J Microbiol, 2004 Dec, 42(4), 285 - 91 Monitoring of soil bacterial community and some inoculated bacteria after prescribed fire in microcosm; Song HG et al.; The soil bacterial community and some inoculated bacteria were monitored to assess the microbial responses to prescribed fire in their microcosm . An acridine orange direct count of the bacteria in the unburned control soil were maintained at a relatively stable level (2.0~2.7 x 10(9) cells/g(-1).soil) during the 180 day study period . The number of bacteria in the surface soil was decreased by fire, but was restored after 3 months . Inoculation of some bacteria increased the number of inoculated bacteria several times and these elevated levels lasted several months . The ratios of eubacteria detected by a fluorescent in situ hybridization (FISH) method to direct bacterial count were in the range of 60~80% during the study period, with the exception of some lower values at the beginning, but there were no definite differences between the burned and unburned soils or the inoculated and uninoculated soils . In the unburned control soil, the ratios of alpha-, beta- and gamma-subgroups of the proteobacteria, Cytophaga-Flavobacterium and other eubacteria groups to that of the entire eubacteria were 13.7, 31.7, 17.1, 16.8 and 20.8%, respectively, at time 0 . The overall change on the patterns of the ratios of the 5 subgroups of eubacteria in the uninoculated burned and inoculated soils were similar to those of the unburned control soil, with the exception of some minor variations during the initial period . The proportions of each group of eubacteria became similar in the different microcosms after 6 months, which may indicate the recovery of the original soil microbial community structure after fire or the inoculation of some bacteria . The populations of Azotobacter vinelandii, Bacillus megaterium and Pseudomonas fluorescens, which had been inoculated to enhance the microbial activities, and monitored by FISH method, showed similar changes in the microcosms, and maintained high levels for several months. J Microbiol, 2004 Dec, 42(4), 278 - 84 Monitoring of bacterial community in a coniferous forest soil after a wildfire; Kim OS et al.; Changes in the soil bacterial community of a coniferous forest were analyzed to assess microbial responses to wildfire . Soil samples were collected from three different depths in lightly and severely burned areas, as well as a nearby unburned control area . Direct bacterial counts ranged from 3.3-22.6 x 10(8) cells/(g.soil) . In surface soil, direct bacterial counts of unburned soil exhibited a great degree of fluctuation . Those in lightly burned soil changed less, but no significant variation was observed in the severely burned soil . The fluctuations of direct bacterial count were less in the middle and deep soil layers . The structure of the bacterial community was analyzed via the fluorescent in situ hybridization method . The number of bacteria detected with the eubacteria-targeted probe out of the direct bacterial count varied from 30.3 to 84.7%, and these ratios were generally higher in the burned soils than in the unburned control soils . In the surface unburned soil, the ratios of alpha-, beta- and gamma-proteobacteria, Cytophaga-Flavobacterium group, and other eubacteria groups to total eubacteria were 9.9, 10.6, 15.5, 9.0, and 55.0%, respectively, and these ratios were relatively stable . The ratios of alpha-, beta- and gamma-proteobacteria, and Cytophaga-Flavobacterium group to total eubacteria increased immediately after the wildfire, and the other eubacterial proportions decreased in the surface and middle layer soils . By way of contrast, the composition of the 5 groups of eubacteria in the subsurface soil exhibited no significant fluctuations during the entire period . The total bacterial population and bacterial community structure disturbed by wildfire soon began to recover, and original levels seemed to be restored 3 months after the wildfire. J Clin Periodontol, 2005 Jan, 32(1), 33 - 9 Differences in the subgingival microbiota of Swedish and USA subjects who were periodontally healthy or exhibited minimal periodontal disease; Haffajee AD et al.; Haffajee AD, Japlit M, Bogren A, Kent Jr RL, Goodson JM, Socransky SS: Differences in the subgingival microbiota of Swedish and USA subjects who were periodontally healthy or exhibited minimal periodontal disease . J Clin Periodontol 2005; 32: 33-39 . doi: 10.1111/j.1600-051X.2004.00624.x . (c) Blackwell Munksgaard, 2005 . Abstract Background: Previous studies have shown differences in the mean proportions of subgingival species in samples from periodontitis subjects in different countries, which may relate to differences in diet, genetics, disease susceptibility and manifestation . The purpose of the present investigation was to determine whether there were differences in the subgingival microbiotas of Swedish and American subjects who exhibited periodontal health or minimal periodontal disease . Method: One hundred and fifty eight periodontally healthy or minimally diseased subjects (N Sweden=79; USA=79) were recruited . Subjects were measured at baseline for plaque, gingivitis, BOP, suppuration, pocket depth and attachment level at 6 sites per tooth . Subgingival plaque samples taken from the mesial aspect of each tooth at baseline were individually analyzed, in one laboratory, for their content of 40 bacterial species using checkerboard DNA-DNA hybridization (total samples=4345) . % DNA probe counts comprised by each species was determined for each site and averaged across sites in each subject . Significance of differences in proportions of each species between countries was determined using ancova adjusting for age, mean pocket depth, gender and smoking status . p values were adjusted for multiple comparisons . Cluster analysis was performed to group subjects based on their subgingival microbial profiles using a chord coefficient and an average unweighted linkage sort . Results: On average, all species were detected in samples from subjects in both countries . After adjusting for multiple comparisons, 5 species were in significantly higher adjusted mean percentages in Swedish than American subjects: Actinomyces naeslundii genospecies 1 (9.7, 3.3); Streptococcus sanguis (2.5, 1.2); Eikenella corrodens (1.7, 1.0); Tannerella forsythensis (3.5, 2.3) and Prevotella melaninogenica (6.3, 1.8) . Leptotrichia buccalis was in significantly higher adjusted mean percentages in American (5.5) than Swedish subjects (3.0) . Cluster analysis grouped 121 subjects into 8 microbial profiles . Twenty four of the 40 test species examined differed significantly among cluster groups . Five clusters were dominated by American subjects and 2 clusters by Swedish subjects . Fifty eight of 79 (73%) of the Swedish subjects fell into 1 cluster group dominated by high proportions of A . naeslundii genospecies 1, Prevotella nigrescens, T . forsythensis and P . melaninogenica . Other clusters were characterized by high proportions of Actinomyces gerencseriae, Veillonella parvula, Capnocytophaga gingivalis, Prevotella intermedia, Eubacterium saburreum, L . buccalis and Neisseria mucosa . Conclusions: The microbial profiles of subgingival plaque samples from Swedish and American subjects who exhibited periodontal health or minimal disease differed . The heterogeneity in subgingival microbial profiles was more pronounced in the American subjects, possibly because of greater genetic and microbiologic diversity in the American subjects sampled. J Synchrotron Radiat, 2005 Jan 1, 12(Pt 1), 111 - 4 Epub 2004 Dec 23. Investigation of zinc-containing peptide deformylase from Leptospira interrogans by X-ray absorption near-edge spectroscopy; Li S; Peptide deformylase (PDF, EC 3.5.1.27) is essential for the normal growth of eubacterium but not for mammalians . Recently, PDF has been studied as a target for new antibiotics . Its activity is strongly dependent on the bound metal ion . The crystallographic studies did not show any significant structural difference upon various bound metal ions . In this paper, X-ray absorption spectroscopy was employed to determine the local structure around the zinc ion of PDF from Leptospira interrogans in dry powder . XANES (X-ray absorption near-edge structure) calculations were performed and the local geometry of the active center was reconstructed successfully . By comparing with the crystal structure of an enzyme-product complex, the results from calculations show that a water molecule has moved towards the zinc ion and lies in the distance range to coordinate with the zinc ion weakly. Poult Sci, 2004 Dec, 83(12), 1964 - 72 Effects of deoxynivalenol on general performance and electrophysiological properties of intestinal mucosa of broiler chickens; Awad WA et al.; A feeding trial was conducted to evaluate the effects of diets contaminated with deoxynivalenol (DON on the performance of broilers and on the electro-physiological parameters of the gut . The control group was fed the starter and finisher diets without addition of DON . Another group of broilers was fed the starter and finisher diets with 10 mg/kg DON, whereas another group was fed the DON-contaminated diets supplemented with a microbial feed additive (Eubacterium sp.) . The diets were provided ad libitum for 6 wk . DON had no effect (P > 0.05) on feed consumption, feed conversion, or body weight . The effect of DON on the electrophysiological parameters of the jejunum was studied in vitro using isolated gut mucosa in Ussing chambers . At the end of the feeding period, 7 birds from each group were killed, and the basal and glucose stimulated transmural potential difference (PD), short-circuit current (Isc), and electrical resistance (R) were measured in the isolated gut mucosa to characterize the electrical properties of the gut . The transmural PD did not differ (P > 0.05) among groups . The tissue resistance was greater (P < 0.05) in birds receiving DON and the microbial feed additive than in the controls and DON group . Addition of D-glucose on the luminal side of the isolated mucosa increased (P < 0.05) Isc in the control and DON-probiotic (Eubacterium sp.; PB) groups, whereas it decreased (P < 0.05) in the DON group indicating that the glucose-induced Isc was altered by DON . Addition of the eubacteria to the DON contaminated feed of the broilers led to electrophysiological properties in the gut that were comparable with those of the control group . It could be concluded that 10 mg/kg DON in the diet impaired the Na(+)-D-glucose cotransport in the jejunum of broilers . In the absence of clinical signs, and without impaired performance, DON appeared to alter the gut function of broilers . The addition of Eubacterium sp . may be useful in counteracting the toxic effects of DON on intestinal glucose transport. RNA, 2005 Jan, 11(1), 99 - 106 Analysis of 2'-phosphotransferase (Tpt1p) from Saccharomyces cerevisiae: evidence for a conserved two-step reaction mechanism; Steiger MA et al.; Tpt1p is an essential protein responsible for the 2'-phosphotransferase step of tRNA splicing in Saccharomyces cerevisiae, in which the splice junction 2'-phosphate of ligated tRNA is transferred to NAD to form mature tRNA and ADP-ribose 1''-2'' cyclic phosphate . We showed previously that Tpt1p is a member of a family of functional 2'-phosphotransferases found in eukaryotes, eubacteria, and archaea, that the Escherichia coli protein (KptA) is highly specific for 2'-phosphorylated RNAs despite the lack of obvious natural substrates, and that KptA acts on a trinucleotide substrate through an intermediate in which RNA is ADP-ribosylated at the 2'-phosphate . This mechanism is similar to a proposed mechanism of NAD-dependent histone deacetylases . We present evidence here that this mechanism is conserved in S . cerevisiae, and we identify residues important for the second step of the reaction, during which the intermediate is resolved into products . We examined 21 Tpt1 protein variants mutated in conserved residues or blocks of residues and show that one of them, Tpt1 K69A/R71S protein, accumulates large amounts of intermediate with trinucleotide substrate due to a very slow second step . This intermediate can be trapped on beads when formed with biotin-NAD . We also show that Tpt1 K69A/R71S protein forms an intermediate with the natural ligated tRNA substrate and demonstrate that, as expected, this mutation is lethal in yeast . The high degree of conservation of these residues suggests that the entire Tpt1p family is involved in a similar two-step chemical reaction. Acta Crystallogr D Biol Crystallogr, 2005 Jan, 61(Pt 1), 45 - 52 Epub 2004 Dec 17. The identification of isoprenoids that bind in the intersubunit cavity of Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase by complementary biophysical methods; Kemp LE et al.; The discovery of a distinct metabolic pathway, the non-mevalonate or 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for isoprenoid precursor biosynthesis, in eubacteria and apicomplexan parasites has revealed a new set of potential drug targets . The emphasis of research on this pathway has been on delineating the intermediates and the biochemical and structural characterization of component enzymes . Two new monoclinic crystal forms of recombinant Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate (MECP) synthase cocrystallized with (i) CMP and (ii) CMP and MECP show well defined electron density at the subunit interface suggestive of an isoprenoid-like ligand . (31)P NMR analysis of the recombinant protein sample indicates the presence of bound diphosphate species and electrospray mass spectrometry identifies a mixture of isopentenyl diphosphate (and/or dimethylallyl diphosphate), geranyl diphosphate and farnesyl diphosphate in an approximate ratio of 1:4:2 . The most prevalent species, geranyl diphosphate, was successfully modelled into the electron density, revealing the important protein-ligand interactions that stabilize binding of the isoprenoid . The observation that MECP synthase binds three metabolites that are produced by enzymes two, three and four stages downstream in isoprenoid biosynthesis suggests that feedback regulation of the non-mevalonate pathway is possible. