Eubacter

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 prokaryotes or eukaryotes, this incorporation requires several gene products, among which the specialized elongation factor SelB and the tRNA(Sec) play a pivotal role . While the molecular actors have been discovered and their role elucidated in the eubacterial machinery, recent data from our and other laboratories pointed to a higher degree of complexity in archaea and eukaryotes . These findings also revealed that more needs to be discovered in this area . This review will focus on phylogenetic aspects of the SelB proteins . In particular, we will discuss the concerted evolution that occurred within the SelB/tRNA(Sec) couples, and also the distinctive roles carried out by the SelB C-terminal domains in eubacteria on the one side, and archaea and eukaryotes, on the other.

Anal Biochem, 2001 Oct 1, 297(1), 60 - 70
A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid; Forster AC et al.; The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components . Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E . coli initiation and elongation factors . Highly purified ribosomes are required to yield products with strong dependencies on the translation factors . Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination . Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA . The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates . The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs .

J Bacteriol, 2001 Oct, 183(20), 6085 - 94
Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin; Dairi T et al.; A gene cluster containing the mevalonate pathway genes (open reading frame 2 {ORF2} to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y . Hamano, T . Dairi, M . Yamamoto, T . Kawasaki, K Kaneda, T . Kuzuyama, N . Itoh, and H . Seto, Biosci . Biotech . Biochem . 65:1627-1635, 2001) . Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes . We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions . The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP . The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter . The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP . The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene . To the best of our knowledge, this is the first report of a eubacterial DC.

RNA, 2001 Sep, 7(9), 1227 - 38
CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin; Till B et al.; Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions . The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene . Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product . Several observations support the notion that CRS1 is a bona fide group II intron splicing factor . First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA . Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain . This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea . crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene . In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation . The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.

Int Endod J, 2001 Sep, 34(6), 463 - 70
Detection of Slackia exigua, Mogibacterium timidum and Eubacterium saphenum from pulpal and periradicular samples using the Polymerase Chain Reaction (PCR) method; Hashimura T et al.; AIM: The purpose of this study was to detect Slackia exigua from root canal samples using a sensitive PCR amplification method . Mogibacterium timidum and Eubacterium saphenum were also included because of their culture-difficult properties . METHODOLOGY: The species-specific PCR primers were prepared according to 16S rDNA sequence analysis data, and confirmed to be effective for PCR amplification as species-specific, respectively . A total of 36 clinical samples were obtained during the first visit of root canal treatment . RESULTS: The sensitivity of detection was a minimum of 10 organisms for S . exigua and five organisms for M . timidum and E . saphenum, respectively . In seven cases of pulpitis, Sexigua was detected in two cases (29%), and M . timidum in two cases (29%), but E . saphenum was not detected . In 17 cases of root canal treatment, S . exigua was detected in seven cases (41%), M . timidum in 12 cases (71%) and E . saphenum in four cases (24%) . In 12 cases of root canal retreatment, S . exigua was detected in three cases (25%), M . timidum in three cases (25%) and E . saphenum in two cases (17%) . CONCLUSIONS: S . exigua, M . timidum and E . saphenum were present in root canal systems, and may be associated with pulpal and periradicular pathosis.

Mol Microbiol, 2001 Sep, 41(5), 1159 - 72
A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress; Hyyrylainen HL et al.; The Gram-positive eubacterium Bacillus subtilis is well known for its high capacity to secrete proteins into the environment . Even though high-level secretion of proteins is an efficient process, it imposes stress on the cell . The present studies were aimed at the identification of systems required to combat this so-called secretion stress . A two-component regulatory system, named CssR-CssS, was identified, which bears resemblance to the CpxR-CpxA system of Escherichia coli . The results show that the CssR/S system is required for the cell to survive the severe secretion stress caused by a combination of high-level production of the alpha-amylase AmyQ and reduced levels of the extracytoplasmic folding factor PrsA . As shown with a prsA3 mutation, the Css system is required to degrade misfolded exported proteins at the membrane-cell wall interface . This view is supported by the observation that transcription of the htrA gene, encoding a predicted membrane-bound protease of B . subtilis, is strictly controlled by CssS . Notably, CssS represents the first identified sensor for extracytoplasmic protein misfolding in a Gram-positive eubacterium . In conclusion, the results show that quality control systems for extracytoplasmic protein folding are not exclusively present in the periplasm of Gram-negative eubacteria, but also in the Gram-positive cell envelope.

Biochem Biophys Res Commun, 2001 Sep 21, 287(2), 462 - 7
Chloroplast targeting of chloroplast division FtsZ2 proteins in Arabidopsis; Fujiwara M et al.; Plant nuclear genomes encode chloroplast division proteins homologous to the eubacterial cell division protein FtsZ . In higher plants, FtsZ genes constitute a small gene family that consists of two subgroups, FtsZ1 and FtsZ2 . It was previously hypothesized that members of one family (FtsZ1) targeted chloroplasts, while members of the other family (FtsZ2) localized in the cytoplasm . We determined the full-length cDNA sequences of two FtsZ2 genes from Arabidopsis thaliana (AtFtsZ2-1 and AtFtsZ2-2) and found that the genes encode polypeptides of 478 and 473 amino acids, respectively, and both contain N-terminal extensions beyond what have previously been predicted . The N-terminal regions of both AtFtsZ2-1 and AtFtsZ2-2 were expressed as green fluorescent protein (GFP) fusions under the cauliflower mosaic virus 35S promoter in bombarded tobacco cells . Confocal laser scanning microscopy revealed both fusions exclusively localized to chloroplasts, demonstrating that the N-terminal regions function as chloroplast-targeting signals in vivo . Thus, FtsZ2 proteins function within chloroplasts .

Plant Physiol, 2001 Sep, 127(1), 97 - 107
Eukaryotic peptide deformylases . Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis; Dirk LM et al.; Arabidopsis (ecotype Columbia-0) genes, AtDEF1 and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase . Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences . Radiolabeled full-length AtDEF1 was imported and processed by isolated pea (Pisum sativum L . Laxton's Progress No . 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts . The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed in Escherichia coli and purified using C-terminal hexahistidyl tags . Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E . coli enzyme . Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively . Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development . The results suggest an essential role for peptide deformylase in protein processing in all plant plastids.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2001 Sep, 92(3), 329 - 34
Identification of oral spirochetes at the species level and their association with other bacteria in endodontic infections; Jung IY et al.; OBJECTIVES: Recent molecular approaches have revealed that fastidious organisms such as Bacteroides forsythus and oral treponemes were frequently found in root canals with apical periodontitis . The purpose of this study was to identify the isolates of oral spirochetes at the species level in endodontic infections and to determine their association with B forsythus and Porphyromonas gingivalis . STUDY DESIGN: Seventy-nine teeth with apical periodontitis were selected for this study . After sampling from the root canals aseptically, polymerase chain reaction amplification for the 16S rRNA gene was performed with eubacterial universal primers . Subsequently, dot-blot hybridization was performed with 8 species-specific oligonucleotide probes . The microbial associations were analyzed by using the odds ratio . RESULTS: The most frequently found species was P gingivalis (27.4%), followed by Treponema maltophilum (26%), B forsythus (16.4%), and Treponema socranskii (2.7%) . Other treponemes, including Treponema denticola, were not detected in our samples . Significant microbial associations were identified between T maltophilum, B forsythus, and P gingivalis by performing analysis with the odds ratio . CONCLUSION: Our results indicate that T maltophilum should be included in etiologic studies of endodontic diseases.

J Biol Chem, 2001 Nov 9, 276(45), 41850 - 5 Epub 2001 Sep 10.
Alternate paradigm for intrinsic transcription termination in eubacteria; Unniraman S et al.; Intrinsic transcription terminators are functionally defined as sites that bring about termination in vitro with purified RNA polymerase alone . Based on studies in Escherichia coli, intrinsic termination requires a palindromic stretch followed by a trail of T (or U) residues in the coding strand . We have developed a highly efficient algorithm to identify hairpin potential sequences in bacterial genomes in order to build a general model for intrinsic transcription termination . The algorithm was applied to analyze the Mycobacterium tuberculosis genome . We find that hairpin potential sequences are concentrated in the immediate downstream of stop codons . However, most of these structures either lack the U trail entirely or have a mixed A/U trail reflecting an evolutionarily relaxed requirement for the U trail in the mycobacterial genome . Predicted atypical structures were shown to work efficiently as terminators both inside the mycobacterial cell and in vitro with purified RNA polymerase . The results are discussed in light of the kinetic competition models for transcription termination . The algorithm identifies >90% of experimentally tested terminators in bacteria and is an invaluable tool in identifying transcription units in whole genomes.

Genome Res, 2001 Sep, 11(9), 1484 - 502
A proteomic view on genome-based signal peptide predictions; Antelmann H et al.; The availability of complete genome sequences has allowed the prediction of all exported proteins of the corresponding organisms with dedicated algorithms . Even though numerous studies report on genome-based predictions of signal peptides and cell retention signals, they lack a proteomic verification . For example, 180 secretory and 114 lipoprotein signal peptides were predicted recently for the Gram-positive eubacterium Bacillus subtilis . In the present studies, proteomic approaches were used to define the extracellular complement of the B . subtilis secretome . Using different growth conditions and a hyper-secreting mutant, approximately 200 extracellular proteins were visualized by two-dimensional (2D) gel electrophoresis, of which 82 were identified by mass spectrometry . These include 41 proteins that have a potential signal peptide with a type I signal peptidase (SPase) cleavage site, and lack a retention signal . Strikingly, the remaining 41 proteins were predicted previously to be cell associated because of the apparent absence of a signal peptide (22), or the presence of specific cell retention signals in addition to an export signal (19) . To test the importance of the five type I SPases and the unique lipoprotein-specific SPase of B . subtilis, the extracellular proteome of (multiple) SPase mutants was analyzed . Surprisingly, only the processing of the polytopic membrane protein YfnI was strongly inhibited in Spase I mutants, showing for the first time that a native eubacterial membrane protein is a genuine Spase I substrate . Furthermore, a mutation affecting lipoprotein modification and processing resulted in the shedding of at least 23 (lipo-)proteins into the medium . In conclusion, our observations show that genome-based predictions reflect the actual composition of the extracellular proteome for approximately 50% . Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.

Int J Syst Bacteriol, 1989 Jan, 39(1), 78 - 84
5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general; Van den Eynde H et al.; The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium . A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses . In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses . Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses . Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

J Mol Evol, 1987, 25, 81 - 8
The evolution of glutathione metabolism in phototrophic microorganisms; Fahey RC et al.; Of the many roles ascribed to glutathione (GSH) the one most clearly established is its role in the protection of higher eucaryotes against oxygen toxicity through destruction of thiol-reactive oxygen byproducts . If this is the primary function of GSH then GSH metabolism should have evolved during or after the evolution of oxygenic photosynthesis . That many bacteria do not produce GSH is consistent with this view . In the present study we have examined the low-molecular-weight thiol composition of a variety of phototrophic microorganisms to ascertain how evolution of GSH production is related to evolution of oxygenic photosynthesis . Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols to fluorescent derivatives, which were analyzed by high-pressure liquid chromatography . Significant levels of GSH were not found in the green bacteria (Chlorobium thiosulfatophilum and Chloroflexus aurantiacus) . Substantial levels of GSH were present in the purple bacteria (Chromatium vinosum, Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodocyclus gelatinosa), the cyanobacteria {Anacystis nidulans, Microcoleus chthonoplastes S.G., Nostoc muscorum, Oscillatoria amphigranulata, Oscillatoria limnetica, Oscillatoria sp . (Stinky Spring, Utah), Oscillatoria terebriformis, Plectonema boryanum, and Synechococcus lividus}, and eucaryotic algae (Chlorella pyrenoidsa, Chlorella vulgaris, Euglena gracilis, Scenedesmus obliquus, and Chlamydomonas reinhardtii) . Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide; several unidentified thiols were also detected . Many of the organisms examined also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability that was quenched by treatment with 2-pyridyl disulfide or 5,5'-bisdithio-(2-nitrobenzoic acid) prior to reaction with mBBr . These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of reactive disulfides . The distribution of GSH in phototrophic eubacteria indicates that GSH synthesis evolved at or around the time that oxygenic photosynthesis evolved.

Syst Appl Microbiol, 1985, 6, 143 - 51
A phylogenetic definition of the major eubacterial taxa; Woese CR et al.; Through oligonucleotide signature analysis of 16S ribosomal RNAs, it is possible to define ten major groups of eubacteria . These are: (1) the Gram positive bacteria, (2) the purple photosynthetic bacteria and their relatives, (3) the spirochetes and their relatives, (4) the sulfur-dependent eubacteria and their relatives, (5) the bacteroides, flavobacteria and cytophagas and their relatives, (6) the cyanobacteria, (7) the green sulfur bacteria, (8) the green non-sulfur bacteria and their relatives, (9) the radio-resistant micrococci, and (10) the planctomyces and their relatives . Although no consensus exists as regards the taconomic terminology, these ten groupings are appropriately termed eubacterial Phyla or Divisions . The major subdivisions of those Phyla or Divisions that have been extensively characterized can also be defined by characteristic oligonucleotide signatures.

Syst Biol, 1996 Dec, 45(4), 568 - 75
Constraints on protein evolution and the age of the eubacteria/eukaryote split; Miyamoto MM et al.; NASA: This article discusses research done by Doolittle et al . to determine the evolutionary distance between eubacteria and eukaryotes . Several equations for estimating evolutionary distances and divergence times are considered . Molecular clock calculations and their effects on these estimates are discussed . The authors conclude that their research supports the range of dates for the cenancestor of modern eubacteria, archaebacteria, and eukaryotes estimated by Doolittle et al .

Trans R Soc Edinb Earth Sci, 1989, 80, 209 - 23
The evolution of ecological tolerance in prokaryotes; Knoll AH et al.; The ecological ranges of Archaeobacteria and Eubacteria are constrained by a requirement for liquid water and the physico-chemical stability limits of biomolecules, but within this broad envelope, prokaryotes have evolved adaptations that permit them to tolerate a remarkable spectrum of habitats . Laboratory experiments indicate that prokaryotes can adapt rapidly to novel environmental conditions, yet geological studies suggest early diversification and long-term stasis within the prokaryotic kingdoms . These apparently contradictory perspectives can be reconciled by understanding that, in general, rates and patterns of prokaryotic evolution reflect the developmental history of the Earth's surface environments . Our understanding of modern microbial ecology provides a lens through which our accumulating knowledge of physiology, molecular phylogeny and the Earth's history can be integrated and focussed on the phenomenon of prokaryotic evolution.

Science, 1985, 229, 762 - 5
Gram-positive bacteria: possible photosynthetic ancestry; Woese CR et al.; A 16S ribosomal RNA gene has been sequenced from Heliobacterium chlorum, the recently discovered photosynthetic bacterium that contains a novel form of chlorophyll . Comparisons with other 16S ribosomal RNA sequences show that the organism belongs to the Gram-positive bacteria (one of ten eubacterial "phyla")--more precisely to the so-called low G + C (G, guanine; C, cytosine) subdivision thereof . This brings to five the number of such phyla that contain photosynthetic species, the other four being the purple bacteria and relatives, the green sulfur bacteria, the green nonsulfur bacteria, and the cyanobacteria . The finding suggests that Gram-positive bacteria may be of photosynthetic ancestry, and it strengthens the case for a common photosynthetic ancestry for all eubacteria.

Syst Appl Microbiol, 1990 Mar, 13(1), 19 - 23
Phylogenetic placement of the Spirosomaceae; Woese CR et al.; Comparative analysis of 16S rRNA sequences shows that the family Spirosomaceae belongs within the eubacterial phylum defined by the flavobacteria and bacteriodes . Its constituent genera, Spirosoma, Flectobacillus, and Runella form a monophyletic grouping therein . The phylogenetic assignment is based not only upon evolutionary distance analysis, but also upon sequence signatures and higher order structural synapomorphies in 16S rRNA . Another genus peripherally associated with the Spirosomaceae, Ancylobacter ("Microcyclus"), does not cluster with the flavobacteria and their relatives, but rather belongs to the alpha subdivision of the purple bacteria.

Syst Appl Microbiol, 1990, 13, 258 - 62
The case for relationship of the flavobacteria and their relatives to the green sulfur bacteria; Woese CR et al.; Analysis of 16S rRNA sequences suggests, but does not convincingly demonstrate a specific relationship between the eubacterial phylum defined by the flavobacteria and their relatives and that defined by the green sulfur bacteria . Consequently, we have sequenced the 23S rRNA from several representatives of the former group and one of the latter in order to bring more data to bear upon this point . The 23S rRNA data alone strongly suggest a specific relationship between the two phyla, and, together with the 16S rRNA results, provides what we consider now to be a convincing case for this specific relationship.

Adv Space Res, 1992, 12(4), 207 - 16
The origin and early evolution of nucleic acid polymerases; Lazcano A et al.; The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA polymerase beta' subunit and its homologues is discussed . We show that in the DNA-dependent RNA polymerases from the three cellular lineages a very conserved sequence of eight amino acids also found in a small RNA-binding site previously described for the E . coli polynucleotide phosphorylase and the S1 ribosomal protein is present . The optimal conditions for the replicase activity of the avian myeloblastosis virus reverse transcriptase are presented . The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is also discussed.

Adv Space Res, 1986, 6(12), 181 - 6
Hypotheses on the appearance of life on Earth (review); Dose K; It is generally accepted within the natural sciences that life emerged on Earth by a kind of proto-Darwinian evolution from molecular assemblies that were predominantly formed from the various constituents of the primitive atmosphere and hydrosphere . Evolutionary stages under discussion are: the self-organization of spontaneously formed biomolecules into early precursors of life (protobionts), their stepwise evolution via (postulated) protocells to (postulated) progenotes and the Darwinian evolution from progenotes to the three kingdoms of contemporary organisms (archaebacteria, eubacteria and eukaryotes) . Considerable discrepancies between scientists have arisen because all evolutionary stages from prebiotic molecules to progenotes are entirely hypothetical and so are the postulated environmental conditions . We can only theorize that all those environmental conditions that allow the existence of the various forms of contemporary life might have allowed also the development of their precursors . Because of all these difficulties the hypothesis that life came to our planet from a remote place of our universe (panspermia) has been revived . But experimental evidence only supports the view that spores can--under favorable circumstances--survive a relatively short journey within our solar system (interplanetary transfer of life) . It is extremely unlikely that spores can survive a journey of hundreds or thousands of years through interstellar space.

Microbiology, 2001 Sep, 147(Pt 9), 2425 - 35
Production of the Gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli; Talarico LA et al.; Sarcina ventriculi grows in a remarkable range of mesophilic environments from pH 2 to pH 10 . During growth in acidic environments, where acetate is toxic, expression of pyruvate decarboxylase (PDC) serves to direct the flow of pyruvate into ethanol during fermentation . PDC is rare in bacteria and absent in animals, although it is widely distributed in the plant kingdom . The pdc gene from S . ventriculi is the first to be cloned and characterized from a Gram-positive bacterium . In Escherichia coli, the recombinant pdc gene from S . ventriculi was poorly expressed due to differences in codon usage that are typical of low-G+C organisms . Expression was improved by the addition of supplemental codon genes and this facilitated the 136-fold purification of the recombinant enzyme as a homo-tetramer of 58 kDa subunits . Unlike Zymomonas mobilis PDC, which exhibits Michaelis-Menten kinetics, S . ventriculi PDC is activated by pyruvate and exhibits sigmoidal kinetics similar to fungal and higher plant PDCs . Amino acid residues involved in the allosteric site for pyruvate in fungal PDCs were conserved in S . ventriculi PDC, consistent with a conservation of mechanism . Cluster analysis of deduced amino acid sequences confirmed that S . ventriculi PDC is quite distant from Z . mobilis PDC and plant PDCs . S . ventriculi PDC appears to have diverged very early from a common ancestor which included most fungal PDCs and eubacterial indole-3-pyruvate decarboxylases . These results suggest that the S . ventriculi pdc gene is quite ancient in origin, in contrast to the Z . mobilis pdc, which may have originated by horizontal transfer from higher plants.

J Biol Chem, 2001 Nov 23, 276(47), 43924 - 31 Epub 2001 Aug 31.
Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals differences in thermostability, cofactor specificity, oligomeric state, and phylogenetic affiliation; Steen IH et al.; With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus . An optimum for activity at 90 degrees C or above was found for each IDH . PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9 degrees C, respectively, compared with 98.3 and 98.5 degrees C for TmIDH and AfIDH, respectively . Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH . TmIDH and ApIDH were NADP-dependent (K(m)((NADP)) of 55.2 and 44.4 microm, respectively) whereas PfIDH was NAD-dependent (K(m)((NAD)) of 68.3 microm) . These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea . The homodimeric NADP-IDH present in A . pernix is the most common form of IDH known so far . The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di- and multimeric IDHs are described and discussed.

Acta Crystallogr D Biol Crystallogr, 2001 Sep, 57(Pt 9), 1290 - 2 Epub 2001 Aug 23.
Overexpression, purification, crystallization and data collection of a single-stranded DNA-binding protein from Sulfolobus solfataricus; Kerr ID et al.; Single-stranded DNA-binding proteins are recruited when single-stranded DNA is exposed by disruption of the duplex . Many important biological processes such as DNA replication can only occur when the two strands of the duplex are separated . A defining trait of these proteins is the presence of the so-called OB fold . The single-stranded DNA-binding protein of the crenarchaeote Sulfolobus solfataricus has a number of interesting differences and similarities to both the eubacterial and eukaryotic homologues . It has an extended C-terminal tail with significant sequence identity to a similar region in the eubacterial protein . However, the sequence of the OB fold is much more like the eukaryotic and euryarchaeal proteins . The S . solfataricus protein remains a monomer in the absence of DNA but rapidly polymerizes upon binding - a behaviour not seen in the Escherichia coli protein . The protein has been overexpressed, purified and crystallized . The protein crystallizes in two related forms, both having space group P6(1) (or P6(5)) with approximate unit-cell parameters a = b = 75, c = 69 A, but the crystals are distinguished by their size and morphology . The larger crystals are hexagonal bipyramids and are merohedrally twinned, diffracting to 1.34 A with diffraction observed to 1.2 A . Smaller needle-like crystals diffract to about 2.0 A but are not twinned . Molecular-replacement attempts have failed owing to low identity with available search models . The structure will be determined by multiple-wavelength methods.

