700924T = JCM 11204T) is the type strain for the latter. Pharmazie, 2000 May, 55(5), 380 - 4 Effect of Australian tea tree oil on the viability of the wall-less bacterium Mycoplasma pneumoniae; Harkenthal M et al.; In vitro assays using a variety of essential oils revealed a particularly high antibacterial effect of Australian tea tree oil (TTO) on a great number of gram-negative and gram-positive bacteria of unrelated phylogenetic origin . In the present study, the susceptibility of cell wall-less bacteria such as the human pathogenic bacterium Mycoplasma pneumoniae to Australian tea tree oil was examined . The minimum inhibitory concentration (MIC) was determined to be 0.006% (v/v) TTO for the wild type and to 0.003% (v/v) TTO for mutants of M . pneumoniae which lost the ability to adhere to host cells (cytadherence-negative) . The MIC and the MBC (minimum bactericidal concentration) for M . pneumoniae are 100 times lower than those for all other eubacteria tested . Electron microscopy with negatively stained cells as well as with ultrathin sections revealed a tendency to ovoid or round cells after oil treatment whereas the untreated cells of the wild type exhibit a flask-shaped morphology with a tip-like structure at one pole of the cell . The integrity of the mycoplasmal membrane seems not to be affected by TTO since no leakage of the Mycoplasma cell was observed after oil treatment . In the HET-CAM test TTO did not show any visible signs of irritation in concentrations less than 25% . Although the active component in TTO that has anti-mycoplasmal activity is not known, it seems very promising to use TTO tentatively for mouth washing and inhalation in case of Mycoplasma-pneumoniae-infection. Appl Environ Microbiol, 2002 Feb, 68(2), 999 - 1004 Novel clade of Rickettsia spp . from leeches; Kikuchi Y et al.; Intracellular rickettsia-like structures were found in the tissues of a glossiphoniid leech, Torix tagoi, by transmission electron microscopy . Diagnostic PCR analysis using specific primers suggested that of the nine glossiphoniid species examined, two species, T . tagoi and Hemicrepsis marginata, harbored bacteria of the genus Rickettsia . A 1.5-kb eubacterial 16S rRNA gene segment obtained from each of these species was amplified by PCR, cloned, and sequenced . Phylogenetic analysis of the 16S rRNA gene demonstrated that the Rickettsia species found in the leeches constituted a novel clade that is distinct from the clade of arthropod-associated Rickettsia species . In natural populations, 97.7% (43 of 44) of T . tagoi leeches and 100% (9 of 9) of H . marginata leeches carried Rickettsia, suggesting that infection with Rickettsia is prevalent in these leeches . This is the first report of Rickettsia found in annelids. J Biol Chem, 2002 Apr 5, 277(14), 12137 - 43 Epub 2002 Jan 30. Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation; Alber BE et al.; The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO(2) fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria . The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to propionyl-CoA . The specific activity of the 3-hydroxypropionate-, CoA-, K(+)-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 micromol min(-1) mg(-1) protein, which was 2-fold down-regulated in heterotrophically grown cells . Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H(+) --> propionyl-CoA + MgAMP + PP(i) + NADP(+) + H(2)O . The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure . Upon incubation of this new enzyme, termed propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA . SDS-PAGE revealed that the subunits of propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined . Two parts of the gene encoding propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C . aurantiacus, and the sequence of the pcs gene was completed . Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site . Similar polyfunctional large enzymes are common in secondary metabolism (e.g . polyketide synthases) but rare in primary metabolism (e.g . eukaryotic type I fatty acid synthase) . These results lend strong support to the operation of the proposed pathway in autotrophic CO(2) fixation. Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1158 - 63 Epub 2002 Jan 29. Studies on the nonmevalonate terpene biosynthetic pathway: metabolic role of IspH (LytB) protein; Rohdich F et al.; Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes . Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy-D-xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria . Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy-D-xylulose can be converted into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes . This article describes the transformation of exogenous {U-(13)C(5)}1-deoxy-D-xylulose into a 5:1 mixture of {U-(13)C(5)}isopentenyl diphosphate and {U-(13)C(5)}dimethylallyl diphosphate by an E . coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes. Nucleic Acids Res, 2002 Feb 1, 30(3), 675 - 84 Conserved economics of transcription termination in eubacteria; Unniraman S et al.; A secondary structure in the nascent RNA followed by a trail of U residues is believed to be necessary and sufficient to terminate transcription . Such structures represent an extremely economical mechanism of transcription termination since they function in the absence of any additional protein factors . We have developed a new algorithm, GeSTer, to identify putative terminators and analysed all available complete bacterial genomes . The algorithm classifies the structures into five classes . We find that potential secondary structure sequences are concentrated downstream of coding regions in most bacterial genomes . Interestingly, many of these structures are not followed by a discernible U-trail . However, irrespective of the nature of the trail sequence, the structures show a similar distribution, indicating that they serve the same purpose . In contrast, such a distribution is absent in archaeal genomes, indicating that they employ a distinct mechanism for transcription termination . The present algorithm represents the fastest and most accurate algorithm for identifying terminators in eubacterial genomes without being restricted by the classical Escherichia coli paradigm. Arch Microbiol, 2001 Dec, 177(1), 113 - 6 Epub 2001 Oct 03. Selenium-dependent growth of Treponema denticola: evidence for a clostridial-type glycine reductase; Rother M et al.; Assessment of the nutritional requirements of Treponema denticola disclosed a strict growth dependence on selenium . In vivo labeling of cells of this organism with (75)Se and electrophoretic analysis revealed three labeled bands, two of which were selenoproteins correlating in size with subunits A and B of glycine reductase . Antibodies directed against glycine- or betaine-reductase subunits of Eubacterium acidaminophilum specifically also reacted with proteins from cell lysates of T . denticola . Moreover, ORFs within the T . denticola genome sequence were found whose products display high sequence similarity to glycine-reductase subunits . These findings strongly support the notion that T . denticola ferments amino acids via the activity of glycine reductase, an enzyme previously thought to be restricted to gram-positive bacteria. J Biol Chem, 2002 Mar 22, 277(12), 10642 - 6 Epub 2002 Jan 15. Crystal structure of human L-isoaspartyl methyltransferase; Ryttersgaard C et al.; The enzyme l-isoaspartyl methyltransferase initiates the repair of damaged proteins by recognizing and methylating isomerized and racemized aspartyl residues in aging proteins . The crystal structure of the human enzyme containing a bound S-adenosyl-l-homocysteine cofactor is reported here at a resolution of 2.1 A . A comparison of the human enzyme to homologs from two other species reveals several significant differences among otherwise similar structures . In all three structures, we find that three conserved charged residues are buried in the protein interior near the active site . Electrostatics calculations suggest that these buried charges might make significant contributions to the energetics of binding the charged S-adenosyl-l-methionine cofactor and to catalysis . We suggest a possible structural explanation for the observed differences in reactivity toward the structurally similar l-isoaspartyl and d-aspartyl residues in the human, archael, and eubacterial enzymes . Finally, the human structure reveals that the known genetic polymorphism at residue 119 (Val/Ile) maps to an exposed region away from the active site. Genome Biol . 2001;2(12):RESEARCH0054 . Epub 2001 Nov 14. The process of genome shrinkage in the obligate symbiont Buchnera aphidicola; Moran NA et al.; BACKGROUND: Very small genomes have evolved repeatedly in eubacterial lineages that have adopted obligate associations with eukaryotic hosts . Complete genome sequences have revealed that small genomes retain very different gene sets, raising the question of how final genome content is determined . To examine the process of genome reduction, the tiny genome of the endosymbiont Buchnera aphidicola was compared to the larger ancestral genome, reconstructed on the basis of the phylogenetic distribution of gene orthologs among fully sequenced relatives of Escherichia coli and Buchnera . RESULTS: The reconstructed ancestral genome contained 2,425 open reading frames (ORFs) . The Buchnera genome, containing 564 ORFs, consists of 153 fragments of 1-34 genes that are syntenic with reconstructed ancestral regions . On the basis of this reconstruction, 503 genes were eliminated within syntenic fragments, and 1,403 genes were lost from the gaps between syntenic fragments, probably in connection with genome rearrangements . Lost regions are sometimes large, and often span functionally unrelated genes . In addition, individual genes and regulatory regions have been lost or eroded . For the categories of DNA repair genes and rRNA genes, most lost loci fall in regions between syntenic fragments . This history of gene loss is reflected in the sequences of intergenic spacers at positions where genes were once present . CONCLUSIONS: The most plausible interpretation of this reconstruction is that Buchnera lost many genes through the fixation of large deletions soon after the acquisition of an obligate endosymbiotic lifestyle . An implication is that final genome composition may be partly the chance outcome of initial deletions and that neighboring genes influence the likelihood of loss of particular genes and pathways. J Ind Microbiol Biotechnol, 2001 Sep, 27(3), 163 - 9 Cross-genomic analysis of the translational systems of various organisms; Fujita K et al.; We have characterized the genes encoding ribosomal proteins (r-proteins) as well as other translation-related factors of 15 eubacteria and four archaebacteria, and the genes for the mitochondrial r-proteins of Saccharomyces cerevisiae by using the complete genomic nucleotide sequence data of these organisms . In eubacteria, including two species of Mycoplasma, the operon structure of the r-protein genes is well conserved, while their relative orientation and chromosomal location are quite divergent . The operon structure of the r-protein genes in archaebacteria, on the other hand, is quite different from eubacteria and