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D317 - 20 BacMap: an interactive picture atlas of annotated bacterial genomes; Stothard P et al.; BacMap is an interactive visual database containing fully labeled, zoomable and searchable chromosome maps from more than 170 bacterial (archaebacterial and eubacterial) species . It uses a recently developed visualization tool (CGView) to generate high-resolution circular genome maps from sequence feature information . Each map includes an interface that allows the image to be expanded and rotated . In the default view, identified genes are drawn to scale and colored according to coding directions . When a region of interest is expanded, gene labels are displayed . Each label is hyperlinked to a custom 'gene card' which provides several fields of information concerning the corresponding DNA and protein sequences . Each genome map is searchable via a local BLAST search and a gene name/synonym search . BacMap is freely available at http://wishart.biology.ualberta.ca/BacMap/. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D308 - 10 The PEDANT genome database in 2005; Riley ML et al.; The PEDANT genome database contains pre-computed bioinformatics analyses of publicly available genomes . Its main mission is to provide robust automatic annotation of the vast majority of amino acid sequences, which have not been subjected to in-depth manual curation by human experts in high-quality protein sequence databases . By design PEDANT annotation is genome-oriented, making it possible to explore genomic context of gene products, and evaluate functional and structural content of genomes using a category-based query mechanism . At present, the PEDANT database contains exhaustive annotation of over 1,240,000 proteins from 270 eubacterial, 23 archeal and 41 eukaryotic genomes. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D112 - 5 NONCODE: an integrated knowledge database of non-coding RNAs; Liu C et al.; NONCODE is an integrated knowledge database dedicated to non-coding RNAs (ncRNAs), that is to say, RNAs that function without being translated into proteins . All ncRNAs in NONCODE were filtered automatically from literature and GenBank, and were later manually curated . The distinctive features of NONCODE are as follows: (i) the ncRNAs in NONCODE include almost all the types of ncRNAs, except transfer RNAs and ribosomal RNAs . (ii) All ncRNA sequences and their related information (e.g . function, cellular role, cellular location, chromosomal information, etc.) in NONCODE have been confirmed manually by consulting relevant literature: more than 80% of the entries are based on experimental data . (iii) Based on the cellular process and function, which a given ncRNA is involved in, we introduced a novel classification system, labeled process function class, to integrate existing classification systems . (iv) In addition, some 1100 ncRNAs have been grouped into nine other classes according to whether they are specific to gender or tissue or associated with tumors and diseases, etc . (v) NONCODE provides a user-friendly interface, a visualization platform and a convenient search option, allowing efficient recovery of sequence, regulatory elements in the flanking sequences, secondary structure, related publications and other information . The first release of NONCODE (v1.0) contains 5339 non-redundant sequences from 861 organisms, including eukaryotes, eubacteria, archaebacteria, virus and viroids . Access is free for all users through a web interface at http://noncode.bioinfo.org.cn. J Inorg Biochem, 2005 Jan, 99(1), 97 - 109 Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins; Milani M et al.; Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily . TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity . The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions . Despite their 'minimal' polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions . Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O(2), CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters . Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed. J Med Microbiol, 2004 Dec, 53(Pt 12), 1247 - 53 Anaerobic, non-sporulating, Gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing; Lau SK et al.; Owing to the difficulties in identifying anaerobic, non-sporulating, Gram-positive bacilli in clinical microbiology laboratories, the epidemiology and clinical spectrum of disease of many of these bacteria have been poorly understood . The application of 16S rRNA gene sequencing in characterizing bacteraemia due to anaerobic, non-sporulating Gram-positive bacilli during a 4-year period is described . The first case of Olsenella uli bacteraemia, in a patient with acute cholangitis, is also reported . Among 165 blood culture isolates of anaerobic, Gram-positive bacilli, 75 were identified as Propionibacterium acnes by phenotypic tests and 21 as members of other anaerobic, non-sporulating Gram-positive bacilli by 16S rRNA gene sequencing . Of these 96 isolates, 16 (17 %) were associated with cases of clinically significant bacteraemia, among which 10 (63 %) were caused by Eggerthella, four (25 %) by Lactobacillus and one (6 %) by each of Eubacterium tenue and O . uli . Five of the 10 Eggerthella isolates were Eggerthella lenta, whereas the other five belonged to two novel Eggerthella species, with Eggerthella hongkongensis being almost as prevalent as Eggerthella lenta . Underlying disease in the gastrointestinal tract, isolation of Eggerthella and Lactobacillus, and monomicrobial bacteraemia were associated with clinically significant bacteraemia, whereas isolation of P . acnes and polymicrobial bacteraemia were associated with pseudobacteraemia . Most patients with clinically significant bacteraemia had underlying diseases, with diseases in the gastrointestinal tract being most common . The overall mortality rate was 31 % . Immunocompromised patients with clinically significant bacteraemia due to anaerobic, non-sporulating, Gram-positive bacilli other than P . acnes should be treated with appropriate antibiotics . The unexpected frequency of isolation of Eggerthella from blood cultures and its association with clinically significant disease suggest that this genus is probably of high pathogenicity . Further studies to look for specific virulence factors are warranted. Plant Cell Physiol, 2004 Nov, 45(11), 1633 - 9 Identification of chloroplast signal recognition particle RNA genes; Rosenblad MA et al.; The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts . In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome . The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43 . An RNA component has not been identified and does not seem to be required for the post-translational cpSRP . The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified . Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible . We have analysed chloroplast genomes from 27 organisms . In higher plant chloroplasts, no SRP RNA genes were identified . However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene . These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP. J Biol Chem . 2004 Nov 29; {Epub ahead of print} Mitochondrial trans-2-enoyl-CoA reductase of wax ester fermentation from euglena gracilis defines a new family of enzymes involved in lipid synthesis; Hoffmeister M et al.; Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation . An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyses reduction of enoyl-CoA to acyl-CoA . Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor . The active enzyme is a monomer with an Mr of 44 kDa . The amino acid sequence of tryptic peptides determined by electrospray ionisation mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in E . coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena . Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions . Using NADH, the recombinant enzyme accepted crotonyl-CoA (k(m) = 68 microM) and trans-2-hexenoyl-CoA (k(m) = 91 microM) . In the crotonyl-CoA dependent reaction, both NADH (k(m) = 109 microM) or NADPH (k(m) = 119 microM) were accepted, with 2-3 fold higher specific activities for NADH relative to NADPH . Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis . Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H dependent acyl-CoA synthesis from enoyl-CoA, to one member of this gene family of previously unknown function. Annu Rev Genet, 2004, 38, 477 - 524 Mitochondria of protists; Gray MW et al.; Over the past several decades, our knowledge of the origin and evolution of mitochondria has been greatly advanced by determination of complete mitochondrial genome sequences . Among the most informative mitochondrial genomes have been those of protists (primarily unicellular eukaryotes), some of which harbor the most gene-rich and most eubacteria-like mitochondrial DNAs (mtDNAs) known . Comparison of mtDNA sequence data has provided insights into the radically diverse trends in mitochondrial genome evolution exhibited by different phylogenetically coherent groupings of eukaryotes, and has allowed us to pinpoint specific protist relatives of the multicellular eukaryotic lineages (animals, plants, and fungi) . This comparative genomics approach has also revealed unique and fascinating aspects of mitochondrial gene expression, highlighting the mitochondrion as an evolutionary playground par excellence. Lancet Infect Dis, 2004 Dec, 4(12), 751 - 60 New developments in the diagnosis of bloodstream infections; Peters RP et al.; New techniques have emerged for the detection of bacteria in blood, because the blood culture as gold standard is slow and insufficiently sensitive when the patient has previously received antibiotics or in the presence of fastidious organisms . DNA-based techniques, hybridisation probes, and PCR-based detection or protein-based detection by mass spectroscopy are aimed at rapid identification of bacteria and provide results within 2 h after the first signal of growth in conventional blood cultures . Also, detection of microorganisms directly in blood by pathogen-specific or broad-range PCR assays (eubacterial or panfungal) shows promising results . Interpretation is complex, however, because of detection of DNA rather than living pathogens, the risk of interfering contamination, the presence of background DNA in blood, and the lack of a gold standard . As these techniques are emerging, clinical value and cost-effectiveness have to be assessed . Nevertheless, molecular assays are expected eventually to replace the current conventional microbiological techniques for detection of bloodstream infections. Nat Prod Rep, 2004 Dec, 21(6), 695 - 721 Epub 2004 Dec. Biosynthesis of pantothenate; Webb