J Clin Microbiol, 2001 Sep, 39(9), 3332 - 8
Multiplex PCR protocol for the diagnosis of staphylococcal infection; Mason WJ et al.; We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection . The protocol was designed to (i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding to Staphylococcus-specific regions of the 16S rRNA genes), (ii) distinguish between S . aureus and the coagulase-negative staphylococci (CNS) (based on amplification of the S . aureus-specific clfA gene), and (iii) provide an indication of the likelihood that the staphylococci present in the specimen are resistant to oxacillin (based on amplification of the mecA gene) . The expected fragments were amplified from each of 60 staphylococcal isolates (13 oxacillin-resistant S . aureus isolates, 23 oxacillin-sensitive S . aureus isolates, 17 oxacillin-resistant CNS, and 7 oxacillin-sensitive CNS) . No amplification products were observed with template DNA from nonstaphylococcal species, and the efficiency of amplification of staphylococcal targets was not adversely affected by the presence of DNA from other bacterial species in the same sample . The utility of the protocol for the analysis of clinical samples was verified by analysis of aliquots taken directly from BacT/Alert blood culture bottles . Of 77 blood cultures tested, only 7 yielded results inconsistent with those of conventional methods of diagnosis and susceptibility testing . Of those, one was identified as a CNS species by PCR and S . aureus by conventional methods . We also identified two isolates that were mecA positive but were oxacillin sensitive according to conventional methods . The other four samples failed to yield any amplification product even with a control set of primers corresponding to a conserved region of the eubacterial rRNA genes.

J Clin Microbiol, 2001 Sep, 39(9), 3282 - 9
Molecular identification of microorganisms from endodontic infections; Rolph HJ et al.; A relatively wide range of bacteria have been isolated from root canals using standard culture techniques . However, only 50% of the bacteria in the oral cavity are cultivable (S . S . Socransky et al., Arch . Oral Biol . 8:278-280, 1963); hence, bacterial diversity in endodontic infections is underestimated . This study used a PCR-based 16S rRNA gene assay, followed by cloning and sequencing of 16S rRNA amplicons from a small subset of samples to assess the diversity of bacteria present in infected root canals . A total of 41 clinical samples from 15 de novo and 26 refractory cases of endodontic infections were assessed . Of these samples, 44% were positive by culture and 68% were positive by PCR . Eight samples were selected for further analysis . Of these, the two de novo cases yielded sequences related to those of the genera Enterococcus, Lactobacillus, Propionibacterium, and Streptococcus and two clones were related to previously uncultivated bacteria, while the sinus-associated, de novo case yielded sequences related to those of the genera Lactobacillus, Pantoea, Prevotella, and Selenomonas . The five refractory cases produced clones which were related to the genera Capnocytophaga, Cytophaga, Dialister, Eubacterium, Fusobacterium, Gemella, Mogibacterium, Peptostreptococcus, Prevotella, Propionibacterium, Selenomonas, Solobacterium, Streptococcus, and Veillonella and two clones representing previously uncultivated bacteria . The phylogenetic positions of several clones associated with the Clostridiaceae and Sporomusa subgroups of the Firmicutes grouping are also shown . This study demonstrates that molecular techniques can detect the presence of bacteria in endodontic infections when culture techniques yield a negative result and can be used to identify a wider range of endodontic-infection-related bacteria including the presence of previously unidentified or unculturable bacteria.

J Biol Chem, 2001 Nov 2, 276(44), 41161 - 4 Epub 2001 Aug 28.
Alteration of product specificity of Rhodobacter sphaeroides phytoene desaturase by directed evolution; Wang CW et al.; Phytoene desaturases occurring in nature convert phytoene to either neurosporene or lycopene in most eubacteria . Approximately 10% of known phytoene desaturases, as in Rhodobacter, produce neurosporene, whereas the rest produce lycopene . These two types of enzymes, although similar in function, have relatively low similarity (below 60%) in terms of nucleotide or amino acid sequence . The mechanism controlling the product specificity of these enzymes is unclear . Here we used directed evolution to change the product of Rhodobacter sphaeroides phytoene desaturase (crtI gene product), a neurosporene-producing enzyme, to lycopene . Two generations of random mutagenesis were performed, from which three positive mutants were isolated and sequenced . We then used site-directed mutagenesis to determine the effect of each amino acid change . Gathering information from random mutagenesis, we further recombined the beneficial mutations by site-directed mutagenesis and increased the percent of lycopene production to 90%.

Structure (Camb), 2001 Apr 4, 9(4), 299 - 310
The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein gephyrin; Xiang S et al.; BACKGROUND: Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in archaea, eubacteria, and eukaryotes . In humans, genetic abnormalities in the biosynthetic pathway result in Moco deficiency, which is accompanied by severe neurological symptoms and death shortly after birth . The Escherichia coli MoeA and MogA proteins are involved in the final step of Moco biosynthesis: the incorporation of molybdenum into molybdopterin (MPT), the organic pyranopterin moiety of Moco . RESULTS: The crystal structure of E . coli MoeA has been refined at 2 A resolution and reveals that the highly elongated MoeA monomer consists of four clearly separated domains, one of which is structurally related to MogA, indicating a divergent evolutionary relationship between both proteins . The active form of MoeA is a dimer, and a putative active site appears to be localized to a cleft formed between domain II of the first monomer and domains III and IV of the second monomer . CONCLUSIONS: In eukaryotes, MogA and MoeA are fused into a single polypeptide chain . The corresponding mammalian protein gephyrin has also been implicated in the anchoring of glycinergic receptors to the cytoskeleton at inhibitory synapses . Based on the structures of MoeA and MogA, gephyrin is surmised to be a highly organized molecule containing at least five domains . This multidomain arrangement could provide a structural basis for its functional diversity . The oligomeric states of MoeA and MogA suggest how gephyrin could assemble into a hexagonal scaffold at inhibitory synapses.

Nat Struct Biol, 2001 Sep, 8(9), 789 - 94
Crystal structure and functional analysis of the SurE protein identify a novel phosphatase family; Lee JY et al.; Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea . Despite the evolutionary conservation, little information is available on the structure and function of their gene products . We have determined the crystal structure of the SurE protein from Thermotoga maritima . The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion . We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate . In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion . Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.

Mol Genet Genomics, 2001 Jul, 265(5), 778 - 90
Structure, expression and products of the ribosomal RNA operons of Rhodopseudomonas palustris No . 7; Zahn K et al.; Rhodopseudomonas palustris strains carry one or two ribosomal rRNA operons, and those with duplicated rrn operons grow faster . The two rrn operons in R . palustris No . 7 are virtually identical over a 54,70-bp stretch containing the genes for 16S rRNA, tRNAile, tRNAala, 23S rRNA and 5S rRNA, as well as the intergenic spacers and part of the extragenic spacer . In R . palustris, unlike most bacteria with multiple rrn operons, the putative promoter sequences of the two operons are highly diverged, suggesting possible functional differentiation . By simultaneous primer-extension analysis of both pre-rRNAs, we detected a two-fold higher level of expression from rrnA under photoautotrophic conditions . Alteration of the conditions of growth leads to changes in the relative levels of expression of the two operons . Within the 5,470-bp segment, only two sequence differences are found between the 23S rRNA genes; one is at the center of the 23S rRNA molecule and affects a site of unknown function, and the other is within or immediately adjacent to sequences involved in processing of the 5' 23S rRNA IVS . In vitro processing of 5' IVS-containing 23S rRNA precursors from each operon does not reveal any detectable difference between them . The 5' ends of the mature 16S, 23S, and 5S rRNAs were determined by primer-extension analysis, and the 3' end of 23S rRNA was determined by RNA linker ligation-mediated cDNA cloning . The 5' and 3' ends of the R . palustris 23S rRNA molecule are extensively processed, suggesting that, unlike the situation in the established eubacterial model, these ends cannot basepair.

Genome Biol . 2000;1(3):REVIEWS1020.
Where does DNA replication start in archaea?
Vas A, Leatherwood J.
Genome-wide measures of DNA strand composition have been used to find archaeal DNA replication origins . Archaea seem to replicate using a single origin (as do eubacteria) even though archaeal replication factors are more like those of eukaryotes.

J Mol Biol, 2001 Aug 24, 311(4), 761 - 76
Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate; Sivaraman J et al.; The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants . The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9) . Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution . This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa . Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain . An N-terminal arm contributes to the dimerization of the two monomers . The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes . Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases . The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis.Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure . Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups . We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site .

Biosci Biotechnol Biochem, 2001 Jul, 65(7), 1627 - 35
Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain; Hamano Y et al.; A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation . By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order . Heterologous expression of these genes in E . coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment . The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E . coli . Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities . This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.

Chem Biol, 2001 Aug, 8(8), 817 - 29
Analysis of the prodiginine biosynthesis gene cluster of Streptomyces coelicolor A3(2): new mechanisms for chain initiation and termination in modular multienzymes; Cerdeno AM et al.; BACKGROUND: Prodiginines are a large family of pigmented oligopyrrole antibiotics with medicinal potential as immunosuppressants and antitumour agents that are produced by several actinomycetes and other eubacteria . Recently, a gene cluster in Streptomyces coelicolor encoding the biosynthesis of undecylprodiginine and butyl-meta-cycloheptylprodiginine has been sequenced . RESULTS: Using sequence comparisons, functions have been assigned to the majority of the genes in the cluster, several of which encode homologues of enzymes involved in polyketide, non-ribosomal peptide, and fatty acid biosynthesis . Based on these assignments, a complete pathway for undecylprodiginine and butyl-meta-cycloheptylprodiginine biosynthesis in S . coelicolor has been deduced . Gene knockout experiments have confirmed the deduced roles of some of the genes in the cluster . CONCLUSIONS: The analysis presented provides a framework for a general understanding of the genetics and biochemistry of prodiginine biosynthesis, which should stimulate rational approaches to the engineered biosynthesis of novel prodiginines with improved immunosuppressant or antitumour activities . In addition, new mechanisms for chain initiation and termination catalysed by hitherto unobserved domains in modular multienzyme systems have been deduced.

Biosci Rep, 2001 Feb, 21(1), 1 - 17
Rickettsiaceae, rickettsia-like endosymbionts, and the origin of mitochondria; Emelyanov VV; Accumulating evolutionary data point to a monophyletic origin of mitochondria from the order Rickettsiales . This large group of obligate intracellular alpha-Proteobacteria includes the family Rickettsiaceae and several rickettsia-like endosymbionts (RLEs) . Detailed phylogenetic analysis of small subunit (SSU) rRNA and chaperonin 60 (Cpn60) sequences testify to polyphyly of the Rickettsiales, and consistently indicate a sisterhood of Rickettsiaceae and mitochondria that excludes RLEs . Thus RLEs are considered as the nearest extant relatives of an extinct last common ancestor of mitochondria and rickettsiae . Phylogenetic inferences prompt the following assumptions . (1) Mitochondrial origin has been predisposed by the long-term endosymbiotic relationship between rickettsia-like bacteria and proto-eukaryotes, in which many endosymbiont genes have been lost while some indispensable genes have been transferred to the host genome . (2) The obligate dependence of rickettsiae upon a eukaryotic host rests on the import of proteins encoded by these transferred genes . The nature of a proto-eukaryotic cell still remains elusive . The divergence of Rickettsiaceae and mitochondria based on Cpn60, and the evolutionary history of two aminoacyl-tRNA synthetases favor the hypothesis that it was a chimera created by fusion of an archaebacterium and a eubacterium not long before an endosymbiotic event . These and other, mostly biochemical data suggest that all the mitochondrion-related organelles, i.e., both aerobically and anaerobically respiring mitochondria and hydrogenosomes, have originated from the same RLE, while hydrogenosomal energy metabolism may have a separate origin resulting from a eubacterial fusion partner.

J Biol Chem, 2001 Oct 26, 276(43), 40041 - 9 Epub 2001 Aug 14.
Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases; Nagaike T et al.; The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC )) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3' terminus of tRNA molecules . It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops . However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops . To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis . The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes . The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several mt tRNAs as substrates . The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E . coli enzyme although both enzymes work equally well on cytoplasmic tRNAs . This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.

Biol Chem, 2001 Jun, 382(6), 979 - 86
A selenocysteine-containing peroxiredoxin from the strictly anaerobic organism Eubacterium acidaminophilum; Sohling B et al.; A strongly 75Se-labeled 22 kDa protein detected previously showed in its N-terminal sequence the highest similarity to the family of thiol-dependent peroxidases, now called peroxiredoxins . The respective gene prxU was cloned and analyzed . prxU encodes a protein of 203 amino acids (22,470 Da) and contains an in-frame UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47 . The second conserved cysteine present in 2-Cys peroxiredoxins was replaced by alanine . Heterologous expression of the Eubacterium acid-aminophilum PrxU as a recombinant selenoprotein in Escherichia coli was not possible . A cysteine-encoding mutant gene, prxU47C, containing UGC instead of UGA was strongly expressed . This recombinant PrxU47C mutant protein was purified to homogeneity by its affinity tag, but was not active as a thiol-dependent peroxidase . The identification of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously only known as non-selenoproteins containing catalytic cysteine residues.

Folia Microbiol (Praha), 2001, 46(2), 99 - 106
Phylogenetic analysis of the rplA genes encoding ribosomal protein L1; Klucar L et al.; Previously we have identified the rplA gene encoding ribosomal protein L1 in Streptomyces aureofaciens . Sequence comparison of ribosomal protein L1 among several bacterial genera revealed a high level of conservation . Based on this conservation, these proteins were used as a phylogenetic tool to compare evolutionary relationships among eubacteria and archaebacteria . This phylogenetic analysis of L1 ribosomal proteins including the S . aureofaciens rplA gene product revealed, except similar bacterial groupings, some new evolutionary relationships.

J Cell Biochem, 2001, 82(4), 573 - 82
Chromatin and histones from Giardia lamblia: a new puzzle in primitive eukaryotes; Triana O et al.; The three deepest eukaryote lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microsporidia, Metamonada, and Parabasalia . They are followed by either the Euglenozoa (e.g., Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eukaryotes . Considering the great divergence of histone proteins in protozoa we have extended our studies of histones from Trypanosomes (Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana) to the Metamonada Giardia lamblia, since Giardia is thought to be one of the most primitive eukaryotes . In the present work, the structure of G . lamblia chromatin and the histone content of the soluble chromatin were investigated and compared with that of higher eukaryotes, represented by calf thymus . The chromatin is present as nucleosome filaments which resemble the calf thymus array in that they show a more regular arrangement than those described for Trypanosoma . SDS-polyacrylamide gel electrophoresis and protein characterization revealed that the four core histones described in Giardia are in the same range of divergence with the histones from other lower eukaryotes . In addition, G . lamblia presented an H1 histone with electrophoretic mobility resembling the H1 of higher eukaryotes, in spite of the fact that H1 has a different molecular mass in calf thymus . Giardia also presents a basic protein which was identified as an HU-like DNA-binding protein usually present in eubacteria, indicating a chimaeric composition for the DNA-binding protein set in this species . Finally, the phylogenetic analysis of selected core histone protein sequences place Giardia divergence before Trypanosoma, despite the fact that Trypanosoma branch shows an acceleration in the evolutionary rate pointing to an unusual evolutionary behavior in this lineage .

J Clin Periodontol, 2001 Sep, 28(9), 860 - 4
Yeasts in periodontal pockets; Reynaud AH et al.; OBJECTIVES: The presence of yeasts in periodontal pockets has been described in a few studies . The association between yeasts and putative periodontal pathogens is not well described . This study aims at assessing the prevalence of yeasts in periodontal pockets and possible associations with the clinical conditions of the sampled sites and other micro-organisms present . MATERIAL AND METHODS: 2 subject groups form the basis for this study . The 1st comprises results from microbiological samples from periodontal pockets of 128 subjects . The 2nd originates from 126 periodontal patients with untreated pockets . Microbiological identification was performed after cultivation on blood and Sabouraud agar plates, and "checkerboard" DNA-DNA hybridisation . RESULTS: The prevalence of subjects with yeasts in the pockets was 15.6% and 17.5% in the 2 groups respectively and was inconsistent according to gender . No correlation was found between age and the presence of yeasts . Eubacterium saburreum was weakly correlated with presence of yeasts (r=0.194 p=0.03) . Yeasts were rarely found in both samples from the same individual . CONCLUSIONS: Our results indicate that yeasts can be expected to be present in periodontal pockets in one out of 6 periodontal patients independent of gender and age . Eubacterium saburreum seems to occur frequently together with yeasts.

J Mol Biol, 2001 Aug 17, 311(3), 491 - 502
High-affinity DNA binding of HU protein from the hyperthermophile Thermotoga maritima; Grove A et al.; Prokaryotic genomes are compacted by association with small basic proteins, generating what has been termed bacterial chromatin . The ubiquitous DNA-binding protein HU serves this function . DNA-binding properties of HU from the hyperthermophilic eubacterium Thermotoga maritima are shown here to differ significantly from those characteristic of previously described HU homologs . Electrophoretic mobility shift analyses show that T . maritima HU (TmHU) binds double-stranded DNA with high affinity (K(d)=5.6(+/-0.7) nM for 37 bp DNA) . Equivalent affinity is observed between 4 degrees C and 45 degrees C . TmHU has higher affinity for DNA containing a set of 4 nt loops separated by 9 bp (K(d)=1.4(+/-0.3) nM), consistent with its introduction of two DNA kinks . Using DNA probes of varying length, the optimal binding site for TmHU is estimated at 37 bp, in sharp contrast to the 9-10 bp binding site reported for other HU homologs . Alignment of >60 HU sequences demonstrates significant sequence conservation: A DNA-intercalating proline residue is almost universally conserved, and it is preceded by arginine and asparagine in most sequences, generating a highly conserved RNP motif; V substitutes for R only in HU from Thermotoga, Thermus and Deinococcus . A fivefold increase in DNA-binding affinity is observed for TmHU in which V is replaced with R (TmHU-V61R; K(d)=1.1(+/-0.2) nM), but a change in the trajectory of DNA flanking the sites of DNA intercalation is inferred from analysis of TmHU-V61R binding to DNA modified with 4 nt loops or with substitutions of 5-hydroxymethyluracil for thymine . Survival in extreme environments places unique demands on protection of genomic DNA from thermal destabilization and on access of DNA to the cellular machinery, demands that may be fulfilled by the specific DNA-binding properties of HU and by the fine structure of the bacterial chromatin .

J Bacteriol, 2001 Sep, 183(17), 5134 - 44
Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans; Svetlitchnyi V et al.; Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans . Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities . Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane . Both enzymes catalyze the reaction CO + H(2)O --> CO(2) + 2 e(-) + 2 H(+) . Oxidized viologen dyes are effective electron acceptors . The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) micromol of CO oxidized min(-1) mg(-1) of protein (methyl viologen, pH 8.0, 70 degrees C) . The two enzymes oxidize CO very efficiently, as indicated by k(cat)/K(m) values at 70 degrees C of 1.3 . 10(9) M(-1) CO s(-1) (CODH I) and 1.7 . 10(9) M(-1) CO s(-1) (CODH II) . The apparent K(m) values at pH 8.0 and 70 degrees C are 30 and 18 microM CO for CODH I and CODH II, respectively . Acetyl coenzyme A synthase activity is not associated with the enzymes . CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers containing 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, respectively . Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of {4Fe-4S} clusters . Ni was EPR silent under any conditions tested . It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.

J Chromatogr A, 2001 Jul 13, 922(1-2), 219 - 24
Chromatographic method for diaminopimelic acid detection in calcareous rocks . Presence of a bacterial biomarker in stromatolites; Borruat G et al.; The presence in the environment of diaminopimelic acid (DAP), a specific eubacterial marker, can be attributed to that of bacteria . We report a reliable and highly sensitive method for the quantification of DAP in calcareous rocks . It consists of acid hydrolysis of rock powder, purification of DAP by chromatography on Dowex 50W and Spherogel AA-NA+ columns, and quantitative analysis by high-performance liquid chromatography . Addition of tritiated DAP, the internal standard, allows one to follow the relevant fractions throughout the purification procedure and to determine their yield . The analytical step consists in pre-column derivatization with ortho-phthaldialdehyde of purified samples, and separation through a reversed-phase C18 column . Chemical controls, i.e., oxidation of samples to rule out the presence of co-eluting lanthionine and cystathionine, as well as mass spectrometry, confirm the presence of DAP in analyzed samples . Our method allows the separation of meso- from L- and/or D-stereoisomers of DAP, and reveals their presence in the examined rocks, two stromatolites of different age and geographic origin.

Genome Res, 2001 Aug, 11(8), 1404 - 9
Capturing whole-genome characteristics in short sequences using a naïve Bayesian classifier; Sandberg R et al.; Bacterial genomes have diverged during evolution, resulting in clearcut differences in their nucleotide composition, such as their GC content . The analysis of complete sequences of bacterial genomes also reveals the presence of nonrandom sequence variation, manifest in the frequency profile of specific short oligonucleotides . These frequency profiles constitute highly specific genomic signatures . Based on these differences in oligonucleotide frequency between bacterial genomes, we investigated the possibility of predicting the genome of origin for a specific genomic sequence . To this end, we developed a naive Bayesian classifier and systematically analyzed 28 eubacterial and archaeal genomes . We found that sequences as short as 400 bases could be correctly classified with an accuracy of 85% . We then applied the classifier to the identification of horizontal gene transfer events in whole-genome sequences and demonstrated the validity of our approach by correctly predicting the transfer of both the superoxide dismutase (sodC) and the bioC gene from Haemophilus influenzae to Neisseria meningitis, correctly identifying both the donor and recipient species . We believe that this classification methodology could be a valuable tool in biodiversity studies.

J Biol Chem, 2001 Oct 5, 276(40), 37076 - 85 Epub 2001 Jul 27.
Escherichia coli SecA helicase activity is not required in vivo for efficient protein translocation or autogenous regulation; Schmidt MO et al.; SecA is an essential ATP-driven motor protein that binds to preproteins and the translocon to promote protein translocation across the eubacterial plasma membrane . Escherichia coli SecA contains seven conserved motifs characteristic of superfamily II of DNA and RNA helicases, and it has been shown previously to possess RNA helicase activity . SecA has also been shown to be an autogenous repressor that binds to its translation initiation region on secM-secA mRNA, thereby blocking and dissociating 30 S ribosomal subunits . Here we show that SecA is an ATP-dependent helicase that unwinds a mimic of the repressor helix of secM-secA mRNA . Mutational analysis of the seven conserved helicase motifs in SecA allowed us to identify mutants that uncouple SecA-dependent protein translocation activity from its helicase activity . Helicase-defective secA mutants displayed normal protein translocation activity and autogenous repression of secA in vivo . Our studies indicate that SecA helicase activity is nonessential and does not appear to be necessary for efficient protein secretion and secA autoregulation.

Bioinformatics, 2001, 17 Suppl 1, S83 - 9
An insight into domain combinations; Apic G et al.; Domains are the building blocks of all globular proteins, and are units of compact three-dimensional structure as well as evolutionary units . There is a limited repertoire of domain families, so that these domain families are duplicated and combined in different ways to form the set of proteins in a genome . Proteins are gene products . The processes that produce new genes are duplication and recombination as well as gene fusion and fission . We attempt to gain an overview of these processes by studying the structural domains in the proteins of seven genomes from the three kingdoms of life: Eubacteria, Archaea and Eukaryota . We use here the domain and superfamily definitions in Structural Classification of Proteins Database (SCOP) in order to map pairs of adjacent domains in genome sequences in terms of their superfamily combinations . We find 624 out of the 764 superfamilies in SCOP in these genomes, and the 624 families occur in 585 pairwise combinations . Most families are observed in combination with one or two other families, while a few families are very versatile in their combinatorial behaviour . This type of pattern can be described by a scale-free network . Finally, we study domain repeats and we compare the set of the domain combinations in the genomes to those in PDB, and discuss the implications for structural genomics.

