J Med Microbiol, 2003 Jul, 52(Pt 7), 591 - 7 Molecular analysis of bacterial flora associated with chronically inflamed maxillary sinuses; Paju S et al.; Chronic maxillary sinusitis is a chronic inflammatory condition in which the role of microbial infection remains undefined . Bacteria have been isolated from chronically inflamed sinuses; however, their role in the chronicity of inflammation is unknown . The objective of this study was to determine whether bacteria are present in clinical samples from chronic maxillary sinusitis and to assess the diversity of the flora present . Washes and/or tissue samples from endoscopic sinus surgery on 11 patients with chronic maxillary sinusitis were subjected to PCR amplification of bacterial 16S rDNA using three universal primer pairs, followed by cloning and sequencing . The samples were also assessed for the presence of bacteria and fungi by conventional culture methods . Viable bacteria and/or bacterial 16S rDNA were detected from maxillary sinus samples of five of the 11 patients examined (45 %) . Three sinus samples were positive by both PCR and culture methods, one was positive only by PCR, and one only by culture . Thirteen bacterial species were identified: Abiotrophia defectiva, Enterococcus avium, Eubacterium sp., Granulicatella elegans, Neisseria sp., Prevotella sp., Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcus gordonii, Streptococcus mitis/Streptococcus oralis and Streptococcus sp . Fungi were not detected . In one patient Streptococcus mitis/Streptococcus oralis, and in another patient Pseudomonas aeruginosa, were detected from both the sinus and the oral cavity using species-specific PCR primers . These results suggest that both aerobic and anaerobic bacteria can be detected in nearly half of chronic maxillary sinusitis cases. Plant Physiol, 2003 Jun, 132(2), 739 - 47 Epub 2003 Apr 24. AtSig5 is an essential nucleus-encoded Arabidopsis sigma-like factor; Yao J et al.; Transcription of chloroplast genes is subject to control by nucleus-encoded proteins . The chloroplast-encoded RNA polymerase (PEP) is a eubacterial-type RNA polymerase that is presumed to assemble with nucleus-encoded sigma-factors mediating promoter recognition . Recently, families of sigma-factor genes have been identified in several plants including Arabidopsis . One of these genes, Arabidopsis SIG5, encodes a sigma-factor, AtSig5, which is phylogenetically distinct from the other family members . To investigate the role of this plant sigma-factor, two different insertional alleles of the SIG5 gene were identified and characterized . Heterozygous mutant plants showed no visible leaf phenotype, but exhibited siliques containing aborted embryos and unfertilized ovules . Our inability to recover plants homozygous for a SIG5 gene disruption indicates that SIG5 is an essential gene . SIG5 transcripts accumulate in flower tissues, consistent with a role for AtSig5 protein in reproduction . Therefore, SIG5 encodes an essential member of the Arabidopsis sigma-factor family that plays a role in plant reproduction in addition to its previously proposed role in leaf chloroplast gene expression. J Dent, 2003 Jul, 31(5), 333 - 9 PCR methodology as a valuable tool for identification of endodontic pathogens; Siqueira JF Jr et al.; OBJECTIVES: This paper reviews the principles of polymerase chain reaction (PCR) methodology, its application in identification of endodontic pathogens and the perspectives regarding the knowledge to be reached with the use of this highly sensitive, specific and accurate methodology as a microbial identification test.DATA SOURCES: Studies published in the medical, dental and biological literature . STUDY SELECTION: Evaluation of published epidemiological studies examining the endodontic microbiota through PCR methodology.CONCLUSIONS: PCR technology has enabled the detection of bacterial species that are difficult or even impossible to culture as well as cultivable bacterial strains showing a phenotypically divergent or convergent behaviour . Moreover, PCR is more rapid, much more sensitive, and more accurate when compared with culture . Its use in endodontics to investigate the microbiota associated with infected root canals has expanded the knowledge on the bacteria involved in the pathogenesis of periradicular diseases . For instance, Tannerella forsythensis (formerly Bacteroides forsythus), Treponema denticola, other Treponema species, Dialister pneumosintes, and Prevotella tannerae were detected in infected root canals for the first time and in high prevalence when using PCR analysis . The diversity of endodontic microbiota has been demonstrated by studies using PCR amplification, cloning and sequencing of the PCR products . Moreover, other fastidious bacterial species, such as Porphyromonas endodontalis, Porphyromonas gingivalis and some Eubacterium spp., have been reported in endodontic infections at a higher prevalence than those reported by culture procedures. Res Microbiol, 2003 May, 154(4), 259 - 67 The diversity and evolution of the T4-type bacteriophages; Desplats C et al.; Recent studies suggest that viruses are the most numerous entities in the biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea, constitute a substantial fraction of this population . In spite of their ubiquity, the vast majority of phages in the environment have never been studied and nothing is known about them . For the last 10 years our research has focused on an extremely widespread group of phages, the T4-type . It has now become evident that phage T4 has a myriad of relatives in nature that differ significantly in their host range . The genomes of all these phages have homology to the T4 genes that determine virion morphology . Although phylogenetically related, these T4-type phages can be subdivided into four groups that are increasingly distant from T4: the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens . Genomic comparisons between the various T4-type phages and T4 indicate that these genomes share homology not only for virion structural components but also for most of the essential genes involved in the T4 life cycle . This suggests that horizontal transmission of the genetic information may have played a less general role in the evolution of these phages than has been supposed . Nevertheless, we have identified several regions of the T4-type genome, such as the segment containing the tail fiber genes that exhibit evidence of extensive modular shuffling during evolution . The T4-type genomes appear to be a mosaic containing a large and fixed group of essential genes as well as highly variable set of non-essential genes . These non-essential genes are probably important for the adaptation of these phages to their particular life-style . Furthermore, swapping autonomous domains within the essential proteins may slightly modify their function(s) and contribute to the adaptive ability of the T4-type phage family . Regulatory sequences also display considerable evolutionary plasticity and this too may facilitate the adaptation of phage gene expression to new environments and stresses. Bioresour Technol, 2003 Sep, 89(3), 237 - 43 Effects of glucose overloading on microbial community structure and biogas production in a laboratory-scale anaerobic digester; Sundh I et al.; This study characterizes the response of the microbial communities of a laboratory-scale mesophilic biogas process, fed with a synthetic substrate based on cellulose and egg albumin, to single pulses of glucose overloading (15 or 25 times the daily feed based on VS) . The microbial biomass and community structure were determined from analyses of membrane phospholipids . The ratio between phospholipid fatty acids (PLFAs; eubacteria and eucaryotes) and di-ethers (PLEL; archaea) suggested that methanogens constituted 4-8% of the microbial biomass . The glucose addition resulted in transient increases in the total biomass of eubacteria while there were only small changes in community structure . The total gas production rate increased, while the relative methane content of the biogas and the alkalinity decreased . However, the biomass of methanogens was not affected by the glucose addition . The results show that the microbial communities of biogas processes can respond quickly to changes in the feeding rate . The glucose overload resulted in a transient general stimulation of degradation rates and almost a doubling of eubacterial biomass, although the biomass increase corresponded to only 7% of the glucose C added. Biochemistry, 2003 Jun 17, 42(23), 7216 - 25 Characterization of DNA strand transfer promoted by Mycobacterium smegmatis RecA reveals functional diversity with Mycobacterium tuberculosis RecA; Ganesh N et al.; The RecA-like proteins constitute a group of DNA strand transfer proteins ubiquitous in eubacteria, eukarya, and archaea . However, the functional relationship among RecA proteins is poorly understood . For instance, Mycobacterium tuberculosis RecA is synthesized as a large precursor, which undergoes an unusual protein-splicing reaction to generate an active form . Whereas the precursor was inactive, the active form promoted DNA strand transfer less efficiently compared to EcRecA . Furthermore, gene disruption studies have indicated that the frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis compared to Mycobacterium smegmatis . The mechanistic basis and the factors that contribute to differences in allele exchange remain to be understood . Here, we show that the extent of DNA strand transfer promoted by the M . smegmatis RecA in vitro differs significantly from that of M . tuberculosis RecA . Importantly, M . smegmatis RecA by itself was unable to promote strand transfer, but cognate or noncognate SSBs rendered it efficient even when added prior to RecA . In the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA and varying lengths of linear duplex DNA with distinctly different pH profiles . The factors that were able to suppress the formation of DNA networks greatly stimulated strand transfer reactions promoted by MsRecA or MtRecA . Although the rate and pH profiles of dATP hydrolysis catalyzed by MtRecA and MsRecA were similar, only MsRecA was able to couple dATP hydrolysis to DNA strand transfer . Together, these results provide insights into the functional diversity in DNA strand transfer promoted by RecA proteins of pathogenic and nonpathogenic species of mycobacteria. Microbiol Mol Biol Rev, 2003 Jun, 67(2), 238 - 76, table of contents Prophage genomics; Canchaya C et al.; The majority of the bacterial genome sequences deposited in the National Center for Biotechnology Information database contain prophage sequences . Analysis of the prophages suggested that after being integrated into bacterial genomes, they undergo a complex decay process consisting of inactivating point mutations, genome rearrangements, modular exchanges, invasion by further mobile DNA elements, and massive DNA deletion . We review the technical difficulties in defining such altered prophage sequences in bacterial genomes and discuss theoretical frameworks for the phage-bacterium interaction at the genomic level . The published genome sequences from three groups of eubacteria (low- and high-G+C gram-positive bacteria and gamma-proteobacteria) were screened for prophage sequences . The prophages from Streptococcus pyogenes served as test case for theoretical predictions of the role of prophages in the evolution of pathogenic bacteria . The genomes from further human, animal, and plant pathogens, as well as commensal and free-living bacteria, were included in the analysis to see whether the same principles of prophage genomics apply for bacteria living in different ecological niches and coming from distinct phylogenetical affinities . The effect of selection pressure on the host bacterium is apparently an important force shaping the prophage genomes in low-G+C gram-positive bacteria and gamma-proteobacteria. Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7503 - 8 Epub 2003 Jun 03. A lupus-like syndrome develops in mice lacking the Ro 60-kDa protein, a major lupus autoantigen; Xue D et al.; Antibodies against a conserved RNA-binding protein, the Ro 60-kDa autoantigen, occur in 24-60% of all patients with systemic lupus erythematosus . Anti-Ro antibodies are correlated with photosensitivity and cutaneous lesions in these patients and with neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with complete congenital heart block and photosensitive skin lesions . In higher eukaryotes, the Ro protein binds small RNAs of unknown function known as Y RNAs . Because the Ro protein also binds misfolded 5S rRNA precursors, it is proposed to function in a quality-control pathway for ribosome biogenesis . Consistent with a role in the recognition or repair of intracellular damage, an orthologue of Ro in the radiation-resistant eubacterium Deinococcus radiodurans contributes to survival of this bacterium after UV irradiation . Here, we show that mice lacking the Ro protein develop an autoimmune syndrome characterized by anti-ribosome antibodies, anti-chromatin antibodies, and glomerulonephritis . Moreover, in one strain background, Ro-/- mice display increased sensitivity to irradiation with UV light . Thus, one function of this major human autoantigen may be to protect against autoantibody development, possibly by sequestering defective ribonucleoproteins from immune surveillance . Furthermore, the finding that mice lacking the Ro protein are photosensitive suggests that loss of Ro function could contribute to the photosensitivity associated with anti-Ro antibodies in humans. J Biol Chem, 2003 Aug 22, 278(34), 32313 - 6 Epub 2003 Jun 03. Genome-based identification and analysis of collagen-related structural motifs in bacterial and viral proteins; Rasmussen M et al.; Collagens are extended trimeric proteins composed of the repetitive sequence glycine-X-Y . A collagen-related structural motif (CSM) containing glycine-X-Y repeats is also found in numerous proteins often referred to as collagen-like proteins . Little is known about CSMs in bacteria and viruses, but the occurrence of such motifs has recently been demonstrated . Moreover, bacterial CSMs form collagen-like trimers, even though these organisms cannot synthesize hydroxyproline, a critical residue for the stability of the collagen triple helix . Here we present 100 novel proteins of bacteria and viruses (including bacteriophages) containing CSMs identified by in silico analyses of genomic sequences . These CSMs differ significantly from human collagens in amino acid content and distribution; bacterial and viral CSMs have a lower proline content and a preference for proline in the X position of GXY triplets . Moreover, the CSMs identified contained more threonine than collagens, and in 17 of 53 bacterial CSMs threonine was the dominating amino acid in the Y position . Molecular modeling suggests that threonines in the Y position make direct hydrogen bonds to neighboring backbone carbonyls and thus substitute for hydroxyproline in the stabilization of the collagen-like triple-helix of bacterial CSMs . The majority of the remaining CSMs were either rich in proline or rich in charged residues . The bacterial proteins containing a CSM that could be functionally annotated were either surface structures or spore components, whereas the viral proteins generally could be annotated as structural components of the viral particle . The limited occurrence of CSMs in eubacteria and lower eukaryotes and the absence of CSMs in archaebacteria suggests that DNA encoding CSMs has been transferred horizontally, possibly from multicellular organisms to bacteria. Appl Environ Microbiol, 2003 Jun, 69(6), 3580 - 92 Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney; Schrenk MO et al.; A large, intact sulfide chimney, designated Finn, was recovered from the Mothra Vent Field on the Juan de Fuca Ridge in 1998 . Finn was venting 302 degrees C fluids on the seafloor and contained complex mineralogical zones surrounding a large open central conduit . Examination of microorganisms within these zones, followed by community analysis with oligonucleotide probes, showed that there were variations in the abundance and diversity of eubacteria and archaea from the exterior to the interior of the chimney . The microbial abundance based upon epifluorescence microscopy and quantitative fatty acid analyses varied from >10(8) cells/g of sulfide 2 to 10 cm within the chimney wall to <10(5) cells/g in interior zones . Direct microscopic observation indicated that microorganisms were attached to mineral surfaces throughout the structure . Whole-cell hybridization results revealed that there was a transition from a mixed community of eubacteria and archaea near the cool exterior of the chimney to primarily archaea near the warm interior . Archaeal diversity was examined in three zones of Finn by cloning and sequencing of the 16S rRNA gene . The majority of sequences from the exterior of the chimney were related to marine group I of the Crenarchaeota and uncultured Euryarchaeota from benthic marine environments . In contrast, clone libraries from interior regions of the chimney contained sequences closely related to methanogens, Thermococcales, and Archaeoglobales, in addition to uncultured crenarchaeal phylotypes obtained from deep subsurface sites . These observations of microbial communities within an active hydrothermal chimney provide insight into the microbial ecology within such structures and may facilitate follow-up exploration into expanding the known upper temperature limits of life. Mol Microbiol, 2003 Jun, 48(5), 1289 - 303 Proteomic studies of diauxic lag in the differentiating prokaryote Streptomyces coelicolor reveal a regulatory network of stress-induced proteins and central metabolic enzymes; Novotna J et al.; Bacteria typically undergo intermittent periods of starvation and adaptation, emulated as diauxic growth in the laboratory . In association with growth arrest elicited by metabolic stress, the differentiating eubacterium Streptomyces coelicolor not only adapts its primary metabolism, but can also activate developmental programmes leading to morphogenesis and antibiotic biosynthesis . Here, we report combined proteomic and metabolomic data of S . coelicolor used to analyse global changes in gene expression during diauxic growth in a defined liquid medium . Cultures initially grew on glutamate, providing the nitrogen source and feeding carbon (as 2-oxoglutarate) into the TCA cycle, followed by a diauxic delay allowing reorientation of metabolism and a second round of growth supported by NH4+, formed during prediauxic phase, and maltose, a glycolytic substrate . Cultures finally entered stationary phase as a result of nitrogen starvation . These four physiological states had previously been defined statistically by their distinct patterns of protein synthesis and heat shock responses . Together, these data demonstrated that the rates of synthesis of heat shock proteins are determined not only by temperature increase but also by the patterns and rates of metabolic flux in certain pathways . Synthesis profiles for metabolic- and stress-induced proteins can now be interpreted by the identification of 204 spots (SWICZ database presented at Cluster analysis showed that the activity of central metabolic enzymes involved in glycolysis, the TCA cycle, starvation or proteolysis each displayed identifiable patterns of synthesis that logically underlie the metabolic state of the culture . Diauxic lag was accompanied by a structured regulatory programme involving the sequential activation of heat-, salt-, cold- and bacteriostatic antibiotic (pristinamycin I, PI)-induced stimulons . Although stress stimulons presumably provide protection during environmental- or starvation-induced stress, their identities did not reveal any coherent adaptive or developmental functions . These studies revealed interactive regulation of metabolic and stress response systems including some proteins known to support developmental programmes in S . coelicolor. Cold Spring Harb Symp Quant Biol, 2001, 66, 147 - 59 Pseudouridines and pseudouridine synthases of the ribosome; Ofengand J et al.; psi are ubiquitous in ribosomal RNA . Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most . The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more . Large subunits appear to need at least one psi but can have up to 50-60 . psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs . The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs . All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif . The aspartate is essential for psi formation in all twelve synthases examined so far . When the need for psi in E . coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop . This growth defect was the result of a major failure in assembly of the large ribosomal subunit . The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi . Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi . This result is not without precedent . Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E . coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al . 1995), but the methylation activity of the enzyme is not required (T . Mason, pers . comm.) . The absence of FtsJ, a heat shock protein that makes Um2552 in E . coli, makes the 50S subunit less stable at 1 mM Mg++ (Bugl et al . 2000) and inhibits subunit joining (Caldas et al . 2000), but, in this case, it is not yet known whether the effects are due to the lack of 2'-O-methylation or to the absence of the enzyme itself . Is there any role for the psi residues themselves? First, as noted above, the 3 psi made by RluD which cluster in the end-loop of helix 69 are highly conserved, with one being universal (Fig . 2B) . In the 70S-tRNA structure (Yusupov et al . 2001), the loop of this helix containing the psi supports the anticodon arm of A-site tRNA near its juncture with the amino acid arm . The middle of helix 69 does the same thing for P-site tRNA . Unfortunately, the resolution is not yet sufficient to provide a more precise alignment of the psi residues with the other structural elements of the tRNA-ribosome complex so that one cannot yet determine what role, if any, is played by the N-1 H that distinguishes psi from U . Second, and more generally, some psi residues in the LSU appear to be near the site of peptide-bond formation or tRNA binding but not actually at it (Fig . 2B) (Nissen et al . 2000; Yusupov et al . 2001) . For example, position 2492 is commonly psi and is only six residues away from A2486, the A postulated to catalyze peptide-bond formation . Position 2589 is psi in all the eukaryotes and is next to 2588, which base-pairs with the C75 of A-site tRNA . Residue 2620, which interacts with the A76 of A-site-bound tRNA, is a psi or is next to a psi in eukaryotes and Archaea, and is five residues away from psi 2580 in E . coli . A2637, which is between the two CCA ends of P- and A-site tRNA, is near psi 2639, psi 2640, and psi 2641, found in a number of organisms . Residue 2529, which contacts the backbone of A-site tRNA residues 74-76, is near psi 2527 psi 2528 in H . marismortui . Residues 2505-2507, which contact A-site tRNA residues 50-53, are near psi 2509 in higher eukaryotes, and residues 2517-2519 in contact with A-site tRNA residues 64-65 are within 1-3 nucleotides of psi 2520 in higher eukaryotes and psi 2514 in H . marismortui . A way to rationalize this might be to invoke the concept suggested in the Introduction that psi acts as a molecular glue to hold loose elements in a more rigid configuration . It may well be that this is more important near the site of peptide-bond formation and tRNA binding, accounting for the preponderance of psi in this vicinity . What might be the role of all the other psi in eukaryotes? One can only surmise that cells, having once acquired the ability to make psi with guide RNAs, took advantage of the system to inexpensively place psi wherever an undesirable loose region was found . It might be that in some of these cases, psi performs the role played by proteins in other regions, namely that of holding the rRNA in its proper configuration . Confirmation of this hypothesis will have to await structural determination of eukaryotic ribosomes. J Biochem (Tokyo), 2003 Jan, 133(1), 83 - 91 Interconversion of the product specificity of type I eubacterial farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase through one amino acid substitution; Kawasaki T et al.; Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths . Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases . We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseolosporeus MF730-N6 {Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H . (2001) BIOSCI: Biotechnol . Biochem . 65, 1627-1635} . In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase) . Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes . To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the -50th, -8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed . The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively . On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did . These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity. Microb Ecol, 2003 Jul, 46(1), 106 - 12 Epub 2003 May 21. Detection of eubacterial 3-hydroxy-3-methylglutaryl coenzyme a reductases from natural populations of actinomycetes; Sigmund JM et al.; Three natural populations of actinomycetes were investigated by PCR for the presence of type I 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA), a gene associated with isoprenoid biosynthesis . The populations were obtained from an agricultural site (69 isolates), a coastal salt marsh (220 isolates), and a desert soil (96 isolates) . A set (34) of standard actinomycete reference strains were also investigated . The target gene was only detected in 5 of the 419 actinomycetes screened, which represented 4 from the coastal salt marsh and one reference strain . The isolates that contained the gene were taxonomically diverse (4 Streptomyces spp . and 1 Nocardia sp.) . These results suggest that type I HMG CoA containing pathways are rare in actinomycetes and their distribution within actinomycetes populations is not random. Mol Microbiol, 2003 May, 48(4), 1017 - 28 Antagonism of PII signalling by the AmtB protein of Escherichia coli; Blauwkamp TA et al.; Escherichia coli AmtB is a member of the MEP/Amt family of ammonia transporters found in archaea, eubacteria, fungi, plants and animals . In prokaryotes, AmtB homologues are co-transcribed with a PII paralogue, GlnK, in response to nitrogen limitation . Here, we show that AmtB antagonizes PII signalling through NRII and that co-expression of GlnK with AmtB overcomes this antagonism . In cells lacking GlnK, expression of AmtB during nitrogen starvation prevented deinduction of Ntr gene expression when a nitrogen source became available . The absence of AmtB in cells lacking GlnK allowed rapid reduction of Ntr gene expression during this transition, indicating that one function of GlnK is to prevent AmtB-mediated antagonism of PII signalling after nitrogen starvation . Other roles of GlnK in controlling Ntr gene expression and maintaining viability during nitrogen starvation were unaffected by AmtB . Expression of AmtB from a constitutive promoter under nitrogen-rich conditions induced full expression of glnALG and elevated expression of glnK in wild-type and glnK cells; thus, the ability of AmtB to raise Ntr gene expression did not require a factor found only in nitrogen-starved cells . Experiments with intact cells showed that AmtB acted downstream of a uridylyl transferase uridylyl-removing enzyme (UTase/UR) and upstream of NRII, suggesting that the target was PII . AmtB also slowed the deuridylylation of PII approximately UMP upon ammonia addition, showing that multiple PII interactions were affected by AmtB . Our data are consistent with a hypothesis that AmtB interacts with PII and GlnK, and that co-transcription of glnK and amtB prevents titration of PII when AmtB is highly expressed. Arch Oral Biol, 2003 Jun, 48(6), 423 - 9 Degradation of arginine and other amino acids by butyrate-producing asaccharolytic anaerobic Gram-positive rods in periodontal pockets; Uematsu H et al.; The use of 20 amino acids by butyrate-producing asaccharolytic anaerobic Gram-positive rods (AAGPRs) in periodontal pockets, i.e . Eubacterium minutum, Filifactor alocis, E . infirmum, E . sulci and E . saphenum, was studied . E . minutum used only arginine and lysine, and produced substantial amounts of butyrate and ammonia as the main metabolic products from arginine, and acetate, butyrate and ammonia from lysine . Fi . alocis used arginine alone and produced butyrate and ammonia . E . infirmum, E . sulci and E . saphenum used lysine alone and produced acetate, butyrate and ammonia . The growth of these bacterial species was supported and enhanced by arginine and/or lysine enriched to culture media, but not by the other amino acids . Arginine deiminase, ornithine carbamoyltransferase and carbamate kinase activity were detected in the cell-free extract of E . minutum, suggesting that arginine was metabolised to citrulline initially, and subsequently to ornithine and carbamoyl phosphate . Ornithine and carbamoyl phosphate were further converted to butyrate, and carbon dioxide and ammonia, respectively . Enzymatic activity of arginine deiminase and ornithine carbamoyltransferase was not detected in Fi . alocis, indicating that Fi . alocis converted arginine to ornithine directly, not via citrulline, and further to butyrate. EMBO J, 2003 May 15, 22(10), 2516 - 25 A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity; Lipps G et al.; Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life . Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E . The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity . Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner . Deoxynucleotides are preferentially used for the synthesis of primers approximately 8 nucleotides long . The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length . The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase . ORF904 constitutes a new type of replication enzyme, which could have evolved independently from the eubacterial and archaeal/eukaryal proteins of DNA replication. J Clin Microbiol, 2003 May, 41(5), 2235 - 6 Eubacterium callanderi bacteremia: report of the first case; Thiolas A et al.; Eubacterium callanderi is an environmental anaerobic rod-shaped bacterium first isolated in 1998 from an industrial anaerobic digester . We report on the first clinical isolate of E . callanderi, which was recovered from the blood of a patient with a bladder carcinoma . Identification of the organism was made by cell fatty acid chromatographic analysis and 16S rRNA gene sequencing. J Clin Microbiol, 2003 May, 41(5), 1977 - 86 Microbial population diversity in the urethras of healthy males and males suffering from nonchlamydial, nongonococcal urethritis; Riemersma WA et al.; Nonchlamydial, nongonococcal urethritis (NCNGU) is suggested to be a sexually transmitted disease in men . NCNGU patients were compared to control subjects with regard to the presence of potentially infectious bacteria in the first void urine . Patients' pre- and post-antibiotic-treatment urine samples and two samples obtained 2 weeks apart from healthy volunteers, who did not receive antibiotic therapy, were analyzed with broad-spectrum PCR tests aiming at eubacterial small subunit rRNA genes . Restriction fragment length polymorphism analysis of the amplicons cloned from the mixtures of PCR products revealed that many different species of microorganisms were found to be colonizing the male urethra . We document here clear differences in the composition of the resident urethral flora between samples obtained from various individuals and between samples obtained at various points in time for a single individual . No major changes in population complexity were found upon antimicrobial treatment . In two of five patients a previously suggested pathogen (Mycoplasma genitalium or Haemophilus parainfluenzae) was accurately identified on the basis of DNA sequencing . No ubiquitous, azithromycin-sensitive organism was identified as a common pathogen in all patients, but up to 40% of all clones represented as-yet-unclassified bacterial species . Relatively often Pseudomonas spp . or Pseudomonas-like organisms were identified in the bacterial flora of patients . Interestingly, an as-yet-uncharacterized microbial species was identified as a negative predictor of NCNGU . This species was identified in all control subjects and was absent from all of the patient' samples (5 of 5 versus 0 of 5, P = 0.0079) . This suggests that NCNGU might also be diagnosed by assessing the absence rather than the presence of certain bacterial species. J Biol Chem, 2003 Jul 11, 278(28), 26127 - 34 Epub 2003 May 05. Mechanistic studies of human molybdopterin synthase reaction and characterization of mutants identified in group B patients of molybdenum cofactor deficiency; Leimkuhler S et al.; Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT . The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase . Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively . MOCS2A and MOCS2B were purified after heterologous expression in E . coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer . The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue . To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated . Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z. J Microbiol Methods, 2003 Jul, 54(1), 57 - 74 A comparison of DNA profiling techniques for monitoring nutrient impact on microbial community composition during bioremediation of petroleum-contaminated soils; Mills DK et al.; Amplicon length heterogeneity PCR (LH-PCR) and terminal restriction fragment length polymorphisms (TRFLP) were used to monitor the impact that nutrient amendments had on microbial community dynamics and structural diversity during bioremediation of petroleum-contaminated soils . Slurried soils contaminated with petroleum hydrocarbons were treated in airlift bench-scale bioreactors and were either amended with optimal inorganic nutrients or left unamended . Direct DNA extraction and PCR amplification of whole eubacterial community DNA were performed with universal primers that bracketed the first two or three hypervariable regions of the 16S rDNA gene sequences . The LH-PCR method profiled a more diverse microbial community than did the TRFLP method . The LH-PCR method also tracked differences between the communities due to nutrient amendments . An in silico database search for bacterial genera with amplicon lengths represented in the community fingerprints was performed . It was possible to qualitatively identify different groups in the microbial community based on the amplicon length variations . A similar "virtual" search was performed for the TRFLP fragments using the web-based TAP-TRFLP program . Cloning and sequencing of the PCR products confirmed the in silico database matches . The application of the LH-PCR method as a monitoring tool for bioremediation could greatly enhance and extend the current understanding of the microbial community dynamics during the biodegradation of environmental contaminants. Prog Biophys Mol Biol, 2003 May-Jul, 82(1-3), 11 - 24 Evolutionary origins of mechanosensitive ion channels; Martinac B et al.; According to the recent revision, the universal phylogenetic tree is composed of three domains: Eukarya (eukaryotes), Bacteria (eubacteria) and Archaea (archaebacteria) . Mechanosensitive (MS) ion channels have been documented in cells belonging to all three domains suggesting their very early appearance during evolution of life on Earth . The channels show great diversity in conductance, selectivity and voltage dependence, while sharing the property of being gated by mechanical stimuli exerted on cell membranes . In prokaryotes, MS channels were first documented in Bacteria followed by their discovery in Archaea . The finding of MS channels in archaeal cells helped to recognize and establish the evolutionary relationship between bacterial and archaeal MS channels and to show that this relationship extends to eukaryotic Fungi (Schizosaccharomyces pombe) and Plants (Arabidopsis thaliana) . Similar to their bacterial and archaeal homologues, MS channels in eukaryotic cell-walled Fungi and Plants may serve in protecting the cellular plasma membrane from excessive dilation and rupture that may occur during osmotic stress . This review summarizes briefly some of the recent developments in the MS channel research field that may ultimately lead to elucidation of the biophysical and evolutionary principles underlying the mechanosensory transduction in living cells. Annu Rev Microbiol, 2003, 57, 101 - 23 Epub 2003 May 01. A salvage pathway for protein structures: tmRNA and trans-translation; Withey JH et al.; Transfer-messenger RNA (tmRNA, or SsrA), found in all eubacteria, has both transfer and messenger RNA activity . Relieving ribosome stalling by a process called trans-translation, tmRNAala enters the ribosome and adds its aminoacylated alanine to the nascent polypeptide . The original mRNA is released and tmRNA becomes the template for translation of a 10-amino-acid tag that signals for proteolytic degradation . Although essential in a few bacterial species, tmRNA is nonessential in Escherichia coli and many other bacteria . Proteins known to be associated with tmRNA include SmpB, ribosomal protein S1, RNase R, and phosphoribosyl pyrophosphate . SmpB, having no other known function, is essential for tmRNA activity . trans-translation operates within ribosomes stalled both at the end of truncated mRNAs and at rare codons and some natural termination sites . Both the release of stalled ribosomes and the subsequent degradation of tagged proteins are important consequences of trans-translation. Microbiology, 2003 May, 149(Pt 5), 1095 - 102 Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth; Sunde PT et al.; Whether micro-organisms can live in periapical endodontic lesions of asymptomatic teeth is under debate . The aim of the present study was to visualize and identify micro-organisms within periapical lesions directly, using fluorescence in situ hybridization (FISH) in combination with epifluorescence and confocal laser scanning microscopy (CLSM) . Thirty-nine periapical lesions were surgically removed, fixed, embedded in cold polymerizing resin and sectioned . The probe EUB 338, specific for the domain Bacteria, was used together with a number of species-specific 16S rRNA-directed oligonucleotide probes to identify bacteria . To control non-specific binding of EUB 338, probe NON 338 was used . Alternatively, DAPI (4',6'-diamidino-2-phenylindole) staining was applied to record prokaryotic and eukaryotic DNA in the specimens . Hybridization with NON 338 gave no signals despite background fluorescence of the tissue . The eubacterial probe showed bacteria of different morphotypes in 50 % of the lesions . Rods, spirochaetes and cocci were spread out in areas of the tissue while other parts seemed bacteria-free . Bacteria were also seen to co-aggregate inside the tissue, forming microcolonies . Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis and treponemes of phylogenetic Group I were detected with specific probes . In addition, colonies with Streptococcus spp . were seen in some lesions . A number of morphotypes occurred that could not be identified with the specific probes used, indicating the presence of additional bacterial species . CLSM confirmed that bacteria were located in different layers of the tissue . Accordingly, the FISH technique demonstrated mixed consortia of bacteria consisting of rods, spirochaetes and cocci in asymptomatic periapical lesions of root-filled teeth. Biosci Biotechnol Biochem, 2003 Mar, 67(3), 490 - 8 Characterization of aspartate aminotransferase from the cyanobacterium Phormidium lapideum; Kim H et al.; Aspartate aminotransferase (AspAT) was purified to homogeneity from cell extracts of the non-N2-fixing cyanobacterium Phormidium lapideum . The NH2-terminal sequence of 25 amino acid residues was different from the sequences of the subfamily Ialpha of AspATs from eukaryotes and Escherichia coli, but it was similar to sequences of the subfamily Igamma of AspATs from archaebacteria and eubacteria . The enzyme was most active at 80 degrees C and was stable at up to 75 degrees C . Thermal inactivation (60-85 degrees C) of the enzyme followed first-order kinetics, with 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures . However, at 25 degrees C the kcat of P . lapideum AspAT was nearly equal to the values of AspATs from mesophilic organisms . The enzyme used L-aspartate and L-cysteine sulfinate as amino donors and 2-oxoglutarate as an amino acceptor . The Km values were 5.0 mM for L-aspartate, 5.7 mM for L-glutamate, 0.2 mM for 2-oxoglutarate, and 0.032 mM for oxaloacetate. Biochemistry, 2003 May 6, 42(17), 4787 - 99 Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, and initiation factor 2 on activity in initiation; Mayer C et al.; Anticodon sequence mutants of Escherichia coli initiator tRNA initiate protein synthesis with codons other than AUG and amino acids other than methionine . Because the anticodon sequence is, in many cases, important for recognition of tRNAs by aminoacyl-tRNA synthetases, the mutant tRNAs are aminoacylated in vivo with different amino acids . The activity of a mutant tRNA in initiation in vivo depends on (i) the level of expression of the tRNA, (ii) the extent of aminoacylation of the tRNA, (iii) the extent of formylation of the aminoacyl-tRNA to formylaminoacyl-tRNA (fAA-tRNA), and (iv) the affinity of the fAA-tRNA for the initiation factor IF2 and the ribosome . Previously, using E . coli overproducing aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, or IF2, we identified the steps limiting the activity in initiation of mutant tRNAs aminoacylated with glutamine and valine . Here, we have identified the steps limiting the activity of mutant tRNAs aminoacylated with isoleucine and phenylalanine . The combined results of experiments involving a variety of initiation codons (AUG, UAG, CAG, GUC, AUC, and UUC) provide support to the hypothesis that the ribosome.fAA-tRNA complex can act as an intermediate in initiation of protein synthesis . Comparison of binding affinities of various fAA-tRNAs (fMet-, fGln-, fVal-, fIle-, and fPhe-tRNAs) to IF2 using surface plasmon resonance supports the idea that IF2 can act as a carrier of fAA-tRNA to the ribosome . Other results suggest that the C1xA72 base pair mismatch, unique to eubacterial and organellar initiator tRNAs, may also be important for the binding of fAA-tRNA to IF2. Mol Biol Evol, 2003 Jun, 20(6), 901 - 6 Epub 2003 Apr 25. The effects of selection against spurious transcription factor binding sites; Hahn MW et al.; Most genomes contain nucleotide sequences with no known function; such sequences are assumed to be free of constraints, evolving only according to the vagaries of mutation . Here we show that selection acts to remove spurious transcription factor binding site motifs throughout 52 fully sequenced genomes of Eubacteria and Archaea . Examining the sequences necessary for polymerase binding, we find that spurious binding sites are underrepresented in both coding and noncoding regions . The average proportion of spurious binding sites found relative to the expected is 80% in eubacterial genomes and 89% in archaeal genomes . We also estimate the strength of selection against spurious binding sites in the face of the constant creation of new binding sites via mutation . Under conservative assumptions, we estimate that selection is weak, with the average efficacy of selection against spurious binding sites, Nes, of -0.12 for eubacterial genomes and -0.06 for archaeal genomes, similar to that of codon bias . Our results suggest that both coding and noncoding sequences are constrained by selection to avoid specific regions of sequence space. Trends Biochem Sci, 2003 Apr, 28(4), 167 - 9 Phototaxis, chemotaxis and the missing link; Oprian DD; Phototaxis in Archaea employs an integral membrane complex composed of a photoreceptor that is similar to the light-driven proton pump bacteriorhodopsin, and a transducer protein that is similar to the familiar eubacterial chemotaxis receptors . Recent structural studies have revealed how these proteins are assembled in the membrane, and provide a heuristic framework for future work on the mechanism of signal transduction by this important class of molecules. J Dent Res, 2003 May, 82(5), 338 - 44 New bacterial species associated with chronic periodontitis; Kumar PS et al.; Recent investigations of the human subgingival oral flora based on ribosomal 16S cloning and sequencing have shown many of the bacterial species present to be novel species or phylotypes . The purpose of the present investigation was to identify potential periodontal pathogens among these newly identified species and phylotypes . Species-specific ribosomal 16S primers for PCR amplification were developed for detection of new species . Associations with chronic periodontitis were observed for several new species or phylotypes, including uncultivated clones D084 and BH017 from the Deferribacteres phylum, AU126 from the Bacteroidetes phylum, Megasphaera clone BB166, clone X112 from the OP11 phylum, and clone I025 from the TM7 phylum, and the named species Eubacterium saphenum, Porphyromonas endodontalis, Prevotella denticola, and Cryptobacterium curtum . Species or phylotypes more prevalent in periodontal health included two uncultivated phylotypes, clone W090 from the Deferribacteres phylum and clone BU063 from the Bacteroidetes, and named species Atopobium rimae and Atopobium parvulum. Antimicrob Agents Chemother, 2003 May, 47(5), 1496 - 502 Role of 16S rRNA Helix 44 in Ribosomal Resistance to Hygromycin B; Pfister P et al.; Hygromycin B is an aminoglycoside antibiotic active against prokaryotic and eukaryotic ribosomes . Ribosomal alterations in bacteria conferring resistance to hygromycin B have not been described, prompting us to use a single rRNA allelic derivative of the gram-positive bacterium Mycobacterium smegmatis for investigation of the molecular mechanisms involved in ribosomal resistance to hygromycin B in eubacteria . Resistance mutations were found to localize exclusively in 16S rRNA . The mutations observed, i.e., 16S rRNA U1406C, C1496U, and U1498C (E . coli numbering), are in close proximity to the hygromycin B binding site located in conserved helix 44 of 16S rRNA . The 16S rRNA positions involved in hygromycin B resistance are highly conserved in all three domains of life, explaining the lack of specificity and general toxicity of hygromycin B. J Clin Microbiol, 2003 Apr, 41(4), 1600 - 8 Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and other eubacteria in ticks from the Thai-Myanmar border and Vietnam; Parola P et al.; A total of 650 ticks, including 13 species from five genera, were collected from animals, from people, or by flagging of the vegetation at sites on the Thai-Myanmar border and in Vietnam . They were tested by PCR to detect DNA of bacteria of the order RICKETTSIALES: Three Anaplasma spp . were detected in ticks collected in Thailand, including (i) Anaplasma sp . strain AnDa465, which was considered a genotype of Anaplasma platys (formerly Ehrlichia platys) and which was obtained from Dermacentor auratus ticks collected from dogs; (ii) Anaplasma sp . strain AnAj360, which was obtained from Amblyomma javanense ticks collected on a pangolin; and (iii) Anaplasma sp . strain AnHl446, which was closely related to Anaplasma bovis and which was detected in Haemaphysalis lagrangei ticks collected from a bear . Three Ehrlichia spp . were identified, including (i) Ehrlichia sp . strain EBm52, which was obtained from Boophilus microplus ticks collected from cattle from Thailand; (ii) Ehrlichia sp . strain EHh324, which was closely related to Ehrlichia chaffeensis and which was detected in Haemaphysalis hystricis ticks collected from wild pigs in Vietnam; and (iii) Ehrlichia sp . strain EHh317, which was closely related to Ehrlichia sp . strain EBm52 and which was also detected in H . hystricis ticks collected from wild pigs in Vietnam . Two Rickettsia spp . were detected in Thailand, including (i) Rickettsia sp . strain RDla420, which was detected in Dermacentor auratus ticks collected from a bear, and (ii) Rickettsia sp . strain RDla440, which was identified from two pools of Dermacentor larvae collected from a wild pig nest . Finally, two bacteria named Eubacterium sp . strain Hw124 and Eubacterium sp . strain Hw191 were identified in Haemaphysalis wellingtoni ticks collected from chicken in Thailand; these strains could belong to a new group of bacteria. J Bacteriol, 2003 May, 185(9), 2901 - 9 Atypical 16S rRNA gene copies in Ochrobactrum intermedium strains reveal a large genomic rearrangement by recombination between rrn copies; Teyssier C et al.; Ochrobactrum intermedium is an opportunistic human pathogen belonging to the alpha 2 subgroup of proteobacteria . The 16S rDNA sequences of nine O . intermedium isolates from a collection of clinical and environmental isolates exhibited a 46-bp insertion at position 187, which was present in only one sequence among the 82 complete or partial 16S rDNA sequences of Ochrobactrum spp . available in data banks . Reverse transcription-PCR experiments showed that the 46-bp insertion remained in the 16S rRNA . The inserted sequence folded into a stem-loop structure, which took place in and prolonged helix H184 of the 16S rRNA molecule . Helix H184 has been described as conserved in length among eubacteria, suggesting the idiosyncratic character of the 46-bp insertion . Pulsed-field gel electrophoresis experiments showed that seven of the clinical isolates carrying the 46-bp insertion belonged to the same clone . Insertion and rrn copy numbers were determined by hybridization and I-CeuI digestion . In the set of clonal isolates, the loss of two insertion copies revealed the deletion of a large genomic fragment of 150 kb, which included one rrn copy; deletion occurred during the in vivo evolution of the clone . Determination of the rrn skeleton suggested that the large genomic rearrangement occurred during events involving homologous recombination between rrn copies . The loss of insertion copies suggested a phenomenon of concerted evolution among heterogeneous rrn copies. Eur J Biochem, 2003 Apr, 270(8), 1599 - 618 Mitochondrial connection to the origin of the eukaryotic cell; Emelyanov VV; Phylogenetic evidence is presented that primitively amitochondriate eukaryotes containing the nucleus, cytoskeleton, and endomembrane system may have never existed . Instead, the primary host for the mitochondrial progenitor may have been a chimeric prokaryote, created by fusion between an archaebacterium and a eubacterium, in which eubacterial energy metabolism (glycolysis and fermentation) was retained . A Rickettsia-like intracellular symbiont, suggested to be the last common ancestor of the family Rickettsiaceae and mitochondria, may have penetrated such a host (pro-eukaryote), surrounded by a single membrane, due to tightly membrane-associated phospholipase activity, as do present-day rickettsiae . The relatively rapid evolutionary conversion of the invader into an organelle may have occurred in a safe milieu via numerous, often dramatic, changes involving both partners, which resulted in successful coupling of the host glycolysis and the symbiont respiration . Establishment of a potent energy-generating organelle made it possible, through rapid dramatic changes, to develop genuine eukaryotic elements . Such sequential, or converging, global events could fill the gap between prokaryotes and eukaryotes known as major evolutionary discontinuity. Infection, 2003 Mar, 31(2), 86 - 91 Marginal and subgingival plaque--a natural habitat of Tropheryma whipplei? Zinkernagel AS, Gmur R, Fenner L, Schaffner