ME et al.; Pantothenate is an essential metabolite for all biological systems, however, the biosynthetic pathway is limited to plants, eubacteria and archaea . This suggests that the pathway is a strong candidate for the discovery of novel antibiotic and herbicidal compounds . The enzymology of this short pathway in both bacteria and plants is discussed in detail . In addition a short survey of studies of the whole pathway, and a discussion of both the metabolism and the transport of pantothenate are included. Biosci Biotechnol Biochem, 2004 Nov, 68(11), 2215 - 23 Roles of chloroplast RNA polymerase sigma factors in chloroplast development and stress response in higher plants; Kanamaru K et al.; Chloroplast transcription in higher plants is performed by two types of RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP) . PEP is a eubacteria-type multisubunit enzyme whose catalytic core subunits are encoded by the chloroplast genome, whereas NEP is the nuclear encoded T7 phage-type single subunit enzyme . PEP is critical for the biogenesis and maintenance of chloroplasts, and is finely tuned by the nuclear encoded sigma subunits . Of the six Arabidopsis sigma subunits, SIG2 is involved in the transcription of several chloroplast tRNA genes, including trnE encoding tRNA-Glu . SIG2 possibly couples translation and pigment synthesis in chloroplasts . On the other hand, SIG5 is induced by various stresses and contributes to repair of damaged photosystem II (PSII) through transcription of the psbD and psbC genes . Thus target genes and the physiological role of each sigma subunit are becoming clearer. Mol Biol (Mosk), 2004 Sep-Oct, 38(5), 823 - 33 {Antimutagenic role of autonomous 3'-->5'-exonucleases}; Type III flagellar protein export and flagellar assembly; Department of Molecular Biophysics and Biochemistry, Yale University, 0734, 266 Whitney Avenue, P.O . Box 208114, New Haven, CT 06520-8114, USABacterial flagella, unlike eukaryotic flagella, are largely external to the cell and therefore many of their subunits have to be exported . Export is ATP-driven . In Salmonella, the bacterium on which this chapter largely focuses, the apparatus responsible for flagellar protein export consists of six membrane components, three soluble components and several substrate-specific chaperones . Other flagellated eubacteria have similar systems . The membrane components of the export apparatus are housed within the flagellar basal body and deliver their substrates into a channel or lumen in the nascent structure from which point they diffuse to the far end and assemble . Both on the basis of sequence similarities of several components and structural similarities, the flagellar protein export systems clearly belong to the type III superfamily, whose other members are responsible for secretion of virulence factors by many species of pathogenic bacteria. J Mol Biol, 2004 Dec 3, 344(4), 1123 - 33 Structural stability of oligomeric chaperonin 10: the role of two beta-strands at the N and C termini in structural stabilization; Sakane I et al.; Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family . GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure . From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible . In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES . From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e . two-stage unfolding reactions that include formation of a partially folded monomer . Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions . We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein . From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein. J Chem Phys, 2004 Nov 15, 121(19), 9648 - 54 Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers; Shinoda K et al.; The lipid membranes found in archaea have high bilayer stability and low permeability . The molecular structure of their constituent lipids is characterized by ether-linked, branched hydrophobic chains, whereas the conventional lipids obtained from eukaryotic or eubacterial sources have ester linked straight chains . In order to elucidate the influence of the ether linkage, instead of an ester one, on the physical properties of the lipid bilayers, we have carried out comparative 10 ns molecular dynamics simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayers in water, respectively . We analyze bilayer structures, hydration of the lipids, membrane dipole potentials, and free energy profiles of water and oxygen across the bilayers . We observe that the membrane dipole potential for the ether-DPhPC bilayer, which arises mainly from the ether linkage, is about half of that of the ester-DPhPC . The calculated free energy barrier for a water molecule in the ether-DPhPC bilayer system is slightly higher than that in the ester-DPhPC counterpart, which is in accord with experimental data. BMC Evol Biol . 2004 Nov 9;4(1):44. A genomic timescale of prokaryote evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land; Battistuzzi FU et al.; BACKGROUND: The timescale of prokaryote evolution has been difficult to reconstruct because of a limited fossil record and complexities associated with molecular clocks and deep divergences . However, the relatively large number of genome sequences currently available has provided a better opportunity to control for potential biases such as horizontal gene transfer and rate differences among lineages . We assembled a data set of sequences from 32 proteins (approximately 7600 amino acids) common to 72 species and estimated phylogenetic relationships and divergence times with a local clock method . RESULTS: Our phylogenetic results support most of the currently recognized higher-level groupings of prokaryotes . Of particular interest is a well-supported group of three major lineages of eubacteria (Actinobacteria, Deinococcus, and Cyanobacteria) that we call Terrabacteria and associate with an early colonization of land . Divergence time estimates for the major groups of eubacteria are between 2.5-3.2 billion years ago (Ga) while those for archaebacteria are mostly between 3.1-4.1 Ga . The time estimates suggest a Hadean origin of life (prior to 4.1 Ga), an early origin of methanogenesis (3.8-4.1 Ga), an origin of anaerobic methanotrophy after 3.1 Ga, an origin of phototrophy prior to 3.2 Ga, an early colonization of land 2.8-3.1 Ga, and an origin of aerobic methanotrophy 2.5-2.8 Ga . CONCLUSIONS: Our early time estimates for methanogenesis support the consideration of methane, in addition to carbon dioxide, as a greenhouse gas responsible for the early warming of the Earths' surface . Our divergence times for the origin of anaerobic methanotrophy are compatible with highly depleted carbon isotopic values found in rocks dated 2.8-2.6 Ga . An early origin of phototrophy is consistent with the earliest bacterial mats and structures identified as stromatolites, but a 2.6 Ga origin of cyanobacteria suggests that those Archean structures, if biologically produced, were made by anoxygenic photosynthesizers . The resistance to desiccation of Terrabacteria and their elaboration of photoprotective compounds suggests that the common ancestor of this group inhabited land . If true, then oxygenic photosynthesis may owe its origin to terrestrial adaptations. Genome Biol . 2004;5(11):248 . Epub 2004 Nov 01. The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases; Friesen JA et al.; The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids . The enzyme is found in eukaryotes and prokaryotes; and phylogenetic analysis has revealed two classes of HMG-CoA reductase, the Class I enzymes of eukaryotes and some archaea and the Class II enzymes of eubacteria and certain other archaea . Three-dimensional structures of the catalytic domain of HMG-CoA reductases from humans and from the bacterium Pseudomonas mevalonii, in conjunction with site-directed mutagenesis studies, have revealed details of the mechanism of catalysis . The reaction catalyzed by human HMG-CoA reductase is a target for anti-hypercholesterolemic drugs (statins), which are intended to lower cholesterol levels in serum . Eukaryotic forms of the enzyme are anchored to the endoplasmic reticulum, whereas the prokaryotic enzymes are soluble . Probably because of its critical role in cellular cholesterol homeostasis, mammalian HMG-CoA reductase is extensively regulated at the transcriptional, translational, and post-translational levels. Genome Biol . 2004;5(11):R88 . Epub 2004 Oct 19. Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum; Huang J et al.; BACKGROUND: The apicomplexan parasite Cryptosporidium parvum is an emerging pathogen capable of causing illness in humans and other animals and death in immunocompromised individuals . No effective treatment is available and the genome sequence has recently been completed . This parasite differs from other apicomplexans in its lack of a plastid organelle, the apicoplast . Gene transfer, either intracellular from an endosymbiont/donor organelle or horizontal from another organism, can provide evidence of a previous endosymbiotic relationship and/or alter the genetic repertoire of the host organism . Given the importance of gene transfers in eukaryotic evolution and the potential implications for chemotherapy, it is important to identify the complement of transferred genes in Cryptosporidium . RESULTS: We have identified 31 genes of likely plastid/endosymbiont (n = 7) or prokaryotic (n = 24) origin using a phylogenomic approach . The findings support the hypothesis that Cryptosporidium evolved from a plastid-containing lineage and subsequently lost its apicoplast during evolution . Expression analyses of candidate genes of algal and eubacterial origin show that these genes are expressed and developmentally regulated during the life cycle of C . parvum . CONCLUSIONS: Cryptosporidium is the recipient of a large number of transferred genes, many of which are not shared by other apicomplexan parasites . Genes transferred from distant phylogenetic sources, such as eubacteria, may be potential targets for therapeutic drugs owing to their phylogenetic distance or the lack of homologs in the host . The successful integration and expression of the transferred genes in this genome has changed the genetic and metabolic repertoire of the parasite. Proc Natl Acad Sci U S A, 2004 Nov 16, 101(46), 16262 - 7 Epub 2004 Nov 16. RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation; Kouzminova EA et al.; RecA- and RecBC-catalyzed repair in eubacteria assembles chromosomes fragmented by double-strand breaks . We propose that recA mutants, being unable to repair fragmented chromosomes, depend on various strategies designed to avoid chromosomal fragmentation . To identify chromosomal fragmentation-avoidance strategies, we screened for Escherichia coli mutants synthetically inhibited in combination with recA inactivation by identifying clones unable to lose a plasmid carrying the recA(+) gene . Using this screen, we have isolated several RecA-dependent mutants and assigned them to three distinct areas of metabolism . The tdk and rdgB mutants affect synthesis of DNA precursors . The fur, ubiE, and ubiH mutants are likely to have increased levels of reactive oxygen species . The seqA, topA mutants and an insertion in smtA perturbing the downstream mukFEB genes affect nucleoid administration . All isolated mutants show varying degree of SOS induction, indicating elevated levels of chromosomal lesions . As predicted, mutants in rdgB, seqA, smtA, topA, and fur show increased levels of chromosomal fragmentation in recBC mutant conditions . Future characterization of these RecA-dependent mutants will define mechanisms of chromosomal fragmentation avoidance. Structure (Camb), 2004 Nov, 12(11), 1977 - 88 Structural basis of CTP-dependent peptide bond formation in coenzyme A biosynthesis catalyzed by Escherichia coli PPC synthetase; Stanitzek S et al.; Phosphopantothenoylcysteine (PPC) synthetase forms a peptide bond between 4'-phosphopantothenate and cysteine in coenzyme A biosynthesis . PPC synthetases fall into two classes: eukaryotic, ATP-dependent and eubacterial, CTP-dependent enzymes . We describe the first crystal structure of E . coli PPC synthetase as a prototype of bacterial, CTP-dependent PPC synthetases . Structures of the apo-form and the synthetase complexed with CTP, the activated acyl-intermediate, 4'-phosphopantothenoyl-CMP, and with the reaction product CMP provide snapshots along the reaction pathway and detailed insight into