also among themselves . In addition, many archaebacterial r-proteins show similarity to rat cytoplasmic r-proteins . Nonetheless, characteristic features of several genes encoding proteins of functional importance are well conserved throughout the bacterial species including archaebacteria, as well as in S . cerevisiae . We searched for the genes encoding mitochondrial r-proteins in yeast by combining informatics and genetic experiments . Furthermore, we characterized some of the r-proteins genes by exchanging portions between Escherichia coli and S . cerevisiae and performed functional analysis of some of the genes from different evolutionary points of view . Our work may be extended towards phylogenetic analysis of organisms producing secondary metabolites of various sorts. Int Microbiol, 2001 Mar, 4(1), 13 - 9 Classification and mode of action of membrane-active bacteriocins produced by gram-positive bacteria; Oscariz JC et al.; Bacteriocins are ribosomally synthesized antimicrobial peptides produced by microorganisms belonging to different eubacterial taxonomic branches . Most of them are small cationic membrane-active compounds that form pores in the target cells, disrupting membrane potentials and causing cell death . The production of small cationic peptides with antibacterial activity is a defense strategy found not only in bacteria, but also in plants and animals . Bacteriocins are classified according to different criteria by different authors; in this review, we will summarize the principal bacteriocin classifications, highlight their main physical and chemical characteristics, and describe the mechanism of some selected bacteriocins that act at the membrane level. Chem Pharm Bull (Tokyo), 2001 Dec, 49(12), 1640 - 3 The heterocyclic ring fission and dehydroxylation of catechins and related compounds by Eubacterium sp . strain SDG-2, a human intestinal bacterium; Wang LQ et al.; A human intestinal bacterium, Eubacterium (E.) sp . strain SDG-2, was tested for its ability to metabolize various (3R)- and (3S)-flavan-3-ols and their 3-O-gallates . This bacterium cleaved the C-ring of (3R)- and (3S)-flavan-3-ols to give 1,3-diphenylpropan-2-ol derivatives, but not their 3-O-gallates . Furthermore, E . sp . strain SDG-2 had the ability of p-dehydroxylation in the B-ring of (3R)-flavan-3-ols, such as (-)-catechin, (-)-epicatechin, (-)-gallocatechin and (-)-epigallocatechin, but not of (3S)-flavan-3-ols, such as (+)-catechin and (+)-epicatechin. J Mol Microbiol Biotechnol, 2002 Jan, 4(1), 77 - 91 An inventory of genes encoding RNA polymerase sigma factors in 31 completely sequenced eubacterial genomes; Mittenhuber G; Sigma factors are important elements involved in transcriptional regulation of gene expression by conferring promoter specificity to RNA polymerase . The number of sigma factor encoding genes in 31 completely sequenced bacterial genomes were compared . Two unrelated families of sigma factors, the sigma70- and the sigma54-family were identified previously . The sigma70-family can be further subdivided into two distantly related groups: the sigma70 subfamily and the poorly characterized ECF subfamily . A total of 215 sigma factors could be attributed to these subfamilies . The construction of phylogenetic trees allows subclassifications of sigma factor encoding genes within the subfamilies . With the exception of Deinococcus radiodurans, all species possess a housekeeping primary sigma factor . Free-living species possess a higher number of both sigma70-type and ECF alternative sigma factors than pathogens or symbionts associated with animals . Different bacterial species exhibit large differences in the number of alternative sigma factor encoding genes and consequently huge flexibility in their transcriptional regulatory patterns . Transcriptional regulation in terms of regulons controlled by alternative sigma factors is a late evolving phenomenon . The current nomenclature for sigma factor encoding genes is confusing and should be revised. Am J Ophthalmol, 2002 Jan, 133(1), 142 - 4 Reliability of nested polymerase chain reaction in the diagnosis of bacterial endophthalmitis; Sharma S et al.; PURPOSE: To test the utility of DNA Taq polymerase (Taq) treated with 8-methoxypsoralen with ultraviolet irradiation and Taq treated with restriction endonuclease in a nested polymerase chain reaction test for the diagnosis of bacterial endophthalmitis . METHODS: Prospective, comparative study . Vitreous biopsy fluid from 32 cases of clinically diagnosed bacterial endophthalmitis and 10 noninfective controls were cultured for aerobic/anaerobic bacteria and tested by nested polymerase chain reaction using two sets of universal eubacterial primers in triplicate with untreated Taq, Taq treated with 8-methoxypsoralen with ultraviolet irradiation, and Taq treated with Sau 3A1 . RESULTS: Using untreated Taq, false-positive results were obtained in nested polymerase chain reaction with all 10 control samples, which were not seen with the other two methods of nested polymerase chain reaction . However, the sensitivity of nested polymerase chain reaction using Sau 3A1 was the same sensitivity as the conventional culture (34.4%), whereas the sensitivity of the nested polymerase chain reaction using 8-methoxypsoralen was 46.9% higher than in the conventional culture . CONCLUSION: To eliminate the problem of false positives in bacterial nested polymerase chain reaction, we recommend the routine utilization of Taq treated with 8- methoxypsoralen and ultraviolet irradiation. Neuroimmunomodulation, 2001, 9(3), 141 - 7 Effect of Mycoplasma fermentans on brain PGE(2): role of glucocorticoids and their receptors; Wohlman A et al.; OBJECTIVES: Mycoplasmas are a group of eubacteria, which cause various diseases in animals and in humans, and can contribute to diseases produced by other infectious agents, particularly HIV . We have recently reported that intracerebral administration of Mycoplasma fermentans (MF) produces both neuroendocrine and behavioral alterations . Some of these responses were mediated by MF-induced production of prostaglandin E(2 )(PGE(2)) . The aim of this study was to examine the role of glucocorticoids (GC) in regulating MF-induced brain prostaglandin production . METHODS: Male rats were injected intracerebroventricularly with various doses of heat-inactivated MF, LPS or IL-1 beta and the following parameters were measured: (1) ex vivo production of hippocampal PGE(2), (2) serum levels of ACTH and corticosterone, and (3) binding capacity of {(3)H}-dexamethasone (DEX) to hippocampal cytosol . RESULTS: MF caused a small increase in hippocampal PGE(2) production, but higher doses failed to produce a further increase . In contrast, the effects of LPS or IL-1 beta on PGE(2) were dose-dependent . Removal of circulating GC by bilateral adrenalectomy significantly enhanced MF-induced brain PGE(2) production . The three immune stimulators increased serum levels of ACTH and corticosterone to the same extent . Finally, MF, but not IL-1 beta increased the specific binding of {(3)H}-DEX to hippocampal cytosol . CONCLUSIONS: Brain PGE(2) induced by MF is regulated by endogenous GC . These hormones have an attenuating effect on PGE(2 )production, probably through an MF-induced increase in GC binding by brain tissue . This mechanism may be important in the pathological effect of MF within the brain of AIDS patients . Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 154 - 6 Epub 2001 Dec 21. Cloning, expression and preliminary X-ray analysis of the dihydroorotase from the hyperthermophilic eubacterium Aquifex aeolicus; Purcarea C et al.; Dihydroorotase (DHOase) catalyzes the formation of dihydroorotate in the de novo pyrimidine biosynthetic pathway . The gene encoding the type I DHOase from the hyperthermophilic bacterium Aquifex aeolicus has been cloned in Escherichia coli with a polyhistidine affinity tag appended to the amino-terminal end and sequenced . The recombinant protein was expressed at high levels and could be purified readily in a single step by Ni(2+) affinity chromatography . Both native and selenomethionine-labeled proteins were crystallized using the hanging-drop vapor-diffusion technique . Screens of the purified protein identified several conditions that yielded crystals; however, the best crystals were obtained using 1 M Li(2)SO(4), 10 mM NiCl(2), 100 mM Tris acetate pH 8.5 as the precipitant . Well formed diamond-shaped crystals appeared within 1 d and continued to grow over several weeks to about 0.5 mm in the largest dimension . The crystals diffract to 1.7 A and belong to space group C2, with unit-cell parameters a = 119.8, b = 88.0, c = 55.2 A, beta = 99.0 degrees and a mosaic spread of 0.6 degrees . There is one DHOase monomer in the asymmetric unit. Plant Cell, 2001 Dec, 13(12), 2823 - 39 The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing; Schroda M et al.; In eubacteria and mitochondria, Hsp70 chaperone activity is controlled by the nucleotide exchange factor GrpE . We have identified the chloroplastic GrpE homolog of Chlamydomonas, CGE1, as an approximately 26-kD protein coimmunoprecipitating with the stromal HSP70B protein . When expressed in Escherichia coli, CGE1 can functionally replace GrpE and interacts physically with DnaK . CGE1 is encoded by a single-copy gene that is induced strongly by heat shock and slightly by light . Alternative splicing generates two isoforms that differ only by two residues in the N-terminal part . The larger form is synthesized preferentially during heat shock, whereas the smaller one dominates at lower temperatures . Fractions of both HSP70B and CGE1 associate with chloroplast membranes in an ATP-sensitive manner . By colorless native PAGE and pulse labeling, CGE1 monomers were found to assemble rapidly into dimers and tetramers . In addition, CGE1 was found to form ATP-sensitive complexes with HSP70B of approximately 230 and approximately 120 kD, the latter increasing dramatically after heat shock. Plant Cell, 2001 Dec, 13(12), 2719 - 30 The Arabidopsis HUELLENLOS gene, which is essential for normal ovule development, encodes a mitochondrial ribosomal protein; Skinner DJ et al.; The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule . We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes . The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins . Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins . HLL and HLP fusions to green fluorescent protein were localized to mitochondria . Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions . We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes . HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves . This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules . Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning. Nucleic Acids Res, 2002 Jan 1, 30(1), 176 - 8 5S Ribosomal RNA Database; Szymanski M et al.; Ribosomal 5S RNA (5S rRNA) is an integral component of the large ribosomal subunit in all known organisms with the exception only of mitochondrial ribosomes of fungi and animals . It is thought to enhance protein synthesis by stabilization of a ribosome structure . This paper presents the updated database of 5S rRNA and their genes (5S rDNA) . Its short characteristics are presented in the Introduction . The database contains 2280 primary structures of 5S rRNA and 5S rRNA genes . These include 536 eubacterial, 61 archaebacterial, 1611 eukaryotic and 72 organelle sequences . The database is available on line through the World Wide Web at http://biobases.ibch.poznan.pl/5SData/. Trends Genet, 2002 Jan, 18(1), 1 - 5 Eubacterial phylogeny based on translational apparatus proteins; Brochier C et al.; Lateral gene transfers are frequent among prokaryotes, although their detection remains difficult . If all genes are equally affected, this questions the very existence of an organismal phylogeny . The complexity hypothesis postulates the existence of a core of genes (those involved in numerous interactions) that are unaffected by transfers . To test the hypothesis, we studied all the proteins involved in translation from 45 eubacterial taxa, and developed a new phylogenetic method to detect transfers . Few of the genes studied show evidence for transfer . The phylogeny based on the genes devoid of transfer is very consistent with the ribosomal RNA tree, suggesting that an eubacterial phylogeny does exist. Gene, 2001 Dec 27, 281(1-2), 123 - 31 Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis; Henze K et al.; Glucokinase (GK) and glucosephosphate isomerase (GPI), the first two enzymes of the glycolytic pathway of the diplomonads Giardia intestinalis and Spironucleus barkhanus, Type I amitochondriate eukaryotes, were sequenced . GPI of the parabasalid Trichomonas vaginalis was also sequenced . The diplomonad GKs belong to a family of specific GKs present in cyanobacteria, in some proteobacteria and also in T . vaginalis, a Type II amitochondriate protist . These enzymes are not part of the hexokinase family, which is broadly distributed among eukaryotes, including the Type I amitochondriate parasite Entamoeba histolytica . G . intestinalis GK expressed in Escherichia coli was specific for glucose and glucosamine, as are its eubacterial homologs . The sequence of diplomonad and trichomonad GPIs formed a monophyletic group more closely related to cyanobacterial and chloroplast sequences than to cytosolic GPIs of other eukaryotes and prokaryotes . The findings show that certain enzymes of the energy metabolism of these amitochondriate protists originated from sources different than those of other eukaryotes . The observation that the two diplomonads and T . vaginalis share the same unusual GK and GPI is consistent with gene trees that suggest a close relationship between diplomonads and parabasalids . The intriguing relationships of these enzymes to cyanobacterial (and chloroplast) enzymes might reflect horizontal gene transfer between the common ancestor of the diplomonad and parabasalid lineages and the ancestor of cyanobacteria. Arch Biochem Biophys, 2001 Dec 15, 396(2), 162 - 70 Characterization of an eukaryotic peptide deformylase from Plasmodium falciparum; Bracchi-Ricard V et al.; Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides . Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins . Until recently, PDF has been thought as an enzyme unique to the bacterial kingdom . Searches of the genomic DNA databases identified several genes that encode proteins of high sequence homology to bacterial PDF from eukaryotic organisms . The cDNA encoding Plasmodium falciparum PDF (PfPDF) has been cloned and overexpressed in Escherichia coli . The recombinant protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is inhibited by specific PDF inhibitors . Western blot analysis indicated expression of mature PfPDF in trophozoite, schizont, and segmenter stages of intraerythrocytic development . These results provide strong evidence that a functional PDF is present in P . falciparum . In addition, PDF inhibitors inhibited the growth of P . falciparum in the intraerythrocytic culture . (c)2001 Elsevier Science. J Biol Chem, 2002 Mar 1, 277(9), 6994 - 7001 Epub 2001 Dec 13. The bdbDC operon of Bacillus subtilis encodes thiol-disulfide oxidoreductases required for competence development; Meima R et al.; The development of genetic competence in the Gram-positive eubacterium Bacillus subtilis is a complex postexponential process . Here we describe a new bicistronic operon, bdbDC, required for competence development, which was identified by the B . subtilis Systematic Gene Function Analysis program . Inactivation of either the bdbC or bdbD genes of this operon results in the loss of transformability without affecting recombination or the synthesis of ComK, the competence transcription factor . BdbC and BdbD are orthologs of enzymes known to be involved in extracytoplasmic disulfide bond formation . Consistent with this, BdbC and BdbD are needed for the secretion of the Escherichia coli disulfide bond-containing alkaline phosphatase, PhoA, by B . subtilis . Similarly, the amount of the disulfide bond-containing competence protein ComGC is severely reduced in bdbC or bdbD mutants . In contrast, the amounts of the competence proteins ComGA and ComEA remain unaffected by bdbDC mutations . Taken together, these observations imply that in the absence of either BdbC or BdbD, ComGC is unstable and that BdbC and BdbD catalyze the formation of disulfide bonds that are essential for the DNA binding and uptake machinery. Plant Physiol, 2001 Dec, 127(4), 1644 - 55 A chloroplast protein homologous to the eubacterial topological specificity factor minE plays a role in chloroplast division; Itoh R et al.; We report the identification of a nucleus-encoded minE gene, designated AtMinE1, of Arabidopsis . The encoded AtMinE1 protein possesses both N- and C-terminal extensions, relative to the eubacterial and algal chloroplast-encoded MinE proteins . The N-terminal extension functioned as a chloroplast-targeting transit peptide, as revealed by a transient expression assay using an N terminus:green fluorescent protein fusion . Histochemical beta-glucuronidase staining of transgenic Arabidopsis lines harboring an AtMinE1 promoter::uidA reporter fusion unveiled specific activation of the promoter in green tissues, especially at the shoot apex, which suggests a requirement for cell division-associated AtMinE1 expression for proplastid division in green tissues . In addition, we generated transgenic plants overexpressing a full-length AtMinE1 cDNA and examined the subcellular structures of those plants . Giant heteromorphic chloroplasts were observed in transgenic plants, with a reduced number per cell, whereas mitochondrial morphology remained similar to that of wild-type plants . Taken together, these observations suggest that MinE is the third conserved component involved in chloroplast division. Nature, 2001 Dec 13, 414(6865), 776 - 9 Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore; Bhoo SH et al.; Phytochromes comprise a principal family of red/far-red light sensors in plants . Although phytochromes were thought originally to be confined to photosynthetic organisms, we have recently detected phytochrome-like proteins in two heterotrophic eubacteria, Deinococcus radiodurans and Pseudomonas aeruginosa . Here we show that these form part of a widespread family of bacteriophytochromes (BphPs) with homology to two-component sensor histidine kinases . Whereas plant phytochromes use phytochromobilin as the chromophore, BphPs assemble with biliverdin, an immediate breakdown product of haem, to generate photochromic kinases that are modulated by red and far-red light . In some cases, a unique haem oxygenase responsible for the synthesis of biliverdin is part of the BphP operon . Co-expression of this oxygenase with a BphP apoprotein and a haem source is sufficient to assemble holo-BphP in vivo . Both their presence in many diverse bacteria and their simplified assembly with biliverdin suggest that BphPs are the progenitors of phytochrome-type photoreceptors. J Biol Chem, 2002 Feb 22, 277(8), 6230 - 9 Epub 2001 Dec 10. The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a . Analysis of its glycosylation; Schaffer C et al.; Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall . In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques . The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids . The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1 . Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry . Glycopeptides obtained after Pronase digestion revealed the glycan structure {-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->}(n = 13-18), with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains . The glycan chains are bound via the disaccharide core -->3)-alpha-l-Rhap-(1-->3)-alpha-L-Rhap-(L--> and the linkage glycose beta-D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794 . This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized. Eur J Biochem, 2001 Dec, 268(24), 6417 - 25 Cys359 of GrdD is the active-site thiol that catalyses the final step of acetyl phosphate formation by glycine reductase from Eubacterium acidaminophilum; Kohlstock UM et al.; In the amino-acid-fermenting anaerobe Eubacterium acidaminophilum, acetyl phosphate is synthesized by protein C of glycine reductase from a selenoprotein A-bound carboxymethyl-selenoether . We investigated specific thiols present in protein C for responsibility for acetyl phosphate liberation . After cloning of the genes encoding the large and the small subunit (grdC1, grdD1), they were expressed separately in Escherichia coli and purified as Strep-tag proteins . GrdD was the only subunit that catalysed arsenate-dependent hydrolysis of acetyl phosphate (up to 274 U.mg-1), whereas GrdC was completely inactive . GrdD contained two cysteine residues that were exchanged by site-directed mutagenesis . The GrdD(C98S) mutant enzyme still catalysed the hydrolysis of acetyl phosphate, but the GrdD(C359A) mutant enzyme was completely inactive . Next, these thiols were analysed further by chemical modification . After iodoacetate treatment of GrdD, the enzyme activity was lost, but in the presence of acetyl phosphate enzyme activity was protected . Subsequently, the inactivated carboxymethylated enzyme and the protected enzyme were both denatured, and the remaining thiols were pyridylethylated . Peptides generated by proteolytic cleavage were separated and subjected to mass spectrometry . Cys98 was not accessible to carboxymethylation by iodoacetate in the native enzyme in the presence or absence of the substrate, but could be alkylated after denaturation . Cys359, in contrast, was protected from carboxymethylation in the presence of acetyl phosphate, but became accessible to pyridylethylation upon prior denaturation of the protein . This clearly confirmed the catalytic role of Cys359 as the active site thiol of GrdD responsible for liberation of acetyl phosphate. J Mol Biol, 2001 Dec 7, 314(4), 695 - 708 Distinctive features of the two classes of eukaryotic peptide deformylases; Serero A et al.; Peptide deformylases (PDFs) are essential enzymes of the N-terminal protein processing pathway of eubacteria . The recent discovery of two types of PDFs in higher plants, PDF1A and PDF1B, and the detection of PDF1A in humans, have raised questions concerning the importance of deformylation in eukaryotes . Here, we have characterized fully in vitro and compared the properties of the two classes of eukaryotic PDFs, PDF1A and PDF1B, using the PDFs from Arabidopsis thaliana and Lycopersicon esculentum . We have shown that the PDFs of a given class (1A or 1B) all display similar features, independently of their origin . We also observed similar specificity of all plant PDFs for natural substrate peptides, but identified a number of biochemical differences between the two classes (1A or 1B) . The main difference lies at the level of the bound cofactor, iron for PDF1B-like bacterial PDFs, and zinc for PDF1A . The nature of the metal cation has important consequences concerning the relative sensitivity to oxygen of the two plant PDFs . Investigation of the specificity of these enzymes with unusual substrates revealed additional differences between the two types of PDFs, enabling us to identify specific inhibitors with a lower affinity against PDF1As . However, the two plant PDFs were inhibited equally strongly in vitro by actinonin, an antibiotic that specifically acts on bacterial PDFs . Uptake of actinonin by A . thaliana seedlings was used to investigate the function of PDFs in the plant . Because it induces an albino phenotype, we conclude that deformylation is likely to play an essential role in the chloroplast . Cell, 2001 Nov 30, 107(5), 679 - 88 High resolution structure of the large ribosomal subunit from a mesophilic eubacterium; Harms J et al.; We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands . The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins . Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA. Protoplasma, 2001, 217(4), 177 - 84 Characterization of intracellular bacteria in the freshwater dinoflagellate Peridinium cinctum; Schweikert M et al.; Intracellular bacteria belonging to two phylogenetically different groups of eubacteria were found in cultures of the freshwater dinoflagellate Peridinium cinctum (O.F . Muller) Ehrenberg isolated from the eutrophic lake Plusssee (Federal Republic of Germany) . The phylogenetic relationships of the bacteria were studied with fluorochrome-conjugated oligonucleotides specific for archaebacteria, eubacteria, alpha-, beta- and gamma-proteobacteria, complementary to 16S rRNA and 23S rRNA sequences, respectively . The bacteria are members of the eubacterial alpha- and gamma-subgroups of proteobacteria. Genes Dev, 2001 Dec 1, 15(23), 3183 - 92 Global analysis of growth phase responsive gene expression and regulation of antibiotic biosynthetic pathways in Streptomyces coelicolor using DNA microarrays; Huang J et al.; The eubacterial species Streptomyces coelicolor proceeds through a complex growth cycle in which morphological differentiation/development is associated with a transition from primary to secondary metabolism and the production of antibiotics . We used DNA microarrays and mutational analysis to investigate the expression of individual genes and multigene antibiotic biosynthetic pathways during these events . We identified expression patterns in biosynthetic, regulatory, and ribosomal protein genes that were associated highly specifically with particular stages of development . A knowledge-based algorithm that correlates temporal changes in expression with chromosomal position identified groups of contiguous genes expressed at discrete stages of morphological development, inferred the boundaries of known antibiotic synthesis gene loci, and revealed novel physical clusters of coordinately regulated genes . Microarray analysis of RNA from cells mutated in genes regulating synthesis of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) identified proximate and distant sites that contain putative ABC transporter and two-component system genes expressed coordinately with genes of specific biosynthetic pathways and indicated the existence of two functionally and physically discrete regulons in the Red pathway. Appl Environ Microbiol, 2001 Dec, 67(12), 5558 - 67 Degradation of quercetin and luteolin by Eubacterium ramulus; Braune A et al.; The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied . Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively . The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra . The structure of the second intermediate, alphitonin, was additionally elucidated by (1)H and (13)C nuclear magnetic resonance analysis . In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid . Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid . The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation of E . ramulus . The degradation of luteolin by resting cells of E . ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis . The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E . ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure. Environ Microbiol, 2001 Oct, 3(10), 630 - 7 Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine; Johnson DB et al.; The biodiversity of culturable acidophilic microbes in three acidic (pH 2.7-3.7), metal-rich waters at an abandoned subarctic copper mine in central Norway was assessed . Acidophilic bacteria were isolated by plating on selective solid media, and dominant isolates were identified from their physiological characteristics and 16S rRNA gene sequences . The dominant iron-oxidizing acidophile in all three waters was an Acidithiobacillus ferrooxidans-like eubacterium, which shared 98% 16S rDNA identity with the type strain . A strain of Leptospirillum ferrooxidans was obtained from one of the waters after enrichment in pyrite medium, but this iron oxidizer was below detectable levels in the acidic waters themselves . In two sites, there were up to six distinct heterotrophic acidophiles, present at 10(3) ml(-1) . These included Acidiphilium-like isolates (one closely related to Acidiphilium rubrum, a second to Acidiphilium cryptum and a third apparently novel isolate), an Acidocella-like isolate (96% 16S rDNA identity to Acidocella facilis) and a bacterium that shared 94.5% 16S rDNA identity to Acidisphaera rubrifaciens . The other numerically significant heterotrophic isolate was not apparently related to any known acidophile, with the closest match (96% 16S rDNA sequence identity) to an acetogen, Frateuria aurantia . The results indicated that the biodiversity of acidophilic bacteria, especially heterotrophs, in acidic mine waters may be much greater than previously recognized. Mol Biol Evol, 2001 Dec, 18(12), 2240 - 9 GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity; Figge RM et al.; In this study we have determined gap sequences from nine different spirochetes . Phylogenetic analyses of these sequences in the context of all other available eubacterial and a selection of eukaryotic Gap sequences demonstrated that the eubacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene diversity encompasses at least five highly distinct gene families . Within these gene families, spirochetes show an extreme degree of sequence divergence that is probably the result of several lateral gene transfer events between spirochetes and other eubacterial phyla, and early gene duplications in the eubacterial ancestor . A Gap1 sequence from the syphilis spirochete Treponema pallidum has recently been shown to be closely related to GapC sequences from Euglenozoa . Here we demonstrate that several other spirochetal species are part of this cluster, supporting the conclusion that an interkingdom gene transfer from spirochetes to Euglenozoa must have occurred . Furthermore, we provide evidence that the GAPDH genes present in the protists Parabasalia may also be of spirochetal descent. J Bacteriol, 2001 Dec, 183(24), 7397 - 402 The mere lack of rT modification in initiator tRNA does not facilitate formylation-independent initiation in Escherichia coli; Thanedar S et al.; Formylation of initiator methionyl-tRNA is essential for normal growth of eubacteria . However, under special conditions, it has been possible to initiate protein synthesis with unformylated initiator tRNA even in eubacteria . Earlier studies suggested that the lack of ribothymidine (rT) modification in initiator tRNA may facilitate initiation in the absence of formylation . In this report we show, by using trmA strains of Escherichia coli (defective for rT modification) and a sensitive in vivo initiation assay system, that the lack of rT modification in the initiators is not sufficient to effect formylation-independent initiation of protein synthesis. J Microbiol Methods, 2001 Dec, 47(3), 281 - 92 Identification of indicator microorganisms using a standardized PNA FISH method; Perry-O'Keefe H et al.; A standardized fluorescent in situ hybridization (FISH) method using Peptide Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed . Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya . These PNA probes were evaluated by PNA FISH using 27 bacterial and 1 yeast species, representing both phylogenetically closely related species, as well as species important to both clinical and industrial settings . The S . aureus and P . aeruginosa PNA probes did not cross react with any of the organisms tested, whereas the E . coli PNA probe, as expected from sequence data, also detected Shigella species . The Salmonella PNA probe reacted with all of the 13 Salmonella strains, representing the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species . The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively . The general applicability of the PNA FISH method made simultaneous identification of multiple species, both gram-negative and gram-positive, in a mixed population an attractive possibility never accomplished using DNA probes . Four color images using differently labeled PNA probes showed simultaneous identification of E . coli, P . aeruginosa, S . aureus and Salmonella, thereby demonstrating the potential of multiplex FISH for various diagnostic applications within both clinical and industrial microbiology. Biochemistry, 2001 Nov 27, 40(47), 14123 - 33 tRNA-guanine transglycosylase from Escherichia coli: molecular mechanism and role of aspartate 89; Kittendorf JD et al.; The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a posttranscriptional transglycosylation reaction involved in the incorporation of the modified base queuine {Q, 7-(4,5-cis-dihydroxy-2-cyclopenten-1-ylaminomethyl)-7-deazaguanine} into tRNA . Previously, the crystal structure of the TGT from Zymomonas mobilis was solved in complex with preQ(1) (the substrate for the eubacterial TGT) {Romier et al . (1996) EMBO J . 15, 2850-2857} . An aspartate residue at position 102 (position 89 in the Escherichia coli TGT) was proposed to play a nucleophilic role in an associative catalytic mechanism . Although this is an attractive and precedented mechanism, a dissociative mechanism is equally plausible . In a dissociative mechanism, aspartate 89 would provide electrostatic stabilization of an oxocarbenium ion intermediate that is formed by dissociation of guanine . To clarify the nature of the catalytic mechanism of TGT, we have generated and characterized four mutations of aspartate 89 in the E . coli TGT (alanine, asparagine, cysteine, and glutamate) . All four mutant TGTs were able to noncovalently bind tRNA, but only the glutamate mutant was able to form a stable complex with the RNA substrate under denaturing conditions that was comparable to wild type . Furthermore, the glutamate mutant was the only mutant TGT that demonstrated significant activity . Kinetic parameters were determined for this enzyme and shown to be comparable to wild type, revealing that the enzyme is considerably tolerant of the positioning of the carboxylate . Under conditions of high enzyme concentrations and long time courses, the alanine, asparagine, and cysteine mutants showed very low levels (ca . 