A, Schoedon G, Schneemann M. BACKGROUND: DNA of Tropheryma whipplei, the etiologic agent of Whipple's disease, has recently been detected in the saliva of healthy subjects . In this pilot study we searched for the habitat of T . whipplei within the oral cavity . MATERIALS AND METHODS: Samples from different oral sites were obtained from periodontically healthy volunteers, patients with progressive periodontitis and Chinese subjects with necrotizing ulcerative gingivitis or gingivitis . Quantitative real-time PCR was performed using T . whippleispecific primers, human beta-globin-specific primers to control for tissue DNA extraction and PCR reaction and broad-range eubacterial primers to control for bacterial DNA extraction . T . whipplei specificity of multiple amplicons was confirmed by sequencing . The detection limit of the method was 10 ag of T . whipplei DNA, corresponding to one to five bacteria under reference assay conditions . RESULTS: T . whipplei was found in the oral cavity of four out of ten healthy individuals from hospital staff and in three out of nine periodontitis patients, but in none of the individuals from China . All positive samples derived from subgingival and gingival sulcus plaque containing between 10(3) and 5 x 10(5) cells ml(-1) of plaque suspension, whereas saliva, smooth surface plaque and samples from the tongue or cheeks were negative . CONCLUSION: Our results suggest that T . whipplei colonizes the human body via the oral cavity and that bacterial plaques of the gingival crevice and the gingival sulcus/pocket may serve as a natural primary habitat. Carbohydr Res, 2003 Apr 22, 338(9), 923 - 30 The structure of the antigenic polysaccharide produced by Eubactrium saburreum T15; Sato N et al.; The antigenic polysaccharide was obtained from the cell wall of Eubacterium saburreum strain T15 by trypsin digestion followed by gel permeation and ion-exchange chromatography . Its structure was determined using acid hydrolysis, methylation analysis, and 1D and 2D NMR spectroscopy . It contained L-threo-pent-2-ulose (Xul), D-fucose (Fuc), and D-glycero-D-galacto-heptose (Hep) in 2:3:3 ratio . Methylation analysis indicated an octasaccharide repeating-unit containing five branches . The 1H and 13C signals in NMR spectra of the sugar residues were assigned by COSY, HOHAHA, and HMQC 2D experiments, and the sequence of sugar residues in the repeating unit was determined by NOESY and HMBC experiments . The polysaccharide also contains two O-acetyl groups in the repeating unit, located on the Hep residue . The repeating structure can be written as: {see text for equation} . This is a novel structure in bacterial cell-wall polysaccharides from Gram-positive bacteria. Proteomics, 2003 Apr, 3(4), 363 - 79 Bacterial glycoproteins: functions, biosynthesis and applications; Upreti RK et al.; Although widely distributed in eukaryotic cells glycoproteins appear to be rare in prokaryotic organisms . The prevalence of the misconception that bacteria do not glycosylate their proteins has been a subject matter of discussion for a long time . Glycoconjugates that are linked to proteins or peptides, generated by the ribosomal translational mechanism have been reported only in the last two to three decades in a few prokaryotic organisms . Most studied prokaryotic glycoproteins are the S-layer glycoproteins of Archeabacteria . Apart from these, membrane-associated, surface-associated, secreted glycoproteins and exoenzymes glycoproteins are also well documented in both, Archea and Eubacteria . From the recent literature, it is now clear that prokaryotes are capable of glycosylating proteins . In general, prokaryotes are deprived of the cellular organelles required for glycosylation . In prokaryotes many different glycoprotein structures have been observed that display much more variation than that observed in eukaryotes . Besides following similar mechanisms in the process of glycosylation, prokaryotes have also been shown to use mechanisms that are different from those found in eukaryotes . The knowledge pertaining to the functional aspects of prokaryotic glycoproteins is rather scarce . This review summarizes developments and understanding relating to characteristics, synthesis, and functions of prokaryotic glycoproteins . An extensive summary of glycosylation that has been reported to occur in bacteria has also been tabulated . Various possible applications of these diverse biomolecules in biotechnology, vaccine development, pharmaceutics and diagnostics are also touched upon. J Biol Chem, 2003 Jun 20, 278(25), 22388 - 95 Epub 2003 Apr 09. A story of chelatase evolution: identification and characterization of a small 13-15-kDa "ancestral" cobaltochelatase (CbiXS) in the archaea; Brindley AA et al.; The cobaltochelatase required for the synthesis of vitamin B12 (cobalamin) in the archaeal kingdom has been identified as CbiX through similarity searching with the CbiX from Bacillus megaterium . However, the CbiX proteins in the archaea are much shorter than the CbiX proteins found in eubacteria, typically containing less than half the number of amino acids in their primary structure . For this reason the shorter CbiX proteins have been termed CbiXS and the longer versions CbiXL . The CbiXS proteins from Methanosarcina barkeri and Methanobacter thermoautotrophicum were overproduced in Escherichia coli as recombinant proteins and characterized . Through complementation studies of a defined chelatase-deficient strain of E . coli and by direct in vitro assays the function of CbiXS as a sirohydrochlorin cobaltochelatase has been demonstrated . On the basis of sequence alignments and conserved active site residues we suggest that CbiXS may represent a primordial chelatase, giving rise to larger chelatases such as CbiXL, SirB, CbiK, and HemH through gene duplication and subsequent variation and selection . A classification scheme for chelatases is proposed. J Invertebr Pathol, 2003 Mar, 82(3), 152 - 61 Extraordinary proliferation of microorganisms in aposymbiotic pea aphids, Acyrthosiphon pisum; Nakabachi A et al.; Aposymbiotic pea aphids, which were deprived of their intracellular symbiotic bacterium, Buchnera, exhibit growth retardation and no fecundity . High performance liquid chromatographic (HPLC) analysis revealed that these aposymbiotic aphids, when reared on broad bean plants, accumulated a large amount of histamine . To assess the possibility of extraordinary proliferation of microorganisms other than Buchnera, we enumerated eubacteria and fungi in aphids using the real-time quantitative PCR method that targets genes encoding small-subunit rRNAs . The result showed that these microorganisms were extremely abundant in the aposymbiotic aphids reared on plants . Microbial communities in aposymbiotic aphids were further profiled by phylogenetic analysis of small-subunit rDNAs . Of 172 nonchimeric sequences of fungal 18S rDNAs, 138 (80.2%) belonged to the phylum Ascomycota . Among them, 21 clustered within a monophyletic group consisting of insect-pathogenic fungi and yeast-like symbionts of homopteran insects . Thirty-one (18.0%), two (1.2%), and one (0.6%) clones were clustered within the Basidiomycota, Zygomycota, and Oomycota, respectively . Of 167 nonchimeric sequences of eubacterial 16S rDNAs, 84 (50.3%) belonged to the gamma-subdivision of Proteobacteria to which most primary endosymbionts of insects and prolific histamine producers belong . Forty (24.0%), 25 (15.0%), 10 (6.0%), and five (3.0%) clones were clustered within alpha-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Actinobacteria, and beta-Proteobacteria, respectively . Three had no phylogenetic association with known taxonomic divisions . None of the sequences studied in this study coincided exactly with those deposited in GenBank. J Endod, 2003 Mar, 29(3), 194 - 200 An immunohistological study of the localization of bacteria invading root pulpal walls of teeth with periapical lesions; Matsuo T et al.; We immunohistologically examined the prevalence and localization of bacteria invading dentinal tubules of the roots of teeth with infected canals . Forty extracted teeth with apical lesions were selected and divided into two groups: a group of untreated teeth and a group of canal-enlarged teeth . The bacteria in the specimens were detected by Brown-Brenn stain and the labeled-streptavidin-biotin method with specific antisera for 16-bacteria . Seventy percent of the examined teeth showed bacteria invading the dentinal tubules of the roots . Fusobacterium nucleatum, Eubacterium alactolyticum, E . nodatum, Lactobacillus casei, and Peptostreptococcus micros were abundant . Even in the canal-enlarged group, invasion of bacteria was observed in 65% of teeth . This study revealed the actual condition of bacteria in infected root dentin and suggested that the canal-enlargement procedure could not completely remove all the bacteria in the infected dentinal tubules of the root. Helicobacter, 2003 Apr, 8(2), 149 - 57 Profiling and identification of eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection; Sun YQ et al.; BACKGROUND: Mongolian gerbils are frequently used to study Helicobacter pylori-induced gastritis and its consequences . The presence of an indigenous bacterial flora with suppressive effect on H . pylori may cause difficulties with establishing this experimental model . AIM: The aim of the present study was to determine bacterial profiles in the stomach of Mongolian gerbils with and without (controls) H . pylori infection . METHODS: Gastric tissue from H . pylori ATCC 43504 and CCUG 17874 infected and control animals were subjected to microbial culturing and histology . In addition, gastric mucosal samples from H . pylori ATCC 43504 infected and control animals were analyzed for bacterial profiling by temporal temperature gradient gel electrophoresis (TTGE), cloning and pyrosequencing of 16S rDNA variable V3 region derived PCR amplicons . RESULTS: Oral administration of H . pylori ATCC 43504, but not CCUG 17874, induced colonization and gastric inflammation in the stomach of Mongolian gerbils . Temporal temperature gradient gel electrophoresis (TTGE) and partial 16S rDNA pyrosequencing revealed the presence of DNA representing a mixed bacterial flora in the stomach of both H . pylori ATCC 43504 infected and control animals . In both cases, lactobacilli appeared to be dominant . CONCLUSION: These findings suggest that indigenous bacteria, particularly lactobacilli, may have an impact on the colonization and growth of H . pylori strains in the stomach of Mongolian gerbils. Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 165 - 72 Bacillus nealsonii sp . nov., isolated from a spacecraft-assembly facility, whose spores are gamma-radiation resistant; Venkateswaran K et al.; One of the spore-formers isolated from a spacecraft-assembly facility, belonging to the genus Bacillus, is described on the basis of phenotypic characterization, 16S rDNA sequence analysis and DNA-DNA hybridization studies . It is a Gram-positive, facultatively anaerobic, rod-shaped eubacterium that produces endospores . The spores of this novel bacterial species exhibited resistance to UV, gamma-radiation, H2O2 and desiccation . The 18S rDNA sequence analysis revealed a clear affiliation between this strain and members of the low G+C Firmicutes . High 16S rDNA sequence similarity values were found with members of the genus Bacillus and this was supported by fatty acid profiles . The 16S rDNA sequence similarity between strain FO-92T and Bacillus benzoevorans DSM 5391T was very high . However, molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus, but DNA-DNA hybridization data support the proposal of FO-92T as Bacillus nealsonii sp . nov . (type strain is FO-92T =ATCC BAAM-519T =DSM 15077T). J Am Soc Mass Spectrom, 2003 Mar, 14(3), 253 - 61 Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry; Ge Y et al.; Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates . The search for these has been slow, even though the entire genome sequence of M . tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized . For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS . The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M(r)) values, for a total of 689 different values . However, only approximately 10% of these values matched (+/-1 Da) the DNA predicted M(r) values, but these identifications were unreliable . Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein . MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously . The low success of M(r) matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation . Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments . For MS/MS, electron capture dissociation was especially effective. J Med Chem, 2003 Mar 27, 46(7), 1133 - 43 Virtual screening for submicromolar leads of tRNA-guanine transglycosylase based on a new unexpected binding mode detected by crystal structure analysis; Brenk R et al.; Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hypermodification of cognate tRNAs, leading to the exchange of G34 by preQ1 at the wobble position in the anticodon loop . Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium due to inefficient translation of a virulence protein mRNA . Herein, we describe the discovery of a ligand with an unexpected binding mode . On the basis of this binding mode, three slightly deviating pharmacophore hypotheses have been derived . Virtual screening based on this composite pharmacophore model retrieved a set of potential TGT inhibitors belonging to several compound classes . All nine tested inhibitors being representatives of these classes showed activity in the micromolar range, two of them even in the submicromolar range. Mol Biol Evol, 2003 Mar, 20(3), 371 - 80 Sequence complexity of histone H1 subtypes; Ponte I et al.; H1 subtypes are involved in chromatin higher-order structure and gene regulation . H1 has a characteristic three-domain structure . We studied the length variation of the available H1 subtypes and showed that the length of the N-terminal and C-terminal domains was more variable than that of the central domain . The N-terminal and C-terminal domains were of low sequence complexity both at the nucleotide and at the amino acid level, whereas the globular domain was of high complexity . In most subtypes, low complexity was due only to cryptic simplicity, which reflects the clustering of a number of short and often imperfect sequence motifs . However, a subset of subtypes from eubacteria, plants, and invertebrates contained tandem repeats of short amino acid motifs (four to 12 residues), which could amount to a large proportion of the terminal domains . In addition, some other subtypes, such as those of Drosophila and mammalian H1t, were only marginally simple . The coexistence of these three kinds of subtypes suggests that the terminal domains could have originated in the amplification of short sequence motifs, which would then have evolved by point mutation and further slippage. J Biomol Struct Dyn, 2003 Apr, 20(5), 657 - 68 A novel complexity measure for comparative analysis of protein sequences from complete genomes; Nandi T et al.; Analysis of sequence complexities of proteins is an important step in the characterization and classification of new genomes . A new measure has been proposed to compute sequence complexity in protein sequences based on linguistic complexity . The algorithm requires a single parameter, is computationally simple and provides a framework for comparative genomic analysis . Protein sequences were classified into groups of high or low complexity based on a quantitative measure termed F(c), which is proportional to the fraction of low complexity sequence present in the protein . The algorithm was tested on sequences of 196 non-homologous proteins whose crystal structures are available at </=2.0 A resolution . Protein sequences of high complexity had 'globular' structures (95% agreement), whereas those of low complexity had non-globular structures (80% agreement) . Application of this measure to proteins of unknown structure/function from different genomes revealed that the sequences of high complexity constitute the majority in all genomes (about 90% in Archaea, about 93% in Eubacteria, 89% in Saccharomyces cerevisiae and 90% in Caenorhabditis elegans) . Aeropyrum pernix among Archaeae and Deinococcus radiodurans among Eubacteria have the lowest fraction of high complexity proteins (75% and 80% respectively) . Further, it was observed that a few bacterial pathogens (Mycobacterium tuberculosis, Pseudomonas aeruginosa) have high fraction of low complexity proteins . The program ScanCom is available from the authors as a PERL script (UNIX system). J Appl Microbiol, 2003, 94(4), 655 - 64 Growth and molecular characterization of dental plaque microcosms; McBain AJ et al.; AIMS: (i) To compare the effects of feeding protocols upon the composition and stability of dental plaque microcosms formed in constant-depth film fermenters (CDFF) . (ii) To evaluate the utility of denaturing gradient gel electrophoresis (DGGE) and culture methodologies for the investigation of such models . METHODS AND RESULTS: Microcosms were established anaerobically in the CDFFs from freshly collected saliva . These were fed either with artificial saliva alone (famine) or combined with discontinuous feeding (feast-famine) . Culture and 16s rDNA sequencing indicated that supplemental feeding gave ca . 2 log increases in Lactobacillus rhamnosus and Prevotella buccae . Feast-famine microcosms were then further characterized by DGGE using primers specific for the V2-V3 region of eubacterial rDNA . These gave single major bands with pure cultures (eight species) and resolved all strains apart from Lact . rhamnosus and Actinomyces naeslundii . Whilst culture with selective media indicated a degree of stability and reproducibility between replicate microcosms, DGGE showed a considerable degree of variability that related to several putatively uncultured bacteria . CONCLUSIONS: Feast-famine regimes altered community composition . DGGE analyses identified putatively unculturable species and demonstrated variability between replicate fermenters . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the utility of DGGE for the analysis of dental plaque, especially with respect to unculturable bacteria . Results question the assumptions of reproducibility of plaque microcosms established in non-replicated CDFFs made on the basis of selective media . Feeding regimes, particularly those involving complex nutrients, will dramatically affect population dynamics. Biochemistry, 2003 Mar 18, 42(10), 3032 - 9 Isolation, characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus; Peng G et al.; The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence . The purified detergent solubilized enzyme is highly active above 50 degrees C . The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degrees C . The A . aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine . SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits . N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G . The isolated complex is highly stable and active in a temperature range from 50 to 90 degrees C, with a half-life of about 10 h at 80 degrees C . The activity shows a linear Arrhenius plot at 50-85 degrees C with an activation energy at 31.92 J/mol K . Single particle electron microscopy shows that the A . aeolicus complex I has the typical L-shape . However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources . In addition, the angle (90 degrees ) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant. Nucleic Acids Res, 2003 Mar 15, 31(6), 1735 - 43 An NMR study on the interaction of Escherichia coli DinI with RecA-ssDNA complexes; Yoshimasu M et al.; The SOS response, a set of cellular phenomena exhibited by eubacteria, is initiated by various causes that include DNA damage-induced replication arrest, and is positively regulated by the co- protease activity of RecA . Escherichia coli DinI, a LexA-regulated SOS gene product, shuts off the initiation of the SOS response when overexpressed in vivo . Biochemical and genetic studies indicated that DinI physically interacts with RecA to inhibit its co-protease activity . Using nuclear magnetic resonance (NMR) spectroscopy, we show that DinI tightly binds to the central region of RecA (between the N- and C-terminal domains) and that this interaction is enhanced upon the oligomerisation of RecA . On the other hand, DinI did not inhibit the interaction between 4mer single-stranded (ss)DNA and RecA- ATPgammaS, but had a slight effect on the structure of ssDNA-RecA-ATPgammaS complexes involving 8mer and 12mer ssDNA . We hypothesise that prevention of repressor binding to the intermolecular cleft region of RecA protomers by DinI, with the possibility of a slight conformational change induced in the DinI-bound ssDNA-RecA-ATPgammaS complex, together function to inhibit the co-protease activity of RecA. Microbiol Mol Biol Rev, 2003 Mar, 67(1), 52 - 65, table of contents Cytokinesis in bacteria; Errington J et al.; Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria . These proteins are directed to the division site by the combination of two negative regulatory systems . Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell . The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms . The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E . coli and B . subtilis . They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization . FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell . FtsA is another cytosolic protein that is related to actin, but its precise function is unclear . The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E . coli and possibly EzrA of B . subtilis, which have a single membrane span but a cytoplasmic C-terminal domain . The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell . The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum. Mol Microbiol, 2003 Mar, 47(6), 1513 - 22 Demonstration of a sensory rhodopsin in eubacteria; Jung KH et al.; We report the first sensory rhodopsin observed in the eubacterial domain, a green light-activated photoreceptor in Anabaena (Nostoc) sp . PCC7120, a freshwater cyanobacterium . The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon . The opsin expressed heterologously in Escherichia coli membranes bound all-trans retinal to form a pink pigment (lambda max 543 nm) with a photochemical reaction cycle of 110 ms half-life (pH 6.8, 18 degrees C) . Co-expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane-embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction . The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity . We detected retinal binding to the protein in Anabaena membranes by SDS-PAGE and autofluorography of 3H-labelled all-trans retinal of reduced membranes from the organism . We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor . Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix-helix interactions with membrane-embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments. Appl Environ Microbiol, 2003 Mar, 69(3), 1710 - 20 Role of Rhodobacter sp . strain PS9, a purple non-sulfur photosynthetic bacterium isolated from an anaerobic swine waste lagoon, in odor remediation; Do YS et al.; Temporal pigmentation changes resulting from the development of a purple color in anaerobic swine waste lagoons were investigated during a 4-year period . The major purple photosynthetic bacterium responsible for these color changes and the corresponding reductions in odor was isolated from nine photosynthetic lagoons . By using morphological, physiological, and phylogenetic characterization methods we identified the predominant photosynthetic bacterium as a new strain of Rhodobacter, designated Rhodobacter sp . strain PS9 . Rhodobacter sp . strain PS9 is capable of photoorganotrophic growth on a variety of organic compounds, including all of the characteristic volatile organic compounds (VOC) responsible for the odor associated with swine production facilities (J . A . Zahn, A . A . DiSpirito, Y . S . Do, B . E . Brooks, E . E . Copper, and J . L . Hatfield, J . Environ . Qual . 30:624-634, 2001) . The seasonal variations in airborne VOC emitted from waste lagoons showed that there was a 80 to 93% decrease in the concentration of VOC during a photosynthetic bloom . During the height of a bloom, the Rhodobacter sp . strain PS9 population accounted for 10% of the total community and up to 27% of the eubacterial community based on 16S ribosomal DNA signals . Additional observations based on seasonal variations in meteorological, biological, and chemical parameters suggested that the photosynthetic blooms of Rhodobacter sp . strain PS9 were correlated with lagoon water temperature and with the concentrations of sulfate and phosphate . In addition, the photosynthetic blooms of Rhodobacter sp . strain PS9 were inversely correlated with the concentrations of protein and fluoride. Appl Environ Microbiol, 2003 Mar, 69(3), 1492 - 8 Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase; Bergendahl V et al.; The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth . Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery . A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled sigma70 and an IC5-labeled fragment of the beta' subunit of RNA polymerase (amino acid residues 100 through 309) . Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission . The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library . We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery. J Biol Chem, 2003 May 16, 278(20), 18271 - 80 Epub 2003 Feb 26. The trypanosomatid signal recognition particle consists of two RNA molecules, a 7SL RNA homologue and a novel tRNA-like molecule; Liu L et al.; Trypanosomatids are ancient eukaryotic parasites affecting humans and livestock . Here we report that the trypanosomatid signal recognition particle (SRP), unlike all other known SRPs in nature, contains, in addition to the 7SL RNA homologue, a short RNA molecule, termed sRNA-85 . Using conventional chromatography, we discovered a small RNA molecule of 85 nucleotides co-migrating with the Leptomonas collosoma 7SL RNA . This RNA molecule was isolated, sequenced, and used to clone the corresponding gene . sRNA-85 was identified as a tRNA-like molecule that deviates from the canonical tRNA structure . The co-existence of these RNAs in a single complex was confirmed by affinity selection using an antisense oligonucleotide to sRNA-85 . The two RNA molecules exist in a particle of approximately 14 S that binds transiently to ribosomes . Mutations were introduced in sRNA-85 that disrupted its putative potential to interact with 7SL RNA by base pairing; such mutants were unable to bind to 7SL RNA and to ribosomes and were aberrantly distributed within the cell . We postulate that sRNA-85 may functionally replace the truncated Alu domain of 7SL RNA . The discovery of sRNA-85 raises the intriguing possibility that sRNA-85 functional homologues may exist in other lower eukaryotes and eubacteria that lack the Alu domain. Chem Biol Interact, 2003 Feb 1, 143-144, 5 - 22 Aldehyde dehydrogenase gene superfamily: the 2002 update; Sophos NA et al.; The aldehyde dehydrogenase (ALDH) superfamily represents a divergently related group of enzymes that metabolize a wide variety of endogenous and exogenous aldehydes . With the advent of megabase genome sequencing, the ALDH superfamily is continuously expanding on many fronts . The presence of ALDH encoding genes in the vast majority of archaeal, eubacterial and eukaryotic genomes supports the notion that these enzymes are important components of metabolic processes in living organisms and that the ALDH superfamily is ancient in origin . As of July 2002, the ALDH superfamily consists of 555 distinct genes: 32 in archaea, 351 in eubacteria, and 172 in eukaryota . Complete sequencing of individual genomes reveals the number of ALDH genes found per organism ranges from 1 to 5 in archaeal species, 1-26 genes in eubacterial species, and 8-17 genes in eukaryotic species . In the human genome, 17 functional genes and 3 pseudogenes have been identified to date . A standardized ALDH gene nomenclature system has been developed based on multiple alignment analysis of eukaryotic ALDH amino acid sequences . Both Human and Mouse Genome Projects have accepted this nomenclature system . In this report, we present a complete listing of all ALDH sequences known to date, along with the evolutionary analysis of the eukaryotic ALDHs . Thus far, the eukaryotic ALDHs comprise 20 gene families . Detailed information on ALDH gene superfamily is also available at http://www.uchsc.edu/sp/sp/alcdbase/aldhcov.html. Mol Microbiol, 2003 Mar, 47(5), 1419 - 32 Axe-Txe, a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant, clinical isolate of Enterococcus faecium; Grady R et al.; Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections . However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria . Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium . A novel proteic-based toxin-antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species . Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli . The toxic effect of Txe was alleviated by co-expression of the antitoxin protein, Axe . Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria . These homologues (yefM-yoeB) present in the E . coli chromosome function as a toxin-antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo . Axe-Txe is one of the first functional proteic toxin-antitoxin systems to be accurately described for Gram-positive bacteria. Mol Biol Evol, 2003 Feb, 20(2), 267 - 77 Parallel evolution of ligand specificity between LacI/GalR family repressors and periplasmic sugar-binding proteins; Fukami-Kobayashi K et al.; The bacterial LacI/GalR family repressors such as lactose operon repressor (LacI), purine nucleotide synthesis repressor (PurR), and trehalose operon repressor (TreR) consist of not only the N-terminal helix-turn-helix DNA-binding domain but also the C-terminal ligand-binding domain that is structurally homologous to periplasmic sugar-binding proteins . These structural features imply that the repressor family evolved by acquiring the DNA-binding domain in the N-terminal of an ancestral periplasmic binding protein (PBP) . Phylogenetic analysis of the LacI/GalR family repressors and their PBP homologues revealed that the acquisition of the DNA-binding domain occurred first in the family, and ligand specificity then evolved . The phylogenetic tree also indicates that the acquisition occurred only once before the divergence of the major lineages of eubacteria, and that the LacI/GalR and the PBP families have since undergone extensive gene duplication/loss independently along the evolutionary lineages . Multiple alignments of the repressors and PBPs furthermore revealed that repressors and PBPs with the same ligand specificity have the same or similar residues in their binding sites . This result, together with the phylogenetic relationship, demonstrates that the repressors and the PBPs individually acquired the same ligand specificity by homoplasious replacement, even though their genes are encoded in the same operon. Biosci Biotechnol Biochem, 2002 Dec, 66(12), 2578 - 86 Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8; Rajashekhara E et al.; The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail . The maximal enzyme activity was observed at pH 6.2 and 80 degrees C . The energy of activation was 74 kJ/mol . The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8 . The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate . The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1) . In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors . Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases . D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)) . The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors . D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations. J Mol Biol, 2003 Mar 7, 326(5), 1559 - 75 Structure and function of an archaeal homolog of survival protein E (SurEalpha): an acid phosphatase with purine nucleotide specificity; Mura C et al.; The survival protein E (SurE) family was discovered by its correlation to stationary phase survival of Escherichia coli and various repair proteins involved in sustaining this and other stress-response phenotypes . In order to better understand this ancient and well-conserved protein family, we have determined the 2.0A resolution crystal structure of SurEalpha from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (Pae) . This first structure of an archaeal SurE reveals significant similarities to and differences from the only other known SurE structure, that from the eubacterium Thermatoga maritima (Tma) . Both SurE monomers adopt similar folds; however, unlike the Tma SurE dimer, crystalline Pae SurEalpha is predominantly non-domain swapped . Comparative structural analyses of Tma and Pae SurE suggest conformationally variant regions, such as a hinge loop that may be involved in domain swapping . The putative SurE active site is highly conserved, and implies a model for SurE bound to a potential substrate, guanosine-5'-monophosphate (GMP) . Pae SurEalpha has optimal acid phosphatase activity at temperatures above 90 degrees C, and is less specific than Tma SurE in terms of metal ion requirements . Substrate specificity also differs between Pae and Tma SurE, with a more specific recognition of purine nucleotides by the archaeal enzyme . Analyses of the sequences, phylogenetic distribution, and genomic organization of the SurE family reveal examples of genomes encoding multiple surE genes, and suggest that SurE homologs constitute a broad family of enzymes with phosphatase-like activities. Philos Trans R Soc Lond B Biol Sci, 2003 Jan 29, 358(1429), 59 - 83; discussion 83-5 On the origins of cells: a hypothesis for the evolutionary transitions from abiotic geochemistry to chemoautotrophic prokaryotes, and from prokaryotes to nucleated cells; Martin W et al.; All life is organized as cells . Physical compartmentation from the environment and self-organization of self-contained redox reactions are the most conserved attributes of living things, hence inorganic matter with such attributes would be life's most likely forebear . We propose that life evolved in structured iron monosulphide precipitates in a seepage site hydrothermal mound at a redox, pH and temperature gradient between sulphide-rich hydrothermal fluid and iron(II)-containing waters of the Hadean ocean floor . The naturally arising, three-dimensional compartmentation observed within fossilized seepage-site metal sulphide precipitates indicates that these inorganic compartments were the precursors of cell walls and membranes found in free-living prokaryotes . The known capability of FeS and NiS to catalyse the synthesis of the acetyl-methylsulphide from carbon monoxide and methylsulphide, constituents of hydrothermal fluid, indicates that pre-biotic syntheses occurred at the inner surfaces of these metal-sulphide-walled compartments, which furthermore restrained reacted products from diffusion into the ocean, providing sufficient concentrations of reactants to forge the transition from geochemistry to biochemistry . The chemistry of what is known as the RNA-world could have taken place within these naturally forming, catalyticwalled compartments to give rise to replicating systems . Sufficient concentrations of precursors to support replication would have been synthesized in situ geochemically and biogeochemically, with FeS (and NiS) centres playing the central catalytic role . The universal ancestor we infer was not a free-living cell, but rather was confined to the naturally chemiosmotic, FeS compartments within which the synthesis of its constituents occurred . The first free-living cells are suggested to have been eubacterial and archaebacterial chemoautotrophs that emerged more than 3.8 Gyr ago from their inorganic confines . We propose that the emergence of these prokaryotic lineages from inorganic confines occurred independently, facilitated by the independent origins of membrane-lipid biosynthesis: isoprenoid ether membranes in the archaebacterial and fatty acid ester membranes in the eubacterial lineage . The eukaryotes, all of which are ancestrally heterotrophs and possess eubacterial lipids, are suggested to have arisen ca . 2 Gyr ago through symbiosis involving an autotrophic archaebacterial host and a heterotrophic eubacterial symbiont, the common ancestor of mitochondria and hydrogenosomes . The attributes shared by all prokaryotes are viewed as inheritances from their confined universal ancestor . The attributes that distinguish eubacteria and archaebacteria, yet are uniform within the groups, are viewed as relics of their phase of differentiation after divergence from the non-free-living universal ancestor and before the origin of the free-living chemoautotrophic lifestyle . The attributes shared by eukaryotes with eubacteria and archaebacteria, respectively, are viewed as inheritances via symbiosis . The attributes unique to eukaryotes are viewed as inventions specific to their lineage . The origin of the eukaryotic endomembrane system and nuclear membrane are suggested to be the fortuitous result of the expression of genes for eubacterial membrane lipid synthesis by an archaebacterial genetic apparatus in a compartment that was not fully prepared to accommodate such compounds, resulting in vesicles of eubacterial lipids that accumulated in the cytosol around their site of synthesis . Under these premises, the most ancient divide in the living world is that between eubacteria and archaebacteria, yet the steepest evolutionary grade is that between prokaryotes and eukaryotes. FEMS Microbiol Lett, 2003 Feb 14, 219(1), 47 - 52 Possible quorum sensing in the rumen microbial community: detection of quorum-sensing signal molecules from rumen bacteria; Mitsumori M et al.; The bioluminescence assay using Vibrio harveyi BB170 was used to examine quorum-sensing autoinducer 2 (AI-2) activity from cell-free culture fluids of rumen bacteria . The assay showed that the culture fluids of four species of rumen bacteria, Butyrivibrio fibrisolvens, Eubacterium ruminantium, Ruminococcus flavefaciens, and Succinimonas amylolytica, contained AI-2-like molecules . Furthermore, homologues for luxS genes were detected in rumen fluids collected from three cows and in bacterial cells of P . ruminicola subsp . ruminicola and R . flavefaciens . These findings suggest that the quorum-sensing system mediated by AI-2 is present in the rumen. RNA, 2003 Mar, 9(3), 287 - 92 Discovery and characterization of Acanthamoeba castellanii mitochondrial 5S rRNA; Bullerwell CE et al.; Although 5S rRNA is a highly conserved and universal component of eubacterial, archaeal, chloroplast, and eukaryotic cytoplasmic ribosomes, a mitochondrial DNA-encoded 5S rRNA has so far been identified only in land plants and certain protists . This raises the question of whether 5S