Appl Environ Microbiol, 2001 Aug, 67(8), 3683 - 92
Cluster structure of anaerobic aggregates of an expanded granular sludge bed reactor; Gonzalez-Gil G et al.; The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described . The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH(4)/g of volatile suspended solids {VSS}.day or 1.1 g of CH(4) chemical oxygen demand/g of VSS.day) . Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively . The spherical aggregates were black but contained numerous whitish spots on their surfaces . Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix . The white clusters were found to develop simultaneously with the increase in diameter . Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates . The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging . High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix . A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.

Mol Biol Evol, 2001 Aug, 18(8), 1574 - 84
Phylogenetic relationships of class II fumarase genes from trichomonad species; Gerbod D et al.; Class II fumarase sequences were obtained by polymerase chain reaction from five trichomonad species . All residues known to be highly conserved in this enzyme were present . Nuclear run-on assays showed that one of the two genes identified in Tritrichomonas foetus was expressed, whereas no fumarase transcripts were detected in the related species Trichomonas vaginalis . These findings corroborate previous biochemical data . Fumarase genes were also expressed in Monocercomonas sp . and Tetratrichomonas gallinarum but not in Pentatrichomonas hominis, Trichomonas gallinae, Trichomonas tenax, and Trichomitus batrachorum under the culture conditions used . Molecular trees inferred by likelihood methods reveal that trichomonad sequences have no affinity to described class II fumarase genes from other eukaryotes . The absence of functional mitochondria in protists such as trichomonads suggests that they diverged from other eukaryotes prior to the alpha-proteobacterial symbiosis that led to mitochondria . Furthermore, they are basal to other eukaryotes in rRNA analyses . However, support for the early-branching status of trichomonads and other amitochondriate protists based on phylogenetic analyses of multiple data sets has been equivocal . Although the presence of hydrogenosomes suggests that trichomonads once had mitochondria, their class II iron-independent fumarase sequences differ markedly from those of other mitochondriate eukaryotes . All of the class II fumarase genes described from other eukaryotes are of apparent alpha-proteobacterial origin and hence a marker of mitochondrial evolution . In contrast, the class II fumarase from trichomonads emerges among other eubacterial homologs . This is intriguing evidence for an independent acquisition of these genes in trichomonads apart from the mitochondrial endosymbiosis event that gave rise to the form present in other eukaryotes . The ancestral trichomonad class II fumarase may represent a prokaryotic form that was replaced in other eukaryotes after the divergence of trichomonads with the movement of endosymbiont genes into the nucleus . Alternatively, it may have been acquired via a separate endosymbiotic event or lateral gene transfer.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1187 - 8 Epub 2001 Jul 23.
Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance A (NusA) from Mycobacterium tuberculosis; Gopal B et al.; N-utilizing substance A (NusA) is a protein which performs several roles as a cofactor of DNA-dependent RNA polymerase . Its acts as an elongation factor and facilitates pausing, termination and the formation of a complex assembly that mediates transcription antitermination in eubacteria . Biochemical and biophysical data in the literature suggest that this protein performs these functions by binding to the core RNA polymerase, other protein factors and certain RNA fragments having specific signal sequences . The NusA of Mycobacterium tuberculosis has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method . The space group is P3(1)21, with unit-cell parameters a = b = 78.1, c = 180.3 A . A native data set complete to 1.7 A resolution has been collected from a single crystal.

Biochemistry, 2001 Jul 31, 40(30), 8918 - 29
Identification of the high affinity Mn2+ binding site of bacteriophage lambda phosphoprotein phosphatase: effects of metal ligand mutations on electron paramagnetic resonance spectra and phosphatase activities; White DJ et al.; Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin . PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus . Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands . Most highly similar to lambdaPP are E . coli PrpA and PrpB . Using the crystal structure of lambdaPP {Voegtli, W . C., et al . (2000) Biochemistry 39, 15365-15374} as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics . Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and {(Mn(2+))(2)} binding . The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8189 - 95
Circles: the replication-recombination-chromosome segregation connection; Barre FX et al.; Crossing over by homologous recombination between monomeric circular chromosomes generates dimeric circular chromosomes that cannot be segregated to daughter cells during cell division . In Escherichia coli, homologous recombination is biased so that most homologous recombination events generate noncrossover monomeric circular chromosomes . This bias is lost in ruv mutants . A novel protein, RarA, which is highly conserved in eubacteria and eukaryotes and is related to the RuvB and the DnaX proteins, gamma and tau, may influence the formation of crossover recombinants . Those dimeric chromosomes that do form are converted to monomers by Xer site-specific recombination at the recombination site dif, located in the replication terminus region of the E . coli chromosome . The septum-located FtsK protein, which coordinates cell division with chromosome segregation, is required for a complete Xer recombination reaction at dif . Only correctly positioned dif sites present in a chromosomal dimer are able to access septum-located FtsK . FtsK acts by facilitating a conformational change in the Xer recombination Holliday junction intermediate formed by XerC recombinase . This change provides a substrate for XerD, which then completes the recombination reaction.

J Biol Chem, 2001 Sep 28, 276(39), 36100 - 9 Epub 2001 Jul 17.
NAD+-dependent DNA ligase encoded by a eukaryotic virus; Sriskanda V et al.; We report the production, purification, and characterization of an NAD(+)-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD(+) ligase from a source other than eubacteria . AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD(+) ligases . Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD(+) . Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD(+) . Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD(+) ligases . Deletion of domain Ia of AmEPV ligase abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick . Alanine substitutions at residues within domain Ia either reduced (Tyr(39), Tyr(40), Asp(48), and Asp(52)) or abolished (Tyr(51)) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of DNA-adenylate . We conclude that: (i) NAD(+)-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD(+).

Mol Genet Genomics, 2001 Jun, 265(4), 663 - 72
Structural and functional characterization of the recR gene of Streptomyces; Pelaez AI et al.; The recR gene product is necessary for homologous recombination and recombinational DNA repair in eubacteria . We report the isolation and sequencing of the recR gene from Streptomyces coelicolor . It encodes a protein of 198 amino acids, with a predicted molecular mass of 22 kDa . The deduced amino acid sequence shows significant similarity to that of RecR proteins from other bacteria, including Escherichia coli and Bacillus subtilis . Like these, Streptomyces RecR contains potential helix-hairpin-helix, zinc finger and ATP-binding motifs, as well as the Toprim domain which is present also in topoisomerases of Types IA and II, primases and nucleases of the OLD family . The recR genes of Escherichia coli and Bacillus subtilis are immediately preceded by a small ORF (orf12 and orf107, respectively) . An equivalent ORF (orf1) is also found in Streptomyces . S . lividans recR mutants, obtained either by insertional inactivation of recR or by deletion of the gene together with the preceding ORF, displayed increased sensitivity to DNA-damaging agents (such as UV light and methylmethanesulfonate), when compared with the wild-type strain . Both mutants could be complemented by the wild-type orflrecR genes and also by the recR gene alone . Based on these results, orf1 appears to be dispensable for the repair function of Streptomyces RecR . In studies of heterologous complementation, the B . subtilis recR region (orf107recR) was found to complement the S . lividans deltaorflrecR mutant, but the equivalent region from E . coli (orf12recR) could not . However, in the absence of orf107, B . subtilis recR was unable to restore the wild-type phenotype to the Streptomyces deletion mutant.

J Eukaryot Microbiol, 2001 Jul-Aug, 48(4), 493 - 7
Archaebacterial relationships of the phosphoenolpyruvate carboxykinase gene reveal mosaicism of Giardia intestinalis core metabolism; Suguri S et al.; A gene encoding a putative GTP-specific phosphoenolpyruvate carboxykinase has been cloned and sequenced from the type I amitochondriate protist Giardia intestinalis . The deduced amino acid sequence is related most closely to homologs from hyperthermophilic archaebacteria and only more distantly to homologs from Eubacteria and Metazoa . Most enzymes of Giardia core metabolism, however, are related more closely to eubacterial and metazoan homologs . An archaebacterial relationship has been noted previously for the unusual acetyl-CoA synthetase (ADP-forming) of this organism . The results suggest that phosphoenolpyruvate carboxykinase and acetyl-CoA synthetase have been acquired from different sources than most enzymes of Giardia core metabolism.

FEMS Microbiol Ecol, 2001 Jul, 36(2-3), 223 - 234
Comparison of paralytic shellfish toxin (PST) production by the dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 in the presence and absence of bacteria; Hold GL et al.; The ability of two Alexandrium species to produce paralytic shellfish toxins (PST) in laboratory culture following the generation of bacteria-free cultures was investigated . The dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 were cultured in the presence of antibiotics and tested for residual bacteria . After treatment with a cocktail of streptomycin, ciprofloxacin, gentamicin and penicillin G, bacteria could not be detected in either of the treated Alexandrium cultures using 17 different solid and broth bacterial growth media, by epifluorescence microscopy with the dye Sybr green 1, or polymerase chain reaction amplification using universal eubacterial primers designed to target the 16S rRNA gene . Subsequent analysis of A . lusitanicum for PST using high performance liquid chromatography demonstrated that the growth rate and toxin profile remained similar in both bacteria-free and control cultures, although the quantity of toxins produced differed with the bacteria-free culture producing generally more of each compound and also having a greater toxin content in terms of saxitoxin equivalents . A . tamarense also retained similarities between the bacteria-free and control cultures in terms of growth rates and toxin profile, although in this instance, depending on the growth stage and the toxin, the control culture produced more of some toxins than the bacteria-free culture . The control culture was also more toxic in terms of saxitoxin equivalents than the axenic culture . These results suggest that bacteria can influence toxin production in laboratory cultures of Alexandrium species although the mechanisms remain unknown.

Mol Microbiol, 2001 Jun, 40(6), 1403 - 13
Glycosylation with heptose residues mediated by the aah gene product is essential for adherence of the AIDA-I adhesin; Benz I et al.; The diffuse adherence of Escherichia coli strain 2787 (O126:H27) is mediated by the autotransporter adhesin AIDA-I (adhesin-involved-in-diffuse-adherence) encoded by the plasmid-borne aidA gene . AIDA-I exhibits an aberrant mobility in denaturing gel electrophoresis . Deletion of the open reading frame (ORF) A immediately upstream of aidA restores the predicted mobility of AIDA-I, but the adhesin is no longer functional . This indicates that the mature AIDA-I adhesin is post-translationally modified and the modification is essential for adherence function . Labelling with digoxigenin hydrazide shows AIDA-I to be glycosylated . Using carbohydrate composition analysis, AIDA-I contains exclusively heptose residues (ratio heptose:AIDA-I approximately 19:1) . The deduced amino acid sequence of the cytoplasmic open reading frame (ORF) A gene product shows homologies to heptosyltransferases . In addition, the modification was completely abolished in an ADP-glycero-manno-heptopyranose mutant . Our results provide direct evidence for glycosylation of the AIDA-I adhesin by heptoses with the ORF A gene product as a specific (mono)heptosyltransferase generating the functional mature AIDA-I adhesin . Consequently, the ORF A gene has been denoted 'aah' (autotransporter-adhesin-heptosyltransferase) . Glycosylation by heptoses represents a novel protein modification in eubacteria.

Mol Microbiol, 2001 Jun, 40(6), 1241 - 8
Bacterial DNA ligases; Wilkinson A et al.; DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell . All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor . All Bacteria (eubacteria) contain NAD+-dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics . In addition to their NAD+-dependent enzymes, some Bacteria contain genes for putative ATP-dependent DNA ligases . The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism . The putative ATP-dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses . Phylogenetic analysis suggests that all NAD+-dependent DNA ligases are closely related, but the ATP-dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.

Genes Cells, 2001 Jun, 6(6), 507 - 17
A member of the YER057c/yjgf/Uk114 family links isoleucine biosynthesis and intact mitochondria maintenance in Saccharomyces cerevisiae; Kim JM et al.; BACKGROUND: Two paralogs, YIL051c and YER057c, in the Saccharomyces cerevisiae genome are members of the YER057c/Yigf/Uk114 family, which is highly conserved among Eubacteria, Archaea and Eukarya . Although the molecular function of this protein family is not clear, previous studies suggest that it plays a role in the regulation of metabolic pathways and cell differentiation . RESULTS: Yil051cp is 70% identical in amino acid sequence to Yer057cp, and differs in that the former is longer by 16 amino acids containing, in part, the mitochondrial targeting signal at the N-terminus of the protein . An HA-tagged protein of Yil051cp is localized strictly in mitochondria, while that of Yer057cp is found in both cytoplasm and nucleus . Disruption of YIL051c (yil051cDelta) resulted in severe growth retardation in glucose medium due to isoleucine auxotroph, and no growth in glycerol medium due to the loss of mitochondria . An extract prepared from yil051cDelta cells showed no transaminase activity for isoleucine, while that for valine or leucine was intact . Haploid yil051cDelta cells newly isolated from the YIL051c/yil051cDelta hetero-diploids gradually lost mitochondrial DNA within 24 h in the absence of, but not in the presence of, an isoleucine . Mutants either requiring leucine (leu2-112) or isoleucine-valine (bat1Delta, bat2Delta) in a YIL051c background showed no changes in mitochondrial DNA maintenance in the absence of requirements . CONCLUSIONS: Based on these results, we named Yil051c as Ibm1 (Isoleucine Biosynthesis and Mitochondria maintenance1) and concluded that: (i) Ibm1p determines the specificity of isoleucine biosynthesis, probably at the transamination step, (ii) Ibm1p is required for the maintenance of mitochondrial DNA when isoleucine is deficient, and (iii) Isoleucine compensates for the lack of Ibm1p . Taken together, Ibm1p may act as a sensor for isoleucine deficiency as well as a regulator determining the specificity for branched amino acid transaminase.

Genome Res, 2001 Jul, 11(7), 1187 - 97
Phylogenetic analysis of ribonuclease H domains suggests a late, chimeric origin of LTR retrotransposable elements and retroviruses; Malik HS et al.; We have conducted a phylogenetic analysis of the Ribonuclease HI (RNH) domains present in Eubacteria, Eukarya, all long-term repeat (LTR)-bearing retrotransposons, and several late-branching clades of non-LTR retrotransposons . Analysis of this simple yet highly conserved enzymatic domain from these disparate sources provides surprising insights into the evolution of eukaryotic retrotransposons . First, it indicates that the lineage of elements leading to vertebrate retroviruses acquired a new RNH domain either from non-LTR retrotransposons or from a eukaryotic host genome . The preexisting retroviral RNH domain degenerated to become the tether (connection) domain of the reverse transcriptase (RT)-RNH complex . Second, it indicates that all LTR retrotransposons arose in eukaryotes well after the origin of the non-LTR retrotransposons . Because of the younger age of the LTR retrotransposons, their complex structure, and the absence of any prokaryotic precursors, we propose that the LTR retrotransposons originated as a fusion between a DNA-mediated transposon and a non-LTR retrotransposon . The resulting two-step mechanism of LTR retrotransposition, in which RNA is reverse transcribed away from the chromosomal target site, rather than directly onto the target site, was probably an adaptation to the uncoupling of transcription and translation in eukaryotic cells.

Nucleic Acids Res, 2001 Jul 1, 29(13), 2747 - 56
Ribosomal protein gene cluster analysis in eubacterium genomics: homology between Sinorhizobium meliloti strain 1021 and Bacillus subtilis; Barloy-Hubler F et al.; The first whole genome sequence of a symbiotic soil bacterium, Sinorhizobium meliloti (formely named Rhizobium meliloti) strain 1021, is due in 2001 . As an active participant in the European and North American consortium that has completed this work, our group has sequenced a region on the chromosome containing clusters rpoBC, str, S10, spc and alpha corresponding to 30 protein genes . The structural organization and function of these genes were compared with those of orthologs in another 15 complete eubacterial genomes available in databases . This study, involving the DNA and amino acid sequences as well as the organization of the whole region (gene order, cluster order, etc.), has shown that the phylogenetic tree resulting from a comparison of the amino acid sequence is rather similar to that derived from 16S rRNA sequence data . However, the tree achieved by aligning DNA sequences groups the organisms with a high GC content (>60% GC), while that based on a comparison of gene cluster orientation and organization reveals a greater level of correspondence between the alpha-proteobacteria S.meliloti and the firmicute Bacillus subtilis.

Microbiology, 2001 Jul, 147(Pt 7), 1783 - 91
YkrB is the main peptide deformylase in Bacillus subtilis, a eubacterium containing two functional peptide deformylases; Haas M et al.; Peptide deformylation is an essential process in eubacteria . The peptide deformylase Def has been suggested to be an attractive target for antibacterial drug discovery . Some eubacteria including medically important pathogens possess two def-like genes . Until now, the functionality of both genes has been tested only in Staphylococcus aureus with the result that one gene copy was functional . Here, expression of two functional def-like gene products in Bacillus subtilis is demonstrated . Besides the def gene, which is chromosomally located close to the formyltransferase gene fmt and which was overexpressed and biochemically tested previously, B . subtilis possesses a second def-like gene, called ykrB . The encoded protein is 32% identical to the def gene product . It was shown that either def or ykrB had to be present for growth of B . subtilis in rich medium (each was individually dispensable) . Studies with a def/ykrB double deletion strain with xylose-inducible ykrB copy demonstrated that, besides def, the gene ykrB is a second cellular target of deformylase inhibitors such as the antibiotic actinonin . The gene products exhibited similar enzymic properties, exemplified by similar inhibition efficacy of actinonin in biochemical assays . Antibiotic susceptibility tests with different deletion strains and Northern analyses indicated that YkrB is probably the predominant deformylase in B . subtilis . It was shown that duplication of the deformylase function does not lead to an increased actinonin-resistance frequency in comparison to B . subtilis mutants carrying only one deformylase gene.

J Mol Biol, 2001 Jul 6, 310(2), 311 - 25
Domain combinations in archaeal, eubacterial and eukaryotic proteomes; Apic G et al.; There is a limited repertoire of domain families that are duplicated and combined in different ways to form the set of proteins in a genome . Proteins are gene products, and at the level of genes, duplication, recombination, fusion and fission are the processes that produce new genes . We attempt to gain an overview of these processes by studying the evolutionary units in proteins, domains, in the protein sequences of 40 genomes . The domain and superfamily definitions in the Structural Classification of Proteins Database are used, so that we can view all pairs of adjacent domains in genome sequences in terms of their superfamily combinations . We find 783 out of the 859 superfamilies in SCOP in these genomes, and the 783 families occur in 1307 pairwise combinations . Most families are observed in combination with one or two other families, while a few families are very versatile in their combinatorial behaviour; 209 families do not make combinations with other families . This type of pattern can be described as a scale-free network . We also study the N to C-terminal orientation of domain pairs and domain repeats . The phylogenetic distribution of domain combinations is surveyed, to establish the extent of common and kingdom-specific combinations . Of the kingdom-specific combinations, significantly more combinations consist of families present in all three kingdoms than of families present in one or two kingdoms . Hence, we are led to conclude that recombination between common families, as compared to the invention of new families and recombination among these, has also been a major contribution to the evolution of kingdom-specific and species-specific functions in organisms in all three kingdoms . Finally, we compare the set of the domain combinations in the genomes to those in the RCSB Protein Data Bank, and discuss the implications for structural genomics .

J Clin Microbiol, 2001 Jul, 39(7), 2425 - 30
rpoB sequence analysis of cultured Tropheryma whippelii; Drancourt M et al.; Until recently no isolate of Tropheryma whippelii was available, and therefore genetic studies were limited to those based on PCR amplification of conserved genes . In this study we determined the nucleotide sequence of rpoB (encoding the beta-subunit of RNA polymerase) from a cultured strain of T . whippelii using degenerate consensus PCR and genome walking . The T . whippelii rpoB consists of 3,657 bp with a 50.4% GC content and encodes 1,218 amino acids with a calculated molecular mass of 138 kDa . Comparison of T . whippelii RpoB with other eubacterial RpoB proteins indicated sequence similarity ranging from 57.19 (Mycoplasma pneumoniae) to 74.63% (Mycobacterium tuberculosis) . Phylogenetic analysis of T . whippelii based on comparison of its RpoB sequence with sequences available for other bacteria was consistent with that previously derived from the 16S ribosomal DNA (rDNA) sequence, indicating that it belongs to the actinomyces clade . The sequence comparison allowed the design of a primer pair, TwrpoB.F and TwrpoB.R, specific for T . whippelii rpoB . When incorporated into a PCR, this primer pair allowed the detection of T . whippelii rpoB in three of three 16S rDNA PCR-positive biopsy specimens and zero of seven negative controls . rpoB could therefore be targeted in PCR-mediated detection and identification of this emerging bacterial species . This approach has previously been shown useful for the identification of related mycobacteria . This study underscores that a method involving isolation and then propagation of emerging bacteria is a useful way to quickly achieve extensive molecular knowledge of these pathogens.

Genome Biol . 2001;2(6):REVIEWS1018 . Epub 2001 Jun 05.
The origin and early evolution of mitochondria; Gray MW et al.; Complete sequences of numerous mitochondrial, many prokaryotic, and several nuclear genomes are now available . These data confirm that the mitochondrial genome originated from a eubacterial (specifically alpha-proteobacterial) ancestor but raise questions about the evolutionary antecedents of the mitochondrial proteome.

Clin Microbiol Infect, 2001 Apr, 7(4), 213 - 7
Microbiological characteristics of subgingival microbiota in adult periodontitis, localized juvenile periodontitis and rapidly progressive periodontitis subjects; Nonnenmacher C et al.; OBJECTIVE: To describe the prevalence of the cultivable subgingival microbiota in periodontal diseases and to draw attention to the polymicrobial nature of periodontic infections . METHODS: The study population consisted of 95 patients, 51 females and 44 males, aged 14-62 years . Twenty-nine patients exhibited adult periodontitis (AP), six localized juvenile periodontitis (LJP), and 60 rapidly progressive periodontitis (RPP) . Two to four pooled bacterial samples were obtained from each patient . Samples were collected with sterile paper points from the deepest periodontal pockets . The samples were cultured under anaerobic and microaerophilic conditions using selective and non-selective media . Isolates were characterized to species level by conventional biochemical tests and by a commercial rapid test system . RESULTS: Prevotella intermedia and Capnocytophaga spp . were the most frequently detected microorganisms in all diagnostic groups . Porphyromonas gingivalis and Peptostreptococcus micros were found more frequently in AP and RPP patients, while Actinobacillus actinomycetemcomitans and Eikenella corrodens were associated with AP, LJP and RPP patients . The other bacterial species, including Actinomyces spp., Streptococcus spp . and Eubacterium spp., were detected at different levels in the three disease groups . CONCLUSIONS: The data show the complexity of the subgingival microbiota associated with different periodontal disease groups, indicating that the detection frequency and levels of recovery of some periodontal pathogens are different in teeth affected by different forms of periodontal disease.

