Biosci Biotechnol Biochem, 1994 Dec, 58(12), 2193 - 6 Levoglucosan dehydrogenase involved in the assimilation of levoglucosan in Arthrobacter sp . I-552; Nakahara K et al.; A levoglucosan (1,6-anhydro-beta-D-glucopyranose)-using bacterium, isolated from soil, was identified . It was shown to belong to the genus Arthrobacter and tentatively named Arthrobacter sp . I-552 . A novel enzyme catalyzed the dehydrogenation of levoglucosan to form 1,6-anhydro-beta-D-ribo-hexopyranos-3-ulose (3-keto levoglucosan), using NAD+ as an electron acceptor, i.e . NAD+: 1,6-anhydro-beta-D-glucopyranose oxidoreductase (trivial name: levoglucosan dehydrogenase) . This enzyme was purified and characterized . A possible reaction scheme for the glucose formation was proposed . This pathway for levoglucosan use is distinct from those in yeast and fungi. Biotechnology (N Y), 1994 Dec, 12(13), 1361 - 5 Detoxification of the plant toxin fluoroacetate by a genetically modified rumen bacterium; Gregg K et al.; We isolated the fluoroacetate dehalogenase gene (H1), from Moraxella species strain B, and placed it under the transcriptional control of a 154 bp fragment of the erm gene promoter . The promoter/gene construct was attached to the Butyrivibrio fibrisolvens shuttle vector pBHerm, and the resulting dehalogenase expression plasmid (pBHf) was transferred to B . fibrisolvens OB156 by electroporation . The erm gene promoter directed expression of dehalogenase activity in both E . coli and B . fibrisolvens OB156 . Cell-free lysates of the genetically modified OB156 defluorinated 10.6 nmol fluoroacetate/min/mg protein . Growing cultures of OB156 were able to detoxify fluoroacetate in the culture medium, at the rate of 9.9 nmol/min/mg . Plasmid pBHf was retained by 100% of OB156 cells after 500 generations of non-selective culture . The restriction pattern of pBHf remained unchanged after extensive non-selective growth and host bacteria continued to produce active dehalogenase . The construction of rumen bacteria that are able to detoxify an important natural poison supports the feasibility of using genetically modified rumen bacteria to aid animal production. Behring Inst Mitt, 1994 Dec, (95), 42 - 8 Pathogenesis of Helicobacter pylori and perspectives of vaccine development against an emerging pathogen; Rappuoli R et al.; Infection of the stomach and the duodenum by Helicobacter pylori is the major cause of acute and chronic gastroduodenal pathologies in humans and increases the risk of gastric cancer . The recognition of the infectious nature of the illness is having a major impact in the management of the disease that is shifting from the treatment of symptoms by anti-H2 blockers to the eradication of the bacterial infection by antibiotic regimen . Experience with other bacterial diseases, suggests that antibiotic treatment will select resistant strains that in the long term will make the antibiotics infective . Vaccination that classically is the most effective way to prevent and control infectious diseases in large population, could be used to prevent infection and possibly also to treat the disease . Here we summarize the studies on the identification and characterization of the virulence factors that are important for the pathogenesis of the bacterium and that may be candidate components for a vaccine . Animal models of the infection are also described. Eur J Gastroenterol Hepatol, 1994 Dec, 6 Suppl 1, S67 - 71 The use of a mouse model in the study of Helicobacter sp.-associated gastric cancer; Lee A; HYPOTHESIS: Long-term infection with Helicobacter pylori and the associated gastritis is now thought to cause a predisposition to gastric cancer through cellular changes resulting from inflammatory damage or because of direct effects of the bacterium . MICE AS MODELS FOR H . PYLORI-ASSOCIATED GASTRIC CANCER: Long-term infection of conventional Swiss mice with either H . felis or H . heilmannii results in atrophic gastritis . Infection of specific pathogen-free Balb/c mice results in the development of lesions similar to H . pylori-associated low-grade B-cell gastric lymphomas . CONCLUSION: H . pylori-infected mice appear to be excellent models for the study of tumours induced by this bacterium. Eur J Gastroenterol Hepatol, 1994 Dec, 6 Suppl 1, S57 - 65 Gastric disease in ferrets: effects of Helicobacter mustelae, nitrosamines and reconstructive gastric surgery; Fox JG; PURPOSE: Animal models are being used to study the mechanisms by which Helicobacter spp . induce gastric disease . To assess the effects of a natural gastric pathogen, Helicobacter mustelae, in the development of chronic gastritis, premalignancy and cancer, the ferret model was studied under natural and experimental conditions . ANIMALS AND METHODS: H . mustelae-infected ferrets were used to study the metabolism of nitrates/nitrites, which are dietary and endogenously formed substances that have been linked to gastric cancer . The ferret was also manipulated by performing gastric reconstructive surgery to study the processing of nitrite and nitrate and to assess the effect of surgery on gastric pathology . In addition, the ferret was tested for its suitability as an animal model for the induction of gastric cancer by oral dosing with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The influence of these variables on gastric pathology and/or metabolic outcomes was examined, and the results in ferrets were compared to findings in humans . RESULTS AND CONCLUSIONS: The ferret appears to be an ideal model for studying various gastric parameters and how these factors influence the development of H . mustelae-associated gastric disease . Gastric reconstructive surgery did not effect nitrite processing or overall severity of gastritis in ferrets . However, a single dose of MNNG (50 mg/kg) produced an unprecedented 90% gastric carcinoma in H . mustelae-infected ferrets . This implies that chronic inflammation induced by the bacterium is a cofactor in gastric carcinogenesis. Biol Pharm Bull, 1994 Dec, 17(12), 1676 - 8 A convenient screening test for hypoxic cell radiosensitizers/cytotoxins; Hori H et al.; A convenient in vitro screening test using E . coli B/r for evaluating a variety of hypoxic cell radiosensitizers/hypoxic cell cytotoxins has been developed for the initial selection of candidates in medicinal/organic chemistry laboratories . E . coli cells were used for convenience since: (1) the bacterium is grown using commercially available broths, where it multiplies rapidly, and requires little specialized equipment for growth and handling . (2) More is known about the genetics and biochemistry of the radiation damage to these cells and their repair than any other organism. Rev Sci Tech, 1994 Dec, 13(4), 1175 - 99 Nematocera (Ceratopogonidae, Psychodidae, Simuliidae and Culicidae) and control methods; Braverman Y; The biology, veterinary importance and control of certain Nematocera are described and discussed . Culicoides spp . (family Ceratopogonidae) transmit the arboviruses of bluetongue (BT), African horse sickness (AHS), bovine ephemeral fever (BEF) and Akabane . Some other arboviruses have been isolated from these species, while fowl pox has been transmitted experimentally by Culicoides . These insects are vectors of the parasitic protozoans Leucocytozoon caulleryi and Haemoproteus nettionis, and the parasitic nematodes Onchocerca gutturosa, O . gibsoni and O . cervicalis . They also cause recurrent summer hypersensitivity in horses, ponies, donkeys, cattle and sheep . Farm animals can die as a result of mass attack by Simulium spp., which are also vectors of Leucocytozoon simondi, L . smithi and the filariae O . gutturosa, O . linealis and O . ochengi . Venezuelan equine encephalomyelitis (VEE) and Rift Valley fever (RVF) have been isolated from simuliids, and vesicular stomatitis virus New Jersey strain has been replicated in Simulium vittatum . Simuliids are well known as vectors of O . volvulus, the cause of human onchocercosis (river blindness) . The family Psychodidae includes the genera Phlebotomus and Lutzomyia (subfamily Phlebotominae), vectors of Leishmania spp . in humans, dogs and other mammals . Vesicular stomatitis virus Indiana strain has been regularly isolated from phlebotomine sandflies . Mass attack by mosquitoes can also prove fatal to farm animals . Mosquitoes are vectors of the viruses of Akabane, BEF, RVF, Japanese encephalitis, VEE, western equine encephalomyelitis, eastern equine encephalomyelitis and west Nile meningoencephalitis, secondary vectors of AHS and suspected vectors of Israel turkey meningoencephalitis . The viruses of hog cholera, fowl pox and reticuloendotheliosis, the rickettsiae Eperythrozoon ovis and E . suis, and the bacterium Borrelia anserina are mechanically transmitted by mosquitoes . These insects also induce allergic dermatitis in horses . They transmit several filarial worms of both animals and humans, and are of great medical importance as vectors of major human diseases, including malaria, yellow fever, dengue fever and many more diseases caused by arboviruses. Microbiologia, 1994 Dec, 10(4), 395 - 402 Autolytic effect of the antibiotic produced by Myxococcus coralloides D; Montoya MD et al.; Myxococcus coralloides D secretes an antibiotic, named corallolysin, when grown on a rich medium . When a critical concentration is reached, this antibiotic lyses the producer bacterium either during vegetative growth or during morphogenesis . Corallolysin has not effect on resting cells nor on myxospores . The autolytic effect is caused by the early inhibition of RNA synthesis. Experientia, 1994 Nov 30, 50(11-12), 1054 - 60 Heat shock proteins in immune response to cancer: the Fourth Paradigm; Srivastava PK; The involvement of heat shock proteins in immune response is categorized into four distinct paradigms . In the First Paradigm, HSP derived from foreign organisms act as classical foreign antigens, and they elicit immune response to the non-conserved HSP epitopes . The Second Paradigm refers to instances where the host responds to self HSP to which there is no central or peripheral tolerance . The Third Paradigm involves molecular mimicry, where cross-reactivity between an HSP and another protein leads to an immune response to the latter under conditions which elicit an immune response to the former, such as infection with a bacterium whose immunodominant antigen is an HSP . The Fourth Paradigm refers to situations where an HSP-antigen complex elicits an effective response to the antigen and not to the HSP . Thus the HSP acts as a carrier for the antigenic peptide . The role of HSP in recognition by gamma delta T cells may also fall into this paradigm . In this article, the Fourth Paradigm is considered as a crucial element in the development of vaccines against cancers and infectious diseases, and is analyzed through the prism of the observed association of hsp70 species with antigenic