rRNA is actually required for and used in mitochondrial translation . In the protist Acanthamoeba castellanii, BLAST searches fail to reveal a 5S rRNA gene in the complete mitochondrial genome sequence, nor is a 5S-sized RNA species detectable in ethidium bromide-stained gels of highly purified mitochondrial RNA preparations . Here we show that an alternative visualization technique, UV shadowing, readily detects a novel, mitochondrion-specific small RNA in A . castellanii mitochondrial RNA preparations, and that this RNA species is, in fact, a 5S rRNA encoded by the A . castellanii mitochondrial genome . These results emphasize the need for caution when interpreting negative results that suggest the absence of 5S rRNA and/or a mitochondrial DNA-encoded 5S rRNA sequence in other (particularly protist) mitochondrial systems. J Appl Genet, 2003, 44(1), 1 - 19 Noncoding RNA transcripts; Szymanski M et al.; Recent analyses of the human genome and available data about the other higher eukaryotic genomes have revealed that, in contrast to Eubacteria and Archaea, only a small fraction of the genetic material (ca 1.5%) codes for proteins . Most of genomic DNA and its RNA transcripts are involved in regulation of gene expression, which can be exerted at either the transcriptional level, controlling whether a gene is transcribed and to what extent, or at the post-translational level, regulating the fate of the transcribed RNA molecules, including their stability, efficiency of their translation and subcellular localization . Noncoding RNA genes produce functional RNA molecules (ncRNAs) rather than encoding proteins . These stable RNAs act by multiple mechanisms such as RNA-RNA base pairing, RNA-protein interactions and intrinsic RNA activity, as well as regulate diverse cellular functions, including RNA processing, mRNA stability, translation, protein stability and secretion . Non-protein-coding RNAs are known to play significant roles . Along with transfer RNAs, ribosomal RNAs and mRNAs, ncRNAs contribute to gene splicing, nucleotide modification, protein transport and regulation of gene expression. Mol Genet Genomics, 2003 Feb, 268(5), 628 - 36 Epub 2003 Jan 10. Diversity of group II introns in the genome of Sinorhizobium meliloti strain 1021: splicing and mobility of RmInt1; Toro N et al.; The number and diversity of known group II introns in eubacteria are continually increasing with the addition of new data from sequencing projects, but the significance of these introns in the evolution of bacterial genomes is unknown . We analyzed the main features of the group II introns present in the genome of the soil microorganism Sinorhizobium meliloti (strain 1021), the nitrogen-fixing symbiont of alfalfa, the DNA sequence of which was recently determined . Strain 1021 harbors three different classes of group II introns: RmInt1, of bacterial class D; SMb2147/SMb21167, which cluster within bacterial class C; and SMa1875, the phylogenetic class of which is uncertain . The group II introns SMb2147/SMb21167 and SMa1875 are widely distributed in S . meliloti, but are present in lower copy numbers than RmInt1 . Strain 1021 harbors three copies of RmInt1, which is pSym-specific . Although RmInt1 is spliced in strain 1021, mobility assays suggested that, in contrast to other S . meliloti strains, the genetic background of strain 1021 does not support intron homing events. Mol Genet Genomics, 2003 Feb, 268(5), 563 - 9 Epub 2003 Jan 15. The ATPase ClpX is conditionally involved in the morphological differentiation of Streptomyces lividans; Viala J et al.; ATP-dependent proteases of the ClpP type are widespread in eubacteria . These proteolytic complexes are composed of a proteolytic subunit and an ATPase subunit . They are involved in the degradation of denatured proteins, but also play a role in specific regulatory pathways . In Streptomyces lividans strains which lack the proteolytic subunit ClpP1, cell cycle progression has been shown to be blocked at early stages of growth . In this study, we examined the role of the ATPase subunit ClpX, a possible partner of the products of the clpP1 operon . A clpX mutant was obtained and it was shown that its growth was impaired only on acidic medium . Thus, the clpX phenotype differs from the clpP1 phenotype, indicating that these two components have only partially overlapping roles . We also analyzed the expression of clpX . Although clpX expression is increased under heat-shock conditions in many bacteria, we found that this is not the case in S . lividans. Plant Physiol, 2003 Feb, 131(2), 643 - 55 The Arabidopsis STICHEL gene is a regulator of trichome branch number and encodes a novel protein; Ilgenfritz H et al.; Here, we analyze the STICHEL (STI) gene, which plays an important role in the regulation of branch number of the unicellular trichomes in Arabidopsis . We have isolated the STI locus by positional cloning and confirmed the identity by sequencing seven independent sti alleles . The STI gene encodes a protein of 1,218 amino acid residues containing a domain with sequence similarity to the ATP-binding eubacterial DNA-polymerase III gamma-subunits . Because endoreduplication was found to be normal in sti mutants the molecular function of STI in cell morphogenesis is not linked to DNA replication and, therefore, postulated to represent a novel pathway . Northern-blot analysis shows that STI is expressed in all organs suggesting that STI function is not trichome specific . The analysis of sti alleles and transgenic lines overexpressing STI suggests that STI regulates branching in a dosage-dependent manner. J Biol Chem, 2003 May 2, 278(18), 15622 - 32 Epub 2003 Feb 13. Complementary impact of paralogous Oxa1-like proteins of Bacillus subtilis on post-translocational stages in protein secretion; Tjalsma H et al.; In mitochondria, chloroplasts, and Gram-negative eubacteria, Oxa1p(-like) proteins are critical for the biogenesis of membrane proteins . Here we show that the Gram-positive eubacterium Bacillus subtilis contains two functional Oxa1p orthologues, denoted SpoIIIJ and YqjG . The presence of either SpoIIIJ or YqjG is required for cell viability . Whereas SpoIIIJ is required for sporulation, YqjG is dispensable for this developmental process . The stability of two membrane proteins was found to be mildly affected upon SpoIIIJ limitation in the absence of YqjG . Surprisingly, the topology and stability of other membrane proteins remained unaffected under these conditions . In contrast, SpoIIIJ- and YqjG-limiting conditions resulted in a strong post-translocational defect in the stability of secretory proteins . Together, these data indicate that SpoIIIJ and YqjG of B . subtilis are involved in both membrane protein biogenesis and protein secretion . However, the reduced stability of secretory proteins seems to be the most prominent phenotype of SpoIIIJ/YqjG-depleted B . subtilis cells . In conclusion, our observations show that SpoIIIJ and YqjG have different, but overlapping functions in B . subtilis . Most importantly, it seems that different members of the Oxa1p protein family have acquired at least partly distinct, species-specific, functions that are essential for life. J Biol Chem, 2003 Apr 25, 278(17), 14739 - 46 Epub 2003 Feb 12. Active lipoprotein precursors in the Gram-positive eubacterium Lactococcus lactis; Venema R et al.; Lipid-modified proteins play important roles at the interface between eubacterial cells and their environment . The importance of lipoprotein processing by signal peptidase II (SPase II) is underscored by the fact that this enzyme is essential for viability of the Gram-negative eubacterium Escherichia coli . In contrast, SPase II is not essential for growth and viability of the Gram-positive eubacterium Bacillus subtilis . This could be due to alternative amino-terminal lipoprotein processing, which was shown previously to occur in SPase II mutants of B . subtilis . Alternatively, uncleaved lipoprotein precursors might be functional . To explore further the importance of lipoprotein processing in Gram-positive eubacteria, an SPase II mutant strain of Lactococcus lactis was constructed . Although some of the 39 (predicted) lactococcal lipoproteins, such as PrtM and OppA, are essential for growth in milk, the growth of SPase II mutant L . lactis cells in this medium was not affected . Furthermore, the activity of the strictly PrtM-dependent extracellular protease PrtP, which is required for casein degradation, was not impaired in the absence of SPase II . Importantly, no alternative processing of pre-PrtM and pre-OppA was observed in cells lacking SPase II . Taken together, these findings show for the first time that authentic lipoprotein precursors retain biological activity. Lett Appl Microbiol, 2003, 36(3), 145 - 9 Digoxigenin-labelled peptide nucleic acid to detect lactobacilli PCR amplicons immobilized on membranes from denaturing gradient gel electrophoresis; Burton JP et al.; AIMS: To develop a digoxigenin (DIG)-labeled peptide nucleic acid (PNA) probe for the detection of Lactobacillus-related genera amongst eubacterial amplicons obtained from vaginal samples using denaturing gradient gel electrophoresis (DGGE) blots . METHODS AND RESULTS: Part of the 16S rRNA gene sequence was used as a target for the PNA probe . After confirming probe specificity using chromosomal DNA from species and isolates that have been detected in the urogenital tract, it was successfully used to detect lactobacilli amplicons generated using eubacterial-specific 16S rRNA gene-targeted primers from vaginal tract samples immobilized on membranes from DGGE . CONCLUSIONS: The Lactobacillus-specific PNA probe could distinguish between DNA fragments from lactobacilli in a DGGE gel from other bacterial species, including those that migrated to a similar position . SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the DIG-labelled PNA probe on blots of eubacterial PCR products from DGGE gels can be used to specifically detect lactobacilli in complex vaginal samples. Water Sci Technol, 2003, 47(1), 283 - 90 A culture-independent approach for studying microbial diversity in aerobic granules; Yi S et al.; This study reports the use of ribosomal-based molecular techniques to study the microbial diversity in aerobic granules . Aerobic granules at different growth stages (young, mature and old) were obtained from a laboratory scale sequential aerobic sludge blanket (SASB) bioreactor fed with glucose as the main source of carbon and energy . Deoxyribonucleic acid (DNA) was extracted from the young, mature and old granules . The polymerase chain reaction (PCR) was used to amplify the Eubacterial 16S ribosomal DNA (rDNA) and three clone libraries were constructed, corresponding to each of the three growth stages . The microbial diversity in each clone library was assessed by amplified ribosomal DNA restriction analysis (ARDRA) . The results reveal that there was considerable diversity in each clone library and there were variations in microbial diversity among the three different clone libraries . This suggests a shift in the composition of the microbial communities . Microorganisms associated with 5 restriction fragment length polymorphism (RFLP) types (A, B, C, D and E) appear to play an important role in the development of aerobic granules. Microbiology, 2003 Jan, 149(Pt 1), 67 - 75 Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens; Hutter G et al.; Subgingival plaque samples were obtained from 26 subjects with advanced periodontal lesions . Bacterial diversity was analysed by amplification of the 16S rRNA genes with two different primer sets, and subsequent cloning and sequencing . A total of 578 sequences was analysed after the exclusion of chimeras . The authors found 148 phylotypes with the clone library 27f/519r (number of clones n=322; coverage, C=66 %) and 75 phylotypes with the clone library 515f/1525r (n=256; C=84 %) . Comparative sequence analysis revealed that 70 % of all of the analysed sequences showed a similarity of at least 99 % to sequences deposited in public databases . The classes Actinobacteria, Bacilli, Bacteroidetes, Clostridia, Deferribacteres, Flavobacteria, Fusobacteria, Mollicutes, Spirochaetes and all classes of the Proteobacteria were represented . Sequences that were at least 99 % identical to Porphyromonas gingivalis, Filifactor alocis and Treponema socranskii were present in at least one-third of the patients . Libraries generated with the two PCR primer pairs differed significantly in their representation of the families Porphyromonadaceae, Prevotellaceae, Fusobacteriaceae, Eubacteriaceae, Streptococcaceae and ACIDAMINOCOCCACEAE: A total of 14 sequences exhibited less than 97 % identity to sequences published previously and were assigned to six different families within the phyla Bacteroidetes and FIRMICUTES: Twelve of 20 putative pathogens were recovered, which were recently proposed to be associated with periodontitis. J Clin Microbiol, 2003 Feb, 41(2), 558 - 63 Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients; Kazor CE et