Res Microbiol, 2001 Apr-May, 152(3-4), 401 - 9
Role of ATP hydrolysis by UvrA and UvrB during nucleotide excision repair; Goosen N et al.; Nucleotide excision repair in eubacteria is a process that repairs DNA damages by the removal of a 12-13-mer oligonucleotide containing the lesion . Recognition and cleavage of the damaged DNA is a multistep ATP-dependent reaction that requires the UvrA, UvrB and UvrC proteins . Both UvrA and UvrB are ATPases, with UvrA having two ATP binding sites which have the characteristic signature of the family of ABC proteins and UvrB having one ATP binding site that is structurally related to that of helicases.

FEBS Lett, 2001 Jun 15, 499(1-2), 37 - 40
LytB, a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli; Altincicek B et al.; The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite . Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway . Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.

Bioorg Med Chem, 2001 Jun, 9(6), 1467 - 77
Molecular cloning, expression and characterization of the first three genes in the mevalonate-independent isoprenoid pathway in Streptomyces coelicolor; Cane DE et al.; The mevalonate-independent biosynthetic pathway to isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors to the isoprenoids, operates in eubacteria, including Escherichia coli, in algae, and in the plastids of higher plants . A search of the Sanger Centre Streptomyces coelicolor genome database revealed open reading frames with ca . 40--50% identity at the deduced amino acid level to the first three E . coli enzymes of this pathway, corresponding to deoxyxylulose phosphate synthase, deoxyxylulose phosphate reductoisomerase and 2-C-methyl erythritol 4-phosphate cytidylyltransferase . The S . coelicolor genes have been cloned and expressed in E . coli, and the recombinant proteins characterized physically and kinetically . The presence of the corresponding enzyme activities in extracts of S . coelicolor CH999 further supports the operation of the mevalonate-independent pathway in this organism.

Genomics, 2001 Mar 15, 72(3), 223 - 30
A complete map of the human ribosomal protein genes: assignment of 80 genes to the cytogenetic map and implications for human disorders; Uechi T et al.; Mapping of the human ribosomal protein (RP) genes has been completed, and all 80 different genes were placed on a cytogenetic map of the human genome . Because of the existence of processed pseudogenes, the localization of the RP genes was complicated, and five genes had remained to be mapped . Here we developed a novel strategy to identify sequence-tagged sites (STSs) at introns of the RP genes, and we localized RPL14, RPL22, RPL35, RPL36, and RPL39 within the chromosomes by radiation hybrid mapping . Unlike the case of eubacteria or archaebacteria, human RP genes are widely scattered about the genome . Together with the previous results, both sex chromosomes and 20 autosomes (all but chromosomes 7 and 21) were found to carry one or more RP genes . To explore the possible involvement of RP genes in human disorders, all 80 genes were assigned to cytogenetic bands according to a published cytogenetic BAC-STS map of the human genome . We compared the assigned positions with candidate regions for Mendelian disorders and found certain genes that might be involved in particular human disorders .

J Mol Biol, 2001 Jun 8, 309(3), 539 - 42
Does a stretched DNA structure dictate the helical geometry of RecA-like filaments?
Egelman EH.
Proteins in the RecA/Rad51/RadA/UvsX family form helical filaments on DNA in which the DNA is stretched and untwisted . A comparison of the average helical parameters of these filaments from five different proteins, obtained from archaea, eubacteria and eukaryotes, suggests that an intrinsic state of DNA may be responsible for the conservation of these particular filament forms across evolution . In this view, these proteins stabilize this existing state of DNA, rather than induce a novel conformation .

J Biol Chem, 2001 Aug 17, 276(33), 31415 - 21 Epub 2001 May 31.
An essential GTPase, der, containing double GTP-binding domains from Escherichia coli and Thermotoga maritima; Hwang J et al.; A gene encoding a putative GTPase containing two tandemly repeated GTP-binding domains from a hyperthermophilic bacterium, Thermotoga maritima, was cloned and expressed in Escherichia coli . The gene (TM1446) termed der is highly conserved in Eubacteria including E . coli . The purified der product (Tm-Der) has GTPase activity but no ATPase activity . GTP, GDP, and dGTP but not GMP, ATP, CTP, and UTP compete for GTP binding to Tm-Der . An optimal condition for the GTPase assay was determined to be pH 7.5 in 400 mm KCl and 5 mm MgCl(2) at 70 degrees C, where K(m), V(max), and k(cat) values were determined to be 110 microm, 3.46 microm/min, and 0.87 min(-1), respectively . A der deletion strain of E . coli was constructed by replacing the der gene (originally annotated yfgK) with a kanamycin resistance gene . The deletion strain was found to form colonies only if the cells harbored a plasmid containing der, indicating that der is essential for E . coli growth.

Nucleic Acids Res, 2001 Jun 1, 29(11), 2251 - 9
Identification of a putative chromosomal replication origin from Helicobacter pylori and its interaction with the initiator protein DnaA; Zawilak A et al.; The key elements of the initiation of Helicobacter pylori chromosome replication, DnaA protein and putative oriC region, have been characterized . The gene arrangement in the H.pylori dnaA region differs from that found in many other eubacterial dnaA regions (rnpA-rmpH-dnaA-dnaN-recF-gyrB) . Helicobacter pylori dnaA is flanked by two open reading frames with unknown function, while dnaN-gyrB and rnpA-rmpH loci are separated from the dnaA gene by 600 and 90 kb, respectively . We show that the dnaA gene encoding initiator protein DnaA is expressed in H.pylori cells . The H.pylori DnaA protein, like other DnaA proteins, can be divided into four domains . Here we demonstrate that the C-terminal domain of H.pylori DnaA protein is responsible for DNA binding . Using in silico and in vitro studies, the putative oriC region containing five DnaA boxes has been located upstream of the dnaA gene . DNase I and gel retardation analyses show that the C-terminal domain of H.pylori DnaA protein specifically binds each of five DnaA boxes.

Appl Environ Microbiol, 2001 Jun, 67(6), 2766 - 74
Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR; Tajima K et al.; A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica . By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored . The dynamics of two fibrolytic bacteria, F . succinogenes and R . flavefaciens, were in agreement with those of earlier, culture-based experiments . The quantity of F . succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA . The R . flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28 . During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P . ruminicola increased 7-fold and that of P . bryantii increased 263-fold . On day 28, the quantity of P . ruminicola DNA decreased 3-fold, while P . bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet . The DNA specific for another xylanolytic bacterium, E . ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28 . The concentration of a rumen spirochete, T . bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28 . The variations in A . lipolytica DNA were not statistically significant . After an initial slight increase in S . dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment . S . bovis DNA displayed a 67-fold increase during the transition period on day 3 . However, on day 28, it actually declined in comparison with the level in animals on the hay ration . The amount of S . ruminantium-M . multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28 . The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers . This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.

Mol Biol Evol, 2001 Jun, 18(6), 1001 - 13
Bayesian selection of continuous-time Markov chain evolutionary models; Suchard MA et al.; We develop a reversible jump Markov chain Monte Carlo approach to estimating the posterior distribution of phylogenies based on aligned DNA/RNA sequences under several hierarchical evolutionary models . Using a proper, yet nontruncated and uninformative prior, we demonstrate the advantages of the Bayesian approach to hypothesis testing and estimation in phylogenetics by comparing different models for the infinitesimal rates of change among nucleotides, for the number of rate classes, and for the relationships among branch lengths . We compare the relative probabilities of these models and the appropriateness of a molecular clock using Bayes factors . Our most general model, first proposed by Tamura and Nei, parameterizes the infinitesimal change probabilities among nucleotides (A, G, C, T/U) into six parameters, consisting of three parameters for the nucleotide stationary distribution, two rate parameters for nucleotide transitions, and another parameter for nucleotide transversions . Nested models include the Hasegawa, Kishino, and Yano model with equal transition rates and the Kimura model with a uniform stationary distribution and equal transition rates . To illustrate our methods, we examine simulated data, 16S rRNA sequences from 15 contemporary eubacteria, halobacteria, eocytes, and eukaryotes, 9 primates, and the entire HIV genome of 11 isolates . We find that the Kimura model is too restrictive, that the Hasegawa, Kishino, and Yano model can be rejected for some data sets, that there is evidence for more than one rate class and a molecular clock among similar taxa, and that a molecular clock can be rejected for more distantly related taxa.

Nucleic Acids Res, 2001 May 15, 29(10), 2145 - 53
The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis; Chambaud I et al.; Mycoplasma pulmonis is a wall-less eubacterium belonging to the Mollicutes (trivial name, mycoplasmas) and responsible for murine respiratory diseases . The genome of strain UAB CTIP is composed of a single circular 963 879 bp chromosome with a G + C content of 26.6 mol%, i.e . the lowest reported among bacteria, Ureaplasma urealyticum apart . This genome contains 782 putative coding sequences (CDSs) covering 91.4% of its length and a function could be assigned to 486 CDSs whilst 92 matched the gene sequences of hypothetical proteins, leaving 204 CDSs without significant database match . The genome contains a single set of rRNA genes and only 29 tRNAs genes . The replication origin oriC was localized by sequence analysis and by using the G + C skew method . Sequence polymorphisms within stretches of repeated nucleotides generate phase-variable protein antigens whilst a recombinase gene is likely to catalyse the site-specific DNA inversions in major M.pulmonis surface antigens . Furthermore, a hemolysin, secreted nucleases and a glyco-protease are predicted virulence factors . Surprisingly, several of the genes previously reported to be essential for a self-replicating minimal cell are missing in the M.pulmonis genome although this one is larger than the other mycoplasma genomes fully sequenced until now.

Ann N Y Acad Sci, 2001 Apr, 929, 55 - 70
The conscious cell; Margulis L; The evolutionary antecedent of the nervous system is "microbial consciousness." In my description of the origin of the eukaryotic cell via bacterial cell merger, the components fused via symbiogenesis are already "conscious" entities . I have reconstructed an aspect of the origin of the neurotubule system by a hypothesis that can be directly tested . The idea is that the system of microtubules that became neurotubules has as its origin once-independent eubacteria of a very specific kind . Nothing, I claim, has ever been lost without a trace in evolution . The remains of the evolutionary process, the sequence that occurred that produced Cajal's neuron and other cells, live today . By study of obscure protists that we take to be extant decendants of steps in the evolution of cells, we reconstruct the past directly from living organisms . Even remnants of "microbial mind" can be inferred from behaviors of thriving microorganisms . All of the eukaryotes, not just lichens or an animal's neurons, are products of symbiogenesis among formerly free-living bacteria, some highly motile . Eukaryotes have evolved by the inheritance of acquired genomes; they have gained all their new features by ingesting and not digesting whole bacterial cells with complete genomes.

J Bacteriol, 2001 Jun, 183(11), 3428 - 35
Novel type of glucose-6-phosphate isomerase in the hyperthermophilic archaeon Pyrococcus furiosus; Hansen T et al.; Glucose-6-phosphate isomerase (phosphoglucose isomerase {PGI}) (EC 5.3.1.9) from the hyperthermophilic archaeon Pyrococcus furiosus was purified 500-fold to homogeneity . The enzyme had an apparent molecular mass of 43 kDa and was composed of a single type of subunit of 23 kDa indicating a homodimeric (alpha(2)) structure . Kinetic constants of the enzyme were determined at the optimal pH 7 and at 80 degrees C . Rate dependence on both substrates followed Michaelis-Menten kinetics . The apparent K(m) values for glucose-6-phosphate and fructose-6-phosphate were 8.7 and 1.0 mM, respectively, and the corresponding apparent V(max) values were 800 and 130 U/mg . The enzyme had a temperature optimum of 96 degrees C and showed a significant thermostability up to 100 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions . Based on the N-terminal amino acid sequence of the subunit, a single open reading frame (ORF; Pf_209264) was identified in the genome of P . furiosus . The ORF was characterized by functional overexpression in Escherichia coli as a gene, pgi, encoding glucose-6-phosphate isomerase . The recombinant PGI was purified and showed molecular and kinetic properties almost identical to those of the native PGI purified from P . furiosus . The deduced amino acid sequence of P . furiosus PGI did not reveal significant similarity to the conserved PGI superfamily of eubacteria and eucarya . This is the first description of an archaeal PGI, which represents a novel type of PGI.

J Bacteriol, 2001 Jun, 183(11), 3383 - 90
Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria; Ehrenshaft M et al.; In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants . Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E . coli . PDX2 was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms . The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C . nicotianae pyridoxine auxotrophs not mutant in PDX1 . Also, targeted gene replacement of PDX2 in C . nicotianae results in pyridoxine auxotrophy . Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to the E . coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain PDX1 homologues . PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.

FEBS Lett, 2001 May 4, 496(1), 6 - 11
A thermostable K(+)-stimulated vacuolar-type pyrophosphatase from the hyperthermophilic bacterium Thermotoga maritima; Perez-Castineira JR et al.; Current evidence suggests the occurrence of two classes of vacuolar-type H(+)-translocating inorganic pyrophosphatases (V-PPases): K(+)-insensitive proteins, identified in eukaryotes, bacteria and archaea, and K(+)-stimulated V-PPases, identified to date only in eukaryotes . Here, we describe the functional characterization of a thermostable V-PPase from the anaerobic hyperthermophilic bacterium Thermotoga maritima by heterologous expression in Saccharomyces cerevisiae . The activity of this 71-kDa membrane-embedded polypeptide has a near obligate requirement for K(+), like the plant V-PPase, and its thermostability depends on the binding of Mg(2+) . Phylogenetic analysis of protein sequences consistently assigned the T . maritima V-PPase to the K(+)-sensitive class of V-PPases so far only known for eukaryotes . The finding of a K(+)-stimulated V-PPase also in a member of a primitive eubacterial lineage strongly supports an ancient evolutionary origin of this group of pyrophosphate-energized proton pumps.

J Biol Chem, 2001 Jun 8, 276(23), 20286 - 91 Epub 2001 May 07.
Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase; Burke B et al.; Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups . Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon . The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA(Pro) possesses prokaryotic features in the acceptor stem . We show here that, in some respects, recognition of tRNA(Pro) by M . jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem . However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases . Although the human enzyme places a stronger emphasis on G35, the M . jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E . coli ProRS . These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M . jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like . This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine.

Biochemistry, 2001 Feb 13, 40(6), 1510 - 7
Formation of a new buried charge drives a large-amplitude protein quake in photoreceptor activation; Xie A et al.; Photoactive yellow protein (PYP) is a eubacterial photoreceptor and a structural prototype of the PAS domain superfamily of receptor and regulatory proteins . We investigate the activation mechanism of PYP using time-resolved Fourier transform infrared (FTIR) spectroscopy . Our data provide structural, kinetic, and energetic evidence that the putative signaling state of PYP is formed during a large-amplitude protein quake that is driven by the formation of a new buried charge, COO(-) of the conserved Glu46, in a highly hydrophobic pocket at the active site . A protein quake is a process consisting of global conformational changes that are triggered and driven by a local structural "fault" . We show that large, global structural changes take place after Glu46 ionization via intramolecular proton transfer to the anionic p-coumarate chromophore, and are suppressed by the absence of COO(-) formation in the E46Q mutant . Our results demonstrate the significance of buried charge formation in photoreceptor activation . This mechanism may serve as one of the general themes in activation of a range of receptor proteins . In addition, we report the results of time-resolved FTIR spectroscopy of PYP crystals . The direct comparison of time-resolved FTIR spectroscopic data of PYP in aqueous solution and in crystals reveals that the structure of the putative signaling state is not developed in P6(3) crystals . Therefore, when the structural developments during the functional process of a protein are experimentally determined to be very different in crystals and solutions, one must be cautious in drawing conclusions regarding the functional mechanism of proteins based on time-resolved X-ray crystallography.

Eur J Biochem, 2001 May, 268(9), 2678 - 86
Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach . Cyanobacterial genes for chloroplast and cytosolic isoenzymes encoded in eukaryotic chromosomes; Krepinsky K et al.; Previous attempts to purify chloroplast 6-phosphogluconate dehydrogenase (cp6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been unsuccessful due to rapid activity loss . An efficient purification protocol was developed and the enzyme from spinach leaves was purified 1000-fold to apparent homogeneity with a specific activity of 60 U.mg-1 . The enzyme is a homodimer with subunits of 50 kDa . Antibodies raised against the purified cp6PGDH detected a 53-kDa protein from a crude extract, indicating alterations during purification . Purified cp6PGDH was microsequenced and the corresponding spinach cDNA was cloned using PCR techniques and degenerate primers . The cDNA for cytosolic 6PGDH from spinach was cloned for comparison . Phylogenetic analysis in the context of available homologues from eukaryotes and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences are more similar to their homologues from gamma-proteobacteria, whereas plant 6PGDH is more similar to its cyanobacterial homologues . The ancestral gene for higher plant 6PGDH was acquired from the antecedent of plastids through endosymbiosis and gene transfer to the nucleus . A subsequent gene duplication gave rise to higher plant cytosolic 6PGDH, which assumed the function of its pre-existing cytosolic homologue through endosymbiotic gene replacement . The protein phylogeny of both 6PGDH and of the first enzyme of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase, indicate a surprisingly close relationship between the plant and Trypanosoma brucei lineages, suggesting that T . brucei (a relative of Euglena gracilis) may be secondarily nonphotosynthetic.

Microbiology, 2001 May, 147(Pt 5), 1137 - 47
Eubacterial arylamine N-acetyltransferases - identification and comparison of 18 members of the protein family with conserved active site cysteine, histidine and aspartate residues; Payton M et al.; Arylamine N-acetyltransferases (NATs) are enzymes involved in the detoxification of a range of arylamine and hydrazine-based xenobiotics . NATs have been implicated in the endogenous metabolism of p-aminobenzoyl glutamate in eukaryotes, although very little is known about the distribution and function of NAT in the prokaryotic kingdom . Using DNA library screening techniques and the analysis of data from whole-genome sequencing projects, we have identified 18 nat-like sequences from the Proteobacteria and Firmicutes . Recently, the three-dimensional structure of NAT derived from the bacterium Salmonella typhimurium (PDB accession code 1E2T) was resolved and revealed an active site catalytic triad composed of Cys(69)-His(107)-Asp(122) . These residues have been shown to be conserved in all prokaryotic and eukaryotic NAT homologues together with three highly conserved regions which are found proximal to the active site triad . The characterization of prokaryotic NATs and NAT-like enzymes is reported . It is also predicted that prokaryotic NATs, based on gene cluster composition and distribution amongst genomes, participate in the metabolism of xenobiotics derived from decomposition of organic materials.

Microbiology, 2001 May, 147(Pt 5), 1129 - 35
Functional characterization of a microbial aquaglyceroporin; Froger A et al.; The major intrinsic proteins (MIPs) constitute a widespread membrane channel family essential for osmotic cell equilibrium . The MIPs can be classified into three functional subgroups: aquaporins, glycerol facilitators and aquaglyceroporins . Bacterial MIP genes have been identified in archaea as well as in Gram-positive and Gram-negative eubacteria . However, with the exception of Escherichia coli, most bacterial MIPs have been analysed by sequence homology . Since no MIP has yet been functionally characterized in Gram-positive bacteria, we have studied one of these members from Lactococcus lactis . This MIP is shown to be permeable to glycerol, like E . coli GlpF, and to water, like E . coli AqpZ . This is the first characterization of a microbial MIP that has a mixed function . This result provides important insights to reconstruct the evolutionary history of the MIP family and to elucidate the molecular pathway of water and other solutes in these channels.

Mol Biol Evol, 2001 May, 18(5), 710 - 20
Pyruvate : NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists; Rotte C et al.; Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH) . Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes . The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO) . Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain . Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2) . The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E . gracilis, no mitochondrial transit peptide for C . parvum PNO was found . Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction . Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits . Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition . These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO . They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.

Appl Environ Microbiol, 2001 May, 67(5), 2255 - 62
Comparison of nifH gene pools in soils and soil microenvironments with contrasting properties; Poly F et al.; The similarities and differences in the structures of the nifH gene pools of six different soils (Montrond, LCSA-p, Vernon, Dombes, LCSA-c, and Thysse Kaymor) and five soil fractions extracted from LCSA-c were studied . Bacterial DNA was directly extracted from the soils, and a region of the nifH gene was amplified by PCR and analyzed by restriction . Soils were selected on the basis of differences in soil management, plant cover, and major physicochemical properties . Microenvironments differed on the basis of the sizes of the constituent particles and the organic carbon and clay contents . Restriction profiles were subjected to principal-component analysis . We showed that the composition of the diazotrophic communities varied both on a large scale (among soils) and on a microscale (among microenvironments in LCSA-c soil) . Soil management seemed to be the major parameter influencing differences in the nifH gene pool structure among soils by controlling inorganic nitrogen content and its variation . However, physicochemical parameters (texture and total C and N contents) were found to correlate with differences among nifH gene pools on a microscale . We hypothesize that the observed nifH genetic structures resulted from the adaptation to fluctuating conditions (cultivated soil, forest soil, coarse fractions) or constant conditions (permanent pasture soil, fine fractions) . We attempted to identify a specific band within the profile of the clay fraction by cloning and sequencing it and comparing it with the gene databases . Unexpectedly, the nifH sequences of the dominant bacteria were most similar to sequences of unidentified marine eubacteria.

Protein Sci, 2001 May, 10(5), 911 - 22
Crystal structure of E . coli beta-carbonic anhydrase, an enzyme with an unusual pH-dependent activity; Cronk JD et al.; Carbonic anhydrases fall into three distinct evolutionary and structural classes: alpha, beta, and gamma . The beta-class carbonic anhydrases (beta-CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea . We have determined the crystal structure of ECCA, a beta-CA from Escherichia coli, to a resolution of 2.0 A . In agreement with the structure of the beta-CA from the chloroplast of the red alga Porphyridium purpureum, the active-site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues . These results confirm the observation of a unique pattern of zinc ligation in at least some beta-CAS: The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity . ECCA activity is highly pH-dependent in the physiological range, and its expression in yeast complements an oxygen-sensitive phenotype displayed by a beta-CA-deletion strain . The structural and biochemical characterizations of ECCA presented here and the comparisons with other beta-CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.

Mol Microbiol, 2001 Apr, 40(2), 485 - 97
Physiological consequences of blocked Caulobacter crescentus dnaA expression, an essential DNA replication gene; Gorbatyuk B et al.; Caulobacter crescentus chromosome replication is precisely coupled to a developmental cell cycle . Like most eubacteria, C . crescentus has a DnaA homologue that is presumed to initiate chromosome replication . However, the C . crescentus replication origin (Cori) lacks perfect consensus Escherichia coli DnaA boxes . Instead, the Cori strong transcription promoter (Ps) may regulate chromosome replication through the CtrA cell cycle response regulator . We therefore created a conditional dnaA C . crescentus strain . Blocking dnaA expression immediately decreased DNA synthesis, which stopped after approximately one doubling period . Fluorescent flow cytometry confirmed that DNA synthesis is blocked at the initiation stage . Cell division also stopped, but not swarmer to stalked cell differentiation . All cells became stalked cells that grew as long filaments . Therefore, general transcription and protein synthesis continued, whereas DNA synthesis stopped . However, transcription was selectively blocked from the flagellar fliQ and fliL and methyltransferase ccrM promoters, which require CtrA and are blocked by different DNA synthesis inhibitors . Interestingly, transcription from Cori Ps continued unaltered . Therefore, Ps transcription is not sufficient for chromosome replication . Approximately 6-8 h after blocked dnaA expression, cells lost viability exponentially . Coincidentally, beta-galactosidase was induced from one transcription reporter, suggesting an altered physiology . We conclude that C . crescentus DnaA is essential for chromosome replication initiation, and perhaps also has a wider role in cell homeostasis.