peptides. J Biotechnol, 1994 Nov 30, 38(1), 11 - 9 Thermostable N-carbamoyl-D-amino acid amidohydrolase: screening, purification and characterization; Ogawa J et al.; A thermostable N-carbamoyl-D-amino acid amidohydrolase was found in the cells of newly isolated bacterium . Blastobacter sp . A17p-4 . The bacterium also showed D-specific hydantoinase activity . The N-carbamoyl-D-amino acid amidohydrolase activity of the cells exhibited a temperature optimum at 50-55 degrees C, and was stable up to 50 degrees C . The N-carbamoyl-D-amino acid amidohydrolase of Blastobacter sp . A17p-4 was purified to homogeneity and characterized . It has a relative molecular weight of about 120,000 and consists of three identical subunits with a relative molecular weight of about 40,000 . N-Carbamoyl-D-amino acids having hydrophobic groups served as good substrates for the enzyme . It has been suggested that D-amino acid production from DL-5-substituted hydantoin involves the action of a series of enzymes involved in pyrimidine degradation, namely amide-ring opening enzyme, dihydropyrimidinase, and N-carbamoylamide hydrolyzing enzyme, beta-ureidopropionase . However, the purified enzyme did not hydrolyze beta-ureidopropionate; suggesting that the N-carbamoyl-D-amino acid amidohydrolase coexisting with D-specific hydantoinase, probably dihydropyrimidinase, in Blastobacter sp . A17p-4 is different from beta-ureidopropionase. Biochemistry, 1994 Nov 22, 33(46), 13668 - 77 QA binding in reaction centers of the photosynthetic purple bacterium Rhodobacter sphaeroides R26 investigated with electron spin polarization spectroscopy; van den Brink JS et al.; The relation between quinone (QA) binding and electron transport in reaction centers (RCs) of photosynthetic purple bacteria is investigated, using electron spin polarization (ESP) X-band (9 GHz) EPR as a tool to probe for structural changes resulting from charge separation and stabilization and from replacing the native QA molecule with other quinones . We present a study of possible changes in QA-binding that might be responsible for the remarkably prolonged lifetime of the charge-separated state at cryogenic temperatures for RCs of Rhodobacter sphaeroides R26 cooled under illumination {Kleinfeld, D., et al . (1984) Biochemistry 23, 5780-5786} . It is shown that this effect is not caused by a major reorientation of the chromophores . Furthermore, we studied the effects of structurally different quinones functioning as primary electron acceptor in different purple bacteria . With simulations of ESP X-band spectra of the spin-polarized secondary radical pair P.+QA.+- in menaquinone-reconstituted, Zn(2+)-substituted RCs of Rb . sphaeroides R26, we show that quinone reconstitution is highly selective for site and orientation . Furthermore, we find that a very small exchange interaction between P.+ and QA.+- (magnitude of JPQ approximately 1 microT) is needed to account accurately for the observed relative line intensities at X-band, without affecting the accuracy of the simulations of reported ESP K-band spectra {Fuchsle, G., et al . (1993) Biochim . Biophys . Acta 1142, 23-35; Van der Est, A., et al . (1993) Chem . Phys . Lett . 212, 561-568} . This pronounced influence of small values for JPQ on the X-band ESP line shape results from cancellation effects of absorptive and emissive contributions to the spectrum, such that small shifts can be observed . The exchange interaction has opposite sign for the native, ubiquinone-containing RC {viz . JP.UQ = (-0.8 +/- 0.2) microT} and the menaquinone-substituted RC {JP.MK = (+0.3 +/- 0.2) microT} . The implications of these observations for electron-transport theory are discussed. Biochemistry, 1994 Nov 22, 33(46), 13517 - 23 Relationship between rate and free energy difference for electron transfer from cytochrome c2 to the reaction center in Rhodobacter sphaeroides; Lin X et al.; The rate of electron transfer from cytochrome c2 to the bacteriochlorophyll dimer of the reaction center from the photosynthetic bacterium Rhodobacter sphaeroides has been investigated using time-resolved optical spectroscopy . Measurements were performed on a series of mutant reaction centers in which the midpoint potentials of the bacteriochlorophyll dimer vary over a range of 350 mV . Dramatic changes in the characteristic time of electron transfer were observed, with the measured values ranging from 7730 to 80 ns compared to 960 ns for wild type . The binding constants (0.15 to 0.25 microM-1) and the second-order rate constants for the slow component (5.5 x 10(8) to 9.4 x 10(8) M-1 s-1) for the mutants are similar to the corresponding values for wild type (0.35 microM-1 and 11 x 10(8) M-1 s-1), indicating that the binding of the cytochrome to the reaction center is not changed in the mutants . In the mutants with the fastest rates, an additional minor component was resolved that is probably due to formation of a reaction center-cytochrome complex in an unfavorable configuration with a binding constant an order of magnitude weaker than the major component . The altered midpoint potentials in the mutants result in values for the free energy difference for this electron transfer reaction ranging from -65 to -420 meV compared to -160 meV for wild type . The relationship between the rate and free energy difference was well fit by a Marcus equation using a reorganization energy of 500 meV.(ABSTRACT TRUNCATED AT 250 WORDS) J Immunol, 1994 Nov 15, 153(10), 4588 - 95 CD8+ T lymphocyte-mediated lysis of Chlamydia-infected L cells using an endogenous antigen pathway; Beatty PR et al.; Intracellular bacterial pathogens have evolved to either grow in the nutrient-rich cytoplasm or remain sequestered within a vacuole . One potentially important selective advantage for growth within a vacuole may be evasion of cell-mediated detection and cytolysis . To address this question we used the endosomally confined bacterium Chlamydia trachomatis, which naturally infects epithelial cells, to examine CTL-mediated lysis of nonphagocytic cells . CTL-mediated lysis of infected target cells was detected, although the increased expression of ICAM-1 by transfection was required . The elimination of CD8+ T cells or addition of brefeldin A or cycloheximide eliminated specific cytolysis, whereas conversely, treatment with chloroquine or ammonium chloride had only minor effects . These results implicate endogenous Ag processing for Chlamydia-specific cytolysis . This work demonstrates CTL-mediated lysis of cells infected with an intracellular bacterium that inhibits lysosomal fusion and is confined to an endosomal vacuole. Biochemistry, 1994 Nov 15, 33(45), 13329 - 39 Reconstitution of a functional photosynthetic receptor complex with isolated subunits of core light-harvesting complex and reaction centers; Bustamante PL et al.; The B820 subunit form of the core light-harvesting complex LHI, isolated from the photosynthetic bacterium Rhodospirillum rubrum, was combined in a reassociation assay with the reaction center (RC) isolated from the same or related bacteria . This reassociation produced a photoreceptor complex (PRC) which appeared, by absorption spectroscopy, circular dichroism measurements, and kinetic absorption spectroscopy measuring transient photochanges, as analogous to the PRC in the intact bacteria . Energy transfer between the LHI and reaction center progressed with almost 100% efficiency and indicated a cooperative pattern of transfer . Treatment of the RC with proteinase K resulted in peptide cleavages of all three polypeptides of the RC but did not alter the light-induced charge separation in the RC or prevent the reassociation of the LHI and modified RC . Energy transfer efficiency from LHI to RC still approached 100% but the cooperative behavior seen in reconstitutions with the intact RC was not observed . Initial experiments using interspecies reassociations (LHI from Rhodobacter sphaeroides and RC from Rs . rubrum) showed a low efficiency of energy transfer from LHI to RC . Possible association domains for the LHI-RC interaction based on considerations of the comparative amino acid sequences of the RC of each bacteria and the most feasible remaining residues in the proteinase K treated RC are considered. Biochemistry, 1994 Nov 8, 33(44), 13022 - 31 Site-directed mutagenesis of arginine-114 and tryptophan-129 in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides: two highly conserved residues predicted to be near the cytoplasmic surface of putative transmembrane helices B and C; Hacker B et al.; The cytochrome b subunit of the ubiquinol:cytochrome c oxidoreductase (the bc1 complex) contains two heme prosthetic groups, cytochrome bL and cytochrome bH . In addition, this subunit also provides major elements of the quinol oxidation site (Qo) and a separate quinone reductase site (Qi), which are thought to be located on opposite sides of the membrane . Site-directed mutagenesis has been used to explore the role(s) of specific amino acid residues in this subunit from the photosynthetic bacterium Rhodobacter sphaeroides . Previous work identified five residues, Gly48 (Gly33), Ala52 (Gly37), His217 (His202), Lys251 (Lys228), and Asp252 (Asp229), as being either at or near the quinone reductase site (the residue numbers in parentheses designate the equivalent positions in the yeast mitochondrial enzyme) . These residues are predicted to be near the cytoplasmic boundaries of transmembrane helices: helix A (G48, A52), helix D (H217), or helix E (K251, D252) . In the current work, the importance of two additional highly conserved residues, which are also predicted to be near the cytoplasmic boundaries of transmembrane helices, is explored by site-directed mutagenesis . R114 (helix B) has been substituted with K, Q, and A, and W129 (helix C) has been changed to A and F . The results suggest that a positively charged residue at position 114 is important . The R114K mutation causes only subtle effects, which appear to be localized to cytochrome bH and the quinone reductase site . In contrast, R114Q is not assembled, and R114A, although partially assembled, is nonfunctional and appears to have a very low amount of cytochrome b associated with the complex . Both mutants at position 129 (W129A and W129F) are able to support the photosynthetic growth of the organism, but show abnormal characteristics . The defects associated with the W129A mutation appear to be primarily associated with the quinone reductase site and cytochrome bH, whereas the W129F mutation appears to result in more global defects that also perturb the cytochrome bL locus . The results are consistent with the placement of residues R114 and W129 near the cytoplasmic side of the membrane, but suggest that these residues are important for the assembly and overall stability of the complex. Clin Diagn Lab Immunol, 1994 Nov, 1(6), 645 - 9 Serologic diagnosis of human monocytic ehrlichiosis