substrate binding and the reaction mechanism of peptide bond formation . Binding of the phosphopantothenate moiety of the acyl-intermediate in a cleft at the C-terminal end of the central beta sheet of the dinucleotide binding fold is accomplished by an otherwise flexible flap . A second disordered loop may control access of cysteine to the active site . The conservation of functionalities involved in substrate binding and catalysis provides insight into similarities and differences of prokaryotic and eukaryotic PPC synthetases. J Bacteriol, 2004 Nov, 186(22), 7653 - 8 The CorA Mg2+ transporter does not transport Fe2+; Papp KM et al.; corA encodes the constitutively expressed primary Mg2+ uptake system of most eubacteria and many archaea . Recently, a mutation in corA was reported to make Salmonella enterica serovar Typhimurium markedly resistant to Fe2+-mediated toxicity . Mechanistically, this was hypothesized to be from an ability of CorA to mediate the influx of Fe2+ . Consequently, we directly examined Fe2+ transport and toxicity in wild-type versus corA cells . As determined by direct transport assay, CorA cannot transport Fe2+ and Fe2+ does not potently inhibit CorA transport of 63Ni2+ . Mg2+ can, relatively weakly, inhibit Fe2+ uptake, but inhibition is not dependent on the presence of a functional corA allele . Although excess Fe2+ was slightly toxic to S . enterica serovar Typhimurium, we were unable to elicit a significant differential sensitivity in a wild-type versus a corA strain . We conclude that CorA does not transport Fe2+ and that the relationship, if any, between iron toxicity and corA is indirect. Mol Membr Biol, 2004 Sep-Oct, 21(5), 323 - 36 Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family; Xie H et al.; NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man . To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E . coli and in Xenopus laevis oocytes . In E . coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force . Competition experiments in E . coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides . Importantly, using high-level expression in E . coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ({1'-(13)C}uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions . Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved . Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices . These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters. Nature, 2004 Oct 28, 431(7012), 1103 - 7 Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex; Dyall SD et al.; Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism . Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an {Fe}-hydrogenase which produces hydrogen . ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria . PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria . Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor . The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear . Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not . Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones . Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues . These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont. J Biol Chem . 2004 Oct 27; {Epub ahead of print} The glutamine residue of the conserved GGQ motif in S . cerevisiae release factor eRF1 is methylated by the product of the YDR140w gene; Heurgue-Hamard V et al.; Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families . They share one sequence motif : a GGQ tripeptide that is vital to release factor activity in both kingdoms . In bacteria, the Gln residue of the motif in RF1 and RF2 is modified to N5-Me-Gln by the AdoMet-dependent methyltransferase PrmC, and the absence of Gln methylation decreases several-fold the release activity of E . coli RF2 in vitro . We show here that the same modification is made to the GGQ motif of S . cerevisiae release factor eRF1, the first time that N5-Me-Gln has been found outside the bacterial kingdom . The product of the YDR140w gene is required for the methylation of eRF1 in vivo, and for optimal yeast cell growth . YDR140w protein has significant homology to PrmC, but lacks the N-terminal domain thought to be involved in recognition of the bacterial release factors . Overproduced in S . cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using AdoMet as methyl donor, provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1*eRF3*GTP. Proc R Soc Lond B Biol Sci, 2004 Aug 7, 271 Suppl 5, S273 - 6 Four kingdoms on glacier ice: convergent energetic processes boost energy levels as temperatures fall; Napolitano MJ et al.; A diverse group of glacially obligate organisms coexist on temperate glaciers between Washington State and Alaska . A fundamental challenge for these and other cold-adapted species is the necessity to maintain an energy flux capable of sustaining life at low physiological temperatures . We show here that ice-adapted psychrophiles from four kingdoms (Animalia, Eubacteria, Fungi, Protista) respond to temperature fluctuations in a similar manner; namely, ATP levels and the total adenylate pool increase as temperatures fall (within their viable temperature limits, respectively), yet growth rate increases with temperature . By contrast, mesophilic representatives of each kingdom respond in an opposite manner (i.e . adenylates increase with temperature) . These observations suggest that elevated adenylate levels in psychrophiles may offset inherent reductions in molecular diffusion at low physiological temperatures. Res Microbiol, 2004 Nov, 155(9), 710 - 9 Comparative genomics and functional roles of the ATP-dependent proteases Lon and Clp during cytosolic protein degradation; Chandu D et al.; The general pathway involving adenosine triphosphate (ATP)-dependent proteases and ATP-independent peptidases during cytosolic protein degradation is conserved, with differences in the enzymes utilized, in organisms from different kingdoms . Lon and caseinolytic protease (Clp) are key enzymes responsible for the ATP-dependent degradation of cytosolic proteins in Escherichia coli . Orthologs of E . coli Lon and Clp were searched for, followed by multiple sequence alignment of active site residues, in genomes from seventeen organisms, including representatives from eubacteria, archaea, and eukaryotes . Lon orthologs, unlike ClpP and ClpQ, are present in most organisms studied . The roles of these proteases as essential enzymes and in the virulence of some organisms are discussed. RNA, 2004 Nov, 10(11), 1702 - 3 Novel application of sRNA: stimulation of ribosomal frameshifting; Olsthoorn RC et al.; Small RNAs play an important role in regulation of gene expression in eukaryotic and eubacterial cells by modulating gene expression both at the level of transcription and translation . Here, we show that short complementary RNAs can also affect gene expression by stimulating ribosomal frameshifting in vitro . This finding has important implications for understanding the process of ribosomal frameshifting and for the potential application of small RNAs in the treatment of diseases that are due to frameshift mutations. Trends Biotechnol, 2004 Nov, 22(11), 570 - 6 Ribosomal antibiotics: structural basis for resistance, synergism and selectivity; Auerbach T et al.; Various antibiotics bind to ribosomes at functionally relevant locations such as the peptidyl-transferase center (PTC) and the exit tunnel for nascent proteins . High-resolution structures of antibiotics bound to ribosomal particles from a eubacterium that is similar to pathogens and an archaeon that shares properties with eukaryotes are deciphering subtle differences in these highly conserved locations that lead to drug selectivity and thereby facilitate clinical usage . These structures also show that members of antibiotic families with structural differences might bind to specific ribosomal pockets in different modes dominated by their chemical properties . Similarly, the chemical properties of drugs might govern variations in the nature of seemingly identical mechanisms of drug resistance . The observed variability in binding modes justifies expectations for structural design of improved antibiotic properties. Oral Microbiol Immunol, 2004 Dec, 19(6), 379 - 85 Detection frequency of periodontitis-associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects; Mayanagi G et al.; The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects . Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes . Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (> or =50%) in subgingival plaque in both periodontitis and healthy subjects . Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002< or =P<0.05) . Two species (Mogibacterium timidum and Porphyromonas gingivalis) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects (P<0.002), with P . gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis . There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque. J Bacteriol, 2004 Nov, 186(21), 7123 - 33 The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis; Minion FC et al.; We present the complete genome sequence of Mycoplasma hyopneumoniae, an important member of the porcine respiratory disease complex . The genome is composed of 892,758 bp and has an average G+C content of 28.6 mol% . There are 692 predicted protein coding sequences, the average protein size is 388 amino acids, and the mean coding density is 91% . Functions have been assigned to 304 (44%) of the predicted protein coding sequences, while 261 (38%) of the proteins are conserved hypothetical proteins and 127 (18%) are unique hypothetical proteins . There is a single 16S-23S rRNA operon, and there are 30 tRNA coding sequences . The cilium adhesin gene has six paralogs in the genome, only one of which contains the cilium binding site . The companion gene, P102, also has six paralogs . Gene families constitute 26.3% of the total coding sequences, and the largest family is the 34-member ABC transporter family . Protein secretion occurs through a truncated pathway consisting of SecA, SecY, SecD, PrsA, DnaK, Tig, and LepA . Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent . The DnaK-DnaJ-GrpR complex is intact, providing the only control over protein folding . There are several proteases that might serve as virulence factors, and there are 53 coding sequences with prokaryotic lipoprotein lipid attachment sites . Unlike other mycoplasmas, M . hyopneumoniae contains few genes with tandem repeat sequences that could be involved in phase switching or antigenic variation . Thus, it is not clear how M . hyopneumoniae evades the immune response and establishes a chronic infection. Bioresour Technol, 2005 Feb, 96(3), 383 - 7 Fishery by-product as a nutrient source for bacteria and archaea growth media; Martone CB et al.; A highly soluble fish protein hydrolysates (FPH) with an 80% protein (peptide size between 1.5 and 20 kDa) and a low free amino acid content was obtained from hake (Merluccius hubssi) filleting waste {Lat . Am . Appl . Res . 30 (2000) 241} . Assays with Halobacterium salinarum, Escherichia coli, Bacillus subtilis and Staphylococcus epidermidis were performed in order to test that FPH as nutrient source for archaea and eubacteria culture media . Cell growth was evaluated by plate count, and by monitoring turbidity and nucleic acids content in liquid cultures . Neither cell growth nor generation times resulting from control and FPH cultures exhibited statistically significant differences at alpha: 0.05 suggesting that FPH can be used as an alternative substrate for microorganism cultural purposes. J Biomol Struct Dyn, 2004 Dec, 22(3), 315 - 29 Genomic choice of codons in 16 microbial species; Mougel F et al.; We study the codon usage over whole set of ORFs of 16 unicellular microbial species: eight archaebacteria, seven eubacteria, and one eukarya . We first try to define, for each species, the neutral expected codon usage to better approach subsequently the influence of selection . Overlapping triplets counted from the complete DNA genomic sequence and mean amino acid composition of ORFs allow us to build satisfying expected codon usage for each species . Within species deviation from this neutral model is then studied through Correspondence Analysis and characterization with bias index, N(C)' (effective number of codons reported to neutral model) . Our results are compared to previously published ones for three species and let appear good agreement in spite of very different methods . We thus propose set of codons probably preferred by selection for nine other species . In the four last species, no clear preference can be evidenced . Finally, we characterize variation of codon usage over functional categories . We propose that the high degree of bias of proteins involved in translation, ribosomal structure and biogenesis has a positive influence on overexpression of the corresponding genes under optimum growth conditions and is a negative regulator of the same genes when amino acids become limited resources. J Clin Microbiol, 2004 Oct, 42(10), 4759 - 64 Two novel real-time reverse transcriptase PCR assays for rapid detection of bacterial contamination in platelet concentrates; Dreier J et al.; The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion . In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays . Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets . Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed . During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted . Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA . The sensitivity of the assays was greatly influenced by the enzyme system which was used . With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml . With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml . With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E . coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found . Furthermore, the use of mRNA as an indicator of viability was demonstrated . Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services. Biotechnol Bioeng, 2004 Dec 5, 88(5), 575 - 84 Robust control of initiation of prokaryotic chromosome replication: essential considerations for a minimal cell; Browning ST et al.; A genomically and chemically detailed mathematical model of a "minimal cell" would be useful to understand better the "design logic" of cellular regulation . A "minimal cell" will be a prokaryote with the minimum number of genes necessary for growth and replication in an ideal environment (i.e., preformed precursors, constant temperature, etc.) . The Cornell single-cell model of Escherichia coli serves as the basic framework upon which a minimal cell model can be constructed . A critical issue for any cell model is to describe a mechanism for control of initiation of chromosome replication . There is strong evidence that the essence of chromosome replication control is highly conserved in eubacteria and even extends to the archae . A generalized mechanism is possible based on binding of the protein DnaA-ATP to the origin of replication (oriC) as a primary control . Other features, such as regulatory inactivation of DnaA (RIDA) by conversion of DnaA-ATP to DnaA-ADP and titration of DnaA by binding to other DnaA boxes on the chromosome, have emerged as critical elements in obtaining a functional system to control initiation of chromosome synthesis . We describe a biologically realistic model of chromosome replication initiation control embedded in a complete whole-cell model that explicitly links the external environment to the mechanism of replication control . The base model is deterministic and then modified to include stochastic variation in the components for replication control . The stochastic model allows evaluation of the model's robustness, employing a low standard deviation of interinitiation time as a measure of robustness . Four factors were examined: DnaA synthesis rate; DnaA-ATP binding sites at oriC; the binding rate of DnaA-ATP to the nonfunctional DnaA boxes; and the effect of changing the number of nonfunctional binding sites . The observed DnaA synthesis rate (2000 molecules/cell) and the number of DnaA binding sites per origin (30) are close to the values predicted by the model to provide good control (low variance of interinitiation time), with a reasonable expenditure of cell resources . At relatively high binding rates for DnaA-ATP to the DnaA boxes (10(16) M(-1) s(-1)), increasing the number of DnaA binding sites to about 300, improved control (but little further improvement was seen by extension to 1000 boxes); however, at a low binding rate (10(10) M(-1) s(-1)), an increase in DnaA boxes had an adverse effect on control . The combination of all four factors is probably necessary to obtain a robust control system . Although this mechanism of replication initiation control is highly conserved, it is not clear if simpler control in a minimal cell might exist based on experimental observations with Mycoplasma . This issue is discussed in this investigation. Microbiology, 2004 Oct, 150(Pt 10), 3393 - 403 Amino acid residues involved in cold adaptation of isocitrate lyase from a psychrophilic bacterium, Colwellia maris; Watanabe S et al.; To investigate the mechanism of cold adaptation of isocitrate lyase (ICL; EC 4.1.3.1) from the psychrophilic bacterium Colwellia maris, Gln207 and Gln217 of this enzyme were substituted by His and Lys, respectively, by site-directed mutagenesis . His184 and Lys194 of ICL from Escherichia coli, corresponding to the two Gln residues of C . maris ICL, are highly conserved in the ICLs of many organisms and are known to be essential for catalytic function . The mutated ICLs (Cm-Q207H and Cm-Q217K, respectively) and wild-type enzymes of C . maris and E . coli (Cm-WT and Ec-WT) with His-tagged peptides were overexpressed in E . coli cells and purified to homogeneity . Thermolabile Cm-WT and mutated ICLs were susceptible to digestion with trypsin, while relatively thermostable Ec-WT was resistant to trypsin digestion, suggesting that the thermostability and resistance to tryptic digestion of the ICLs are related . Cm-Q207H and Cm-Q217K showed specific activities similar to Cm-WT at temperatures between 30 degrees C and 40 degrees C, but their activities between 10 degrees C and 25 degrees C were decreased, indicating that the two Gln residues of the C . maris ICL play important roles in its cold adaptation . Phylogenetic analysis of ICLs from various organisms revealed that the C . maris ICL can be categorized in a novel group, subfamily 3, together with several eubacterial ICLs. Mol Cell, 2004 Oct 8, 16(1), 11 - 21 Conserved translational frameshift in dsDNA bacteriophage tail assembly genes; Xu J et al.; A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities . In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly . We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages . Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu . Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein . Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry. Appl Environ Microbiol, 2004 Oct, 70(10), 6131 - 7 Cloning and expression of a phloretin hydrolase gene from Eubacterium ramulus and characterization of the recombinant enzyme; Schoefer L et al.; Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus . The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity . The insert of a clone conferring phloretin hydrolase activity was sequenced . Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure . The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens . The phloretin hydrolase was heterologously expressed in Escherichia coli and purified . The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration . The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer . The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid . The optimal temperature and pH of the catalyzed reaction mixture were 37 degrees C and 7.0, respectively . The K(m) for phloretin was 13 +/- 3 microM and the k(cat) was 10 +/- 2 s(-1) . The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one . The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl(2) to 3, 20, 35, and 85%, respectively . Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively. J Bacteriol, 2004 Oct, 186(20), 6915 - 27 Crystal structures of Escherichia coli ATP-dependent glucokinase and its complex with glucose; Lunin VV et al.; Intracellular glucose in Escherichia coli cells imported by phosphoenolpyruvate-dependent phosphotransferase system-independent uptake is phosphorylated by glucokinase by using ATP to yield glucose-6-phosphate . Glucokinases (EC 2.7.1.2) are functionally distinct from hexokinases (EC 2.7.1.1) with respect to their narrow specificity for glucose as a substrate . While structural information is available for ADP-dependent glucokinases from Archaea, no structural information exists for the large sequence family of eubacterial ATP-dependent glucokinases . Here we report the first structure determination of a microbial ATP-dependent glucokinase, that from E . coli O157:H7 . The crystal structure of E . coli glucokinase has been determined to a 2.3-A resolution (apo form) and refined to final Rwork/Rfree factors of 0.200/0.271 and to 2.2-A resolution (glucose complex) with final Rwork/Rfree factors of 0.193/0.265 . E . coli GlK is a homodimer of 321 amino acid residues . Each monomer folds into two domains, a small alpha/beta domain (residues 2 to 110 and 301 to 321) and a larger alpha+beta domain (residues 111 to 300) . The active site is situated in a deep cleft between the two domains . E . coli GlK is structurally similar to Saccharomyces cerevisiae hexokinase and human brain hexokinase I but is distinct from the ADP-dependent GlKs . Bound glucose forms hydrogen bonds with the residues Asn99, Asp100, Glu157, His160, and Glu187, all of which, except His160, are structurally conserved in human hexokinase 1 . Glucose binding results in a closure of the small domains, with a maximal Calpha shift of approximately 10 A . A catalytic mechanism is proposed that is consistent with Asp100 functioning as the general base, abstracting a proton from the O6 hydroxyl of glucose, followed by nucleophilic attack at the gamma-phosphoryl group of ATP, yielding glucose-6-phosphate as the product. Science, 2004 Nov 19, 306(5700), 1390 - 3 Epub 2004 Nov 19. Anabaena sensory rhodopsin: a photochromic color sensor at 2.0 A; Vogeley L et al.; Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms . Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported . We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions . Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein . The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis . Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer. Trends Biochem Sci, 2004 Oct, 29(10), 519 - 22 Aminoacylation of the anticodon stem by a tRNA-synthetase paralog: relic of an ancient code? Grosjean H, de Crecy-Lagard V, Bjork GR. The activation and charging of amino acids onto the acceptor stems of their cognate tRNAs are the housekeeping functions of aminoacyl-tRNA synthetases . The availability of whole genome sequences has revealed the existence of synthetase-like proteins that have other functions linked to different aspects of cell metabolism and physiology . In eubacteria, a paralog of glutamyl-tRNA synthetase, which lacks the tRNA-binding domain, was found to aminoacylate tRNA(Asp) not on the 3'-hydroxyl group of the acceptor stem but on a cyclopentene diol of the modified nucleoside queuosine present at the wobble position of anticodon loop . This modified nucleoside might be a relic of an ancient code. Biochim Biophys Acta, 2004 Sep 1, 1701(1-2), 37 - 48 Unique ligation properties of eukaryotic NAD+-dependent DNA ligase from Melanoplus sanguinipes entomopoxvirus; Lu J et al.; The eukaryotic Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial nicotinamide adenine dinucleotide (NAD(+))-dependent DNA ligases {J . Virol . 73 (1999) 533} . This 522-amino acid open reading frame (ORF) contains all conserved nucleotidyl transferase motifs but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD(+) ligases . Nevertheless, cloned MsEPV ligase seals DNA nicks in a NAD(+)-dependent fashion, while adenosine 5'-monophosphate (ATP) cannot serve as an adenylation cofactor . The ligation activity of MsEPV ligase requires Mg(2+) or Mn(2+) . MsEPV ligase seals sticky ends efficiently, but has little activity on 1-nucleotide gap or blunt-ended DNA substrates even in the presence of polyethylene glycol . In comparison, bacterial NAD(+)-dependent ligases seal blunt-ended DNA substrates in the presence of polyethylene glycol . MsEPV DNA ligase readily joins DNA nicks with mismatches at either side of the nick junction, except for mismatches at the nick junction containing an A base in the template strand (A/A, G/A, and C/A) . MsEPV NAD(+)-dependent DNA ligase can join DNA probes on RNA templates, a unique property that distinguishes this enzyme from other conventional bacterial NAD(+) DNA ligases . T4 ATP-dependent DNA ligase shows no detectable mismatch ligation at the 3' side of the nick but substantial 5' T/G mismatch ligation on an RNA template . In contrast, MsEPV ligase joins mismatches at the 3' side of the nick more frequently than at the 5' side of the nick on an RNA template . The complementary specificities of these two enzymes suggest alternative primer design for genomic profiling approaches that use allele-specific detection directly from RNA transcripts. Plant Physiol, 2004 Oct, 136(2), 3333 - 40 Epub 2004 Sep 24. Starch division and partitioning . A mechanism for granule propagation and maintenance in the picophytoplanktonic green alga Ostreococcus tauri; Ral JP et al.; Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule . In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood . Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes . We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants . At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast . These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery. Curr Microbiol, 2004 Sep, 49(3), 180 - 5 Construction and analyses of mutant ftsH alleles of Bacillus subtilis involving the ATPase- and Zn-binding domains; Kotschwar M et al.; The ftsH gene, present in all eubacterial species, is anchored in the cytoplasmic membrane and contains an ATP- and a Zn-binding domain that are both part of a metalloprotease activity . The Bacillus subtilis ftsH is not essential, but null mutants exhibit a pleiotropic phenotype including filamentous growth; hypersensitivity towards heat and salt stress and a failure to sporulate . To find out whether one or the other functional domain is involved in these different phenotypes, point mutations were introduced into the coding region for both domains leading to a replacement of conserved amino acid residues . The mutant alleles were fused to a xylose-inducible promoter and integrated ectopically into two different strains, one expressing the wild-type ftsH allele and the other carrying a ftsH knockout . While none of the strains exhibited a growth defect in rich medium at 37 degrees C, those strains expressing only the mutant alleles did not resume growth after heat or salt stress challenge . Furthermore, none of the mutant alleles promoted sporulation . While only those purified mutant FtsH proteins with an intact Walker A box exhibited ATPase activity, all of them failed to degrade beta-casein. J Biol Chem, 2004 Dec 10, 279(50), 52262 - 9 Epub 2004 Dec 10. The Pseudomonas aeruginosa initiation factor IF-2 is responsible for formylation-independent protein initiation in P . aeruginosa; Steiner-Mosonyi M et al.; Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli . However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet . Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P . aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet . The E . coli IF-2, which participates exclusively in formylation-dependent protein initiation in E . coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P . aeruginosa . However, the E . coli IF-2 was made to function in formylation-independent protein initiation in P . aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA . Furthermore increasing the positive charge potential of this cleft in the P . aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation . Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation . Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination. J Mol Evol, 2004 Jul, 59(1), 133 - 41 Independent origins of subgroup Bl + B2 and subgroup B3 metallo-beta-lactamases; Hall BG et al.; The metallo-beta-lactamases constitute Class B in the Ambler classification of beta-lactamases and are divided into three subclasses: Bl, B2, and B3 . Bayesian phylogenies of the Subclass B1 + B2 and Subclass B3 metallo-beta-lactamases and their homologs show that the beta-lactam-hydrolyzing function evolved independently within each group . In Subclass B1+B2 that function evolved about 1 billion years ago, and in Subclass B3 it evolved before the divergence of the Gram-positive and Gram-negative eubacteria, about 2 billion years ago . These results lend additional support to the proposal that the metallo-beta-lactamases should be divided into two distinct classes. J Mol Evol, 2004 Jul, 59(1), 20 - 30 A plant orthologue of RNase L inhibitor (RLI) is induced in plants showing RNA interference; Braz AS et al.; RNase L inhibitors (RLIs) correspond to a group of soluble proteins from the large ATP binding cassette (ABC) family of proteins . Structurally, RLIs have an N-terminal Fe-S domain and two nucleotide binding domains . Orthologous RLI sequences with more than 48% identity have been found from Archea to Eukaryota, but have not as yet been identified in Eubacteria . Some organisms, like Arabidopsis thaliana and human, have paralogous genes with differential expression patterns, the function of which remains to be determined . Expression of Arabidopsis RLI2 was slightly increased in transgenic plants showing RNA interference, suggesting a role in this pathway. J Mol Biol, 2004 Oct 8, 343(1), 267 - 78 Evolution of vitamin B2 biosynthesis . A novel class of riboflavin synthase in Archaea; Fischer M et al.; The open reading frame MJ1184 of Methanococcus jannaschii with similarity to riboflavin synthase of Methanothermobacter thermoautotrophicus was cloned into an expression vector but was poorly expressed in an Escherichia coli host strain . However, a synthetic open reading frame that was optimized for expression in E.coli directed the synthesis of abundant amounts of a protein with an apparent subunit mass of 17.5 kDa . The protein was purified to apparent homogeneity . Hydrodynamic studies indicated a relative mass of 88 kDa suggesting a homopentamer structure . The enzyme was shown to catalyze the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 24 nmol mg(-1) min(-1) at 40 degrees C . Divalent metal ions, preferably manganese or magnesium, are required for maximum activity . In contrast to pentameric archaeal type riboflavin synthases, orthologs from plants, fungi and eubacteria are trimeric proteins characterized by an internal sequence repeat with similar folding patterns . In these organisms the reaction is achieved by binding the two substrate molecules in an antiparallel orientation . With the enzyme of M.jannaschii, 13C NMR spectroscopy with 13C-labeled 6,7-dimethyl-8-ribityllumazine samples as substrates showed that the regiochemistry of the dismutation reaction is the same as observed in eubacteria and eukaryotes, however, in a non-pseudo-c2 symmetric environment . Whereas the riboflavin synthases of M.jannaschii and M.thermoautotrophicus are devoid of similarity with those of eubacteria and eukaryotes, they have significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalyzing the penultimate step of riboflavin biosynthesis . 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor . Some Archaea have eubacterial type riboflavin synthases which may have been acquired by lateral gene transfer. Bioorg Chem, 2004 Oct, 32(5), 292 - 308 Perspectives in anti-infective drug design . The late steps in the biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate; Rohdich F et al.; A mevalonate-independent pathway for the biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that has been elucidated during the last decade is essential in plants, many eubacteria and apicomplexan parasites, but is absent in Archaea and animals . The enzymes of the pathway are potential targets for the development of novel antibiotic, antimalarial and herbicidal agents . This review is focused on the late steps of this pathway . The intermediate 2C-methyl-D-erythritol 2,4-cyclodiphosphate is converted into IPP and DMAPP via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of the iron-sulfur proteins IspG and IspH . IPP and DMAPP can be interconverted by IPP isomerase which is essential in microorganisms using the mevalonate pathway, whereas its presence is optional in microorganisms using the non-mevalonate pathway . A hitherto unknown family of IPP isomerases using FMN as coenzyme has been discovered recently in Archaea and certain eubacteria. Infect Genet Evol, 2004 Dec, 4(4), 301 - 8 The extracytoplasmic function sigma factors: role in bacterial pathogenesis; Bashyam MD et al.; Bacteria utilize a distinct subfamily of sigma factors to regulate extra cytoplasmic function (thus termed as ECF subfamily) . Eubacteria appear to have evolved to incorporate extensive genetic diversity into their repertoire of ECF sigma factors (some species have more than 60 ECF sigma factors), while maintaining three major themes common to all members including: (1) they regulate and respond to extracytoplasmic functions; (2) they are themselves regulated by anti-sigma and/or anti-anti-sigma factors; and (3) most of them control a relatively small regulon . The cell wall is the first bacterial structure that comes in contact with the host during infection by pathogenic bacteria . The cell wall components are often associated with functions related to host cell invasion . It is therefore, likely that the ECF sigma factors regulate the bacterial response to host insult . Moreover, in some cases, virulence factors have been shown to be regulated directly by the ECF sigma factors . Unfortunately, this facet of the ECF sigma factors has not been an important area of study by researchers . The present review attempts to highlight the important role played by ECF sigma factors in bacterial pathogenesis and highlights several areas of future study involving the genetics of ECF sigma factors vis-a-vis bacterial pathogenesis. J Med Chem, 2004 Sep 23, 47(20), 4941 - 9 Macrocyclic inhibitors for peptide deformylase: a structure-activity relationship study of the ring size; Hu X et al.; Peptide deformylase (PDF) catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria . Its essential role in bacterial cells but not in mammalian cells makes it an attractive target for antibacterial drug design . We have previously reported an N-formylhydroxylamine-based, metal-chelating macrocyclic PDF inhibitor, in which the P(1)' and P(3)' side chains are covalently joined . In this work, we have carried out a structure-activity relationship study on the size of the macrocycle and found that 15-17-membered macrocycles are optimal for binding to the PDF active site . Unlike the acyclic compounds, which are simple competitive inhibitors, the cyclic compounds all act as slow-binding inhibitors . As compared to their acyclic counterparts, the cyclic inhibitors displayed 20-50-fold higher potency against the PDF active site (K(I) as low as 70 pM), improved selectivity toward PDF, and improved the metabolic stability in rat plasma . Some of the macrocyclic inhibitors had potent, broad spectrum antibacterial activity against clinically significant Gram-positive and Gram-negative pathogens . These results suggest that the macrocyclic scaffold provides an excellent lead for the development of a new class of antibiotics. Nature, 2004 Sep 9, 431(7005), 152 - 5 The ring of life provides evidence for a genome fusion origin of eukaryotes; Rivera MC et al.; Genomes hold within them the record of the evolution of life on Earth . But genome fusions and horizontal gene transfer seem to have obscured sufficiently the gene sequence record such that it is difficult to reconstruct the phylogenetic tree of life . Here we determine the general outline of the tree using complete genome data from representative prokaryotes and eukaryotes and a new genome analysis method that makes it possible to reconstruct ancient genome fusions and phylogenetic trees . Our analyses indicate that the eukaryotic genome resulted from a fusion of two diverse prokaryotic genomes, and therefore at the deepest levels linking prokaryotes and eukaryotes, the tree of life is actually a ring of life . One fusion partner branches from deep within an ancient photosynthetic clade, and the other is related to the archaeal prokaryotes . The eubacterial organism is either a proteobacterium, or a member of a larger photosynthetic clade that includes the Cyanobacteria and the Proteobacteria. Annu Rev Genet . 