Mol Microbiol, 2001 Apr, 40(2), 347 - 60
A set of ftsZ mutants blocked at different stages of cell division in Caulobacter; Wang Y et al.; FtsZ is required throughout the cell division process in eubacteria and in archaea . We report the isolation of novel mutants of the FtsZ gene in Caulobacter crescentus . Clusters of charged amino acids were changed to alanine to minimize mutations that affect protein folding . Molecular modelling indicated that all the clustered-charged-to-alanine mutations had altered amino acids at the surface of the protein . Of 13 such mutants, four were recessive-lethal, three were dominant-lethal, and six had no discernible phenotype . An FtsZ depletion strain of Caulobacter was constructed to analyse the phenotype of the recessive-lethal mutations and used to show that they blocked cell division at distinct stages . One mutation blocked the initiation of cell division, two mutations blocked cell division randomly, and one mutation blocked both early and late stages of cell division . The effect of the recessive mutations on the subcellular localization of FtsZ was determined . Models to explain the various mutant phenotypes are discussed . This is the first set of recessive alleles of ftsZ blocked at different stages of cell division.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 499 - 525
The aldo-keto reductase (AKR) superfamily: an update; Jez JM et al.; The aldo-keto reductases (AKRs) are one of three enzyme superfamilies encompassing a range of oxidoreductases . Members of the AKR superfamily are monomeric (alpha/beta)(8)-barrel proteins, about 320 amino acids in length, which bind NAD(P)(H) to metabolize an array of substrates . AKRs have been identified in vertebrates, invertebrates, plants, protozoa, fungi, eubacteria, and archaebacteria, implying that this is an ancient superfamily of enzymes . Earlier, in an attempt to clarify the confusion caused by multiple names for particular AKRs, we proposed a systematic and expandable nomenclature system to assign consistent designations to unique members of the AKR superfamily . Since then, the number of characterized AKRs has expanded to 105 proteins in 12 families . In addition, molecular cloning and genome sequencing projects have identified 125 potential AKR genes, many of which have no assigned function . The nomenclature system for the AKR superfamily is accepted by the Human Genome Project . Using the earlier described nomenclature system, we now provide an updated listing of AKRs and potential superfamily members.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 323 - 37
Aldehyde dehydrogenase gene superfamily: the 2000 update; Sophos NA et al.; Aldehyde dehydrogenase (ALDH) superfamily represents a group of NAD(P)(+)-dependent enzymes that catalyze the oxidation of a wide spectrum of endogenous and exogenous aldehydes . With the advent of megabase genome sequencing, the ALDH superfamily is expanding rapidly on many fronts . As expected, ALDH genes are found in virtually all genomes analyzed to date, indicating the importance of these enzymes in biological functions . Complete genome sequences of various species have revealed additional ALDH genes . As of July 2000, the ALDH superfamily consists of 331 distinct genes, of which eight are found in archaea, 165 in eubacteria, and 158 in eukaryota . The number of ALDH genes in some species with their genomes completely sequenced and annotated, Escherichia coli and Caenorhabditis elegans, ranges from 10 to 17 . In the human genome, 17 functional genes and three pseudogenes have been identified to date . Divergent evolution, based on multiple alignment analysis of 86 eukaryotic ALDH amino-acid sequences, was the basis of the standardized ALDH gene nomenclature system (Pharmacogenetics 9: 421-434, 1999) . Thus far, the eukaryotic ALDHs comprise 20 gene families . A complete list of all ALDH sequences known to date is presented here along with the evolution analysis of the eukaryotic ALDHs.

Mol Microbiol, 2001 Apr, 40(1), 115 - 25
Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis; Feucht A et al.; The actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division . Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells . As in other organisms FtsA is probably recruited immediately after FtsZ . In sporulating cells of B . subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole . We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system . FtsA is present in much higher quantities in B . subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ . This means that there is sufficient FtsA to form a complete circumferential ring at the division site . Therefore, FtsA may have a direct structural role in cell division . We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities . This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B . subtilis (and possibly in most eubacteria).

J Gen Virol, 2001 May, 82(Pt 5), 1027 - 41
The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences; Donnelly ML et al.; The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini . We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame . Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and {GFP2A} . Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect . To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed . Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted -- with the exception of aspartate-12 . This residue is not, however, conserved amongst all functional '2A-like' sequences . '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp . and a eubacterial alpha-glucosiduronasesequence(Thermatoga maritima aguA) . All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha-glucosiduronase 2A-like sequence . This method of control of protein biogenesis may well not, therefore, be confined to members of the PICORNAVIRIDAE: Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements.

J Mol Biol, 2001 Apr 13, 307(5), 1181 - 212
Protein cofactor-dependent acquisition of novel catalytic activity by the RNase P ribonucleoprotein of E . coli; Cole KB et al.; Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity . Ribonucleoproteins sampled after ten generations of selection show a >400-fold increase in the first-order rate constant (k(cat)) on a DNA substrate, reflecting a significant improvement in the chemical cleavage step . This increase is offset by a reduction in substrate binding, as measured by K(M) . We trace the catalytic enhancement to two ubiquitous A-->U sequence changes at positions 136 and 333 in the M1 RNA component, positions that are phylogenetically conserved in the Eubacteria . Furthermore, although the mutations are located in different folding domains of the catalytic RNA, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by the presence of the C5 protein . The activity of the evolved ribonucleoproteins on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type levels . In contrast, improved DNA cleavage activity is accompanied by a 500-fold decrease in pre-tRNA cleavage efficiency (k(cat)/K(M)) . The presence of the C5 component does not buffer this tradeoff in catalytic activities, despite the in vivo role played by the C5 protein in enhancing the substrate versatility of RNase P . The change at position 136, located in the J11/12 single-stranded region, likely alters the geometry of the pre-tRNA-binding cleft and may provide a functional explanation for the observed tradeoff . These results thus shed light both on structure/function relations in E . coli RNase P and on the crucial role of proteins in enhancing the catalytic repertoire of RNA .

Phys Rev Lett, 2001 Mar 12, 86(11), 2471 - 4
Long-range correlations in genomic DNA: a signature of the nucleosomal structure; Audit B et al.; We use the "wavelet transform microscope" to carry out a comparative statistical analysis of DNA bending profiles and of the corresponding DNA texts . In the three kingdoms, one reveals on both signals a characteristic scale of 100-200 bp that separates two different regimes of power-law correlations (PLC) . In the small-scale regime, PLC are observed in eukaryotic, in double-strand DNA viral, and in archaeal genomes, which contrasts with their total absence in the genomes of eubacteria and their viruses . This strongly suggests that small-scale PLC are related to the mechanisms underlying the wrapping of DNA in the nucleosomal structure . We further speculate that the large scale PLC are the signature of the higher-order structure and dynamics of chromatin.

Protein Eng, 2001 Jan, 14(1), 17 - 25
Post-translational GPI lipid anchor modification of proteins in kingdoms of life: analysis of protein sequence data from complete genomes; Eisenhaber B et al.; To investigate the occurrence of glycosylphosphatidylinositol (GPI) lipid anchor modification in various taxonomic ranges, potential substrate proteins have been searched for in completely sequenced genomes . We applied the big-pi predictor for the recognition of propeptide cleavage and anchor attachment sites with a new, generalized analytical form of the extreme-value distribution for evaluating false-positive prediction rates . (i) We find that GPI modification is present among lower and higher Eukaryota (approximately 0.5% of all proteins) but it seems absent in all eubacterial and three archaeobacterial species studied . Four other archaean genomes appear to encode such a fraction of substrate proteins (in the range of eukaryots) that they cannot be explained as false-positive predictions . This result supports the possible existence of GPI anchor modification in an archaean subgroup . (ii) The frequency of GPI-modified proteins on various chromosomes of a given eukaryotic species is different . (iii) Lists of potentially GPI-modified proteins in complete genomes with their predicted cleavage sites are available at (iv) Orthologues of known transamidase subunits have been found only for EUKARYA: Inconsistencies in domain structure among homologues some of which may indicate sequencing errors are described . We present a refined model of the transamidase complex.

J Biol Chem, 2001 May 25, 276(21), 18052 - 9 Epub 2001 Mar 09.
Chorismate synthase from the hyperthermophile Thermotoga maritima combines thermostability and increased rigidity with catalytic and spectral properties similar to mesophilic counterparts; Fitzpatrick TB et al.; Chorismate synthase, the last enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate, a biochemically unique reaction in that it requires reduced FMN as a cofactor . Here we report on the cloning, expression, and characterization of the protein for the first time from an extremophilic organism Thermotoga maritima which is also one of the oldest and most slowly evolving eubacteria . The protein is monofunctional in that it does not have an intrinsic ability to reduce the FMN cofactor and thereby reflecting the nature of the ancestral enzyme . Circular dichroism studies indicate that the melting temperature of the T . maritima protein is above 92 degrees C compared with 54 degrees C for the homologous Escherichia coli protein while analytical ultracentrifugation showed that both proteins have the same quaternary structure . Interestingly, UV-visible spectral studies revealed that the dissociation constants for both oxidized FMN and 5-enolpyruvylshikimate 3-phosphate decrease 46- and 10-fold, respectively, upon heat treatment of the T . maritima protein . The heat treatment also results in the trapping of the flavin cofactor in an apolar environment, a feature which is enhanced by the presence of the substrate 5-enolpyruvylshikimate 3-phosphate . Nevertheless, stopped-flow spectrophotometric evidence suggests that the mechanism of the T . maritima protein is similar to that of the E . coli protein . In essence, the study shows that T . maritima chorismate synthase exhibits considerably higher rigidity and thermostability while it has conserved features relevant to its catalytic function.

Biochimie, 2001 Feb, 83(2), 243 - 9
The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants; Sonnenfield JM et al.; The StpA protein is closely related to H-NS, the well-characterised global regulator of gene expression which is a major component of eubacterial chromatin . Despite sharing a very high degree of sequence identify and having biochemical properties in common with H-NS, the physiological function of StpA remains unknown . We show that StpA exhibits similar DNA-binding activities to H-NS . Although both display a strong preference for binding to curved DNA, StpA binds DNA with a four-fold higher affinity than H-NS, with K(d)s of 0.7 microM and 2.8 microM, respectively . It has previously been reported that expression of stpA is derepressed in an hns mutant . We have quantified the amount of StpA protein produced under this condition and find it to be only one-tenth the level of H-NS protein in wild-type cells . Our findings explain why the presence of StpA does not compensate for the lack of H-NS in an hns mutant, and why the characteristic pleiotropic hns mutant phenotype is observed.

Genome Biol . 2001;2(3):RESEARCH0009 . Epub 2001 Mar 06.
The cohesin complex: sequence homologies, interaction networks and shared motifs; Jones S et al.; BACKGROUND: Cohesin is a macromolecular complex that links sister chromatids together at the metaphase plate during mitosis . The links are formed during DNA replication and destroyed during the metaphase-to-anaphase transition . In budding yeast, the 14S cohesin complex comprises at least two classes of SMC (structural maintenance of chromosomes) proteins - Smc1 and Smc3 - and two SCC (sister-chromatid cohesion) proteins - Scc1 and Scc3 . The exact function of these proteins is unknown . RESULTS: Searches of protein sequence databases have revealed new homologs of cohesin proteins . In mouse, Mmip1 (Mad member interacting protein 1) and Smc3 share 99% sequence identity and are products of the same gene . A phylogenetic tree of SMC homologs reveals five families: Smc1, Smc2, Smc3, Smc4 and an ancestral family that includes the sequences from the Archaea and Eubacteria . This ancestral family also includes sequences from eukaryotes . A cohesion interaction network, comprising 17 proteins, has been constructed using two proteomic databases . Genes encoding six proteins in the cohesion network share a common upstream region that includes the MluI cell-cycle box (MCB) element . Pairs of the proteins in this network share common sequence motifs that could represent common structural features such as binding sites . Scc2 shares a motif with Chk1 (kinase checkpoint protein), that comprises part of the serine/threonine protein kinase motif, including the active-site residue . CONCLUSIONS: We have combined genomic and proteomic data into a comprehensive network of information to reach a better understanding of the function of the cohesin complex . We have identified new SMC homologs, created a new SMC phylogeny and identified shared DNA and protein motifs . The potential for Scc2 to function as a kinase - a hypothesis that needs to be verified experimentally - could provide further evidence for the regulation of sister-chromatid cohesion by phosphorylation mechanisms, which are currently poorly understood.

J Biol Chem, 2001 Jun 8, 276(23), 20064 - 8 Epub 2001 Mar 23.
Recognition of tRNAs by Methionyl-tRNA transformylase from mammalian mitochondria; Takeuchi N et al.; Protein synthesis involves two methionine-isoaccepting tRNAs, an initiator and an elongator . In eubacteria, mitochondria, and chloroplasts, the addition of a formyl group gives its full functional identity to initiator Met-tRNA(Met) . In Escherichia coli, it has been shown that the specific action of methionyl-tRNA transformylase on Met-tRNA(f)(Met) mainly involves a set of nucleotides in the acceptor stem, particularly a C(1)A(72) mismatch . In animal mitochondria, only one tRNA(Met) species has yet been described . It is admitted that this species can engage itself either in initiation or elongation of translation, depending on the presence or absence of a formyl group . In the present study, we searched for the identity elements of tRNA(Met) that govern its formylation by bovine mitochondrial transformylase . The main conclusion is that the mitochondrial formylase preferentially recognizes the methionyl moiety of its tRNA substrate . Moreover, the relatively small importance of the tRNA acceptor stem in the recognition process accounts for the protection against formylation of the mitochondrial tRNAs that share with tRNA(Met) an A(1)U(72) motif.

J Bacteriol, 2001 Apr, 183(8), 2411 - 6
GcpE is involved in the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli; Altincicek B et al.; In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway . Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated . In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway . E . coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway . These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.

Microbiol Immunol, 2001, 45(1), 29 - 37
Intraperitoneal immune cell responses to Eubacterium saphenum in mice; Salam MA et al.; Oral asaccharolytic Eubacterium saphenum, which are newly isolated gram-positive rods and one of the predominant microorganisms in human periodontal pockets, were injected intraperitoneally in mice to elucidate their pathogenicity in periodontal diseases . Infiltrating immune cells in the peritoneal exudate were quantitated and intracellular T cell (CD4+/CD8+/gammadelta+) production of cytokines IL-4 and IFN-gamma which are related to cellular and humoral immunity, respectively, was determined . Neutrophils appeared first in peritoneal exudates, followed by macrophages and lymphocytes, after the injection of either E . saphenum or Porphyromonas gingivalis . Intracellular IL-4+ and IFN-gamma+ gammadelta T cells were detected in the exudates after the injection of E . saphenum (4.6 +/- 0.8% and 10.1 +/- 1.4%, respectively) and P . gingivalis (5.3 +/- 1.6% and 10.1 +/- 2.1%, respectively) . The intracellular production of IL-4/IFN-gamma in CD4+/CD8+ T cells was rather low indicating that the main response was from gammadelta T cells which initiated the immune reactions in mouse peritoneal cavities after injection of E . saphenum or P . gingivalis . Serum IgG and IgM levels were elevated in animals injected with E . saphenum and similarly with P . gingivalis . The present study showed that with slight differences, similar modes of cell response and cytokine and Ig production were observed after intraperitoneal injection of both E . saphenum and P . gingivalis, indicating that E . saphenum may play just as important a role in periodontal diseases as P . gingivalis.

Acta Crystallogr D Biol Crystallogr, 2001 Apr, 57(Pt 4), 612 - 3
Crystallization and preliminary X-ray crystallographic analysis of the surE protein from Thermotoga maritima; Kwak JE et al.; The surE protein from Thermotoga maritima is a 247-residue protein of unknown function . Its homologues are well conserved among both the eubacteria and the archaea . It has been overexpressed in soluble form in Escherichia coli . The protein has been crystallized at 296 K using 2-propanol as a precipitant . X-ray diffraction data have been collected to 1.9 A resolution using synchrotron radiation . The crystals belong to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 115.96, c = 78.60 A, alpha = beta = 90, gamma = 120 degrees . The asymmetric unit contains two monomers of the surE protein, with a corresponding V(M) of 2.72 A(3) Da(-1) and a solvent content of 54.7%.

Mol Biol Evol, 2001 Apr, 18(4), 593 - 605
Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins; Khuri S et al.; The cupin superfamily is a group of functionally diverse proteins that are found in all three kingdoms of life, Archaea, Eubacteria, and Eukaryota . These proteins have a characteristic signature domain comprising two histidine- containing motifs separated by an intermotif region of variable length . This domain consists of six beta strands within a conserved beta barrel structure . Most cupins, such as microbial phosphomannose isomerases (PMIs), AraC- type transcriptional regulators, and cereal oxalate oxidases (OXOs), contain only a single domain, whereas others, such as seed storage proteins and oxalate decarboxylases (OXDCs), are bi-cupins with two pairs of motifs . Although some cupins have known functions and have been characterized at the biochemical level, the majority are known only from gene cloning or sequencing projects . In this study, phylogenetic analyses were conducted on the conserved domain to investigate the evolution and structure/function relationships of cupins, with an emphasis on single- domain plant germin-like proteins (GLPs) . An unrooted phylogeny of cupins from a wide spectrum of evolutionary lineages identified three main clusters, microbial PMIs, OXDCs, and plant GLPs . The sister group to the plant GLPs in the global analysis was then used to root a phylogeny of all available plant GLPs . The resulting phylogeny contained three main clades, classifying the GLPs into distinct subfamilies . It is suggested that these subfamilies correlate with functional categories, one of which contains the bifunctional barley germin that has both OXO and superoxide dismutase (SOD) activity . It is proposed that GLPs function primarily as SODs, enzymes that protect plants from the effects of oxidative stress . Closer inspection of the DNA sequence encoding the intermotif region in plant GLPs showed global conservation of thymine in the second codon position, a character associated with hydrophobic residues . Since many of these proteins are multimeric and enzymatically inactive in their monomeric state, this conservation of hydrophobicity is thought to be associated with the need to maintain the various monomer- monomer interactions . The type of structure-based predictive analysis presented in this paper is an important approach for understanding gene function and evolution in an era when genomes from a wide range of organisms are being sequenced at a rapid rate.

Mol Biol Evol, 2001 Apr, 18(4), 514 - 22
Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies; Edgcomb VP et al.; Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists . They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes . To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis . These are the first reports of nuclear genes from these organisms . Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids . While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J . libera and R . americana, the two other jakobids, M . jakobiformis and J . incarcerata, had unclear affinities . In all three analyses, J . libera, R . americana, and M . jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads . A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J . libera-R . americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists . Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies . In alpha- tubulin trees, J . incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed . Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the "jakobids" may be polyphyletic . Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.

Plant J, 2001 Feb, 25(4), 375 - 87
Quinone oxidoreductase message levels are differentially regulated in parasitic and non-parasitic plants exposed to allelopathic quinones; Matvienko M et al.; Allelopathic chemicals released by plants into the rhizosphere have effects on neighboring plants ranging from phytoxicity to inducing organogenesis . The allelopathic activity of naturally occurring quinones and phenols is primarily a function of reactive radicals generated during redox cycling between quinone and hydroquinone states . We isolated cDNAs encoding two distinct quinone oxidoreductases from roots of the parasitic plant Triphysaria treated with the allelopathic quinone 2,6-dimethoxybenzoquinone (DMBQ) . TvQR1 is a member of the zeta-crystallin quinone oxidoreductase family that catalyzes one-electron quinone reductions, generating free radical semiquinones . TvQR2 belongs to a family of detoxifying quinone oxidoreductases that catalyze bivalent redox reactions which avoid the radical intermediate . TvQR1 and TvQR2 message levels are rapidly upregulated in Triphysaria roots as a primary response to treatment with various allelopathic quinones . Inhibition of quinone oxidoreductase enzymatic activity with dicumarol prior to quinone treatment resulted in increased transcript levels . While TvQR2 homologs were upregulated by DMBQ in roots of all plants examined, TvQR1 homologs were upregulated only in roots of parasitic plants . Phylogenetic trees constructed of TvQR1 and TvQR2 protein homologs in Archea, Eubacteria and Eukaryotes indicated that both gene families are ancient, yet the families have dissimilar evolutionary histories in angiosperms . We hypothesize that TvQR2-like proteins function to detoxify allelopathic quinones in the rhizosphere, while TvQR1 has specific functions associated with haustorium development in parasitic plants.

Gene, 2001 Mar 7, 265(1-2), 95 - 101
Molecular evolution of the AMP-forming Acetyl-CoA synthetase; Karan D et al.; Acetyl-CoA-Synthetase (ACS) is involved in the production of acetate, a major metabolite in numerous organisms . There are two forms of this enzyme: ADP-forming ACS and ATP-forming ACS . We focus mainly on the AMP-forming ACS gene, which is relatively well conserved in eubacteria, archeaebacteria, and eukaryotes . BLAST searches in databases showed 30 protein sequences significantly related to the ACS . Most of these sequences were identified as ACS but three of them, belonging to the mammalian species, were annotated as another gene named: the SA gene, which is involved in the essential hypertension . The ACS and SA genes probably derived from a duplication of an ancestral gene but have acquired different functions . Six conserved regions of the ACS protein were defined across the three domains of life . While the precise function of the conserved regions remains unknown, they are probably involved in the enzymatic activity . Among eukaryotes, we found a high variability with respect to the number and the position of introns . However, some positions are conserved between fungi and a nematode . A maximum likelihood tree based upon the conserved regions showed that all sequences except the one from B . subtilis, belong to two basic groups: one the SA-like group including sequences from Archaeoglobus fulgidus and Streptomyces coelicolor, and second, the ACS group . The later can be further divided in two parts: a prokaryotic one including eubacteria and an archaebacterium, and a eukaryotic group within which two proteobacterial sequences branch including ACS from the alpha-proteobacterium Rhodobacter capsulatus . Within the eukaryotic group, bootstrap support is very low, but overall the data are consistent with the view that eukaryotes acquired their ACS gene from the ancestors of mitochondria . The localization of this enzyme in eukaryotic mitochondria is the additional evidence in favor of this interpretation.