by immunoblot analysis; Brouqui P et al.; Human monocytic ehrlichiosis is caused by Ehrlichia chaffeensis, an intracellular bacterium probably transmitted by the tick Amblyomma americanum in the United States . Despite its lack of specificity in discriminating among infections by closely related Ehrlichia spp., immunofluorescence assay (IFA) is the most frequently used serological diagnostic method . To improve the specificity of the serological diagnosis, we compared antigenic profile of E . canis and E . chaffeensis antigen with homologous and heterologous sera, searching for the specificity of the presence of low-molecular-weight proteins . Western immunoblot analysis of IFA-positive human sera revealed 27- and 29-kDa proteins which are not found in E . canis IFA-positive sera from dogs . IFA-positive sera from dogs revealed a low-molecular-weight group of proteins (20 to 28 kDa) which were not found in human E . chaffeensis-positive sera except for a weak band at 22 kDa . The presence o antibodies directed against the 27- and 29-kDa proteins on Western blots is specific for E . chaffeensis infection, and we suggest that the Western blot might complete IFA in cases with low positive predictive value. Am J Med, 1994 Nov, 97(5), 451 - 8 Immunohistologic demonstration of Coxiella burnetii in the valves of patients with Q fever endocarditis; Brouqui P et al.; PURPOSE: Cardiac valves that were resected from patients with Q fever endocarditis were examined by immunohistologic methods to correlate the presence of Coxiella burnetii in the valves with the histopathologic, serologic, microbiologic, and clinical findings . PATIENTS: Seventeen patients with serologic and microbiologic or clinical evidence of Q fever endocarditis who presented with cardiac failure secondary to valvular dysfunction and required valve replacement surgery were selected from the clinical records of the Unite des Rickettsies, Marseille, France . METHODS: Clinical data were collected by questionnaire . Serologic characterization was performed by indirect immunofluorescent antibody testing; shell vial cultivation of C burnetii was performed from resected valves and blood when available; and pathologic and immunohistologic testing for localization of C burnetti in resected valves were performed by standard methods using both polyclonal and monoclonal C burnetti antibodies . RESULTS: Demographic and clinical findings were typical of patients with Q fever endocarditis . Pure chronic inflammation or mixtures of acute and chronic inflammation were the most frequent inflammatory patterns present and were associated with fibrin deposition, necrosis, and fibrosis . Well-formed granulomas were not present, but the granulomatous inflammation observed in 6 of these 17 patients was associated with foreign body reactions or with valvular calcifications secondary to preexisting valvular damage and could not be directly attributed to infection . C burnetii were present nearly exclusively in macrophages in sites of inflammation and valvular injury and only in the vegetations . Immunohistologic results confirmed the valve culture results in 10 of 14 cases . CONCLUSION: The pathologic findings in the valves of patients with Q fever endocarditis are nonspecific . The presence of empty or foamy macrophages is suggestive of infection by C burnetii; however, definitive identification rests upon the demonstration of the organism in the tissue by immunohistology . Q fever endocarditis probably results from infection of previously damaged heart valves . The finding of the absence of granulomas in these cases contrasts with the pathologic findings in patients with acute, self-limited Q fever and suggests an aberrant host immune response that permits persistence of the bacterium and chronic, prolonged valvular infection and injury . The pathologic findings and distribution of C burnetii in the damaged valve tissues explain the clinical findings of valve failure and occasional embolic episodes, as well as the frequent ability to isolate C burnetii from the peripheral blood of infected patients . Immunohistology may be a valuable diagnostic tool in places where serology and culture are not available. J Bacteriol, 1994 Nov, 176(22), 6936 - 43 Characterization of a light-responding trans-activator responsible for differentially controlling reaction center and light-harvesting-I gene expression in Rhodobacter capsulatus; Buggy JJ et al.; The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus regulates synthesis of its photosystem in response to two environmental stimuli, oxygen tension and light intensity . Here we describe the identification and characterization of the trans-acting regulatory gene hvrA, which we show is involved in differentially controlling reaction center and light-harvesting gene expression in response to alterations in light intensity . An hvrA mutant strain is shown to lack the capability to trans-activate light-harvesting-I and reaction center gene expression but retain normal light-harvesting-II and photopigment regulation, in response to a reduction in light intensity . As a consequence of altered expression, hvrA mutant strains exhibit reduced photosynthetic growth capabilities under dim-light conditions . The results of this study and additional studies indicate that regulated synthesis of the photosystem involves complex sets of overlapping regulatory circuits that differentially control photosystem gene expression in response to environmental stimuli such as oxygen tension and light intensity. J Bacteriol, 1994 Nov, 176(21), 6776 - 80 A physical map of the hyperthermophilic bacterium Aquifex pyrophilus chromosome; Shao Z et al.; A genomic map of the hyperthermophilic hydrogen-oxidizing bacterium Aquifex pyrophilus was established with NotI (GC/GGCCGC), SpeI (A/CTAGT), and XbaI (T/CTAGA) . Linking clones and cross-hybridization of restriction fragments revealed a single circular chromosome of 1.6 Mbp . A single flagellin gene and six rRNA gene units were located on this map by Southern hybridization. J Bacteriol, 1994 Nov, 176(21), 6636 - 43 Molecular cloning and characterization of outer membrane protein E of Moraxella (Branhamella) catarrhalis; Bhushan R et al.; Outer membrane protein E (OMP E) is a 50-kDa protein of Moraxella (Branhamella) catarrhalis . It is a potential vaccine antigen because it is expressed on the surface of the bacterium and has antigenic determinants which are conserved among most strains of M . catarrhalis . To clone the gene encoding OMP E, an EMBL-3 genomic library of strain 25240 was screened with a family of degenerate oligonucleotides based on the amino-terminal protein sequence . The OMP E gene was identified in one of the six positive clones by Southern blot analysis . An open reading frame of 1,377 bp encoding a protein of 460 amino acids was identified . The calculated molecular mass of the mature protein of 436 amino acid residues was 47.03 kDa, which correlated well with the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The protein product of the OMP E gene had a leader peptide of 25 amino acids and a signal peptidase 1 cleavage site similar to those of known OMPs of Escherichia coli . The transcription initiation site of the OMP E gene was mapped by primer extension to be 78 nucleotides upstream of the ATG start codon . Borderline homology was found to the FadL protein of E . coli (49.1% similarity and 25.6% identity), which is involved in the binding and transport of fatty acids . Analysis of restriction fragment length polymorphisms of the OMP E genes of 19 different strains of M . catarrhalis showed that the OMP E gene is highly conserved . The high degree of conservation of sequences of the OMP E genes of M . catarrhalis from diverse sources, along with earlier observations that the protein contains antigenic determinants on the bacterial surface, indicates that OMP E should be studied further as a potential vaccine antigen. Infect Immun, 1994 Nov, 62(11), 4804 - 10 Abrogation of gamma interferon-induced inhibition of Ehrlichia chaffeensis infection in human monocytes with iron-transferrin; Barnewall RE et al.; Ehrlichia chaffeensis is an obligate intracellular bacterium which infects cells of the macrophage/monocyte lineage . To test whether gamma interferon (IFN-gamma) inhibits infection of monocytes with E . chaffeensis, human peripheral blood monocytes were incubated with recombinant human IFN-gamma for 3 h and then exposed to E . chaffeensis . With 2,000 U of IFN-gamma per ml, maximal inhibition of infection by E . chaffeensis was observed . THP-1 cells, a human monocyte cell line, pretreated with phorbol myristic acetate or not pretreated, were incubated with various concentrations of IFN-gamma . Maximum inhibition was obtained at 1,000 U of IFN-gamma per ml with phorbol myristic acetate-treated THP-1 cells . However, nontreated cells did not achieve a similar level of anti-ehrlichial activity even with 10,000 U of IFN-gamma per ml . IFN-gamma given within 6 h postinfection was effective in inhibiting E . chaffeensis . Nitric oxide production was not demonstrated in the monocyte medium incubated with IFN-gamma and E . chaffeensis . None of the reactive oxygen intermediate scavengers tested blocked the IFN-gamma-induced anti-ehrlichial activity . Deferoxamine, an intracellular iron chelator, at 15 microM completely inhibited the survival of E . chaffeensis . Iron-saturated transferrin at 1.67 mg/ml completely reversed the IFN-gamma-induced ehrlichial killing . These results indicate that (i) E . chaffeensis is sensitive to intracytoplasmic iron depletion, (ii) E . chaffeensis is sensitive to IFN-gamma-induced killing, and (iii) the anti-ehrlichial activity induced in human monocytes by IFN-gamma is mediated by limitation of available cytoplasmic iron and is not due to the generation of reactive oxygen intermediates or nitric oxide. Proteins, 1994 Nov, 20(3), 283 - 6 Crystallization of the reaction center from Rhodobacter sphaeroides in a new tetragonal form; Allen JP; The reaction center from the nonsulfur purple bacterium Rhodobacter sphaeroides has been crystallized in a new form . The crystals grew in the presence of polyethylene glycol 4000, the detergent beta-octyl glucoside, and the amphiphiles heptane triol and benzamidine hydrochloride, using the sitting drop method . The space group of these crystals is tetragonal, P4(1)(4(3))2(1)2, and the cell constants area a = b = 141.5 A and c = 276.7 A with probably 2 proteins per asymmetric unit . A native data set has been set collected to a resolution of 2.8 A consisting of 56,332 unique reflections (50,731 with F > 2 sigma) with an Rsym of 9.5% . Analysis of the diffraction data is underway using molecular and isomorphous replacement. Mol Microbiol, 1994 Nov, 14(4), 785 - 95 Genes required for both gliding motility and development in Myxococcus xanthus; MacNeil SD et al.; Myxococcus xanthus cells can glide both as individual cells, dependent on Adventurous motility (A motility), and as groups of cells, dependent upon Social motility (S motility) . Tn5-lac mutagenesis was used to generate 16 new A- and nine new S- mutations . In contrast with previous results, we find that subsets of A- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation . All S- mutants are defective in fruiting body morphogenesis, consistent with previous results . Whereas some S- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores . Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis . Subsets of both A and S genes are essential for sporulation . Three S::Tn5-lac insertions result in surprising phenotypes . Colonies of two S- mutants glide on 'swim' (0.35% agar) plates to form fractal patterns . These S- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading . An otherwise wild-type strain with one S- insertion resembles the frz- sglA1- mutants upon development, suggesting that this S- gene defines a new chemotaxis component in M . xanthus. Proc Nutr Soc, 1994 Nov, 53(3), 605 - 14 Micronutrients and cancer aetiology: the epidemiological evidence; Key T; Micronutrient deficiencies occur most commonly in poor countries and, therefore, are most likely to be associated with cancers common in these countries . Epidemiological studies are hampered by inaccurate measurement of micronutrient intake and by the correlations between intakes of many nutrients . The strongest evidence for a protective effect of micronutrients is for oesophageal cancer . The identity of the micronutrients is not certain, but may include retinol, riboflavin, ascorbic acid and Zn; alcohol, smoking and dietary nitrosamines increase the risk for oesophageal cancer . For stomach cancer there is good evidence that fruit and vegetables are protective . The protective effect of these foods might be largely due to ascorbic acid, but other nutrients and non-nutrients may also be important; the risk for stomach cancer is increased by salt, some types of preserved foods, and by infection of the stomach with the bacterium Helicobacter pylori . The risk for lung cancer appears to be reduced by a high intake of fruit and vegetables, but it is not clear which agents are responsible and the major cause of lung cancer is cigarette smoking . Diet is probably the major determinant of the risk for colo-rectal cancer; there is evidence that fruit and vegetables and fibre reduce risk and that meat and animal fat increase risk, but there is no convincing evidence that these relationships are mediated by micronutrients . The risk for cervical cancer is inversely related to fruit and vegetable consumption and, therefore, to consumption of carotenoids and ascorbic acid, but the major cause of this cancer is human papillomavirus and it is not yet clear whether the dietary associations indicate a true protective effect or whether they are due to confounding by other variables . The evidence that micronutrients are important in the aetiology of either breast cancer or prostate cancer is weak, but the possible roles of 1,25-dihydroxycholecalciferol and alpha-tocopherol in prostate cancer require further study. Biofizika, 1994 Nov-Dec, 39(6), 1040 - 5 {Emergence of a quasi-periodic spatial structure in the model of aggregation of moving, dividing cells, possessing chemotactic properties}; Polezhaev AA; It has been analyzed the Keller-Segel model of aggregation of divided cells possessed hemotaxis . During the cell division homogeneous stationary distribution becomes unstable due to increasing of the common cells amount . It is shown that accidental variation of the primary stable homogeneous stationary distribution result to forming of a like periodical space structure with characteristic scale that basically is defined by the cell division rate . This approach allows to explain the arising of complex space structures in the movable bacterium colonies. Scand J Gastroenterol, 1994 Nov, 29(11), 961 - 5 Positive correlation between H,K-adenosine triphosphatase autoantibodies and Helicobacter pylori antibodies in patients with pernicious anemia; Ma JY et al.; BACKGROUND: Helicobacter pylori is a major cause of gastritis, and the parietal cell H,K-adenosine triphosphatase (ATPase) is a major autoantigen in autoimmune atrophic corpus gastritis, which may eventually lead to pernicious anemia and/or neuropathy . Whether the bacterium induces the autoimmune response is unknown . METHODS: By means of enzyme-linked immunosorbent assay the occurrence of antibodies against porcine H,K-ATPase and H . pylori was determined in sera from 30 patients with pernicious anemia . RESULTS: All sera scored positive against H,K-ATPase, and 25 (83%) scored positive against H . pylori . The titers of antibodies against both antigen preparations inversely correlated with the duration of disease . A possible common epitope in the antigen preparations was tested with a competition assay . There was no indication of a common epitope in either human or porcine H,K-ATPase and H . pylori . CONCLUSIONS: There was a positive correlation and a high incidence of antibodies against H,K-ATPase and H . pylori in sera from patients with pernicious anemia . These antibodies recognized different epitopes. J Cell Sci, 1994 Nov, 107 ( Pt 11), 3065 - 76 Fusion between large phagocytic vesicles: targeting of yeast and other particulates to phagolysosomes that shelter the bacterium Coxiella burnetii or the protozoan Leishmania amazonensis in Chinese hamster ovary cells; Veras PS et al.; This report examines the fusion of phagocytic vesicles with the large phagolysosome-like vacuoles induced in Chinese hamster ovary cells by the bacterium Coxiella burnetti or the Protozoan flagellate Leishmania amazonensis . Infection by these organisms is compatible with cell survival and multiplication . Fusion was inferred from the transfer of microscopically identifiable particles from donor to target vesicles . Donor vesicles contained heat-killed yeast, zymosan, beta-glucan or latex beads taken up by the host cells . Yeast and zymosan were also coated with Concanavalin A to increase their uptake by the cells (Goldman, R., Exp . Cell Res . 104, 325-334, 1977) . Particle localization, routinely ascertained by phase-contrast microscopy, was confirmed by confocal laser fluorescence and by transmission electron microscopy . Coxiella vacuoles admitted all the particles tested and transfer took place whether the particles were given to the cells prior to or after infection . Transfer of uncoated or Concanavalin-A-coated yeast or zymosan was dependent on the number of particles ingested and on the incubation period (between 2 and 24 hours) . Furthermore, the transfer step was quite efficient, since over 85% of the particles ingested entered Coxiella vacuoles at all particle to cell ratios examined . The fraction of uncoated or Concanavalin-A-coated yeast or zymosan transferred to Leishmania vacuoles was consistently lower and diminished at higher particle loads . In addition, only rarely did latex beads enter these vacuoles . The models proposed may be useful for the delineation of biochemical and molecular mechanisms involved in the fusion of large phagocytic vesicles and the modulation of the latter by cellular and pathogen-derived signals. Eur J Clin Microbiol Infect Dis, 1994 Nov, 13(11), 1000 - 6 Immunobiology of Mycobacterium avium infection; Bermudez LE; Infection caused by organisms of the Mycobacterium avium complex is diagnosed in 50% to 60% of AIDS patients with the advanced stage of disease . Mycobacterium avium is an environmental bacterium that gains access to the host through both the gastrointestinal tract and the respiratory tract . After crossing the mucosal barrier Mycobacterium avium disseminates, infecting chiefly mononuclear phagocytes of the reticuloendothelial system . A number of cells of the immune system such as CD4+ T cells, natural killer cells and macrophages have been shown to be involved in the host response to Mycobacterium avium . The interaction between Mycobacterium avium and macrophages results in the production of immune-suppressive cytokines that inhibit the effector function of TH1 subtype CD4+ T cells, natural killer cells and macrophages, possibly allowing survival of Mycobacterium avium . Some cytokines such as tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor have been shown to induce mycobacteriostatic activity and mycobactericidal activity in infected macrophages . Over the next few years, much new information will certainly be gleaned about host-pathogen interactions, which will lead to a better understanding of the disease and possibly to the design of new forms of therapy. N Engl J Med, 1994 Oct 27, 331(17), 1110 - 5 A comparison of tacrolimus (FK 506) and cyclosporine for immunosuppression in liver transplantation . The U.S . Multicenter FK506 Liver Study Group; Asymmetric binding of the primary acceptor quinone in reaction centers of the photosynthetic bacterium Rhodobacter sphaeroides R26 et al.; Department of Biophysics, Huygens Laboratory, Leiden University, The NetherlandsThe reaction center (RC)-bound primary acceptor quinone QA of the photosynthetic bacterium Rhodobacter sphaeroides R26 functions as a one-electron gate . The radical anion QA.- is proposed to have an asymmetric electron distribution, induced by the protein environment . We replace the native ubiquinone-10 (UQ10) with specifically 13C-labelled UQ10, and use Q-band (35 GHz) EPR spectroscopy to investigate this phenomenon in closer detail . The direct observation of the 13C-hyperfine splitting of the gz-component of UQ10A.- in the RC and in frozen isopropanol shows that the electron spin distribution is symmetric in the isopropanol glass, and asymmetric in the RC . Our results allow qualitative assessment of the spin and charge distribution for QA.