10(3)-fold lower than wild type) of activity that were linear with respect to enzyme concentration and dependent upon pH in a fashion similar to that of the wild type . However, the observed initial velocities were too low to accurately determine k(cat) and K(m) values . We hypothesize that the activity observed for these mutants is most likely derived from host strain TGT (wt) contamination . These results are most consistent with aspartate 89 acting as a nucleophile in an associative catalytic mechanism. Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 155 - 60 Essential role of residue H49 for activity of Escherichia coli 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme catalyzing the first step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid Synthesis; Querol J et al.; The first step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plant plastids and most eubacteria is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a recently described transketolase-like enzyme . To identify key residues for DXS activity, we compared the amino acid sequence of Escherichia coli DXS with that of E . coli and yeast transketolase (TK) . Alignment showed a previously undetected conserved region containing an invariant histidine residue that has been described to participate in proton transfer during TK catalysis . The possible role of the conserved residue in E . coli DXS (H49) was examined by site-directed mutagenesis . Replacement of this histidine residue with glutamine yielded a mutant DXS-H49Q enzyme that showed no detectable DXS activity . These findings are consistent with those obtained for yeast TK and demonstrate a key role of H49 for DXS activity . Infect Immun, 2001 Dec, 69(12), 7277 - 84 Enzyme degradation and proinflammatory activity in arthritogenic and nonarthritogenic Eubacterium aerofaciens cell walls; Zhang X et al.; Two almost-identical strains of Eubacterium aerofaciens isolated from the normal human gut flora were used . The cell wall (CW) of one strain with a peptidoglycan (PG) type A4alpha induces chronic arthritis in the rat after a single intraperitoneal injection, whereas CW of the other with PG type A4beta induces only a transient acute arthritis . The CW of the arthritogenic E . aerofaciens was a twofold-more-potent stimulator of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1) than the nonarthritogenic CW . After degradation with mutanolysin, the capacity of the arthritogenic PG to stimulate production of TNF-alpha and MCP-1 was significantly increased, whereas that of the nonarthritogenic PG was significantly decreased . In other words, after enzyme degradation the arthritogenic PG had a four- to fivefold-stronger stimulatory capacity than that of the enzyme-treated nonarthritogenic PG . These findings indicate that the arthritogenicity of CW or a PG is not dependent on the enzyme resistance alone but also on how the PG fragments released by enzyme degradation stimulate the production of proinflammatory cytokines. Biochemistry, 2001 Nov 20, 40(46), 13788 - 801 A molecular movie at 1.8 A resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds; Ren Z et al.; The photocycle of the bacterial blue-light photoreceptor, photoactive yellow protein, was stimulated by illumination of single crystals by a 7 ns laser pulse . The molecular events were recorded at high resolution by time-resolved X-ray Laue diffraction as they evolved in real time, from 1 ns to seconds after the laser pulse . The complex structural changes during the photocycle at ambient temperature are displayed in a movie of difference electron density maps relative to the dark state . The step critical to entry into the photocycle is identified as flipping of the carbonyl group of the 4-hydroxycinnamic acid chromophore into an adjacent, hydrophobic environment rather than the concomitant isomerization about the double bond of the chromophore tail . The structural perturbation generated at the chromophore propagates throughout the entire protein as a light-induced "protein quake" with its "epicenter" at the carbonyl moiety of the chromophore. Mol Microbiol, 2001 Oct, 42(2), 503 - 17 GidA is an FAD-binding protein involved in development of Myxococcus xanthus; White DJ et al.; A gene encoding a homologue of the Escherichia coli GidA protein (glucose-inhibited division protein A) lies immediately upstream of aglU, a gene encoding a WD-repeat protein required for motility and development in Myxococcus xanthus . The GidA protein of M . xanthus shares about 48% identity overall with the small (approximately equal to 450 amino acid) form of GidA from eubacteria and about 24% identity overall with the large (approximately equal to 620 amino acid) form of GidA from eubacteria and eukaryotes . Each of these proteins has a conserved dinucleotide-binding motif at the N-terminus . To determine if GidA binds dinucleotide, the M . xanthus gene was expressed with a His6 tag in E . coli cells . Purified rGidA is a yellow protein that absorbs maximally at 374 and 450 nm, consistent with FAD or FMN . Thin-layer chromatography (TLC) showed that rGidA contains an FAD cofactor . Fractionation and immunocytochemical localization show that full length GidA protein is present in the cytoplasm and transported to the periplasm of vegetative-grown M . xanthus cells . In cells that have been starved for nutrients, GidA is found in the cytoplasm . Although GidA lacks an obvious signal sequence, it contains a twin arginine transport (Tat) motif, which is conserved among proteins that bind cofactors in the cytoplasm and are transported to the periplasm as folded proteins . To determine if GidA, like AglU, is involved in motility and development, the gidA gene was disrupted . The gidA- mutant has wild-type gliding motility and initially is able to form fruiting bodies like the wild type when starved for nutrients . However, after several generations, a stable derivative arises, gidA*, which is indistinguishable from the gidA- parent on vegetative medium, but is no longer able to form fruiting bodies . The gidA* mutant releases a heat-stable, protease-resistant, small molecular weight molecule that acts in trans to inhibit aggregation and gene expression of wild-type cells during development. Mol Microbiol, 2001 Oct, 42(2), 427 - 37 Isolation and characterization of mutations in region 1.2 of Escherichia coli sigma70; Baldwin NE et al.; Eubacterial RNA polymerase uses the sigma (sigma) subunit for recognition of and transcription initiation from promoter DNA sequences . One family of sigma factors includes those related to the primary sigma factor from Escherichia coli, sigma70 . Members of the sigma70 family have four highly conserved domains, of which regions 2 to 4 are present in all members . Region 1 can be subdivided into regions 1.1 and 1.2 . Region 1.1 affects DNA binding by sigma70 alone, as well as transcription initiation by holoenzyme . Region 1.2, present and highly conserved in most sigma factors, has not yet been assigned a putative function, although previous work has demonstrated that it is not required for either association with the core subunits of RNA polymerase or promoter-specific binding by holoenzyme . We generated random single amino acid substitutions targeted to region 1.2 of E . coli sigma70 as well as a deletion of region 1.2, and characterized the behaviour of the mutant sigma factors both in vivo and in vitro to investigate the function of region 1.2 during transcription initiation . In this study, we show that mutations in region 1.2 can affect promoter binding, open complex and initiated complex formation and the transition from abortive transcription to elongation. Plant Cell, 2001 Nov, 13(11), 2539 - 51 HCF164 encodes a thioredoxin-like protein involved in the biogenesis of the cytochrome b(6)f complex in Arabidopsis; Lennartz K et al.; To understand the biogenesis of the plastid cytochrome b(6)f complex and to identify the underlying auxiliary factors, we have characterized the nuclear mutant hcf164 of Arabidopsis and isolated the affected gene . The mutant shows a high chlorophyll fluorescence phenotype and is severely deficient in the accumulation of the cytochrome b(6)f complex subunits . In vivo protein labeling experiments indicated that the mutation acts post-translationally by interfering with the assembly of the complex . Because of its T-DNA tag, the corresponding gene was cloned and its identity confirmed by complementation of homozygous mutant plants . HCF164 encodes a thioredoxin-like protein that possesses disulfide reductase activity . The protein was found in the chloroplast, where it is anchored to the thylakoid membrane at its lumenal side . HCF164 is closely related to the thioredoxin-like protein TxlA of Synechocystis sp PCC6803, most probably reflecting its evolutionary origin . The protein also shows a limited similarity to the eubacterial CcsX and CcmG proteins, which are required for the maturation of periplasmic c-type cytochromes . The putative roles of HCF164 for the assembly of the cytochrome b(6)f complex are discussed. J Med Microbiol, 2001 Nov, 50(11), 947 - 51 Characterisation of Eubacterium-like strains isolated from oral infections; Downes J et al.; The genus Eubacterium currently includes a heterogeneous group of gram-positive, non-spore-forming anaerobic bacilli, many of which are slow growing, fastidious and generally unreactive in biochemical tests . As a consequence, cultivation and identification of isolates are difficult and the taxonomy of the group remains indifferent . In this study, 105 isolates from odontogenic infections, infections associated with dental implants or saliva from healthy subjects and provisionally assigned to the genus Eubacterium were subjected to phenotypic and genotypic analysis . Ninety-one of the isolates were identified as belonging to one of 14 previously described species: Atopobium parvulum (5 isolates), A . rimae (29), Bulleidia extructa (2), Cryptobacterium curtum (1), Dialister pneumosintes (1), Eubacterium saburreum (2), E . sulci (8), E . yurii subsp . yurii (1), Filifactor alocis (3), Lactobacillus uli (1), Mogibacterium timidum (13), M . vescum (6), Pseudoramibacter alactolyticus (6) and Slackia exigua (13) . The remaining 14 isolates did not correspond to existing species . This study confirms the diversity of organisms provisionally assigned to the genus Eubacterium by conventional identification methods . This group of organisms is frequently isolated from oral infections but their role in the aetiology of these conditions has yet to be determined. Chemosphere, 2001 Nov, 45(6-7), 835 - 42 Biotransformation of p-toluic acid in anoxic estuarine sediments under a CO2 or N2/H2 atmosphere; Kuo CE et al.; The composition of the headspace gas affected the growth dynamics of microbial populations and the biotransformation pattern of p-toluic acid in anoxic estuarine sediments . Under CO2 atmosphere, p-toluic acid was