Gene, 2001 Feb 21, 264(2), 265 - 71
Evolutionary relationships of the glucokinase from the amitochondriate protist, Trichomonas vaginalis; Wu G et al.; Two genes coding for Trichomonas vaginalis glucokinase were isolated and sequenced . The putative translation products have molecular masses of 41,584 and 41,772 Da, corresponding to 375 and 377 amino acids, respectively . These values agree with data determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the enzyme purified from the organism . The two sequences showed 78% amino acid identity . The sequences and their phylogenetic reconstruction show that they are members of a glucokinase/fructokinase protein family found in eubacteria and also in the eukaryote Giardia lamblia and are only distantly related to typical eukaryotic hexokinases . The results indicate that the evolutionary past of this enzyme, catalyzing the first step of glycolysis in T . vaginalis, is different from that of the enzyme performing this key role in almost all other eukaryotes.

Biometals, 2000 Dec, 13(4), 281 - 8
Role of conserved histidine residues in metalloactivation of the ArsA ATPase; Bhattacharjee H et al.; The ArsA ATPase is the catalytic subunit of a pump that is responsible for resistance to arsenicals and antimonials in Escherichia coli . Arsenite or antimonite allosterically activates the ArsA ATPase activity . ArsA homologues from eubacteria, archaea and eukarya have a signature sequence (DTAPTGHT) that includes a conserved histidine . The ArsA ATPase has two such conserved motifs, one in the NH2-terminal (Al) half and the other in the COOH-terminal (A2) half of the protein . These sequences have been proposed to be signal transduction domains that transmit the information of metal occupancy at the allosteric to the catalytic site to activate ATP hydrolysis . The role of the conserved residues His148 and His453, which reside in the A1 and A2 signal transduction domains respectively, was investigated by mutagenesis to create H148A, H453A or H148A/H453A ArsAs . Each altered protein exhibited a decrease in the Vmax of metalloid-activated ATP hydrolysis, in the order wild type ArsA>H148A>H453A>H148A/H453A . These results suggest that the histidine residues play a role in transmission of the signal between the catalytic and allosteric sites.

Mol Genet Metab, 2001 Mar, 72(3), 273 - 6
Nonorthologous gene displacement of phosphomevalonate kinase; Houten SM et al.; Phosphomevalonate kinase (PMK; EC 2.7.4.2) catalyzes the phosphorylation of 5-phosphomevalonate into 5-diphosphomevalonate, an essential step in isoprenoid biosynthesis via the mevalonate pathway . So far, two nonorthologous genes encoding PMK have been described, the Saccharomyces cerevisiae ERG8 gene and the human PMK gene . Here, we report that orthologues of ERG8 are present in eubacteria, fungi, and plants, while orthologues of human PMK are found only in animals, indicative of a nonorthologous gene displacement early in animal evolution . This also is reflected by different consensus ATP-binding motifs: a protein kinase motif in the ERG8 orthologues versus a P-loop or Walker A motif in the animal orthologues . The fact that ERG8 orthologues are found in pathogenic eubacteria and fungi but not in man makes them attractive targets for the development of antibacterial and/or antifungal drugs .

Insect Mol Biol, 2001 Feb, 10(1), 57 - 67
Psyllid endosymbionts exhibit patterns of co-speciation with hosts and destabilizing substitutions in ribosomal RNA; Spaulding AW et al.; Eubacterial 16S rDNAs were sequenced from endosymbionts of seven psyllids (Psylloidea) and one whitefly (Aleyrodoidea), to investigate the evolution of endosymbionts and their hosts . Primary endosymbionts from all psyllids formed a highly supported clade, tentatively placed as the sister to whitefly primary endosymbionts, and showing several points of congruence with the host morphological phylogeny . Almost all host taxa yielded an additional eubacterial sequence, related either to known psyllid secondary endosymbionts or to other insect endosymbionts or parasites . The relationships of some secondary endosymbionts also suggested cospeciation with psyllid hosts, or ancient horizontal transfers . All primary endosymbionts, and some secondary endosymbionts, exhibited molecular genetic effects of a long-term, intracellular existence in their biased nucleotide content and decreased stability of rRNA secondary structure.

Biochem Biophys Res Commun, 2001 Mar 2, 281(3), 741 - 6
A putative prokaryote voltage-gated Ca(2+) channel with only one 6TM motif per subunit; Durell SR et al.; Until now, voltage-gated Ca(2+) channel proteins have been found only in eukaryotes . Here we report that a gene recently discovered in the eubacterium Bacillus halodurans codes for a protein closely related to eukaryotic Ca(2+) channels, but that has only one 6-transmembrane-segement (6TM) motif, instead of four, in its pore-forming subunit . This is supported by the comparison of consensus sequences, which, along with the patterns of residue conservation, indicates a similar structure in the membrane to voltage-gated K(+) channels . From this we hypothesize that Ca(2+) channels originally evolved in bacteria, and that the specific eubacteria protein highlighted here is an ideal candidate for structure determination efforts.

RNA, 2001 Feb, 7(2), 242 - 53
Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function; Condon C et al.; Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600) . Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria . We propose that the gene be named rnmV . The rnmV gene is nonessential . B . subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function . 5S rRNA precursors can, however, be found in both free and translating ribosomes . In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B . subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step . For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines . A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2497 - 502 Epub 2001 Feb 20.
Estimation of divergence times from multiprotein sequences for a few mammalian species and several distantly related organisms; Nei M et al.; When many protein sequences are available for estimating the time of divergence between two species, it is customary to estimate the time for each protein separately and then use the average for all proteins as the final estimate . However, it can be shown that this estimate generally has an upward bias, and that an unbiased estimate is obtained by using distances based on concatenated sequences . We have shown that two concatenation-based distances, i.e., average gamma distance weighted with sequence length (d(2)) and multiprotein gamma distance (d(3)), generally give more satisfactory results than other concatenation-based distances . Using these two distance measures for 104 protein sequences, we estimated the time of divergence between mice and rats to be approximately 33 million years ago . Similarly, the time of divergence between humans and rodents was estimated to be approximately 96 million years ago . We also investigated the dependency of time estimates on statistical methods and various assumptions made by using sequence data from eubacteria, protists, plants, fungi, and animals . Our best estimates of the times of divergence between eubacteria and eukaryotes, between protists and other eukaryotes, and between plants, fungi, and animals were 3, 1.7, and 1.3 billion years ago, respectively . However, estimates of ancient divergence times are subject to a substantial amount of error caused by uncertainty of the molecular clock, horizontal gene transfer, errors in sequence alignments, etc.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2268 - 73 Epub 2001 Jan 23.
Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria; Kowal AK et al.; Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell . Here we describe two such aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli . The "21st synthetase-tRNA pairs" include E . coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E . coli initiator tRNA, for use in E . coli . The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons . Plasmids carrying the E . coli GlnRS gene can be stably maintained in yeast . However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E . coli . This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E . coli proline tRNA . By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E . coli . These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E . coli proline tRNA by factors of 2.2- to 6.8-fold.

EMBO J, 2001 Jan 15, 20(1-2), 77 - 81
The projection structure of EmrE, a proton-linked multidrug transporter from Escherichia coli, at 7 A resolution; Tate CG et al.; EmrE belongs to a family of eubacterial multidrug transporters that confer resistance to a wide variety of toxins by coupling the influx of protons to toxin extrusion . EmrE was purified and crystallized in two dimensions by reconstitution with dimyristoylphosphatidylcholine into lipid bilayers . Images of frozen hydrated crystals were collected by cryo-electron microscopy and a projection structure of EmrE was calculated to 7 A resolution . The projection map shows an asymmetric EmrE dimer with overall dimensions approximately 31 x 40 A, comprising an arc of highly tilted helices separating two helices nearly perpendicular to the membrane from another two helices, one tilted and the other nearly perpendicular . There is no obvious 2-fold symmetry axis perpendicular to the membrane within the dimer, suggesting that the monomers may have different structures in the functional unit.

J Bacteriol, 2001 Mar, 183(6), 1983 - 9
Identification of an Actinobacillus pleuropneumoniae consensus promoter structure; Doree SM et al.; Actinobacillus pleuropneumoniae promoter-containing clones were isolated from a genomic DNA library constructed in our lVET promoter trap vector pTF86 . The promoter-containing clones were identified by their ability to drive expression of the promoterless luxAB genes of Vibrio harveyi . The degree of expression was quantifiable, and only high-expression or "hot" promoters were used for this study . Nine clones were sequenced, and their transcriptional start sites were determined by primer extension . The sequences upstream of the start site were aligned, and a consensus promoter structure for A . pleuropneumoniae was identified . The consensus promoter sequence for A . pleuropneumoniae was found to be TATAAT and TTG/AAA, centered approximately 10 and 35 bp upstream of the transcriptional start site, respectively . A comparison of the A . pleuropneumoniae consensus with other prokaryotic consensus promoters showed that the A . pleuropneumoniae consensus promoter is similar to that found in other eubacteria in terms of sequence, with an identical -10 element and a similar but truncated -35 element . However, the A . pleuropneumoniae consensus promoter is unique in the spacing between the -10 and -35 elements . The promoter spacing was analyzed by site-directed mutagenesis, which demonstrated that optimal spacing for an A . pleuropneumoniae promoter is shorter than the spacing identified for Escherichia coli and Bacillus subtilis promoters.

J Bacteriol, 2001 Mar, 183(6), 1961 - 73
Essential thioredoxin-dependent peroxiredoxin system from Helicobacter pylori: genetic and kinetic characterization; Baker LM et al.; Helicobacter pylori, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of higher organisms than to most other eubacterial AhpC proteins . Allelic replacement mutagenesis revealed ahpC to be essential, suggesting a critical role for AhpC in defending H . pylori against oxygen toxicity . Characterization of the ahpC promoter region divulged two putative regulatory elements and identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon . No homologue of ahpF, which encodes the dedicated AhpC reductase in most eubacteria, was found in the H . pylori genome . Instead, homologues of Escherichia coli thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system for H . pylori AhpC . A second Trx homologue (Trx2, HP1458) was identified but was incapable of AhpC reduction, although Trx2 exhibited disulfide reductase activity with other substrates {insulin and 5,5'-dithiobis(2-nitrobenzoic acid)} . AhpC interactions with each substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable (infinite V(max) and K(m) values) but rapid enough (at 1 x 10(5) to 2 x 10(5) M(-1) s(-1)) to suggest an important role for AhpC in cellular peroxide metabolism . AhpC also exhibited a wide specificity for hydroperoxide substrates, which, taken together with the above results, suggests a minimal binding site for hydroperoxides composed of little more than the cysteinyl (Cys49) active site . H . pylori AhpC was not reduced by Salmonella typhimurium AhpF and was slightly more active with E . coli TrxR and Trx1 than was S . typhimurium AhpC, demonstrating the specialized catalytic properties of this peroxiredoxin.

J Bacteriol, 2001 Mar, 183(6), 1853 - 61
Degenerative minimalism in the genome of a psyllid endosymbiont; Clark MA et al.; Psyllids, like aphids, feed on plant phloem sap and are obligately associated with prokaryotic endosymbionts acquired through vertical transmission from an ancestral infection . We have sequenced 37 kb of DNA of the genome of Carsonella ruddii, the endosymbiont of psyllids, and found that it has a number of unusual properties revealing a more extreme case of degeneration than was previously reported from studies of eubacterial genomes, including that of the aphid endosymbiont Buchnera aphidicola . Among the unusual properties are an exceptionally low guanine-plus-cytosine content (19.9%), almost complete absence of intergenic spaces, operon fusion, and lack of the usual promoter sequences upstream of 16S rDNA . These features suggest the synthesis of long mRNAs and translational coupling . The most extreme instances of base compositional bias occur in the genes encoding proteins that have less highly conserved amino acid sequences; the guanine-plus-cytosine content of some protein-coding sequences is as low as 10% . The shift in base composition has a large effect on proteins: in polypeptides of C . ruddii, half of the residues consist of five amino acids with codons low in guanine plus cytosine . Furthermore, the proteins of C . ruddii are reduced in size, with an average of about 9% fewer amino acids than in homologous proteins of related bacteria . These observations suggest that the C . ruddii genome is not subject to constraints that limit the evolution of other known eubacteria.

Mol Gen Genet, 2001 Jan, 264(5), 682 - 90
Comparison of psbK operon organization and group III intron content in chloroplast genomes of 12 Euglenoid species; Doetsch NA et al.; A novel mixed operon has been identified in the photosynthetic protist E . gracilis . The genes for psbK, ycf12, psaM, and trnR are co-transcribed . The resulting tetracistronic transcripts are processed through endonucleolytic cleavage of the intergenic spacers and intron splicing to form three mature monocistronic mRNAs and a tRNA . A group III twintron and a group III intron are located in psbK . Another group III intron is found in ycf12 . The psbK operon has been cloned by PCR amplification from nine related Euglenoid species . In each species, the gene order and content of the psbK operon is conserved . The psbK operons contain phylogenetically conserved eubacterial promoter, translational, and 3' processing elements . Intron content varies significantly from species to species . Based on a comparison of the intron content with the results of phylogenetic analysis, group III intron evolution within the Euglenoid lineage is much more complex than previously believed.

Int J Food Microbiol, 2001 Jan 22, 63(1-2), 109 - 16
Nisin-producing organisms during traditional 'Fior di latte' cheese-making monitored by multiplex-PCR and PFGE analyses; Moschetti G et al.; In this work we studied using different molecular methods the population dynamics of nisin-producing organisms and the persistence of such organisms within a complex ecosystem, 'Fior di latte' cheese, a traditional high-moisture pasta filata cheese . Using the primers targeting the eubacterial 16S-23S rRNA spacer region, together with those amplifying the nisA or nisZ gene, we were able to provide a rapid species identification of the isolates . Inhibitors of Lactococcus lactis subsp . lactis DSM 20481T used as indicator occurred during the whole process of cheese manufacture as a significant part of lactic microflora; however, only 12 among 109 isolates of bacteriocin producers were nisin producers . Amplification of the nisA or nisZ gene, using DNA extracted directly from dairy samples as templates, showed that the nisin structural gene was detected during cheese-making from milk samples up to the end of curd ripening but not in the final cheese . In order to monitor nisin-producing strains during cheese manufacturing, the 12 Lactococcus lactis nis+ strains were analysed by low frequency restriction fragment and PFGE . Nine isolates among the 12 nisin-producers exhibited an unique and distinct DNA banding pattern and are considered to be genetically diverse . The other three isolates from curd after ripening showed the same restriction pattern and could be the same strain . In fact, it was also isolated 2 months after the first analysis of cheese-making of 'Fior di latte'.

J Mol Microbiol Biotechnol, 2001 Jan, 3(1), 113 - 22
Organisation and evolution of the tol-pal gene cluster; Sturgis JN; The genomic context and phylogenetic distribution of the tol-pal gene cluster and homologues to its various components have been investigated . The structure of this operon is well conserved across the gram negative bacteria, and the machine encoded by these genes probably evolved with the appearance of gram negative bacteria . Since the evolutionary appearance of the operon some species appear to have lost the genes . These bacteria seem to fall into two classes, namely obligate intracellular parasites and bacteria that produce large numbers of outer membrane vesicles . The evolution of the alphabeta and gamma proteobacteria was accompanied by the association of an additional gene (ybgC) with the operon . Several coincidences of genomic context argue for an important role of the tol-pal operon in cell envelope maintenance . Genes homologous to tolQ and tolR proved to be very widespread being found throughout the eubacteria, and one example in the archea, this distribution argues for an ancient origin of these genes . The genomic context of these genes often suggests a role in micronutrient uptake . Interestingly in all the cases examined the tolQ and tolR genes or their homologues appear to be present as a pair, with a potential for a tight translational regulation.

Environ Microbiol, 1999 Jun, 1(3), 231 - 41
Microbial characterization of a JP-4 fuel-contaminated site using a combined lipid biomarker/polymerase chain reaction--denaturing gradient gel electrophoresis (PCR-DGGE)-based approach; Stephen JR et al.; The impact of pollution on soil microbial communities and subsequent bioremediation can be measured quantitatively in situ using direct, non-culture-dependent techniques . Such techniques have advantages over culture-based methods, which often account for less than 1% of the extant microbial community . In 1988, a JP-4 fuel spill contaminated the glacio-fluvial aquifer at Wurtsmith Air Force Base, Michigan, USA . In this study, lipid biomarker characterization of the bacterial and eukaryotic communities was combined with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the eubacterial community to evaluate correlation between contaminant (JP-4 fuel) concentration and community structure shifts . Vadose, capillary fringe and saturated zone samples were taken from cores within and up- and down-gradient from the contaminant plume . Lipid biomarker analysis indicated that samples from within the plume contained increased biomass, with large proportions of typically gram-negative bacteria . Outside the plume, lipid profiles indicated low-biomass microbial communities compared with those within the initial spill site . 16S rDNA sequences derived from DGGE profiles from within the initial spill site suggested dominance of the eubacterial community by a limited number of phylogenetically diverse organisms . Used in tandem with pollutant quantification, these molecular techniques should facilitate significant improvements over current assessment procedures for the determination of remediation end-points.

J Mol Biol, 2001 Feb 23, 306(3), 455 - 67
A new target for shigellosis: rational design and crystallographic studies of inhibitors of tRNA-guanine transglycosylase; Gradler U et al.; Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hyper-modification of cognate tRNAs leading to the exchange of G34 at the wobble position in the anticodon loop by preQ1 (2-amino-5-(aminomethyl)pyrrolo{2,3-d}pyrimidin-4(3H)-one) as part of the biosynthesis of queuine (Q) . Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium, revealing TGT as a new target for the design of potent drugs against Shigellosis . The X-ray structure of Zymomonas mobilis TGT in complex with preQ1 was used to search for new putative inhibitors with the computer program LUDI . An initial screen of the Available Chemical Directory, a database compiled from commercially available compounds, suggested several hits . Of these, 4-aminophthalhydrazide (APH) showed an inhibition constant in the low micromolar range . The 1.95 A crystal structure of APH in complex with Z . mobilis TGT served as a starting point for further modification of this initial lead.

Nat Genet, 2001 Feb, 27(2), 172 - 80
A candidate prostate cancer susceptibility gene at chromosome 17p; Tavtigian SV et al.; It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity . A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p . We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees . In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer . ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria . The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3' end cleavage and polyadenylation specificity factor (CPSF73).

Ann Rheum Dis, 2001 Mar, 60(3), 269 - 74
Characterisation of Eubacterium cell wall: peptidoglycan structure determines arthritogenicity; Zhang X et al.; OBJECTIVE: To elucidate factors involved in the arthritogenicity of bacterial cell walls . METHODS: For characterisation of an arthritogenic Eubacterium aerofaciens cell wall, peptidoglycan-polysaccharide (PG-PS) polymers were isolated by removing cell wall associated proteins (CWPs), PG and PS moieties were separated, and an attempt was made to de-O-acetylate PG-PS . The cell wall of E limosum was used as a non-arthritogenic control . The chemical composition of these cell wall preparations was analysed by gas chromatography-mass spectrometry . Also, their ability to resist lysozyme degradation and to sustain experimental chronic arthritis was tested . RESULTS: The observations made with the cell wall of E aerofaciens, an anaerobic habitant of the human intestine, were compared with those reported from a pathogenic Streptococcus, showing that in both strains a complex consisting of PG-PS is required for the induction of chronic arthritis . The PS moiety most probably protects PG from enzyme degradation, allowing prolonged tissue persistence and leading to the chronic synovial inflammation . CWPs attached to PG-PS are not necessary for this function . O-Acetylation of PG, which is required for arthritogenicity of the streptococcal cell wall, seems not to be present in the arthritogenic E aerofaciens PG or only occurs to a small degree; attempts to de-O-acylate the E aerofaciens cell wall did not affect its arthritogenicity or lysozyme resistance . CONCLUSION: The results obtained indicate that the source of bacterial cell wall plays no part in the chemical or structural requirements for PG to induce chronic cell wall arthritis in the rats; the chemical structure of the PG moiety is decisive.

Gene, 2000 Dec 30, 261(1), 153 - 9
Evolution of the mitochondrial genetic system: an overview; Saccone C et al.; Mitochondria, semi-autonomous organelles possessing their own genetic system, are commonly accepted to descend from free-living eubacteria, namely hydrogen-producing alpha-proteobacteria . The progressive loss of genes from the primitive eubacterium to the nucleus of the eukaryotic cell is strongly justified by the Muller rachet principle, which postulates that asexual genomes, like mitochondrial ones, accumulate deleterious and sublethal mutations faster than sexual genomes, like the nucleus . According to this principle, the mitochondrial genome would be doomed to death; instead, we observe that the mitochondrial genome has a variable size and structure in the different organisms, though it contains more or less the same set of genes . This is an example of genetic conservation versus structural diversity . From an evolutionary point of view the genetic system of organelles is clearly under strong selective pressure and for its survival it needs to utilize strategies to slow down or halt the ratchet . Anyway, the mitochondrial genome changes with time, and the rate of evolution is different for both diverse regions of the mtDNA and between lineages, as demonstrated in the case of mammalian mt genomes . We report here our data on the evolution of the mitochondrial DNA in mammals which demonstrate the suitability of mtDNA as a molecular tool for evolutionary analyses.

FEBS Lett, 2001 Jan 19, 488(3), 170 - 3
Identification of gcpE as a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis in Escherichia coli; Campos N et al.; The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most eubacteria and plants and has remarkable biotechnological interest . However, only the first steps of this pathway have been determined . Using bioinformatic and genetic approaches, we have identified gcpE as a novel gene of the MEP pathway . The distribution of this gene in bacteria and plants strictly parallels that of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, which catalyses the first committed step of the MEP pathway . Our data demonstrate that the gcpE gene is essential for the MEP pathway in Escherichia coli and indicate that this gene is required for the trunk line of the isoprenoid biosynthetic route.

Nucleic Acids Res, 2001 Feb 15, 29(4), 914 - 20
Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus; Wadsworth RI et al.; Single-stranded DNA binding proteins (SSBs) play central roles in cellular and viral processes involving the generation of single-stranded DNA . These include DNA replication, homologous recombination and DNA repair pathways . SSBs bind DNA using four 'OB-fold' (oligonucleotide/oligosaccharide binding fold) domains that can be organised in a variety of overall quaternary structures . Thus eubacterial SSBs are homotetrameric whilst the eucaryal RPA protein is a heterotrimer and euryarchaeal proteins vary significantly in their subunit compositions . We demonstrate that the crenarchaeal SSB protein is an abundant protein with a unique structural organisation, existing as a monomer in solution and multimerising on DNA binding . The protein binds single-stranded DNA distributively with a binding site size of approximately 5 nt per monomer . Sulfolobus SSB lacks the zinc finger motif found in the eucaryal and euryarchaeal proteins, possessing instead a flexible C-terminal tail, sensitive to trypsin digestion, that is not required for DNA binding . In comparison with Escherichia coli SSB, the tail may play a role in protein-protein interactions during DNA replication and repair.