- in the RC . The carbonyl oxygen of the semiquinone anion nearest to the S = 2 Fe(2+)-ion and QB is shown to acquire the highest (negative) charge density. Science, 1994 Oct 21, 266(5184), 430 - 2 The structure of flavocytochrome c sulfide dehydrogenase from a purple phototrophic bacterium; Chen ZW et al.; The structure of the heterodimeric flavocytochrome c sulfide dehydrogenase from Chromatium vinosum was determined at a resolution of 2.53 angstroms . It contains a glutathione reductase-like flavin-binding subunit and a diheme cytochrome subunit . The diheme cytochrome folds as two domains, each resembling mitochondrial cytochrome c, and has an unusual interpropionic acid linkage joining the two heme groups in the interior of the subunit . The active site of the flavoprotein subunit contains a catalytically important disulfide bridge located above the pyrimidine portion of the flavin ring . A tryptophan, threonine, or tyrosine side chain may provide a partial conduit for electron transfer to one of the heme groups located 10 angstroms from the flavin. J Biol Chem, 1994 Oct 21, 269(42), 26136 - 43 Influence of monovalent cations on the ultraviolet-visible spectrum of tryptophan tryptophylquinone-containing methylamine dehydrogenase from bacterium W3A1; Kuusk V et al.; The influence of the monovalent cations on the UV-visible spectra of the methylamine dehydrogenase (MADH) from bacterium W3A1 was investigated . The spectra for the oxidized and 1- and 2-electron-reduced forms, unperturbed by bound cations, were obtained for the enzyme, and the extinction coefficients for these forms were determined . The binding of the following cations was investigated: Li+, Na+, K+, Rb+, Cs+, NH4+, (CH3)3NH+, and (CH3)4N+ . It was shown that each cation produced unique spectral changes, some of which were pH-dependent . Except for NH4+, all spectral changes produced by binding of the monovalent cations can be explained by assuming two different binding sites in MADH (type I and type II sites) . Na+ and K+ displayed monophasic binding to the type II site, (CH3)3NH+ and (CH3)4N+ displayed monophasic binding to the type I site, and Li+, Rb+, and Cs+ displayed either monophasic or biphasic binding to one or both sites depending on pH . The pH dependence for binding to the two sites is different, i.e . plots of log(Kd) versus pH have negative slopes approximately 1 for the type II site, whereas the negative slope is significantly less than 1 (0.6-0.8) for the type I site . This difference leads to pH-dependent changes in spectral features produced by binding of Li+, Rb+, and Cs+ . The spectral changes seen during titrations with NH+4 were unlike those seen for any other cation . The binding of NH+4 was biphasic, and the spectra produced in each phase were unaffected by pH . It is assumed that this cation binds to the tryptophan tryptophylquinone cofactor to produce the iminoquinone in the first phase and then binds to the type I monovalent cation binding site in the second phase . It is suggested that binding of NH+4 (and CH3NH+3) to the type I site is a prelude to binding to the cofactor. Structure, 1994 Oct 15, 2(10), 925 - 36 Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.65 A resolution: cofactors and protein-cofactor interactions; Ermler U et al.; BACKGROUND: Photosynthetic reaction centres (RCs) catalyze light-driven electron, transport across photosynthetic membranes . The photosynthetic bacterium Rhodobacter, sphaeroides is often used for studies of RCs, and three groups have determined the structure of its reaction centre . There are discrepancies between these structures, however, and to resolve these we have determined the structure to higher resolution than before, using a new crystal form . RESULTS: The new structure provides a more detailed description of the Rb . sphaeroides RC, and allows us to compare it with the structure of the RC from Rhodopseudomonas viridis . We find no evidence to support most of the published differences in cofactor binding between the RCs from Rps . viridis and Rb . sphaeroides . Generally, the mode of cofactor binding is conserved, particularly along the electron transfer pathway . Substantial differences are only found at ring V of one bacteriochlorophyll of the 'special pair' and for the secondary quinone, QB . A water chain with a length of about 23 A including 14 water molecules extends from the QB to the cytoplasmic side of the RC . CONCLUSIONS: The cofactor arrangement and the mode of binding to the protein seem to be very similar among the non-sulphur bacterial photosynthetic RCs . The functional role of the displaced QB molecule, which might be present as quinol, rather than quinone, is not yet clear . The newly discovered water chain to the QB binding site suggests a pathway for the protonation of the secondary quinone QB. Biochemistry, 1994 Oct 11, 33(40), 12077 - 84 Comparative study of reaction centers from photosynthetic purple bacteria: electron paramagnetic resonance and electron nuclear double resonance spectroscopy; Rautter J et al.; Reaction centers (RCs) from four species of purple bacteria, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and the recently discovered bacterium Rhodospirillum centenum, have been characterized by optical spectroscopy {Wang, S., Lin, X., Woodbury, N . W., & Allen, J . P . (1994) Photosynth . Res . (submitted for publication)} and magnetic resonance spectroscopy . All RCs contain a bacteriochlorophyll (BChl) a dimer as the primary donor . For Rb . sphaeroides and Rs . rubrum the donor QY optical band is at approximately 865 nm, compared to approximately 850 nm for Rb . capsulatus and Rs . centenum . The primary donor in the RCs can be converted between these two forms by the addition or removal of charged detergents . The electronic structure of the cation radical of the primary electron donor P+ . was investigated in these species using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and electron nuclear triple resonance (TRIPLE) spectroscopy . The EPR line widths of P+ . vary significantly and the ENDOR and Special TRIPLE spectra reveal drastic differences in the spin density distribution of the dimer for the different species . Reaction centers from Rb . sphaeroides and Rs . rubrum have a slightly asymmetric spin density distribution over the two halves of the dimer . The respective ratios are 2:1 and 1.6:1 in favor of the L-half of the BChl a dimer . In contrast, the spectra of P+ . in reaction centers from Rb . capsulatus and Rs . centenum show an almost complete localization of the unpaired electron on the L-half of the dimer (ratio approximately 5:1).(ABSTRACT TRUNCATED AT 250 WORDS) Int J Syst Bacteriol, 1994 Oct, 44(4), 832 - 5 Comparative analysis of the 16S rRNA gene sequence of the putative agent of proliferative ileitis of hamsters; Peace TA et al.; Proliferative ileitis of hamsters is consistently associated with the presence of intracellular bacteria in affected ileal epithelial cells . The 16S rRNA gene sequence of the putative etiologic agent of proliferative ileitis was determined by using cell culture-maintained organisms . The highest level of relatedness (98.4%) was observed with a newly described obligately intracellular bacterium obtained from porcine intestines, and the level of homology with Desulfovibrio desulfuricans was 87.5%. Am J Gastroenterol, 1994 Oct, 89(10), 1806 - 10 Association of Helicobacter pylori with gastric cancer and observations on the detection of this bacterium in gastric cancer cases; Hu PJ et al.; OBJECTIVE: The aim of the present study was to assess the prevalence of Helicobacter pylori infection in Chinese patients with advanced gastric cancer, to compare this with a matched control population, and to identify factors that may effect the detection of H . pylori in patients with gastric cancer . METHODS: Fifty-one patients with advanced gastric cancer and 102 age/sex-matched controls were included in the study . For the detection of H . pylori, both biopsy specimens and sera were collected from the patients, whereas only sera were collected from the controls . A strong association was shown between H . pylori and both intestinal and diffuse type gastric cancer . Antibiotic intake in the month before endoscopic examination, the site of collection of biopsy specimens, and tumor size were identified as factors that may reduce the histological detection of H . pylori in gastric cancer patients . CONCLUSIONS: These data provide supporting evidence of an association between H . pylori infection and gastric cancer and indicate that, in certain circumstances, histological evaluation of H . pylori infection in gastric cancer cases may be less reliable than serological evaluation. Am J Gastroenterol, 1994 Oct, 89(10), 1801 - 5 Bacterial mucosal infiltration in Helicobacter pylori-associated gastritis: histological and clinical consequences; Neri M et al.; OBJECTIVES: We wished to demonstrate that gastric epithelial cells infiltration by HP is associated with the active inflammatory response and the severity of gastritis in the gastric antrum of patients harboring the bacterium . METHODS: We studied 129 patients with HP-associated gastritis and 60 HP-negative controls with gastritis of different origin . Gastric mucosal biopsies were obtained from all subjects at endoscopy and were examined for histological features of active inflammation and type of gastritis, as well as for electronmicroscopical features of invasion and damage, according to a four-degree classification (range 0-3) . RESULTS: At entry, the presence of acute inflammatory activity, defined according to the presence of a polymorphonuclear cell infiltrate, was significantly greater in HP-positive patients than in controls (p < 0.00001) and was well related to the depth of mucosal invasion (p < 0.001) . Accordingly, the prevalence of chronic atrophic gastritis was higher in HP-positive patients (p < 0.02 vs . controls) and at grade 3 of invasion (p < 0.04 vs . grade 1 and 2) . Peptic ulcers were more frequent in grade 3 patients (p < 0.04) . CONCLUSION: Gastric epithelial cell infiltration and damage by HP, as assessed by electron microscopy, is an important feature of HP-associated gastritis due to its histological and clinical correlates. Infect Immun, 1994 Oct, 62(10), 4682 - 5 Effects of temperature stress on expression of fimbriae and superoxide dismutase by Porphyromonas gingivalis; Amano A et al.; We examined the biosynthesis of fimbriae and superoxide dismutase (SOD) produced by the periodontopathic bacterium Porphyromonas gingivalis in response to elevated temperature . P . gingivalis 2561, grown at 37 degrees C to mid-logarithmic phase, was subsequently incubated at 39, 41, and 43 degrees C, respectively, to stationary phase . There was no difference in the growth of cells at 37 and 39 degrees C . However, at 39 degrees C there was a 54% reduction in the amount of fimbrillin (fimbriae) as well as decreased expression of mRNA for fimA . On the other hand, under the same conditions, a more than twofold increase in the amount of SOD activity, as well as in the levels of SOD mRNA, was observed . Moreover, cells cultured for 20 h at 39 degrees C showed an 86% decrease of fimbrillin protein and a threefold increase in SOD activity . These observations suggest that P . gingivalis may undergo alterations in its virulence and susceptibility to host immune responses as a result of the elevated temperatures found in inflamed periodontal pockets. Antimicrob Agents Chemother, 1994 Oct, 38(10), 2458 - 9 Biocidal action of chlorhexidine is annulled by nicotinic acid; Baker H et al.; An analytical system comprising a bacterium and a protozoan was used to pinpoint the metabolic lesion whereby chlorhexidine (CLX) produced cell death . Nicotinic acid but not nicotinamide annulled the biocidal action of CLX . The results suggest that CLX may not permit bioconversion of nicotinamide to nicotinic acid to annul the growth inhibition