2004 Jul 14; {Epub ahead of print} Mitochondria of Protists; Gray MW et al.; Over the past several decades, our knowledge of the origin and evolution of mitochondria has been greatly advanced by determination of complete mitochondrial genome sequences . Among the most informative mitochondrial genomes have been those of protists (primarily unicellular eukaryotes), some of which harbor the most gene-rich and most eubacteria-like mitochondrial DNAs (mtDNAs) known . Comparison of mtDNA sequence data has provided insights into the radically diverse trends in mitochondrial genome evolution exhibited by different phylogenetically coherent groupings of eukaryotes, and has allowed us to pinpoint specific protist relatives of the multicellular eukaryotic lineages (animals, plants, and fungi) . This comparative genomics approach has also revealed unique and fascinating aspects of mitochondrial gene expression, highlighting the mitochondrion as an evolutionary playground par excellence . Expected online publication date for the Annual Review of Genetics Volume 38 is November 10, 2004 . Please see for revised estimates. Proc Natl Acad Sci U S A, 2004 Sep 14, 101(37), 13436 - 41 Epub 2004 Sep 07. The crystal structure of ribosomal chaperone trigger factor from Vibrio cholerae; Ludlam AV et al.; Trigger factor is a molecular chaperone that is present in all species of eubacteria . It binds to the ribosomal 50S subunit near the translation exit tunnel and is thought to be the first protein to interact with nascent polypeptides emerging from the ribosome . The chaperone has a peptidyl-prolyl cis-trans isomerase (PPIase) activity that catalyzes the rate-limiting proline isomerization in the protein-folding process . We have determined the crystal structure of nearly full-length trigger factor from Vibrio cholerae by x-ray crystallography at 2.5-A resolution . The structure is composed of two trigger-factor molecules related by a noncrystallographic two-fold symmetry axis . The monomer has an elongated shape and is folded into three domains: an N-terminal domain I that binds to the ribosome, a central domain II that contains PPIase activity, and a C-terminal domain III . The active site of the PPIase domain is occupied by a loop from domain III, suggesting that the PPIase activity of the protein could be regulated . The dimer interface is formed between domains I and III and contains residues of mixed properties . Further implications about dimerization, ribosome binding, and other functions of trigger factor are discussed. Proc Natl Acad Sci U S A, 2004 Oct 5, 101(40), 14361 - 6 Epub 2004 Sep 07. Reverse transcriptase activity innate to DNA polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complex; Bao K et al.; Replication of Streptomyces linear chromosomes and plasmids proceeds bidirectionally from a central origin, leaving recessed 5' termini that are extended by a telomere binding complex . This complex contains both a telomere-protecting terminal protein (Tpg) and a telomere-associated protein that interacts with Tpg and the DNA ends of linear Streptomyces replicons . By using histidine-tagged telomere-associated protein (Tap) as a scaffold, we identified DNA polymerase (PolA) and topoisomerase I (TopA) proteins as other components of the Streptomyces telomere complex . Biochemical characterization of these proteins indicated that both PolA and TopA exhibit highly efficient reverse transcriptase (RT) activity in addition to their predicted functions . Although RT activity innate to other DNA-dependent DNA polymerases has been observed previously, its occurrence in a topoisomerase is unprecedented . Deletion mapping and sequence analysis showed that the RT activity of Streptomcyces TopA resides in a peptide region containing motifs that are absent from most bacterial topoisomerases but are highly conserved in a novel subfamily of eubacterial topoisomerases found largely in Actinobacteria . Within one of these motifs, and essential to the RT function of Streptomyces TopA, is an Asp-Asp doublet sequence required also for the RT activities of human immunodeficiency virus and eukaryotic cell telomerases. J Eukaryot Microbiol, 2004 Jul-Aug, 51(4), 456 - 63 Pyruvate formate lyase (PFL) and PFL activating enzyme in the chytrid fungus Neocallimastix frontalis: a free-radical enzyme system conserved across divergent eukaryotic lineages; Gelius-Dietrich G et al.; Fermentative formate production involves the activity of pyruvate formate lyase, an oxygen-sensitive enzyme that employs a glycyl radical in its reaction mechanism . While common among anaerobic prokaryotes, this enzyme has so far been found in only two distantly related eukaryotic lineages, anaerobic chytridiomycetes and chlorophytes . Sequence comparisons of homologues from the chytridiomycetes Piromyces and Neocallimastix, the chlorophyte Chlamydomonas, and numerous prokaryotes suggest a single, eubacterial origin of eukaryotic pyruvate formate lyases . Pyruvate formate lyase activating enzyme introduces the glycyl radical into the pyruvate formate lyase protein chain . We discovered this enzyme, which had not previously been reported from eukaryotes, in the same two eukaryotic lineages and show that it shares a similar evolutionary history to pyruvate formate lyase . Sequences with high homology to pyruvate formate lyase activating enzyme were identified in the genomes of the anaerobic protozoan parasites Trichomonas vaginalis, Entamoeba histolytica, and Giardia intestinalis . While the occurrence of pyruvate formate lyase activating enzyme together with pyruvate formate lyase in fungi and chlorophytes was to be expected, the target protein of a glycyl radical enzyme-activating enzyme in these protozoa remains to be identified. Mol Microbiol, 2004 Sep, 53(6), 1757 - 70 Identification of a gene required for the formation of lyso-ornithine lipid, an intermediate in the biosynthesis of ornithine-containing lipids; Gao JL et al.; Under phosphate-limiting conditions, some bacteria replace their membrane phospholipids by lipids not containing any phosphorus . One of these phosphorus-free lipids is an ornithine-containing lipid (OL) that is widespread among eubacteria . In earlier work, we had identified a gene (olsA) required for OL biosynthesis that probably encodes an O-acyltransferase using acyl-acyl carrier protein (acyl-AcpP) as an acyl donor and that converts lyso-ornithine lipid into OL . We now report on a second gene (olsB) required for OL biosynthesis that is needed for the incorporation of radiolabelled ornithine into OL . Overexpression of OlsB in an olsA-deficient mutant of Sinorhizobium (Rhizobium) meliloti leads to the transient accumulation of lyso-ornithine lipid, the biosynthetic intermediate of OL biosynthesis . Overexpression of OlsB in Escherichia coli is sufficient to cause the in vivo formation of lyso-ornithine lipid in this organism and is the cause for a 3-hydroxyacyl-AcpP-dependent acyltransferase activity forming lyso-ornithine lipid from ornithine . These results demonstrate that OlsB is required for the first step of OL biosynthesis, in which ornithine is N-acylated with a 3-hydroxy-fatty acyl residue in order to obtain lyso-ornithine lipid . OL formation in a wild-type S . meliloti is increased upon growth under phosphate-limiting conditions . Expression of OlsB from a broad host range vector leads to the constitutive formation of relatively high amounts of OL (12-14% of total membrane lipids) independently of whether strains are grown in the presence of low or high concentrations of phosphate, suggesting that in S . meliloti the formation of OlsB is usually limiting for the amount of OL formed in this organism . Open reading frames homologous to OlsA and OlsB were identified in many eubacteria and although in S . meliloti the olsB and olsA gene are 14 kb apart, in numerous other bacteria they form an operon. Acta Crystallogr D Biol Crystallogr, 2004 Sep, 60(Pt 9), 1662 - 4 Epub 2004 Aug 26. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic investigations of a unique editing domain from archaebacteria; Dwivedi S et al.; Threonyl-tRNA synthetase (ThrRS) faces a crucial double-discrimination problem during the translation of genetic code . Most ThrRSs from the archaeal kingdom possess a unique editing domain that differs from those of eubacteria and eukaryotes . In order to understand the structural basis of the editing mechanism in archaea, the editing module of ThrRS from Pyrococcus abyssi comprising of the first 183 amino-acid residues was cloned, expressed, purified and crystallized . The crystals belong to the trigonal space group P3(1(2))21, with one molecule in the asymmetric unit. Oral Microbiol Immunol, 2004 Oct, 19(5), 343 - 6 DNA-DNA relatedness and phylogenetic positions of Slackia exigua, Slackia heliotrinireducens, Eggerthella lenta, and other related bacteria; Nakazawa F et al.; Recently, two asaccharolytic Eubacterium species, Eubacterium exiguum and Eubacterium lentum, and Peptostreptococcus heliotrinreducens have been reclassified as Slackia exigua, Eggerthella lenta and Slackia heliotrinireducens in the novel genera on the basis of 16S rDNA sequence analysis . But DNA-DNA relatedness among these species and other related bacteria have not been reported yet . DNA-DNA relatedness is the standard arbiter and the recommended method for the designation and evaluation of new species, particularly closely related ones . In the present study, DNA-DNA hybridization studies were performed on S . exigua, S . heliotrinireducens and E . lenta together with the other bacterial species in the related genera . The phylogenetic relationships of these species were also investigated by comparison analysis of 16S rDNA sequence data . In the DNA-DNA hybridization studies, S . exigua showed a DNA homology level of 33% to S . heliotrinireducens and 11% to E . lenta . DNA-DNA homology between S . heliotrinireducens and E . lenta was 10% . But these three species showed very low homology (less than 5%) to the related asaccharolytic species such as Eubacterium and Mogibacterium . In conclusion, the DNA-DNA relatedness data together with the evolutionary data in the present paper further support the reclassification of Eubacterium exiguum, Peptostreptococcus heliotrinreducens and Eubacterium lentum as Slackia exigua, Slackia heliotrinireducens and Eggerthella lenta, respectively. J Microbiol Methods, 2004 Oct, 59(1), 117 - 25 Quantitative detection of periodontopathogens by real-time PCR; Nonnenmacher C et al.; Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear . We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis . Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR . This method showed a broad quantification range from 10(2) to 10(8) and accurate sensitivity and specificity . Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined . Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects . Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples. EMBO Rep, 2004 Sep, 5(9), 889 - 94 Dual targeting of plastid division protein FtsZ to chloroplasts and the cytoplasm; Kiessling J et al.; FtsZ is a filament-forming protein that assembles into a ring at the division site of prokaryotic cells . As FtsZ and tubulin share several biochemical and structural similarities, FtsZ is regarded as the ancestor of tubulin . Chloroplasts--the descendants of endosymbiotic bacteria within plant cells--also harbour FtsZ . In contrast to eubacteria, plants have several different FtsZ isoforms . So far, these isoforms have only been implicated with filamentous structures, rings and networks, inside chloroplasts . Here, we demonstrate that a novel FtsZ isoform in the moss Physcomitrella patens is located not only in chloroplasts but also in the cytoplasm, assembling into rings in both cell compartments . These findings comprise the first report on cytosolic localization of a eukaryotic FtsZ isoform, and indicate that this protein might connect cell and organelle division at least in moss. Extremophiles, 2004 Dec, 8(6), 499 - 505 Epub 2004 Jul 30. Archaeal elongation factor 1alpha from Sulfolobus solfataricus interacts with the eubacterial antibiotic GE2270A; Masullo M et al.; The thiazolyl-peptide antibiotic GE2270A, an inhibitor of the elongation factor Tu from Escherichia coli (EcEF-Tu), was used to study the effects produced in the biochemical properties of the archaeal functional analogue elongation factor 1alpha from Sulfolobus solfataricus (SsEF-1alpha) . GE2270A did not substantially affect the poly(U)-directed-poly(Phe) incorporation catalyzed by SsEF-1alpha and the formation of the ternary complex SsEF-1alpha.GTP.Phe-tRNA(Phe) . On the other hand, the antibiotic was able to increase the GDP/GTP exchange rate of SsEF-1alpha; nevertheless, this improvement was not associated with an increase in the catalytic activity of the enzyme . In fact, GE2270A inhibited both the intrinsic GTPase of SsEF-1alpha (GTPase(Na)) and that stimulated by ribosomes . Interestingly, GTPase(Na) of both intact and C-terminal-deleted SsEF-1alpha resulted in a greater sensitivity to the antibiotic with respect to SsEF-1alpha lacking both the M- and C-terminal domains . This result suggested that, similar to what is found for EcEF-Tu, the M domain of SsEF-1alpha is the region of the enzyme most responsible for the interaction with GE2270A . The different behavior observed in the inhibition of protein synthesis with respect to EcEF-Tu can be ascribed to the different adaptive structural changes that have occurred in SsEF-1alpha during evolution. J Biol Chem, 2004 Oct 1, 279(40), 41960 - 5 Epub 2004 Jul 26. Residues Lys-149 and Glu-153 switch the aminoacylation of tRNA(Trp) in Bacillus subtilis; Jia J et al.; Tryptophanyl-tRNA synthetase (TrpRS) consists of two identical subunits that induce the cross-subunit binding mode of tRNA(Trp) . It has been shown that eubacterial and eukaryotic TrpRSs cannot efficiently cross-aminoacylate the corresponding tRNA(Trp) . Although the identity elements in tRNA(Trp) that confer the species-specific recognition have been identified, the corresponding elements in TrpRS have not yet been reported . In this study two residues, Lys-149 and Glu-153, were identified as being crucial for the accurate recognition of tRNA(Trp) . These residues reside adjacent to the binding pocket for Trp-AMP and show phylogenic diversities in the charge on their side chains between eubacteria and eukaryotes . Single mutagenesis at Lys-149 or Glu-153 reduced the activity of TrpRS in the activation of Trp . The reduction was less than that caused by the double mutant WBHA (K149D/E153R) . It is unusual that E153G had no detectable activity in the activation of Trp unless tRNA(Trp) was added to the reaction . In addition, we successfully switched the species specificity of Bacillus subtilis TrpRS recognition of tRNA(Trp) . The affinity of WBHA, K149E and E153K to human tRNA(Trp) was 31-, 13.5-, and 12.9-fold greater than that of wild type B . subtilis TrpRS, respectively . Indeed WBHA and E153K were found to prefer genuine human tRNA(Trp) to their cognate eubacteria tRNA(Trp). Biochem J, 2004 Oct 15, 383(Pt 2), 343 - 51 Substrate specificity of Helicobacter pylori histone-like HU protein is determined by insufficient stabilization of DNA flexure points; Chen C et al.; The histone-like HU protein is ubiquitous in the eubacteria . A role for Escherichia coli HU in compaction of the bacterial genome has been reported, along with regulatory roles in DNA replication, transposition, repair and transcription . We show here that HU from the human pathogen Helicobacter pylori, which has been implicated in the development of ulcers and gastric cancer, exhibits enhanced thermal stability and distinct DNA substrate specificity . Thermal denaturation of HpyHU (H . pylori HU) measured by CD spectroscopy yields a melting temperature (T(m)) of 56.4+/-0.1 degrees C . HpyHU binds linear duplex DNA with a site size of approximately 19 bp and with low affinity, but in striking contrast to E . coli HU, HpyHU has only modest preference for DNA with mismatches, nicks or gaps . Instead, HpyHU binds stably to four-way DNA junctions with half-maximal saturation of 5 nM . Substitution of two residues adjacent to the DNA-intercalating prolines attenuates both the preference for flexible DNA and the ability to bend and supercoil DNA . These observations suggest that proline intercalation generates hinges that must be stabilized by adjacent residues; insufficient stabilization leads to reduced bending and a failure to bind preferably to DNA with flexure points, such as gaps and mismatches. J Biol Chem, 2004 Sep 17, 279(38), 39838 - 45 Epub 2004 Jul 13. A novel phosphoglucose isomerase (PGI)/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum is a member of the PGI superfamily: structural evidence at 1.16-A resolution; Swan MK et al.; The crystal structure of a dual specificity phosphoglucose isomerase (PGI)/phosphomannose isomerase from Pyrobaculum aerophilum (PaPGI/PMI) has been determined in native form at 1.16-A resolution and in complex with the enzyme inhibitor 5-phosphoarabinonate at 1.45-A resolution . The similarity of its fold, with the inner core structure of PGIs from eubacterial and eukaryotic sources, confirms this enzyme as a member of the PGI superfamily . The almost total conservation of amino acids in the active site, including the glutamate base catalyst, shows that PaPGI/PMI uses the same catalytic mechanisms for both ring opening and isomerization for the interconversion of glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate (Fru-6-P) . The lack of structural differences between native and inhibitor-bound enzymes suggests this activity occurs without any of the conformational changes that are the hallmark of the well characterized PGI family . The lack of a suitable second base in the active site of PaPGI/PMI argues against a PMI mechanism involving a trans-enediol intermediate . Instead, PMI activity may be the result of additional space in the active site imparted by a threonine, in place of a glutamine in other PGI enzymes, which could permit rotation of the C-2-C-3 bond of mannose 6-phosphate. Environ Microbiol, 2004 Aug, 6(8), 799 - 808 Bacterial diversity in marine hydrocarbon seep sediments; LaMontagne MG et al.; Marine seeps introduce significant amounts of hydrocarbons into oceans and create unusual habitats for microfauna and -flora . In the vicinity of chronic seeps, microbes likely exert control on carbon quality entering the marine food chain and, in turn, hydrocarbons could influence microbial community composition and diversity . To determine the effects of seep oil on marine sediment bacterial communities, we collected sediment piston cores within an active marine hydrocarbon seep zone in the Coal Oil Point Seep Field, at a depth of 22 m in the Santa Barbara Channel, California . Cores were taken adjacent to an active seep vent in a hydrocarbon volcano, on the edge of the volcano, and at the periphery of the area of active seepage . Bacterial community profiles were determined by terminal restriction fragment length polymorphisms (TRFLPs) of 16S ribosomal genes that were polymerase chain reaction (PCR)-amplified with eubacterial primers . Sediment carbon content and C/N ratio increased with oil content . Terminal restriction fragment length polymorphisms suggested that bacterial communities varied both with depth into sediments and with oil concentration . Whereas the apparent abundance of several peaks correlated positively with hydrocarbon content, overall bacterial diversity and richness decreased with increasing sediment hydrocarbon content . Sequence analysis of a clone library generated from sediments collected at the periphery of the seep suggested that oil-sensitive species belong to the gamma Proteobacteria and Holophaga groups . These sequences were closely related to sequences previously recovered from uncontaminated marine sediments . Our results suggest that seep hydrocarbons exert a strong selective pressure on bacterial communities in marine sediments . This selective pressure could, in turn, control the effects of oil on other biota in the vicinity of marine hydrocarbon seeps. J Exp Bot, 2004 Aug, 55(404), 1809 - 20 Epub 2004 Jul 02. The multi-protein family of Arabidopsis sulphotransferases and their relatives in other plant species; Klein M et al.; All members of the sulphotransferase (SOT, EC 2.8.2.-) protein family use 3'-phosphoadenosine 5'-phosphosulphate (PAPS) as the sulphuryl donor and transfer the sulphonate group to an appropriate hydroxyl group of several classes of substrates . These enzymes have highly conserved domains and can be found in eubacteria and eukaryotes . In mammals, sulphate conjugation catalysed by SOTs constitutes an important reaction in the transformation of xenobiotics, and in the modulation of the biological activity of steroid hormones and neurotransmitters . In plants, sulphate-conjugation reactions seem to play an important role in plant growth, development, and adaptation to stress . To date only a few plant SOTs have been characterized in detail . The flavonol 3- and 4'-SOTs from Flaveria species (Asteraceae), which catalyse the sulphonation of flavonol aglycones and flavonol 3-sulphates, respectively, were the first plant SOTs for which cDNA clones were isolated . The plasma membrane associated gallic acid SOT of Mimosa pudica L . pulvini cells may be intrinsic to signalling events that modify the seismonastic response . In Brassica napus L . a SOT catalyses the O-sulphonation of brassinosteroids and thereby abolishes specifically the biological activity of 24-epibrassinolide . The fully sequenced genome of Arabidopsis thaliana Heynh . contains in total 18 genes that are likely to encode SOT proteins based on sequence similarities of the translated products with an average identity of 51.1% . So far only one SOT from A . thaliana (At5g07000) was functionally characterized: the protein was shown to catalyse the sulphonation of 12-hydroxyjasmonate and thereby inactivate excess jasmonic acid in plants . The substrates and, therefore, the physiological roles of SOTs are very diverse . By using the numerous informative databases and methods available for the model plant A . thaliana, the elucidation of the functional role of the SOT protein family will be accelerated. Environ Sci Technol, 2004 Jun 1, 38(11), 3195 - 202 Enhanced biohydrogen production from sewage sludge with alkaline pretreatment; Cai M et al.; Batch tests were carried out to analyze influences of the alkaline pretreatment and initial pH value on biohydrogen production from sewage sludge . Experimental results of the impact of different initial pH on biohydrogen production showed that both the maximal hydrogen yield occurred and that no methane was detected in the tests of at the initial pH of 11.0 . The final pH decreased at the initial pH of 7.0-12.5 but increased atthe initial pH of 3.0-6.0, probably due to the combination of solubilized protein from sludge and the formation of volatile fatty acids (VFAs) and ammonia during biohydrogen fermentation . The performance of biohydrogen production using the raw sludge and the alkaline pretreated sludge was then compared in batch fermentation tests atthe initial pH of 11.0 . The hydrogen yield was increased from 9.1 mL of H2/g of dry solids (DS) of the raw sludge to 16.6 mL of H2/g of DS of the alkaline pretreated sludge . No methane and less carbon dioxide (0.8% of control) were present in the biohydrogen production from the alkaline pretreated sludge . These results clearly showed that biohydrogen production could be enhanced and maintained stable by the combination of the high initial pH and alkaline pretreatment . The mechanism of biohydrogen production from sewage sludge at high initial pH was therefore investigated because the results of this study were differentfrom previous studies of biohydrogen production . Results showed that protein was the major substrate for biohydrogen production from sewage sludge and that Eubacterium multiforme and Paenibacillus polymyxa were the dominant bacteria in biohydrogen production from alkaline pretreated sludge at an initial pH of 11.0 . The combination of alkaline pretreatment and high initial pH could not only maintain a suitable pH range for the growth of dominant hydrogen-producing anaerobes but also inhibit the growth of hydrogen-consuming anaerobes . In addition, the changes in pH value, oxidation-reduction potential, VFAs and soluble COD during hydrogen fermentation were also discussed. J Ind Microbiol Biotechnol, 2004 Jul, 31(6), 278 - 88 Epub 2004 Jun 19. Studies on the microbial populations of the rhizosphere of big sagebrush ( Artemisia tridentata); Basil AJ et al.; Prolonged use of broad-spectrum antibiotics has led to the emergence of drug-resistant pathogens, both in medicine and in agriculture . New threats such as biological warfare have increased the need for novel and efficacious antimicrobial agents . Natural habitats not previously examined as sources of novel antibiotic-producing microorganisms still exist . One such habitat is the rhizosphere of desert shrubs . Here, we show that one desert shrub habitat, the rhizosphere of desert big sagebrush ( A