Biophys J, 2001 Jan, 80(1), 469 - 79
Multicolored protein conformation states in the photocycle of transducer-free sensory rhodopsin-I; Szundi I et al.; Sensory rhodopsin-I (SRI), a phototaxis receptor of archaebacteria, is a retinal-binding protein that exists in the cell membrane intimately associated with a signal-transducing protein (HtrI) homologous to eubacterial chemotaxis receptors . Transducer-free sensory rhodopsin-I (fSRI), from cells devoid of HtrI, undergoes a photochemical cycle kinetically different from that of native SRI . We report here on the measurement and analysis of the photochemical kinetics of fSRI reactions in the 350-750-nm spectral range and in a 10(-7) s to 1 s time window . The lack of specific intermolecular interactions between SRI and HtrI results in early return of the ground form via distinct branching reactions in fSRI, not evident in the photocycle of native SRI . The chromophore transitions are loosely coupled to protein structural transitions . The coexistence of multiple spectral forms within kinetic intermediates is interpreted within the concept of multicolored protein conformational states.

J Bacteriol, 2001 Feb, 183(4), 1259 - 68
Systematic identification of selective essential genes in Helicobacter pylori by genome prioritization and allelic replacement mutagenesis; Chalker AF et al.; A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H . pylori J99 but highly diverged in other eubacteria . A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H . pylori were identified . Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro . Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged . We propose that the products of a number of these H . pylori-specific essential genes may be suitable targets for novel anti-H . pylori therapies.

Curr Opin Microbiol, 1998 Oct, 1(5), 562 - 6
Findings emerging from complete microbial genome sequences; Clayton RA et al.; Sixteen microorganisms, including one eukaryote, four archaeons, and 11 eubacteria, have been completely sequenced and published . More than 50 genomes are scheduled to be completed by the year 2000 . This explosive growth of information is forcing change in many scientific disciplines (e.g . bioinformatics and molecular genetics), spawning new fields, and even changing the way scientific information is used and shared . Novel, global genome sequence comparisons seem slow to appear but the infrastructure for these projects is being built, and we expect exciting developments in the near future.

J Biol Chem, 2001 Apr 6, 276(14), 10745 - 52 Epub 2001 Jan 08.
A novel member of the bacterial-archaeal regulator family is a nonspecific dna-binding protein and induces positive supercoiling; Napoli A et al.; In hyperthermophilic Archaea genomic DNA is from relaxed to positively supercoiled in vivo because of the action of the enzyme reverse gyrase, and this peculiarity is believed to be related to stabilization of DNA against denaturation . We report the identification and characterization of Smj12, a novel protein of Sulfolobus solfataricus, which is homologous to members of the so-called Bacterial-Archaeal family of regulators, found in multiple copies in Eubacteria and Archaea . Whereas other members of the family are sequence-specific DNA- binding proteins and have been implicated in transcriptional regulation, Smj12 is a nonspecific DNA-binding protein that stabilizes the double helix and induces positive supercoiling . Smj12 is not abundant, suggesting that it is not a general architectural protein, but rather has a specialized function and/or localization . Smj12 is the first protein with the described features identified in Archaea and might participate in control of superhelicity during DNA transactions.

J Mol Evol, 2001 Jan, 52(1), 17 - 28
Detecting changes in the functional constraints of paralogous genes; Marin I et al.; We describe a new procedure to determine whether regional alterations in the evolutionary constraints imposed on paralogous proteins have occurred . We used as models the A and B (alternatively called alpha and beta) subunits of V/F/A-ATPases, originated by a gene duplication more than 3 billion years ago . Changes associated to three major splits (eubacteria versus Archaea-eukaryotes; Archaea versus eukaryotes; and among free-living bacteria and symbiotic mitochondria) were studied . Only in the first case, when we compared eubacterial or mitochondrial F-ATPases versus eukaryotic vacuolar V-ATPases or archaeal A-ATPases, constraint changes were observed . Modifications in the degree of regional constraining were not detected for the other two types of comparisons (V-ATPases versus A-ATPases and within F-ATPases, respectively) . When the rates of evolution of the two subunits were compared, it was found that F-ATPases regulatory subunits evolved faster than catalytic subunits, but the opposite was true for A- and V-ATPases . Our results suggest that, even for universal and essential proteins, selective constraints may be occasionally altered . On the other hand, in some cases no changes were detected after periods of more than 2.2 billion years.

FASEB J, 2001 Jan, 15(1), 34 - 42
Origin of H1 linker histones; Kasinsky HE et al.; In which taxa did H1 linker histones appear in the course of evolution? Detailed comparative analysis of the histone H1 and histone H1-related sequences available to date suggests that the origin of histone H1 can be traced to bacteria . The data also reveal that the sequence corresponding to the 'winged helix' motif of the globular structural domain, a domain characteristic of all metazoan histone H1 molecules, is evolutionarily conserved and appears separately in several divergent lines of protists . Some protists, however, appear to have only a lysine-rich basic protein, which has compositional similarity to some of the histone H1-like proteins from eubacteria and to the carboxy-terminal domain of the H1 linker histones from animals and plants . No lysine-rich basic proteins have been described in archaebacteria . The data presented in this review provide the surprising conclusion that whereas DNA-condensing H1-related histones may have arisen early in evolution in eubacteria, the appearance of the sequence motif corresponding to the globular domain of metazoan H1s occurred much later in the protists, after and independently of the appearance of the chromosomal core histones in archaebacteria.

Plant Cell, 2000 Dec, 12(12), 2455 - 2472
The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton; Schwarz G et al.; Molybdenum (Mo) plays an essential role in the active site of all eukaryotic Mo-containing enzymes . In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism . Mo is bound to a unique metal binding pterin (molybdopterin {MPT}), thereby forming the active Mo cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria . Here, we describe the function of the two-domain protein Cnx1 from Arabidopsis in the final step of Moco biosynthesis . Cnx1 is constitutively expressed in all organs and in plants grown on different nitrogen sources . Mo-repairable cnxA mutants from Nicotiana plumbaginifolia accumulate MPT and show altered Cnx1 expression . Transformation of cnxA mutants and the corresponding Arabidopsis chl-6 mutant with cnx1 cDNA resulted in functional reconstitution of their Moco deficiency . We also identified a point mutation in the Cnx1 E domain of Arabidopsis chl-6 that causes the molybdate-repairable phenotype . Recombinant Cnx1 protein is capable of synthesizing Moco . The G domain binds and activates MPT, whereas the E domain is essential for activating Mo . In addition, Cnx1 binds to the cytoskeleton in the same way that its mammalian homolog gephyrin does in neuronal cells, which suggests a hypothetical model for anchoring the Moco-synthetic machinery by Cnx1 in plant cells.

Plant Cell Physiol, 2000 Oct, 41(10), 1119 - 28
Chloroplast targeting, distribution and transcriptional fluctuation of AtMinD1, a Eubacteria-type factor critical for chloroplast division; Kanamaru K et al.; In Arabidopsis thaliana, a mature mesophyll cell contains approximately 100 chloroplasts . Although 12 arc mutants (accumulation and replication of chloroplasts) and two chloroplast division genes homologous to eubacterial ftsZ have been isolated from A . thaliana, the molecular mechanism underlying the chloroplast division is still unclear . We characterized AtMinD1, a eubacterial minD homolog, for chloroplast division in A . thaliana . AtMinD1-green fluorescent protein targeted to the chloroplasts and possibly associated with the envelope membranes in vivo . During the seed germination, the AtMinD1 transcripts were accumulated twice, just after release from cold treatment and at the beginning of rapid greening, in similar fashion to AtFtsZs . Furthermore the transcript level in a severest chloroplast division mutant, arc6, was 3-5-fold higher than that in wild-type.

Genetica, 2000, 108(1), 1 - 7
Conservation of gene order amongst cell wall and cell division genes in Eubacteria, and ribosomal genes in Eubacteria and Eukaryotic organelles; Nikolaichik YA et al.; Comparison of genome sequences from Eubacteria and Eukaryotic organelles shows that the order of genes in gene clusters encoding certain highly conserved cell division proteins and ribosomal proteins is itself highly conserved . Experiments with a cluster of cell division and related genes of E . coli have shown that this gene order is not essential for function . Comparisons between genomes also show that no pair of genes are necessarily adjacent in all genomes . The reason for the extreme conservation of order is therefore unknown, although one possible explanation might be the lateral exchange of tightly-linked groups of genes coding for co-adapted sets of proteins.

Mem Inst Oswaldo Cruz, 2000, 95 Suppl 1, 123 - 31
Current millennium biotechniques for biomedical research on parasites and host-parasite interactions; Teixeira AR et al.; The development of biotechnology in the last three decades has generated the feeling that the newest scientific achievements will deliver high standard quality of life through abundance of food and means for successfully combating diseases . Where the new biotechnologies give access to genetic information, there is a common belief that physiological and pathological processes result from subtle modifications of gene expression . Trustfully, modern genetics has produced genetic maps, physical maps and complete nucleotide sequences from 141 viruses, 51 organelles, two eubacteria, one archeon and one eukaryote (Saccharomices cerevisiae) . In addition, during the Centennial Commemoration of the Oswaldo Cruz Institute the nearly complete human genome map was proudly announced, whereas the latest Brazilian key stone contribution to science was the publication of the Shillela fastidiosa genomic sequence highlythed on a Nature cover issue . There exists a belief among the populace that further scientific accomplishments will rapidly lead to new drugs and methodological approaches to cure genetic diseases and other incurable ailments . Yet, much evidence has been accumulated, showing that a large information gap exists between the knowledge of genome sequence and our knowledge of genome function . Now that many genome maps are available, people wish to know what are we going to do with them . Certainly, all these scientific accomplishments will shed light on many more secrets of life . Nevertheless, parsimony in the weekly announcements of promising scientific achievements is necessary . We also need many more creative experimental biologists to discover new, as yet un-envisaged biotechnological approaches, and the basic resource needed for carrying out mile stone research necessary for leading us to that "promised land" often proclaimed by the mass media.

J Clin Periodontol, 2001 Jan, 28(1), 103 - 6
Checkerboard assessments of serum antibodies to oral microbiota as surrogate markers of clinical periodontal status; Papapanou PN et al.; BACKGROUND: This study addresses whether checkerboard assessments of serum IgG antibodies to oral bacteria may serve as surrogate markers of clinical periodontal status in epidemiologic studies . METHOD: The analysis involved data from 205 subjects, 132 periodontitis patients and 73 periodontally-intact controls, from whom full-mouth clinical periodontal data and serum IgG titers against 19 periodontal bacterial species were available . RESULTS: A logistic regression model involving titers against 6 species (Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Eubacterium nodatum, Eikenella corrodens, Capnocytophaga ochracea and Actinomyces naeslundii genospecies 2) classified correctly 74.5% of the subjects examined, with 84% sensitivity, 57.5% specificity, 78% positive predictive value and 66.7% negative predictive value . CONCLUSIONS: Checkerboard serology may be useful in providing surrogate markers for clinical periodontal status when such data are not readily available and, thus may serve as a valuable complement in the armamentarium of epidemiologic tools suited for the study of periodontal diseases.

Genetics, 2001 Jan, 157(1), 399 - 411
RNA sequence evolution with secondary structure constraints: comparison of substitution rate models using maximum-likelihood methods; Savill NJ et al.; We test models for the evolution of helical regions of RNA sequences, where the base pairing constraint leads to correlated compensatory substitutions occurring on either side of the pair . These models are of three types: 6-state models include only the four Watson-Crick pairs plus GU and UG; 7-state models include a single mismatch state that combines all of the 10 possible mismatches; 16-state models treat all mismatch states separately . We analyzed a set of eubacterial ribosomal RNA sequences with a well-established phylogenetic tree structure . For each model, the maximum-likelihood values of the parameters were obtained . The models were compared using the Akaike information criterion, the likelihood-ratio test, and Cox's test . With a high significance level, models that permit a nonzero rate of double substitutions performed better than those that assume zero double substitution rate . Some models assume symmetry between GC and CG, between AU and UA, and between GU and UG . Models that relaxed this symmetry assumption performed slightly better, but the tests did not all agree on the significance level . The most general time-reversible model significantly outperformed any of the simplifications . We consider the relative merits of all these models for molecular phylogenetics.

Nat Struct Biol, 2001 Jan, 8(1), 42 - 6
Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation; Rudolph MJ et al.; Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea and eukaryotes, including humans . Genetic deficiencies of enzymes involved in Moco biosynthesis in humans lead to a severe and usually fatal disease . Moco contains a tricyclic pyranopterin, termed molybdopterin (MPT), that bears the cis-dithiolene group responsible for molybdenum ligation . The dithiolene group of MPT is generated by MPT synthase, which consists of a large and small subunits . The 1.45 A resolution crystal structure of MPT synthase reveals a heterotetrameric protein in which the C-terminus of each small subunit is inserted into a large subunit to form the active site . In the activated form of the enzyme this C-terminus is present as a thiocarboxylate . In the structure of a covalent complex of MPT synthase, an isopeptide bond is present between the C-terminus of the small subunit and a Lys side chain in the large subunit . The strong structural similarity between the small subunit of MPT synthase and ubiquitin provides evidence for the evolutionary antecedence of the Moco biosynthetic pathway to the ubiquitin dependent protein degradation pathway.

Appl Environ Microbiol, 2001 Jan, 67(1), 300 - 6
Non-growth-associated demethylation of dimethylsulfoniopropionate by (homo)acetogenic bacteria; Jansen M et al.; The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied . Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662(T), Sporomusa sphaeroides DSM 2875(T), and Acetobacterium woodii DSM 1030(T) were shown to demethylate DMSP stoichiometrically to MTPA . The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass . The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens . In batch cultures of E . limosum PM31 DMSP and glycine betaine were demethylated simultaneously . In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol . In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h(-1)) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min(-1) mg of protein(-1), both after growth in the presence of DMSP and after growth in its absence . In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min(-1) mg of protein(-1) were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower . A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14421 - 6
Widespread occurrence of structurally diverse tetraether membrane lipids: evidence for the ubiquitous presence of low-temperature relatives of hyperthermophiles; Schouten S et al.; Isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs) and branched glycerol dialkyl diethers are main membrane constituents of cultured hyperthermophilic archaea and eubacteria, respectively, and are found in environments with temperatures >60 degrees C . Recently, we developed a new technique for the analysis of intact core tetraether lipids in cell material and sediments . The application of this technique to recent sediments shows that known and newly identified isoprenoid and branched GDGTs are widespread in low-temperature environments (<20 degrees C) and are structurally far more diverse than previously thought . Their distribution indicates the ubiquitous environmental presence of as yet uncultivated, nonthermophilic organisms that may have independently evolved from hyperthermophilic archaea and eubacteria . The structures of some of the new GDGTs point to the hybridization of both typical archaeal and eubacterial biosynthetic pathways in single organisms.

Biochem J, 2001 Jan 1, 353(Pt 1), 59 - 67
Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis; Campos N et al.; Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function . IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway . Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined . We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli . A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E . coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium . The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway . We used this system to show that the ygbP, ychB and ygbB genes are essential in E . coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.

J Bacteriol, 2001 Jan, 183(1), 1 - 11
1-Deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus; Hahn FM et al.; In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway . The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate . The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase . In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase . ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome . The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps . 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits . A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4) . The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E . coli strain FH11.

Gene, 2000 Nov 27, 258(1-2), 147 - 54
Light-modulated NADP-malate dehydrogenases from mossfern and green algae: insights into evolution of the enzyme's regulation; Ocheretina O et al.; Chloroplast NADP-dependent malate dehydrogenase is one of the best-studied light-regulated enzymes . In C3 plants, NADP-MDH is a part of the 'malate valve' that controls the export of reducing equivalents in the form of malate to the cytosol . NADP-MDH is completely inactive in the dark and is activated in the light with reduced thioredoxin . Compared with its permanently active NAD-linked counterparts, NADP-MDH exhibits N- and C-terminal sequence extensions, each bearing one regulatory disulphide . Upon reduction of the C-terminal disulphide, the enzyme active site becomes accessible for the substrate . Reduction of the N-terminal disulphide promotes a conformational change advantageous for catalysis . To trace the evolutionary development of this intricate regulation mechanism, we isolated cDNA clones for NADP-MDH from the mossfern Selaginella and from two unicellular green algae . While the NADP-MDH sequence from Selaginella demonstrates the classic cysteine pattern of the higher plant enzyme, the sequences from the green algae are devoid of the N-terminal regulatory disulphide . Phylogenetic analysis of new sequences and of those available in the databases led to the conclusion that the chloroplast NADP-MDH and the cytosolic NAD-dependent form arose via duplication of an ancestral eubacterial gene, which preceded the separation of plant and animal lineages . Redox-sensitive NADP-MDH activity was detected only in the 'green' plant lineage starting from the primitive prasinophytic algae but not in cyanobacteria, Cyanophora paradoxa, red algae and diatoms . The latter organisms therefore appear to utilize mechanisms other than the light-regulated 'malate valve' to remove from plastids excessive electrons produced by photosynthesis.

Phys Rev Lett, 2000 Dec 11, 85(24), 5246 - 9
Elasticity of the rod-shaped gram-negative eubacteria; Boulbitch A et al.; We report a theoretical calculation of the elasticity of the peptidoglycan network, the only stress-bearing part of rod-shaped Gram-negative eubacteria . The peptidoglycan network consists of elastic peptides and inextensible glycan strands, and it has been proposed that the latter form zigzag filaments along the circumference of the cylindrical bacterial shell . The zigzag geometry of the glycan strands gives rise to nonlinear elastic behavior . The four elastic moduli of the peptidoglycan network depend on its stressed state . For a bacterium under physiological conditions the elasticity is proportional to the bacterial turgor pressure . Our results are in good agreement with recent measurements.

Arch Biochem Biophys, 2000 Nov 1, 383(1), 1 - 16
Catalytic activities of the 20 S proteasome, a multicatalytic proteinase complex; Orlowski M et al.; The proteasome, a multisubunit, multicatalytic proteinase complex, is attracting growing attention as the main intracellular, extralysosomal, proteolytic system involved in ubiquitin-(Ub) dependent and Ub-independent intracellular proteolysis . Its involvement in the mitotic cycle, and control of the half-life of most cellular proteins, functions absolutely necessary for cell growth and viability, make it an attractive target for researchers of intracellular metabolism and an important target for pharmacological intervention . The proteasome belongs to a new mechanistic class of proteases, the N-terminal nucleophile hydrolases, where the N-terminal threonine residue functions as the nucleophile . This minireview focuses on the three classical catalytic activities of the proteasome, designated chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolyzing in eukaryotes and also the activities of the more simple Archaebacteria and Eubacteria proteasomes . Other catalytic activities of the proteasome and their possible origin are also examined . The specificity of the catalytic components toward synthetic substrates, natural peptides, and proteins and their relationship to the catalytic centers are reviewed . Some unanswered questions and future research directions are suggested.

FEBS Lett, 2000 Nov 24, 485(2-3), 178 - 82
Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor sigma B; Shirano Y et al.; Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light . However, little is known of the factors involved in the complex coordination of light-induced plastid gene expression, which must be directed by both nuclear and plastid genomes . We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype . The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial-like plastid RNA polymerase . Our results provide direct evidence that a nuclear-derived prokaryotic-like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.

Structure Fold Des, 2000 Nov 15, 8(11), 1127 - 36
Structure of the molybdate/tungstate binding protein mop from Sporomusa ovata; Wagner UG et al.; BACKGROUND: Transport of molybdenum into bacteria involves a high-affinity ABC transporter system whose expression is controlled by a repressor protein called ModE . While molybdate transport is tightly coupled to utilization in some bacteria, other organisms have molybdenum storage proteins . One class of putative molybdate storage proteins is characterized by a sequence consisting of about 70 amino acids (Mop) . A tandem repeat of Mop sequences also constitutes the molybdate binding domain of ModE . RESULTS: We have determined the crystal structure of the 7 kDa Mop protein from the methanol-utilizing anaerobic eubacterium Sporomusa ovata grown in the presence of molybdate and tungstate . The protein occurs as highly symmetric hexamers binding eight oxyanions . Each peptide assumes a so-called OB fold, which has previously also been observed in ModE . There are two types of oxyanion binding sites in Mo at the interface between two or three peptides . All oxyanion binding sites were found to be occupied by WO(4) rather than MoO(4) . CONCLUSIONS: The biological function of proteins containing only Mop sequences is unknown, but they have been implicated in molybdate homeostasis and molybdopterin cofactor biosynthesis . While there are few indications that the S . ovata Mop binds pterin, the structure suggests that only the type-1 oxyanion binding sites would be sufficiently accessible to bind a cofactor . The observed occupation of the oxyanion binding sites by WO(4) indicates that Mop might also be involved in controlling intracellular tungstate levels.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13172 - 7
Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes; Lange BM et al.; Isopentenyl diphosphate (IPP) is the central intermediate in the biosynthesis of isoprenoids, the most ancient and diverse class of natural products . Two distinct routes of IPP biosynthesis occur in nature: the mevalonate pathway and the recently discovered deoxyxylulose 5-phosphate (DXP) pathway . The evolutionary history of the enzymes involved in both routes and the phylogenetic distribution of their genes across genomes suggest that the mevalonate pathway is germane to archaebacteria, that the DXP pathway is germane to eubacteria, and that eukaryotes have inherited their genes for IPP biosynthesis from prokaryotes . The occurrence of genes specific to the DXP pathway is restricted to plastid-bearing eukaryotes, indicating that these genes were acquired from the cyanobacterial ancestor of plastids . However, the individual phylogenies of these genes, with only one exception, do not provide evidence for a specific affinity between the plant genes and their cyanobacterial homologues . The results suggest that lateral gene transfer between eubacteria subsequent to the origin of plastids has played a major role in the evolution of this pathway.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 483 - 90
Regulation of the pts operon in low G+C Gram-positive bacteria; Vadeboncoeur C et al.; The sugar transport system called phosphoenolpyruvate: sugar phosphotransferase (PTS) is widespread among eubacteria . Its is generally composed of two cytoplasmic proteins, HPr and El, which are found in all bacteria possessing a PTS, and a family of Ells whose number, specificity, and molecular structure in terms of domain arrangement vary from species to species . In low G+C Gram-positive bacteria, the genes coding for the general proteins HPr and El, designated ptsH and ptsl respectively, are organized into the pts operon . In this paper, we summarize current knowledge about the regulation of the pts operon in low G+C Gram-positive bacteria . Physiological data indicate that El and most particularly HPr make up a substantial proportion of cellular proteins . Their synthesis is not coordinated and is influenced by environmental factors . The principal DNA cis-elements involved in the regulation of pts operon transcription are a strong promoter whose sequence and structure are very similar to those of the canonical promoter recognized by the Escherichia coli and Bacillus subtilis major RNA polymerases, a 5'-untranslated region, a rho-dependent terminator located at the 5' end of ptsl, and an intrinsic terminator located downstream from ptsl . Analysis of ptsH and ptsl Shine-Dalgarno sequences as well as experimental results obtained with a Streptococcus salivarius mutant suggest that the expression of HPr and El is also controlled at the translation level.