induced by CLX. Mol Microbiol, 1994 Oct, 14(2), 243 - 54 Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ; McCormick JR et al.; A homologue of the bacterial cell division gene ftsZ was cloned from the filamentous bacterium Streptomyces coelicolor . The gene was located on the physical map of the chromosome at about '11 o'clock' (in the vicinity of glkA, hisA and trpB) . Surprisingly, a null mutant in which the 399-codon ftsZ open reading frame was largely deleted was viable, even though the mutant was blocked in septum formation . This indicates that cell division may not be essential for the growth and viability of S . coelicolor . The ftsZ mutant was able to produce aerial hyphae but was unable to produce spores, a finding consistent with the idea that ftsZ is required in order for aerial hyphae to undergo septation into the uninucleoid cells that differentiate into spores. Intern Med, 1994 Oct, 33(10), 628 - 31 Infective endocarditis caused by an indigenous bacterium (Gemella morbillorum); Terada H et al.; A case of infective endocarditis (IE) caused by a rare pathogen, Gemella morbillorum, is presented . Because of persistent low-grade fever after dental treatment, the patient was given oral antibiotics . Whereas he was diagnosed as having aortic regurgitation by a cardiologist, and IE was not suggested unfortunately . After long-term chemotherapy over five months, he was aware of nocturnal dyspnea and Gemella morbillorum was detected by blood culture . Then, he was treated with intravenous administration of Penicillin-G, and underwent surgical operation for valve replacement . No cases of IE due to this organism have been reported in Japan. Trends Biotechnol, 1994 Oct, 12(10), 420 - 6 Unravelling the pathogenic role of Helicobacter pylori in peptic ulcer: potential new therapies and vaccines; Telford JL et al.; The recognition that peptic ulcer is an infectious disease caused by the bacterium Helicobacter pylori has revolutionized the approach to diagnosis and therapy of this condition . Treatment of the symptoms of peptic ulcer with drugs that block acid secretion is already being replaced by antibiotic eradication of the causative agent . Studies of the molecular events that lead to H . pylori pathogenesis have shown that clinical isolates can be divided into two groups, only one of which produces a cytotoxin and is associated with severe disease . The cloning of the genes coding for molecules specific for disease-associated strains of H . pylori, and the development of animal models that mimic the human pathology, will provide the basis for better strategies to treat and prevent peptic-ulcer disease. Zhonghua Liu Xing Bing Xue Za Zhi, 1994 Oct, 15(5), 292 - 5 {Epidemiology of melioidosis in China}; Li L et al.; From 1975 to 1989, a total of 73 strains of P . pseudomallei was isolated from the water samples and the pathological samples of human and domestic animals in 13 counties and cities located different latitude from four provinces Qiong, Yue, Gui and Xiang in China . Serological investigation demonstrated that the geographical distribution of the organism had a significant correlation with the positive rate of antibodies against P . pseudomallei and the native foci of the organisms distributed over the southern subtropical zone and the edge of tropical zone in Qiong, Yue and Gui . In endemic areas, the positive rates of antibodies against P . pseudomallei in human-beings, horses, oxen and pigs are 3.8%-15.2%, 9.1%-18.4%, 6.6%-33.0% and 35% respectively . The investigation results showed the horses and mules infected by the organism would interfere with quarantine of the animals, meanwhile, the meat contaminated by the bacterium would endanger the public health . In Sept . and Oct . of 1989, three cases in Zhanjiang and Sanya of Hainan were reported, two cases died of acute melioidosis with septicemia, another case was the chronic leg ulcers . So, it was predicated that there could have some cases of melioidosis which were misdiagnosed or missed out. Biochim Biophys Acta, 1994 Sep 27, 1187(3), 347 - 53 Molecular characterization of two spontaneous antimycin A resistant mutants of Rhodospirillum rubrum; Uhrig JF et al.; Antimycin A is an inhibitor of cytochrome bc1 complexes acting at the quinone reducing site (Qi) of the cytochrome b subunit . We report here the isolation and molecular characterization of two spontaneous mutants of the purple non-sulfur bacterium Rhodospirillum rubrum resistant to this inhibitor . In the two mutants antimycin A resistance was found to be conferred by replacement of an aspartate residue at position 243 of the cytochrome b polypeptide chain, in one case by histidine and in the other by glutamate . The mutants exhibit cross-resistance to aurachin C but not to aurachin D . The exchange of Asp-243 does not only diminish the antimycin sensitivity of the isolated cytochrome bc1 complexes but also has effects on the function of the quinone reducing site (Qi) . Oxidant-induced reduction of cytochrome b, requiring addition of antimycin A in the wild type, is already at a maximum in the absence of antimycin A . This indicates a diminished electron flow between heme b-566 and ubiquinone at the quinone reducing site (Qi) of cytochrome b. Gene, 1994 Sep 15, 147(1), 21 - 8 Expression of the division-controlling gene ftsZ during growth and sporulation of the filamentous bacterium Streptomyces griseus; Dharmatilake AJ et al.; The branched, filamentous cells of Streptomyces form two different types of septum: those found infrequently in vegetative mycelia and those that form the boundaries of developing spores . To begin to understand the role of cell septation events in the Streptomyces life cycle, we have isolated the ftsZ locus from Streptomyces griseus, an organism that undergoes sporulation on solid surfaces and in liquid culture . The nucleotide sequence of the cloned DNA indicates that ftsZ in S . griseus lies within a region containing other genes likely to be involved in cell division and cell wall biogenesis . A gene (ORF1) showing significant similarity to ftsQ maps a short distance upstream from ftsZ, but there is no evidence for an ftsA homologue between ftsZ and ORF1 . Transcription analysis suggests that ftsZ is expressed during both vegetative growth and sporulation . Immunoblots of soluble protein preparations from vegetative and sporulating mycelia indicate that FtsZ is present at similar levels during growth and differentiation . There appears to be only one ftsZ gene in S . griseus . We interpret these results to indicate that any temporal regulation of FtsZ that may be necessary for the enhanced synthesis of septa during sporulation of S . griseus is likely to occur predominantly at the level of activity rather than synthesis. FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 19 - 25 The F1 genes of the F1F0 ATP synthase from the acidophilic bacterium Thiobacillus ferrooxidans complement Escherichia coli F1 unc mutants; Brown LD et al.; An atp gene cluster from the extreme acidophile Thiobacillus ferrooxidans was able to complement Escherichia coli F1 unc mutants for growth on minimal medium plus succinate . Complementation with all four E . coli F1 mutants tested was observed and subunits for the F1 portion of the T . ferrooxidans ATP synthase formed a functional association with the F0 subunits of the E . coli enzyme . In addition, a hybrid F1 enzyme in which some units were derived from E . coli and some from T . ferrooxidans was partially functional . No clones capable of complementing E . coli F0 unc mutants were isolated . The nucleotide sequence of the gene cluster was determined and the genes for the F0 and the F1 ATP synthase subunits were found to be physically linked. FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 181 - 5 The restriction endonuclease RflFII, isolated from Ruminococcus flavefaciens FD-1, recognizes the sequence 5'-AGTACT-3', and is inhibited by site-specific adenine methylation; Morrison M et al.; Molecular studies of the rumen bacterium Ruminococcus flavefaciens are constrained by the lack of stable gene transfer systems . We report here on the characterization of RflFII, a restriction endonuclease isolated from R . flavefaciens FD-1 . The enzyme is an isoschizomer of ScaI, and cleavage of the DNA is blunt-ended, between the internal TA dinucleotide sequence of 5'-AGTACT-3' . Chromosomal DNA preparations were used to demonstrate that adenine methylation of DNA within the sequence 5'-GTAC-3' inhibits both RflFII and the restriction endonucleases RsaI and ScaI . Chromosomal DNA from R . flavefaciens FD-1 is also host modified to protect against cleavage by ScaI. Cell, 1994 Sep 9, 78(5), 867 - 76 Antigen diversity in the bacterium B . hermsii through "somatic" mutations in rearranged vmp genes; Restrepo BI et al.; B . hermsii counters host immunity with multiphasic antigenic variation . This is conferred by interplasmidic and intraplasmidic rearrangements of vmp genes . In several independent events, activation of a silent vmp gene through intraplasmidic deletions but not interplasmidic recombinations was followed by the appearance at its 5' end of multiple mutations that were not present in the silent gene . The prevalence of mutant alleles in postwitch populations increased during infections . Differences between the silent and expressed genes were at the same nucleotides at which vmp pseudogenes differed, suggesting these were templates for postswitch gene conversions . The mechanism of this bacterium to generate diversity, namely, intramolecular deletions followed by mutations in the rearranged gene, mirrors the strategy used by vertebrate hosts to eliminate it. J Bacteriol, 1994 Sep, 176(18), 5735 - 52 Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization; Saxena IM et al.; The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-localized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization . To identify the different components involved in this process, we isolated an Acetobacter cellulose-synthesizing (acs) operon from this bacterium . Analysis of DNA sequence shows the presence of three genes in the acs operon, in which the first gene (acsAB) codes for a polypeptide with a molecular mass of 168 kDa, which was identified as the cellulose synthase . A single base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is described in the present paper, along with the sequences of the other two genes (acsC and acsD) . The requirement of the acs operon genes for cellulose production was determined using site-determined TnphoA/Kanr GenBlock insertion mutants . Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the acsD mutant produced reduced amounts of two cellulose allomorphs (cellulose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization . The role of the acs operon genes in determining the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the different mutants; however, this arrangement was observed only in cells that actively produced cellulose microfibrils, suggesting that it may be influenced by the crystallization of the nascent glucan chains. J Bacteriol, 1994 Sep, 176(17), 5350 - 6 Detection and subcellular localization of three Ptl proteins involved in the secretion of pertussis toxin from Bordetella pertussis; Johnson FD et al.; The ptl locus of Bordetella pertussis contains eight open reading frames which are predicted to encode proteins (PtlA to PtlH) that are essential for secretion of pertussis toxin from the bacterium and which are members of a family of transport proteins found in other types of bacteria . We have detected PtlE, PtlF, and PtlG in immunoblots of extracts of B . pertussis by using antibodies raised to fusion proteins consisting of maltose-binding protein and the individual Ptl proteins . These proteins have apparent molecular weights similar to those predicted by DNA sequence analysis . Cell fractionation studies indicated that all three Ptl proteins are associated with the membranes of B . pertussis, suggesting that the Ptl proteins form a gate or channel which facilitates transport of pertussis toxin . Cell extracts of other Bordetella spp . were probed with antibodies to Ptl proteins for the presence of these transport proteins . Neither Bordetella parapertussis nor Bordetella bronchiseptica contained detectable levels of PtlE or PtlF . This lack of detectable Ptl protein may provide an explanation for previous observations which indicated that introduction of the genes encoding pertussis toxin subunits from B . pertussis into other Bordetella spp . results in production of the toxin but not secretion of the toxin. J Bacteriol, 1994 Sep, 176(17), 5341 - 9 Use of a phase variation-specific promoter of Myxococcus xanthus in a strategy for isolating a phase-locked mutant; Laue BE et al.; The bacterium Myxococcus xanthus alternates between two colony types distinguished by colony morphology and pigmentation . Because the two phases are interconvertible, this phenomenon has been termed phase variation . In one phase, the colonies are bright yellow, rough, and swarming . In the alternate phase, the colonies are tan and mucoid with smooth edges . During exponential vegetative growth, the populations within a colony reach an equilibrium of approximately 99% yellow and 1% tan cells . Neither the biological function nor the genetic mechanism of phase variation is currently understood . To investigate phase variation, a yellow-phase-specific promoter was identified by Tn5lac mutagenesis . A tan-phase-locked mutant was isolated by a strategy, described in this study, which involved introducing a selectable marker expressed under phase-regulated expression . This was accomplished by a fusion of the cloned yellow-phase-specific promoter to a promoterless kanamycin resistance gene . The defect in the phase-locked mutant, given the designation var-683, caused the rate of switching from the tan to yellow phase to be reduced by at least 10(3)-fold below the wild-type rate of switching . This strain will provide a stable tan population for genetic and biological analysis . Evidence is presented for the existence of a transcriptional regulator which controls the expression of phase-regulated promoters. Infect Immun, 1994 Sep, 62(9), 3672 - 8 Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line; Mintz KP et al.; Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells . The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A . actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or {3H}thymidine-labeled bacteria . Adhesion was found to be time dependent and increased linearly with increasing numbers of bacteria added . Variation in the level of adhesion was noted among strains of A . actinomycetemcomitans . Adhesion was not significantly altered by changes in pH (from pH 5 to 9) but was sensitive to sodium chloride concentrations greater than 0.15 M . Pooled human saliva was inhibitory for adhesion when bacteria were pretreated with saliva before being added to the cells . Pretreatment of the KB cells with saliva did not inhibit adhesion . Protease treatment of A . actinomycetemcomitans reduced adhesion of the bacteria to KB cells . The data are consistent with the hypothesis that a protein(s) is required for bacterial adhesion and that host components may play a role in modulating adhesion to epithelial cells. Infect Immun, 1994 Sep, 62(9), 3640 - 8 Interaction of lipopolysaccharides of Helicobacter pylori with basement membrane protein laminin; Valkonen KH et al.; The ability of hemagglutinating and poorly hemagglutinating strains of the gastroduodenal pathogen Helicobacter pylori to bind 125I-radiolabelled laminin was quantitated in a liquid phase assay . Although all strains bound laminin, some hemagglutinating strains were good binders of laminin (maximum of 31% binding), whereas poorly hemagglutinating strains bound intermediate to small amounts of laminin (minimum of 6% binding) . Since a hydrophobic component of the bacterium has been reported to be involved in binding of laminin (T . J . Trust, P . Doig, L . Emody, Z . Kienle, T . Wadstrom, and P . O'Toole, Infect . Immun . 59:4398-4404, 1991), we investigated the role of lipopolysaccharide (LPS) in the interaction of both types of strains with laminin . Although the extent of inhibition varied among strains, laminin binding to hemagglutinating and poorly hemagglutinating strains was inhibited with homologous and heterologous smooth-form LPS . The ability of heterologous rough-form LPS to produce inhibition comparable to that shown by smooth-form LPS indicated that the O side chain of H . pylori LPS was not involved in the interaction . Further inhibition experiments with dephosphorylated LPS, isolated core oligosaccharide, and free lipid A suggested that a phosphorylated structure in the core oligosaccharide mediates the interaction of a hemagglutinating strain of H . pylori with laminin, whereas a conserved nonphosphorylated structure in the core oligosaccharide mediates the interaction of a poorly hemagglutinating strain . Furthermore, we showed that the interaction of H . pylori LPS with 125I-radiolabelled laminin in a solid phase assay was saturable, specific, and inhibitable with unlabelled laminin . It was postulated that the initial recognition and binding of laminin by H . pylori may occur through LPS and that subsequently a more specific interaction with a lectin-like adhesin on the bacterial surface occurs. Head Neck, 1994 Sep-Oct, 16(5), 450 - 2 Fusobacterium pneumonia and death following uvulo-palato-pharyngoplasty; Paaske PB et al.; A case with a fatal outcome caused by infection with Fuso-bacterium species was seen in a patient recently operated on for heavy snoring with uvulo-palato-pharyngoplasty (UPPP) . The mechanism of infection is discussed . It is concluded that a febrile episode seen in patients less than 2 weeks postoperatively should be considered a serious symptom and be treated intensively after thorough examination. Can J Microbiol, 1994 Sep, 40(9), 782 - 6 Transformation of Bartonella bacilliformis by electroporation; Grasseschi HA et al.; Bartonella bacilliformis is a member of the order Rickettsiales, family Bartonellaceae . The bacterium is an intracellular parasite of human erythrocytes . To date, members of the family Bartonellaceae have not been transformed by standard chemical methods . We report the first successful transformation of a member of the Bartonellaceae family, B . bacilliformis, by the method of electroporation . The optimal conditions for electroporation of B . bacilliformis include a field strength of 12.5 kV/cm and a time constant of 5 ms using 0.2-cm cuvettes . With these parameters and the cosmid pEST (RK2 replicon), a transformation efficiency of 7.8 x 10(5) was obtained . Transformants were readily cultured on medium containing kanamycin sulfate at concentrations ranging from 15 to 600 micrograms/mL . Bacterial survival was approximately 31% under optimal electroporation conditions, and the maximal number of transformants was obtained with 80 ng of pEST DNA . Bartonella bacilliformis was verified as the transformed organism by a comparison of transformant protein profiles with those of wild-type B . bacilliformis using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and detection of the exogenous plasmid in DNA from the transformed bacteria by DNA hybridization . Transformations using the plasmids pMK20, pML31, and pUCK18 (containing the replicons ColE1, F, and pMB1, respectively) were not successful. Appl Environ Microbiol, 1994 Sep, 60(9), 3396 - 400 Anaerobic degradation of catechol by Desulfobacterium sp . strain Cat2 proceeds via carboxylation to protocatechuate; Gorny N et al.; Under anoxic conditions, most methoxylated mononuclear aromatic compounds are degraded by bacteria, with catechol being formed as an important intermediate . On the basis of our experiments with the sulfate-reducing bacterium Desulfobacterium sp . strain Cat2, we describe for the first time the enzymatic activities involved in the complete anaerobic oxidation of catechol and protocatechuate . Results obtained from experiments with dense cell suspensions of strain Cat2 demonstrated that all enzymes necessary for protocatechuate and benzoate degradation were induced during growth with catechol . In addition, anaerobic oxidation of catechol was found to be a CO2-dependent process . Phenol was not degraded in suspensions of cells grown with catechol . In cell extracts of Desulfobacterium sp . strain Cat2, protocatechuyl-coenzyme A (CoA) was formed from catechol, bicarbonate, and uncombined CoA . This oxygen-sensitive reaction requires high concentrations of both bicarbonate and protein, and only very low levels of enzyme were detected . In a second oxygen-sensitive step, protocatechuyl-CoA was reduced to 3-hydroxybenzoyl-CoA by reductive elimination of the p-hydroxyl group . Further dehydroxylation to benzoyl-CoA was not detectable . Key reactions described for anaerobic degradation of benzoate were catalyzed by cell extracts of strain Cat2, too. J Bacteriol, 1994 Sep, 176(17), 5202 - 9 Sequence divergence in the ORF6 gene of Mycoplasma pneumonia; Ruland K et al.; The ORF6 gene product of Mycoplasma pneumoniae is involved in a yet-unknown manner in the adhesion of the bacterium to its host cell . Part of the ORF6 gene is a repetitive DNA sequence (RepMP5), about 1,900 bp long . Seven additional similar copies of RepMP5 are dispersed on the genome . In the independently isolated strains M . pneumoniae M129 and FH, the RepMP5 copies residing in the ORF6 gene are not identical . Two conserved regions, ranging from nucleotides 1 to 799 and from nucleotide 1795 to the end of the gene, border a variable region, ranging from nucleotides 800 to 1794 . This variable region differs in DNA sequence and by 201 bp . Analysis