transformed by the sediment microorganisms without a lag period, while under N2/H2 atmosphere, p-toluic acid was transformed after a lag period of 55 days . Under the N2/H2 atmosphere, the methanogen population, following a rapid increase of almost two orders of magnitude, remained at a high level until just before the onset of biotransformation . We hypothesize that during the lag period, the hydrogenotrophic methanogens were removing the H2, a step which is essential before the reaction can be exergonic . Acetogenic bacteria did not initiate decarboxylation as the first step of biotransformation under either atmosphere . Neither the methanogens nor the acetogenic bacteria appeared to be directly involved in the biotransformation of p-toluic acid under either atmosphere . Under the CO2 atmosphere, biotransformation of p-toluic acid involved sulfate-reducing bacteria, while under N2/H2, both sulfate-reducing bacteria and other eubacteria were involved. Cell Mol Life Sci, 2001 Sep, 58(10), 1461 - 74 Polyisoprenyl glycolipids as targets of CD1-mediated T cell responses; Moody DB; T cells are well known to recognize peptide antigens presented by major histocompatibility (MHC) class I or class II molecules . More recently, the CD1 family of antigen-presenting molecules has been shown to present both mammalian and microbial glycolipid antigens for specific recognition by T cells . Human CD1c proteins mediate T cell recognition of polyisoprenyl glycolipids, evolutionarily conserved phosphoglycolipids, which function in glycan synthesis pathways . This family of antigenic molecules is particularly attractive for the study of the molecular features that control T cell recognition of self and foreign glycolipids because natural polyisoprenols from mammals, fungi, protozoa, mycobacteria and eubacteria differ in structure . Moreover, these naturally occurring structural differences can influence their recognition by CDlc-restricted T cells . This review of the structural diversity and evolutionary relationships of polyisoprenoid glycolipids emphasizes those features of polyisoprenyl glycolipid biosynthesis that are relevant to their functions as targets of CD1-mediated T cell responses. Mikrobiol Z, 2001 Jul-Aug, 63(4), 3 - 8 {Gram-negative eubacteria isolated from the water, molluscs and algae of the Black Sea}; Smirnov VV et al.; Microorganisms strains (384) have been isolated from water, algae and molluscs collected in the Karadag reserve and the village of Katsiveli (Black Sea shore of the Crimea); 81 of the above strains are gram-negative oxidase-positive rods which require sodium ions for their growth . A group of nonfermenting mobile bacteria deprived of arginine dihydrolase have been distinguished among the latter; as to their physiological and biochemical properties and sensitivity to some antibiotics these bacteria were referred to Alteromonas, Marinomonas, Pseudoalteromonas genera. Nucleic Acids Res, 2001 Nov 1, 29(21), 4310 - 8 Identification of the first eubacterial endonuclease coded by an intein allele in the pps1 gene of mycobacteria; Saves I et al.; A survey of a vast range of mycobacterial strains led us to discover a new Pps1 intein allele in Mycobacterium gastri which differs from those of Mycobacterium tuberculosis and Mycobacterium leprae in both its sequence and insertion site . While little is known about Pps1, except that it belongs to the YC24 family of ABC transporters, we show that, unlike the other inteins described so far from Eubacteria, the MgaPps1 intein possesses a specific endonuclease activity . The intein is the first eubacterial intein to be characterised as an endonuclease . Like other intein endonucleases, its minimal sequence for recognition and cleavage is quite large, with 22 bp spanning the Pps1-c site . The fact that an active endonuclease is found among the mycobacterial inteins supports the concept of a cyclical model of invasion by horizontal transfer of these genes, followed by degeneration and loss until a new invasion event, thus explaining their long-term persistence in closely related eubacterial species. Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13449 - 53 Epub 2001 Oct 30. The plant oncogene rolD encodes a functional ornithine cyclodeaminase; Trovato M et al.; The plant oncogene rolD stimulates the reproductive phase transition in plants . We define here the function of its gene product . We show that the RolD protein bears sequence homology with ornithine cyclodeaminase, an uncommon enzyme of specialized-niche eubacteria and archaea that catalyzes the unusual NAD(+)-dependent conversion of ornithine to proline . To confirm the prediction of the bioinformatic analysis, the RolD protein was expressed in Escherichia coli and purified . An ornithine-dependent NAD(+) reduction that can be ascribed only to ornithine cyclodeaminase (OCD) activity was detected both in bacterial extracts containing RolD and in assays on the purified RolD protein . Furthermore, OCD activity was observed in soluble extracts from plants overexpressing rolD . The role of rolD in plant pathogenesis and its effect on plant reproductive development are discussed in light of the newly demonstrated enzymatic activity of its gene product. J Biochem (Tokyo), 2001 Nov, 130(5), 695 - 701 The structure of the archaebacterial ribosomal protein S7 and its possible interaction with 16S rRNA; Hosaka H et al.; Ribosomal protein S7 is one of the ubiquitous components of the small subunit of the ribosome . It is a 16S rRNA-binding protein positioned close to the exit of the tRNA, and it plays a role in initiating assembly of the head of the 30S subunit . Previous structural analyses of eubacterial S7 have shown that it has a stable alpha-helix core and a flexible beta-arm . Unlike these eubacterial proteins, archaebacterial or eukaryotic S7 has an N-terminal extension of approximately 60 residues . The crystal structure of S7 from archaebacterium Pyrococcus horikoshii (PhoS7) has been determined at 2.1 A resolution . The final model of PhoS7 consists of six major alpha-helices, a short 3(10)-helix and two beta-stands . The major part (residues 18-45) of the N-terminal extension of PhoS7 reinforces the alpha-helical core by well-extended hydrophobic interactions, while the other part (residues 46-63) is not visible in the crystal and is possibly fixed only by interacting with 16S rRNA . These differences in the N-terminal extension as well as in the insertion (between alpha1 and alpha2) of the archaebacterial S7 structure from eubacterial S7 are such that they do not necessitate a major change in the structure of the currently available eubacterial 16S rRNA . Some of the inserted chains might pass through gaps formed by helices of the 16S rRNA. Curr Microbiol, 2001 Dec, 43(6), 424 - 8 Chemical analysis of lipid-modified membrane proteins in Acholeplasma laidlawii; Le Henaff M et al.; The lipid modification of membrane proteins was investigated in Acholeplasma laidlawii by metabolic labeling and by chemical analysis . A S-glycerylcysteine residue was identified from membrane proteins and we reported the strong preference for saturated acyl chains into the lipid modification . Differential release of fatty acids revealed a ratio {(O-ester- + amide-bound acyl chains)/O-ester-linked chains} close to 1.1 which suggests the involvement of only two O-ester linked fatty acids in the acylation process . Present data indicate that acyl proteins in A . laidlawii are true lipoproteins (mainly diacylated) probably processed by a mechanism analogous to that described for eubacteria and other mycoplasmas. Arch Microbiol, 2001 Oct, 176(4), 237 - 42 Negative regulation of the heat shock response in Streptomyces; Servant P et al.; All organisms respond to a sudden increase in temperature by inducing the synthesis of a set of proteins called heat shock proteins (HSPs) . Although the induction of HSPs is a universal response, a diversity of mechanisms control HSP synthesis in different organisms . In Streptomyces, the synthesis of major HSPs, such as the widespread molecular chaperones DnaK, ClpB, GroEL and HSP18, is negatively controlled at the transcriptional level by at least three different repressors . The control of groE gene expression involves an inverted repeat (called the CIRCE element) that is highly conserved among eubacteria, and the HrcA repressor . The dnaK operon and clpB belong to the HspR /HAIR regulon . The HspR repressor-HAIR operator system is used in some bacteria but is not widespread . In particular, it has not been found in gram-positive bacteria with low G+C content . Transcription of hsp18, which encodes a small HSP, is regulated by the RheA repressor . This repressor, which has intrinsic thermosensor activity, has to date been identified only in Streptomyces. FEBS Lett, 2001 Oct 26, 507(2), 123 - 7 In silico analysis of the tRNA:m1A58 methyltransferase family: homology-based fold prediction and identification of new members from Eubacteria and Archaea; Bujnicki JM; The amino acid sequences of Gcd10p and Gcd14p, the two subunits of the tRNA:(1-methyladenosine-58; m(1)A58) methyltransferase (MTase) of Saccharomyces cerevisiae, have been analyzed using iterative sequence database searches and fold recognition programs . The results suggest that the 'catalytic' Gcd14p and 'substrate binding' Gcd10p are related to each other and to a group of prokaryotic open reading frames, which were previously annotated as hypothetical protein isoaspartate MTases in sequence databases . It is predicted that the prokaryotic proteins are genuine tRNA:m(1)A MTases based on similarity of their predicted active site to the Gcd14p family . In addition to the MTase domain, an additional domain was identified in the N-terminus of all these proteins that may be involved in interaction with tRNA . These results suggest that the eukaryotic tRNA:m(1)A58 MTase is a product of gene duplication and divergent evolution of a possibly homodimeric prokaryotic enzyme. Mol Cell, 2001 Oct, 8(4), 733 - 4 Translation termination: a ghost ballet? Yarus M. In the October 5th issue of Cell, clarify the end of translation on a eubacterial mRNA . By elucidating the molecular choreography of class I and class II release factors within the ribosome, they show how the steps around the release of nascent protein are ordered. RNA, 2001 Oct, 7(10), 1432 - 41 tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition; Nonekowski ST et al.; In eubacteria, the biosynthesis of queuine, a modified base found in the wobble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway . One of the key enzymes in this pathway, tRNA-guanine transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1 . Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the second position of its seven base anticodon loop . Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited structural context . To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E . coli tRNA(Tyr) minihelix . The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants . Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU(+1)) . The other analogs were not active . This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem . In E . coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine . We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33 . U33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs . We find that mutation of this base confers on TGT the ability to recognize U34G35U36, and suggests that loop conformation affects recognition . The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate. Nature, 2001 Oct 25, 413(6858), 814 - 21 Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria; Schlunzen F et al.; Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics . Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action . Conversely, drugs are excellent tools for studying the ribosome function . To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin . We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins . Here we report the details of antibiotic interactions with the components of their binding sites . Our results also show the importance of putative Mg+2 ions for the binding of some drugs . This structural analysis should facilitate rational drug design. J Biol Chem, 2001 Dec 28, 276(52), 48915 - 20 Epub 2001 Oct 24. Repair of oxidized proteins . Identification of a new methionine sulfoxide reductase; Grimaud R et al.; Oxidation of methionine residues to methionine sulfoxide can lead to inactivation of proteins . Methionine sulfoxide reductase (MsrA) has been known for a long time, and its repairing function well characterized . Here we identify a new methionine sulfoxide reductase, which we referred to as MsrB, the gene of which is present in genomes of eubacteria, archaebacteria, and eucaryotes . The msrA and msrB genes exhibit no sequence similarity and, in some genomes, are fused . The Escherichia coli MsrB protein (currently predicted to be encoded by an open reading frame of unknown function named yeaA) was used for genetic, enzymatic, and mass spectrometric investigations . Our in vivo study revealed that msrB is required for cadmium resistance of E . coli, a carcinogenic compound that induces oxidative stress . Our in vitro studies, showed that (i) MsrB and MsrA enzymes reduce free methionine sulfoxide with turn-over rates of 0.6 min(-1) and 20 min(-1), respectively, (ii) MsrA and MsrB act on oxidized calmodulin, each by repairing four to six of the eight methionine sulfoxide residues initially present, and (iii) simultaneous action of both MsrA and MsrB allowed full reduction of oxidized calmodulin . A possibility is that these two ubiquitous methionine sulfoxide reductases exhibit different substrate specificity. Arch Virol, 2001 Aug, 146(8), 1465 - 85 An iteron-related domain is associated to Motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach; Arguello-Astorga GR et al.; Geminiviruses encode a replication initiator protein, Rep, which binds in a sequence-specific fashion to iterated DNA motifs (iterons) functioning as essential elements for virus-specific replication . By using the iterons of more than one hundred geminiviruses as heuristic devices, we have identified a Rep subdomain 8 to 10 residues in length, whose primary structure varies among viruses harboring different iterons, but which is similar among viruses with identical iterons, regardless of their differences in host range, insect vector, geographical origin or genome structure . Close analysis of this iteron-related domain (IRD) revealed consistent correlations between specific Rep residues and defined nucleotides of its cognate iteron, thus providing important insights about the molecular code which dictates the Rep preference for specific DNA sequences . A model of potential Rep-iteron contacts is proposed . The identified IRD is adjacent to a conserved motif characteristic of a superfamily of rolling-circle (RC) replication proteins, and secondary structure predictions suggest that those Rep subdomains form together the core of a novel DNA-binding domain possessing a beta-sheet as recognition subdomain, which is apparently conserved in the replication proteins of nanoviruses, circoviruses, microviruses, and a variety of ssDNA plasmids of eubacteria, archaebacteria and red algae . The evolutionary implications of these findings are discussed. J Mol Evol, 2001 Oct-Nov, 53(4-5), 394 - 401 Distinct stages of protein evolution as suggested by protein sequence analysis; Trifonov EN et al.; Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code . The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses . According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first . Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A) . It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes . After the fusion of the minigenes, a mosaic of the alphabets would form . Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units . The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25-30 residues (Berezovsky et al., 2000) . Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25-30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops . The loop closure stage is followed by the formation of typical folds of 100-200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains . This size was apparently dictated by the optimal ring closure for DNA . In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures . Further gene fusions lead to the formation of modern multidomain proteins . Recombinational gene splicing is likely to have appeared after the DNA circularization stage. J Mol Evol, 2001 Oct-Nov, 53(4-5), 364 - 76 Skew of mononucleotide frequencies, relative abundance of dinucleotides, and DNA strand asymmetry; Shioiri C et al.; Based on 152 mitochondrial genomes and 36 bacterial chromosomes that have been completely sequenced, as well as three long contigs for human chromosomes 6, 21, and 22, we examined skews of mononucleotide frequencies and the relative abundance of dinucleotides in one DNA strand . Each group of these genomes has its own characteristics . Regarding mitochondrial genomes, both CpG and GpT are underrepresented, while either GpG or CpC or both are overrepresented . The relative frequency of nucleotide T vs A and of nucleotide G vs C is strongly skewed, due presumably to strand asymmetry in replication errors and unidirectional DNA replication from single origins . Exceptions are found in the plant and yeast mitochondrial genomes, each of which may replicate from multiple origins . Regarding bacterial genomes, the "universal" rule of CpG deficiency is restricted to archaebacteria and some eubacteria . In other eubacteria, the most underrepresented dinucleotide is either TpA or GpT . In general, there are significant T vs A and G vs C skews in each half of the bacterial genome, although these are almost exactly canceled out over the whole genome . Regarding human chromosomes 6, 21, and 22, dinucleotide CpG tends to be avoided . The relative frequency of mononucleotides exhibits conspicuous local skews, suggesting that each of these chromosomal segments contains more than one DNA replication origin . It is concluded that, when there are several replicons in a genomic region, not only the number of DNA replication origins but also the directionality is important and that the observed patterns of nucleotide frequencies in the genome strongly support the hypothesis of strand asymmetry in replication errors. Plant Cell Physiol, 2001 Oct, 42(10), 1034 - 43 An Arabidopsis sigma factor (SIG2)-dependent expression of plastid-encoded tRNAs in chloroplasts; Kanamaru K et al.; A eubacteria-type RNA polymerase (PEP) plays crucial roles for chloroplast development in higher plants . The core subunits are encoded on plastid DNA (rpo genes) while the regulatory sigma factors are encoded on the nuclear DNA (SIG genes) . However, the definite gene specificity of each sigma factor is unknown . We recently identified an Arabidopsis recessive pale-green mutant abc1 in which T-DNA is inserted in SIG2 (sigB) . In this mutant, almost normal etioplasts were developed under dark conditions while the small chloroplasts with poor thylakoid membranes and stacked lamellar were developed under light conditions . The sig2-1 mutant was deficient in accumulating enough photosynthetic and photosynthesis-related proteins as well as chlorophyll . However, mRNAs of their structural genes were not significantly reduced . Further analyses revealed that several plastid-encoded tRNAs including trnE-UUC that has dual function for protein and ALA biosyntheses were drastically reduced in the sig2-1 mutant . In contrast, nucleus-encoded T7 phage-type RNA polymerase (NEP)-dependent gene transcripts were steadily accumulated in the mutant . These results indicate that progress of chloroplast development requires SIG2-dependent expression of plastid genes, particularly some of the tRNA genes. J Bacteriol, 2001 Nov, 183(22), 6714 - 6 Presence of prokaryotic and eukaryotic species in all subgroups of the PP(i)-dependent group II phosphofructokinase protein family; Muller M et al.; Inorganic pyrophosphate-dependent phosphofructokinase (PP(i)-PFK) of the amitochondriate eukaryote Mastigamoeba balamuthi was sequenced and showed about 60% identity to PP(i)-PFKs from two eubacteria, Propionibacterium freudenreichii and Sinorhizobium meliloti . These gene products represent a newly recognized lineage of PFKs . All four lineages of group II PFKs, as defined by phylogenetic analysis, contained both prokaryotic and eukaryotic species, underlining the complex evolutionary history of this enzyme. Proc Natl Acad Sci U S A, 1991 Sep 15, 88(18), 8184 - 8 Membrane-bounded nucleoid in the eubacterium Gemmatata obscuriglobus; Fuerst JA et al.; The freshwater budding eubacterium Gemmata obscuriglobus possesses a DNA-containing nuclear region that is bounded by two nuclear membranes . The membrane-bounded nature of the nucleoid in this bacterium was shown by thin sectioning of chemically fixed cells, thin sectioning of freeze-substituted cells, and freeze-fracture/freeze-etch . The fibrillar nucleoid was surrounded by electron-dense granules that were in turn enveloped by two nuclear membranes separated by an electron-transparent space . Immunogold labeling of thin sections of conventionally fixed cells with anti-double-stranded DNA antibody demonstrated double-stranded DNA associated with fibrillar material within the membrane boundary . The occurrence of a membrane-bounded nucleoid in a eubacterial prokaryote is a significant exception to the evidence supporting the prokaryote/eukaryote dichotomous classification of cell structure. Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1797 - 804 Characterization of novel human oral isolates and cloned 16S rDNA sequences that fall in the family Coriobacteriaceae: description of olsenella gen . nov., reclassification of Lactobacillus uli as Olsenella uli comb . nov . and description of Olsenella profusa sp . nov; Dewhirst FE et al.; The diversity of organisms present in the subgingival pockets of patients with periodontitis and acute necrotizing ulcerative gingivitis (ANUG) were examined previously . The 16S rRNA genes of subgingival plaque bacteria were amplified using PCR with a universal forward primer and a spirochaete-selective reverse primer . The amplified DNA was cloned into Escherichia coli . In one subject with ANUG, 70 clones were sequenced . Seventy-five per cent of the clones were spirochaetal, as expected . Twelve of the remaining clones fell into two clusters that represent novel phylotypes in the family Coriobacteriaceae . The first novel phylotype was most closely related to Atopobium rimae (98% similarity) . The phylotype probably represents a novel Atopobium species, but will not be named until cultivable strains are obtained . The second novel phylotype was only 91% similar to described Atopobium species and 84% similar to Coriobacterium glomerans . The 16S rRNA sequences of the type strain of Lactobacillus uli and a strain representing the Moores' Eubacterium group D52 were determined as part of on ongoing sequence analysis of oral bacteria . The sequence for L . uli was more than 99.8% similar to sequences for the second clone phylotype . It therefore appears that the second clone phylotype and L . uli represent the same species . The sequence for the Eubacterium D52 strain was 95.6% similar to that of L . uli . The G+C content of the DNA of L . uli and Eubacterium D52 is 63-64 mol % . These organisms are thus distinct from the neighbouring genus Atopobium, which has a DNA G+C content of 35-46 mol% . A new genus, Olsenella gen . nov., is proposed for these two species on the basis of phenotypic characteristics and 16S rRNA sequence analysis to include Olsenella uli comb . nov . and Olsenella profusa sp . nov. Structure (Camb), 2001 Aug, 9(8), 647 - 58 Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site; Vicens Q et al.; BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA . RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution . Each RNA fragment contains two A sites inserted between Watson-Crick pairs . The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines . In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site . In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics . Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408 . Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair . The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II . The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex . CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns . The observed contacts rationalize the protection, mutation, and resistance data . The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome. FEBS Lett, 2001 Aug 3, 502(3), 113 - 6 Evolution of tuf genes: ancient duplication, differential loss and gene conversion; Lathe WC 3rd et al.; The tuf gene of eubacteria, encoding the EF-tu elongation factor, was duplicated early in the evolution of the taxon . Phylogenetic and genomic location analysis of 20 complete eubacterial genomes suggests that this ancient duplication has been differentially lost and maintained in eubacteria. BMC Evol Biol . 2001;1(1):4 . Epub 2001 Sep 12. A genomic timescale for the origin of eukaryotes; Hedges SB et al.; BACKGROUND: Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis . However, details of the timing and number of symbiotic events are unclear . A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen . We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes . RESULTS: Eukaryotes were found to evolve faster than prokaryotes, with those eukaryotes derived from eubacteria evolving faster than those derived from archaebacteria . We found an early time of divergence (approximately 4 billion years ago, Ga) for archaebacteria and the archaebacterial genes in eukaryotes . Our analyses support at least two horizontal gene transfer events in the origin of eukaryotes, at 2.7 Ga and 1.8 Ga . Time estimates for the origin of cyanobacteria (2.6 Ga) and the divergence of an early-branching eukaryote that lacks mitochondria (Giardia) (2.2 Ga) fall between those two events . CONCLUSIONS: We find support for two symbiotic events in the origin of eukaryotes: one premitochondrial and a later mitochondrial event . The appearance of cyanobacteria immediately prior to the earliest undisputed evidence for the presence of oxygen (2.4-2.2 Ga) suggests that the innovation of oxygenic photosynthesis had a relatively rapid impact on the environment as it set the stage for further evolution of the eukaryotic cell. Plant Cell Physiol, 2001 Sep, 42(9), 1017 - 23 The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane; Yamada H et al.; Common histidine-to-aspartate (His-->Asp) phosphorelay is a paradigm of signal transduction in both prokaryotes and eukaryotes for the propagation of certain environmental stimuli, in which histidine (His)-kinases play central roles as sensors for environmental signals . For the higher plant, Arabidopsis thaliana, it was recently suggested that the His-kinase (AHK4 / CRE1 / WOL) is a sensor for cytokinins, which are a class of plant hormones important for the regulation of cell division and differentiation . Interestingly, AHK4 is capable of functioning as a cytokinin sensor in the eubacterium, Escherichia coli (Suzuki et al . 2001, Plant Cell Physiol . 42: 107) . Here we further show that AHK4 is a primary receptor that directly binds a variety of natural and synthetic cytokinins (e.g . not only N(6)-substituted aminopurines such as isopentenyl-adenine, trans-zeatin, benzyl-adenine, but also diphenylurea derivatives such as thidiazuron), in a highly specific manner (K(d) = 4.55+/-0.48x10(-9) M) . AHK4 has a presumed extracellular domain, within which a single amino acid substitution (Thr-301 to Ile) was shown to result in loss of its ability to bind cytokinins . This particular mutation corresponds to the previously reported wol allele (wooden leg) that causes a striking phenotype defective in vascular morphogenesis . Collectively, evidence is presented that AHK4 and its homologues (AHK3 and possibly AHK2) are receptor kinases that can transduce cytokinin signals across the plasma membrane of A . thaliana. J Biol Chem, 2001 Dec 7, 276(49), 45959 - 68 Epub 2001 Sep 27. Characterization of single-stranded DNA-binding proteins from Mycobacteria . The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein; Reddy MS et al.; Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination . We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis . Sequence comparison of M . smegmatis SSB revealed that it is homologous to M . tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail . The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB . The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain . Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions . Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo . The carboxyl-terminal domain of M . smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA . These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination. EMBO J, 2001 Oct 1, 20(19), 5305 - 11 The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with GDP reveals novel features in nucleotide binding and exchange; Vitagliano L et al.; The crystal structure of elongation factor 1alpha from the archaeon Sulfolobus solfataricus in complex with GDP (SsEF-1alpha.GDP) at 1.8 A resolution is reported . As already known for the eubacterial elongation factor Tu, the SsEF-1alpha.GDP structure consists of three different structural domains . Surprisingly, the analysis of the GDP-binding site reveals that the nucleotide- protein interactions are not mediated by Mg(2+) . Furthermore, the residues that usually co-ordinate Mg(2+) through water molecules in the GTP-binding proteins, though conserved in SsEF-1alpha, are located quite far from the binding site . {(3)H}GDP binding experiments confirm that Mg(2+) has only a marginal effect on the nucleotide exchange reaction of SsEF-1alpha, although essential to GTPase activity elicited by SsEF-1alpha . Finally, structural comparisons of SsEF- 1alpha.GDP with yeast EF-1alpha in complex with the nucleotide exchange factor EF-1beta shows that a dramatic rearrangement of the overall structure of EF-1alpha occurs during the nucleotide exchange. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11627 - 32 A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems; Dalrymple BP et al.; The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis . This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes . The clamp proteins also participate in a variety of dynamic and competing protein-protein interactions . However, clamp-protein binding sequences have not so far been identified in the eubacteria . Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein-protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL{SD}LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding clamp (the beta subunit of Escherichia coli DNA polymerase III holoenzyme) . Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PolC, PolB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family . The component tripeptide DLF inhibits the binding of the alpha (DnaE) subunit of E . coli DNA polymerase III to beta at microM concentration, identifying key residues . Comparison of the eubacterial, eukaryotic, and archaeal sliding clamp binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape. C R Acad Sci III, 2001 Oct, 324(10), 923 - 8 Mycoplasmas, plants, insect vectors: a matrimonial triangle; Garnier M et al.; Plant pathogenic mycoplasmas were discovered by electron microscopy, in 1967, long after the discovery and culture in 1898 of the first pathogenic mycoplasma of animal origin, Mycoplasma mycoides . Mycoplasmas are Eubacteria of the class Mollicutes, a group of organisms phylogenetically related to Gram-positive bacteria . Their more characteristic features reside in the small size of their genomes, the low guanine (G) plus cytosine (C) content of their genomic DNA and the lack of a cell wall . Plant pathogenic mycoplasmas are responsible for several hundred diseases and belong to two groups: the phytoplasmas and the spiroplasmas . The phytoplasmas (previously called MLOs, for mycoplasma like organisms) were discovered first; they are pleiomorphic, and have so far resisted in vitro cultivation . Phytoplasmas represent the largest group of plant pathogenic Mollicutes . Only three plant pathogenic spiroplasmas are known today . Spiroplasma citri, the agent of citrus stubborn was discovered and cultured in 1970 and shown to be helical and motile . S . kunkelii is the causal agent of corn stunt . S . phoeniceum, responsible for periwinkle yellows, was discovered in Syria . There are many other spiroplasmas associated with insects and ticks . Plant pathogenic mycoplasmas are restricted to the phloem sieve tubes in which circulates the photosynthetically-enriched sap, the food for many phloem-feeding insects (aphids, leafhoppers, psyllids, etc.) . Interestingly, phytopathogenic mycoplasmas are very specifically transmitted by leafhoppers or psyllid species . In this paper, the most recent knowledge on phytopathogenic mycoplasmas in relation with their insect and plant habitats is presented as well as the experiments carried out to control plant mycoplasma diseases, by expression of mycoplasma-directed-antibodies in plants (plantibodies). Biofactors, 2001, 14(1-4), 5 - 10 Distinctive features in the SelB family of elongation factors for selenoprotein synthesis . A glimpse of an evolutionary complexified translation apparatus; Fagegaltier D et al.; The last ten years have seen a dramatic increase in our understanding of the molecular mechanism allowing specific incorporation of selenocysteine into selenoproteins . Whether in pro