J Med Microbiol, 2000 Nov, 49(11), 969 - 75
Competition for glucose between Candida albicans and oral bacteria grown in mixed culture in a chemostat; Basson NJ; The competition for glucose as a growth-limiting substrate between Candida albicans and a mixed community of oral bacteria was investigated . A chemostat was operated under glucose-limiting and glucose excess conditions at a dilution rate of 0.05/h . A mixed population of oral bacteria was established and after a steady state had been reached the chemostat was inoculated with C . albicans . Seven bacterial species Streptococcus sanguis, S . sobrinus, S . mitis, Lactobacillus casei, Veillonella dispar, Eubacterium saburreum and Fusobacterium nucleatum - were able to establish stable populations under glucose-limiting conditions . The yeast was unable to grow with the bacteria under glucose limitation . Only three bacterial species, S . sobrinus, L . casei and E . saburreum, became established under glucose-excess conditions . C . albicans was also able to become established in the glucose-excess chemostat and could grow and maintain a steady state in a mixed culture with these organisms . L . casei, S . mitis and S . sobrinus had faster glucose consumption rates than C . albicans . All the bacteria, except for E nucleatum, had maximum specific growth rates higher than C . albicans . The results suggest that glucose may act as a growth-limiting substrate for C . albicans in the establishment and growth of the yeast in a mixed community of oral bacteria.

Genome Inform Ser Workshop Genome Inform, 1998, 9, 3 - 12
Genomic Analysis of the Genes Encoding Ribosomal Proteins in Eight Eubacterial Species and Saccharomyces cerevisiae; Fujita K et al.; The complete genomic nucleotide sequence data of more than 10 unicellular organisms have become available . During the past years, we have been focusing our attention to the analysis of the structure and function of the ribosome and its protein components . By making use of the genomic sequence data, our work can now be extended to comparative analysis of the ribosomal components at the genomic level . Such analysis will contribute to our understanding of the structure-function relationship of the ribosome that is vital to the expression of genetic information . Bearing these in mind, the ribosomal protein genes of organisms whose genomic sequence data are available were analyzed, which included Aquifex aeolicus; Archaeoglobus fulgidus; Borrelia burgdorferi; Bacillus subtilis; Escherichia coli; Haemophilus influenzae; Helicobacter pylori; Methanococcus jannaschii; Mycoplasma genitalium; Mycoplasma pneumoniae; Synechosystis sp., and Saccharomyces cerevisiae . In addition, the amino acid sequence data of Bacillus stearothermophilus ribosomal proteins were used in the evolutionary evaluation . The results indicate that, in eubacteria including two species of Mycoplasma, the operon structure of ribosomal protein genes is well conserved, while their relative orientation and chromosomal location are diverged into several classes . The operon structure in M . jannaschii on the other hand is quite different from the eubacterial one and we noticed that its many genes show similarity to rat ribosomal protein genes . The degrees of sequence conservation differ from one ribosomal protein gene to another, but several genes encoding proteins that are considered to be of structural importance are conserved throughout the bacterial species including archaebacteria and further in S . cerevisiae.

Mol Biol Evol, 2000 Nov, 17(11), 1695 - 709
Iron hydrogenases and the evolution of anaerobic eukaryotes; Horner DS et al.; Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree . Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis . Here, we show that sequences related to iron-only hydrogenases ({Fe} hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested . Genes encoding small proteins which contain conserved structural features unique to {Fe} hydrogenases were identified on all well-surveyed aerobic eukaryote genomes . Longer sequences encoding {Fe} hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes . We also identified a new {Fe} hydrogenase sequence from Trichomonas vaginalis, bringing the total of {Fe} hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes . Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the {Fe} hydrogenases in anaerobic eukaryotes are not monophyletic . Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for {Fe} hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes . Trees where the {Fe} hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly . In most analyses, the Nyctotherus sequence formed a sister group with a {Fe} hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris . Thus, it is possible that Nyctotherus obtained its hydrogenosomal {Fe} hydrogenase from a different source from Trichomonas for its hydrogenosomes . We find no support for the hypothesis that components of the Nyctotherus {Fe} hydrogenase fusion protein derive from the mitochondrial respiratory chain.

Mol Biol Evol, 2000 Nov, 17(11), 1581 - 8
Nucleotide bias causes a genomewide bias in the amino acid composition of proteins; Singer GA et al.; We analyzed the nucleotide contents of several completely sequenced genomes, and we show that nucleotide bias can have a dramatic effect on the amino acid composition of the encoded proteins . By surveying the genes in 21 completely sequenced eubacterial and archaeal genomes, along with the entire Saccharomyces cerevisiae genome and two Plasmodium falciparum chromosomes, we show that biased DNA encodes biased proteins on a genomewide scale . The predicted bias affects virtually all genes within the genome, and it could be clearly seen even when we limited the analysis to sets of homologous gene sequences . Parallel patterns of compositional bias were found within the archaea and the eubacteria . We also found a positive correlation between the degree of amino acid bias and the magnitude of protein sequence divergence . We conclude that mutational bias can have a major effect on the molecular evolution of proteins . These results could have important implications for the interpretation of protein-based molecular phylogenies and for the inference of functional protein adaptation from comparative sequence data.

Mol Microbiol, 2000 Nov, 38(3), 602 - 12
The clpP multigenic family in Streptomyces lividans: conditional expression of the clpP3 clpP4 operon is controlled by PopR, a novel transcriptional activator; Viala J et al.; The clpP genes are widespread among living organisms and encode the proteolytic subunit of the Clp ATP-dependent protease . These genes are present in a single copy in most eubacteria . However, five clpP genes were identified in Streptomyces coelicolor . The clpP1 clpP2 operon was studied: mutations affected the growth cycle in various Streptomyces . Here, we report studies of the expression of the clpP3 clpP4 operon in Streptomyces lividans . The clpP3 operon was induced in a clpP1 mutant strain, and the regulation of expression was investigated in detail . The product of the putative regulator gene, downstream from clpP4, was purified . Gel migration shift assays and DNase I footprinting showed that this protein binds to the clpP3 promoter and recognizes a tandem 6 bp palindromic repeat (TCTGCC-3N-GGCAGA) . In vivo, this DNA-binding protein, named PopR, acts as an activator of the clpP3 operon . Studies of popR expression indicate that the regulator is probably controlled at the post-transcriptional level.

Mol Microbiol, 2000 Nov, 38(3), 446 - 55
Early lateral transfer of genes encoding malic enzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica; Field J et al.; The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes . Here, genes encoding malic enzyme and acetyl-CoA synthetase (nucleoside diphosphate forming) were cloned from E . histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction . The E . histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin . The E . histolytica acetyl-CoA synthetase, which converts acetyl-CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl-CoA synthetase than to those of archaea . Phylogenetic analyses suggested that the adh1 and adhe genes of E . histolytica and Gram-positive eubacteria share a common ancestor . Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E . histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT-rich codon usage of the parasite.

Lett Appl Microbiol, 2000 Oct, 31(4), 332 - 7
Accumulation of ppGpp and ppGp in Staphylococcus aureus 8325-4 following nutrient starvation; Crosse AM et al.; AIM: To investigate the accumulation of highly phosphorylated guanosine nucleotides in Staphylococcus aureus 8325-4 following nutrient deprivation . METHODS AND RESULTS: Nutrient shiftdown of Staph . aureus, HPLC of nucleotides and Western blotting of cell-free extracts . ppGpp rapidly accumulated when cells were deprived of isoleucine following addition of mupirocin, or after carbon deprivation . In contrast, total amino acid starvation led to delayed production of ppGp, which suggests that Staph . aureus exhibits a unique response to total amino acid deprivation compared with other eubacteria . Intracellular ppGp was observed at high levels under all starvation conditions, which suggests that this nucleotide is linked to nutrient limitation and may therefore be involved in regulating the stringent response in Staph . aureus . pppGpp was not observed under any nutrient-limiting condition . Western blot analysis of whole-cell extracts from Staph . aureus 8325-4, showed that antibodies to RelA and SpoT cross-reacted under conditions that detected these proteins in Escherichia coli . CONCLUSIONS: Staph . aureus produces ppGpp and ppGp following nutrient limitation . Immunological analysis indicates that Staph . aureus contains RelA and SpoT proteins, similar to those produced by E . coli . SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a new example of the diversity of metabolic regulations in bacteria.

J Periodontol, 2000 Oct, 71(10), 1554 - 60
Identification of periodontal pathogens in atheromatous plaques; Haraszthy VI et al.; BACKGROUND: Recent studies suggest that chronic infections including those associated with periodontitis increase the risk for coronary vascular disease (CVD) and stroke . We hypothesize that oral microorganisms including periodontal bacterial pathogens enter the blood stream during transient bacteremias where they may play a role in the development and progression of atherosclerosis leading to CVD . METHODS: To test this hypothesis, 50 human specimens obtained during carotid endarterectomy were examined for the presence of Chlamydia pneumoniae, human cytomegalovirus, and bacterial 16S ribosomal RNA using specific oligonucleotide primers in polymerase chain reaction (PCR) assays . Approximately 100 ng of chromosomal DNA was extracted from each specimen and then amplified using standard conditions (30 cycles of 30 seconds at 95 degrees C, 30 seconds at 55 degrees C, and 30 seconds at 72 degrees C) . Bacterial 16S rDNA was amplified using 2 synthetic oligonucleotide primers specific for eubacteria . The PCR product generated with the eubacterial primers was transferred to a charged nylon membrane and probed with digoxigenin-labeled synthetic oligonucleotides specific for Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, and Prevotella intermedia . RESULTS: Eighty percent of the 50 endarterectomy specimens were positive in 1 or more of the PCR assays . Thirty-eight percent were positive for HCMV and 18% percent were positive for C . pneumoniae . PCR assays for bacterial 16S rDNA also indicated the presence of bacteria in 72% of the surgical specimens . Subsequent hybridization of the bacterial 16S rDNA positive specimens with species-specific oligonucleotide probes revealed that 44% of the 50 atheromas were positive for at least one of the target periodontal pathogens . Thirty percent of the surgical specimens were positive for B . forsythus, 26% were positive for P . gingivalis, 18% were positive for A . actinomycetemcomitans, and 14% were positive for P . intermedia . In the surgical specimens positive for periodontal pathogens, more than 1 species was most often detected . Thirteen (59%) of the 22 periodontal pathogen-positive surgical specimens were positive for 2 or more of the target species . CONCLUSIONS: Periodontal pathogens are present in atherosclerotic plaques where, like other infectious microorganisms such as C . pneumoniae, they may play a role in the development and progression of atherosclerosis leading to coronary vascular disease and other clinical sequelae.

Nat Biotechnol, 2000 Nov, 18(11), 1211 - 3
A hyperthermostable bacterial histone-like protein as an efficient mediator for transfection of eukaryotic cells; Esser D et al.; Gene delivery has shown potential in a variety of applications, including basic research, therapies for inborn genetic defects, cancer, AIDS, tissue engineering, and vaccination . Most available systems have serious drawbacks, such as safety hazards, inefficiency under in vivo-like conditions, and expensive production . When using naked DNA, for instance, a large amount of ultrapure DNA has to be applied as a result of degradation by nucleases . Similarly, the use of eukaryotic histones, synthetic peptides, or peptide nucleic acids may be limited by high production costs . We have demonstrated a biotechnologically feasible and economical approach for gene delivery using the histone-like protein from the hyperthermostable eubacterium Thermotoga maritima, TmHU as an efficient gene transfer reagent . HU can be easily isolated from recombinant Escherichia coli, is extraordinarily stable, and protects dsDNA from thermal denaturation . This study demonstrates its use as an inexpensive tool for gene delivery.

Biochim Biophys Acta, 2000 Oct 18, 1482(1-2), 35 - 45
Evolution of the lipocalin family as inferred from a protein sequence phylogeny; Gutierrez G et al.; The lipocalins constitute a family of proteins that have been found in eubacteria and a variety of eukaryotic cells, where they play diverse physiological roles . It is the primary goal of this review to examine the patterns of change followed by lipocalins through their complex history, in order to stimulate scientists in the field to experimentally contrast our phylogeny-derived hypotheses . We reexamine our previous work on lipocalin phylogeny and update the phylogenetic analysis of the family . Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades . The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer . The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency.

Mutat Res, 2000 Nov 9, 461(3), 169 - 77
A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V; Liu J et al.; Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA . Based on amino acids sequence homology, putative homologs of endonuclease V were identified in several organisms including archaebacteria, eubacteria as well as eukaryotes . The translated amino acid sequence of the Archaeoglobus fulgidus nfi gene shows 39% identity and 55% similarity to the E . coli nfi gene . A . fulgidus endonuclease V was cloned and expressed in E . coli as a C-terminal hexa-histidine fusion protein . The C-terminal fusion protein was purified to apparent homogeneity by a combination of Ni(++) affinity and MonoS cation exchange liquid chromatography . The purified C-terminal fusion protein has a molecular weight of about 25kDa and showed endonuclease activity towards DNA containing deoxyinosine . A . fulgidus endonuclease V has an absolute requirement for Mg(2+) and an optimum reaction temperature at 85 degrees C . However, in contrast to E . coli endonuclease V, which has a wide substrate spectrum, endonuclease V from A . fulgidus recognized only deoxyinosine . These data suggest that the deoxyinosine cleavage activity is a primordial activity of endonuclease V and that multiple enzymatic activities of E . coli endonuclease V were acquired later during evolution.

Appl Environ Microbiol, 2000 Nov, 66(11), 5035 - 42
Phylogenetic characterization and in situ detection of a Cytophaga-Flexibacter-Bacteroides phylogroup bacterium in Tuber borchii vittad . Ectomycorrhizal mycelium; Barbieri E et al.; Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms . In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad . Universal eubacterial primers specific for the 5' and 3' ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses . The 16S rDNA was amplified directly from four pure cultures of T . borchii Vittad . mycelium . A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T . borchii mycelium studied . The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously . In situ detection of the CFB bacterium in the hyphal tissue of the fungus T . borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium . Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue . This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.

J Pathol, 2000 Nov, 192(3), 289 - 92
Intestinal inflammatory pseudotumour with regional lymph node involvement: identification of a new bacterium as the aetiological agent; Cheuk W et al.; Inflammatory pseudotumours are the morphological expression of diverse processes such as reactive/reparative, infective, and neoplastic . This paper reports an example of intestinal inflammatory pseudotumour, with identification of a newly characterized bacterium in the lesion . The patient presented with intestinal obstruction . Laparotomy revealed a tumour in the terminal ileum causing stricture, and multiple enlarged regional lymph nodes . Histologically, the tumour and lymph nodes were composed of plump spindle cells disposed in a vague storiform pattern, and associated with lymphocytes and plasma cells . Immunohistochemical studies showed that most of the spindle cells were histiocytes (CD68 positive), prompting a search for a bacterial aetiology, akin to mycobacterial spindle cell pseudotumour . All histochemical stains for micro-organisms were unrewarding . Ultrastructural studies, however, revealed abundant bacteria within the spindle histiocytes . Polymerase chain reaction, using conserved oligonucleotide primers complementary to the 16S rRNA genes of eubacteria, was employed to amplify 16S rRNA gene fragments directly from the involved lymph node tissue . Phylogenetic analysis of the amplified DNA sequences revealed an organism with 99% sequence conformity to Pseudomonas veronii, a bacterium which has hitherto not been implicated in human infection . The importance of searching for an infective agent in inflammatory pseudotumour in the appropriate setting is re-emphasized .

Gene, 2000 Oct 3, 256(1-2), 169 - 82
Common phylogeny of catalase-peroxidases and ascorbate peroxidases; Zamocky M et al.; Catalase-peroxidases belong to Class I of the plant, fungal, bacterial peroxidase superfamily, together with yeast cytochrome c peroxidase and ascorbate peroxidases . Obviously these bifunctional enzymes arose via gene duplication of an ancestral hydroperoxidase . A 230-residues long homologous region exists in all eukaryotic members of Class I, which is present twice in both prokaryotic and archaeal catalase-peroxidases . The overall structure of eukaryotic Class I peroxidases may be retained in both halves of catalase-peroxidases, with major insertions in several loops, some of which may participate in inter-domain or inter-subunit interactions.Interspecies distances in unrooted phylogenetic trees, analysis of sequence similarities in distinct structural regions, as well as hydrophobic cluster analysis (HCA) suggest that one single tandem duplication had already occurred in the common ancestor prior to the segregation of the archaeal and eubacterial lines . The C-terminal halves of extant catalase-peroxidases clearly did not accumulate random changes, so prolonged periods of independent evolution of the duplicates can be ruled out . Fusion of both copies must have occurred still very early or even in the course of the duplication . We suggest that the sparse representatives of eukaryotic catalase-peroxidases go back to lateral gene transfer, and that, except for several fungi, only single copy hydroperoxidases occur in the eukaryotic lineage.The N-terminal halves of catalase-peroxidases, which reveal higher homology with the single-copy members of the superfamily, obviously are catalytically active, whereas the C-terminal halves of the bifunctional enzymes presumably control the access to the haem pocket and facilitate stable folding . The bifunctional nature of catalase-peroxidases can be ascribed to several unique sequence peculiarities conserved among all N-terminal halves, which most likely will affect the properties of both haem ligands.

Nature, 2000 Oct 12, 407(6805), 757 - 62
The complete sequence of the mucosal pathogen Ureaplasma urealyticum; Glass JI et al.; The comparison of the genomes of two very closely related human mucosal pathogens, Mycoplasma genitalium and Mycoplasma pneumoniae, has helped define the essential functions of a self-replicating minimal cell, as well as what constitutes a mycoplasma . Here we report the complete sequence of a more distant phylogenetic relative of those bacteria, Ureaplasma urealyticum (parvum biovar), which is also a mucosal pathogen of humans . It is the third mycoplasma to be sequenced, and has the smallest sequenced prokaryotic genome except for M . genitalium . Although the U . urealyticum genome is similar to the two sequenced mycoplasma genomes, features make this organism unique among mycoplasmas and all bacteria . Almost all ATP synthesis is the result of urea hydrolysis, which generates an energy-producing electrochemical gradient . Some highly conserved eubacterial enzymes appear not to be encoded by U . urealyticum, including the cell-division protein FtsZ, chaperonins GroES and GroEL, and ribonucleoside-diphosphate reductase . U . urealyticum has six closely related iron transporters, which apparently arose through gene duplication, suggesting that it has a kind of respiration system not present in other small genome bacteria The genome is only 25.5% G+C in nucleotide content, and the G+C content of individual genes may predict how essential those genes are to ureaplasma survival.

Arch Microbiol, 2000 Sep, 174(3), 200 - 12
A selDABC cluster for selenocysteine incorporation in Eubacterium acidaminophilum; Gursinsky T et al.; The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions . The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA) . A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA . SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD . The function of selA, selB, and selC from E . acidaminophilum was investigated by complementation of the respective E . coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate . selA and selC from E . acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid . Complementation of the E . coli selB mutant was only observed when both selB and selC from E . acidaminophilum were present . Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type . Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 21 - 5
Biotransformation of the isoflavonoids biochanin A, formononetin, and glycitein by Eubacterium limosum; Hur H et al.; Eubacterium limosum (ATCC 8486), a strict anaerobe from the human intestinal tract that is capable of O-demethylation of several compounds, was tested for the ability to metabolize three methoxylated isoflavonoids, biochanin A, formononetin, and glycitein . High-performance liquid chromatography elution profiles of metabolites produced from biochanin A, formononetin, and glycitein showed peaks that had identical retention times to authentic genistein, daidzein, and 6,7,4'-trihydroxyisoflavone, respectively . The metabolites were identified, using an on line liquid chromatography-electrospray mass spectrometer . E . limosum produced 61.4 microM of genistein and 13.2 microM of daidzein from 100 microM of biochanin A and formononetin, after 26 days incubation . O-demethylase activity is cell-associated and was not detected in the extracellular fraction of bacterial culture . This is the first study in which conversion of biochanin A, and formononetin to more potent phytoestrogens by a bacterium has been shown.

Genes Dev, 2000 Oct 15, 14(20), 2664 - 75
The alpha subunit of E . coli RNA polymerase activates RNA binding by NusA; Mah TF et al.; The Escherichia coli NusA protein modulates pausing, termination, and antitermination by associating with the transcribing RNA polymerase core enzyme . NusA can be covalently cross-linked to nascent RNA within a transcription complex, but does not bind RNA on its own . We have found that deletion of the 79 carboxy-terminal amino acids of the 495-amino-acid NusA protein allows NusA to bind RNA in gel mobility shift assays . The carboxy-terminal domain (CTD) of the alpha subunit of RNA polymerase, as well as the bacteriophage lambda N gene antiterminator protein, bind to carboxy-terminal regions of NusA and enable full-length NusA to bind RNA . Binding of NusA to RNA in the presence of alpha or N involves an amino-terminal S1 homology region that is otherwise inactive in full-length NusA . The interaction of the alpha-CTD with full-length NusA stimulates termination . N may prevent termination by inducing NusA to interact with N utilization (nut) site RNA rather than RNA near the 3' end of the nascent transcript . Sequence analysis showed that the alpha-CTD contains a modified helix-hairpin-helix motif (HhH), which is also conserved in the carboxy-terminal regions of some eubacterial NusA proteins . These HhH motifs may mediate protein-protein interactions in NusA and the alpha-CTD.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 247 - 53
Characterization of the chemotaxis fli Y and che A genes in Bacillus cereus; Celandroni F et al.; This paper describes the first identification of chemotaxis genes in Bacillus cereus . We sequenced and studied the genomic organization and the expression of the cheA and fliY genes in two different B . cereus strains, ATCC 14579 and ATCC 10987 . While cheA encodes a highly conserved protein acting as the main regulator of the chemotactic response in flagellated eubacteria, fliY, which has been previously described only in B . subtilis, is one of the three genes encoding proteins of the flagellar switch complex . Although the sequences and relative position of cheA and fliY were found to be identical in the two B . cereus strains analyzed, the restriction fragment containing both genes was located differently on the physical maps of B . cereus ATCC 14579 and ATCC 10987 . Evidence is shown that the genomic organization and the expression of fliY and cheA in B . cereus differ significantly from that described for B . subtilis, which is considered a model microorganism for chemotaxis in gram-positive bacteria.

Annu Rev Cell Dev Biol, 2000, 16, 51 - 87
Evolutionarily related insertion pathways of bacterial, mitochondrial, and thylakoid membrane proteins; Dalbey RE et al.; The inner membranes of eubacteria and mitochondria, as well as the chloroplast thylakoid membrane, contain essential proteins that function in oxidative phosphorylation and electron transport processes or in photosynthesis . Because most of the organellar proteins are nuclear encoded, they are synthesized in the cytoplasm and subsequently imported into the organelle before they are inserted into the membrane . This review focuses on the pathways of protein insertion into the inner membrane of eubacteria and mitochondria and into the chloroplast thylakoid membrane . In many respects, insertion of proteins into the inner membrane of bacteria is a process similar to that used by proteins of the thylakoid membrane . In both of these systems a signal recognition particle (SRP) and a SecYE-translocase are involved, as in translocation into the endoplasmic reticulum . The pathway of proteins into the mitochondrial membranes appears to be different in that it involves no SecYE-like components . A conservative pathway, recently identified in mitochondria, involves the Oxa1 protein for the insertion of proteins from the matrix . The presence of Oxa1 homologues in eubacteria and chloroplasts suggests that this pathway is evolutionarily conserved.

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