of RepMP5 copies outside the ORF6 gene showed that both M . pneumoniae M129 and M . pneumoniae FH carry a RepMP5 copy on a 6-kbp EcoRI fragment which has the same DNA sequence as the variable region of RepMP5 in the M . pneumoniae FH ORF6 gene . According to these data, a switch from the M . pneumoniae M129 ORF6 gene to the M . pneumoniae FH ORF6 gene could take place by gene conversion. Prev Med, 1994 Sep, 23(5), 712 - 6 Benefits from elimination of Helicobacter pylori infection include major reduction in the incidence of peptic ulcer disease, gastric cancer, and primary gastric lymphoma; Graham DY; BACKGROUND . It has recently been recognized that gastritis, gastric ulcer, duodenal ulcer, gastric carcinoma, and primary gastric B-cell lymphoma are all associated with gastroduodenal infection with the bacterium Helicobacter pylori . Ten percent of Americans develop a peptic ulcer: one in six of those is infected . Four to five million Americans have peptic ulcer disease and ulcer disease is responsible for $43 to $44 billion in annual health care costs . METHODS . We review the accumulated data showing that successful treatment of H . pylori infection results in healing of gastritis and cure of peptic ulcer disease . Current data suggest that by elimination of H . pylori infection it may be possible to prevent most gastric carcinomas and primary gastric lymphomas . CONCLUSIONS . H . pylori infection is currently considered primarily as a cause of peptic ulcer . H . pylori infection is a major public health problem and elimination or prevention of the H . pylori infection will result in a tremendous reduction in medical costs, morbidity, and mortality. Gen Pharmacol, 1994 Sep, 25(5), 833 - 41 Gastroprotective agents in mucosal defense against Helicobacter pylori; Slomiany BL et al.; 1 . Convincing evidence now exists that infection with H . pylori plays a major role in the pathogenesis of gastric disease . Having a niche bordering two major perimeters of mucosal defenses, the bacterium apparently exerts its detrimental effect on the mucus layer as well as the gastric epithelium . Therefore, gastroprotective agents capable of counteracting these detrimental effects of H . pylori are gaining importance in the treatment of gastric disease . 2 . The colonization of gastric mucosa by H . pylori involves specific glycolipid receptors bearing acidic substituents, a process inhibited by gastric sulfomucins . Two antiulcer agents bearing sulfated sugar groups have been demonstrated to possess the ability to interfere with H . pylori colonization process . These are sucralfate and sulglycotide . The two agents are also potent inhibitors of H . pylori glycosulfatase activity directed against indigenous mucosal defenses . 3 . A variety of extracellular enzymes such as proteases, lipases and phospholipases, elaborated by H . pylori cause the weakening of the integrity of gastric mucus coat and render the underlying epithelium vulnerable to noxious luminal contents . Among the most potent agents capable of countering the proteolytic activity of H . pylori are nitecapone, ebrotidine and sulglycotide, while ebrotidine and sulglycotide were found to be most effective inhibitors of H . pylori lipolytic activities . 4 . The gastric epithelial integrity is compromized by the H . pylori cell-wall lipopolysaccharide untoward effect on the epithelial surface receptors . The interference of the lipopolysaccharide with the laminin receptor was found to be most efficiently countered by ebrotidine, sulglycotide and sucralfate, whereas sulglycotide is the most potent in the reversal of the inhibitory effect of the lipopolysaccharide on mucin receptor binding. Radiologe, 1994 Sep, 34(9), 537 - 41 {Mediastinal actinomycosis with formation of an esophagotracheal fistula . A case report}; Dux M et al.; Before antibiotics were available, actinomycosis was the most commonly diagnosed "fungal disease" of the lung because of its morphological similarity to true fungi . At that time actinomycosis presented a fairly typical clinical picture of empyema thoracis and sinus tracts in the chest wall . Nowadays it has become a rare infectious disease that is usually caused by the bacterium Actinomyces israelii and is amenable to treatment by most antibiotics available today . The following report describes the case of a 59-year-old man with an uncommon mediastinal actinomycosis that caused an oesophagotracheal fistula . This complication may develop due to the necrotizing inflammatory process that is typical for actinomycosis . With regard to the literature, the clinical manifestations of the disease and diagnostic and therapeutic considerations are discussed. Biosci Biotechnol Biochem, 1994 Sep, 58(9), 1733 - 7 Cloning and sequence of the L2 form of RubisCO from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110; Yaguchi T et al.; A form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene was isolated and sequenced from a marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110 . To our knowledge, this is the first report for the sequence of a form II (L2-form) RubisCO gene derived from a chemolithoautotrophic bacterium . The form II RubisCO gene coded for 463 amino acid residues (1389 bp, M(r) = 50,655) . The deduced amino acid sequence had high homology with those of the L2-form RubisCO of Rhodospirillum rubrum (63%) and the L4-form RubisCO from Rhodobacter sphaeroides (62%), but low similarity to other L8S8 form RubisCOs (under 40%). Nat Struct Biol, 1994 Sep, 1(9), 591 - 6 Structure of a pertussis toxin-sugar complex as a model for receptor binding; Stein PE et al.; Pertussis toxin is an exotoxin from the bacterium Bordetella pertussis which is important the pathogenesis of whooping cough and the generation of a protective immune response . The diverse biological activities of the toxin depend on its ability to recognize carbohydrate-containing receptors on a wide variety of eukaryotic cells . We present here the crystal structure of pertussis toxin complexed with a soluble oligosaccharide from transferrin . Binding sites for the terminal sialic acid-galactose moiety are revealed on both subunits S2 and S3 of the B-oligomer . Identification of amino acid residues involved in receptor binding will improve the design of genetically inactivated toxins for use in new acellular whooping cough vaccines. Microb Pathog, 1994 Sep, 17(3), 159 - 66 Chlamydia trachomatis does not bind to alpha beta 1 integrins to colonize a human endometrial epithelial cell line cultured in vitro; Wyrick PB et al.; Chlamydia trachomatis is the leading cause of bacterially acquired sexually transmitted diseases in the United States and Europe . As an obligate intracellular pathogen, this bacterium must invade epithelial cells in order to survive and grow . Thus, multiple strategies probably exist for initial binding of chlamydiae to their target cells . Since a variety of bacteria have exploited integrins to colonize tissues, and a precedent existed for the involvement of extracellular matrix components in chlamydial attachment, this study first analyzed, by flow cytometry, integrins expressed by the human endometrial epithelial cell line HEC-1B . The genital cells were then exposed to monoclonal antibodies directed against those integrins and assayed for chlamydial attachment and inclusion development . Monoclonal antibodies bound to the alpha and/or beta 1 subunit of classic integrin receptors displayed by HEC-1B cells were not able to prevent colonization and infection of the epithelial cells by a genital isolate of C . trachomatis. Appl Environ Microbiol, 1994 Sep, 60(9), 3437 - 9 A phylogenetic tree of 16S rRNA sequences from sulfate-reducing bacteria in a sandy marine sediment; Devereux R et al.; The divergence of 16S rDNA sequences in marine sediment was investigated . Twenty unique partial sequences were found among 33 cloned following PCR . Thirteen shared 82 to 91% similarity with sequences of delta subclass sulfate-reducing bacteria . Three contained the target sequence for a sulfate-reducing bacterium-specific oligonucleotide probe designed from pure-culture studies. Mutat Res, 1994 Sep 1, 309(2), 175 - 84 Defective transformation of chromosomal markers in DNA polymerase I mutants of the radioresistant bacterium Deinococcus radiodurans; Fuchs P et al.; The transformation efficiency of six independently selected chromosomal markers (four for rifampicin resistance and two for acriflavine resistance) was found to be reduced by about 3 logs in a Deinococcus radiodurans strain that was isogenic with wild type except for an insertional mutation in the pol gene that eliminated DNA polymerase I activity (strain 6R1A) . D . radiodurans strains UV17 and 303, previously obtained by chemical mutagenesis, were determined to be partially deficient in DNA Pol I activity as assessed in a permeabilized cell system . Both UV17 and 303 demonstrated intermediate transforming efficiencies that correlated with their levels of residual polymerase activity . The transformation efficiency of strain 6R1A could be greatly restored by expression of cloned E . coli DNA Pol I, but not to wild-type levels . Plasmid transfer and chromosomal duplication insertion were not substantially affected by lack of DNA Pol I activity . D . radiodurans is known to possess extraordinarily efficient repair pathways for DNA damage, and is refractory to DNA damage-induced mutagenesis caused by numerous agents, including several that cause base mispairing . We suggest that D . radiodurans may differ from other naturally transformable bacteria in that DNA Pol I is needed to efficiently convert most drug-resistance markers . This unusual mechanism may be required to accomplish chromosomal conversion prior to correction of donor DNA by this organism's efficient repair pathways. Gene, 1994 Aug 19, 146(1), 111 - 5 Gene sequence, purification and characterization of N-acetyl-beta-glucosaminidase from a marine bacterium, Alteromonas sp . strain O-7; Tsujibo H et al.; The gene (cht60) encoding N-acetyl-beta-glucosaminidase (Cht; EC 3.2.1.30) from the marine bacterium Alteromonas sp . strain O-7 was cloned into pUC18 in Escherichia coli JM109 . The nucleotide (nt) sequence of cht60 was determined . A 1797-bp open reading frame encoded a polypeptide of 598 amino acids (aa) (M(r) 64,535) . The aa sequence of the cloned enzyme (Cht60) deduced from the nt sequence showed no significant sequence homologies with available aa sequences from databases . Cht60 was purified from the periplasmic fraction of E . coli cells carrying pCHT982 . The enzyme was most active towards p-nitrophenyl-N-acetyl-beta-D-glucosaminide(PNP-beta-GlcNAc) and diacetylchitobiose . The optimum pH and temperature of the enzyme were pH 7.5 and 37 degrees C, respectively . The N-terminal 11 aa residues of Cht60 were sequenced, and the location of the signal peptide cleavage site was clarified. Eur J Biochem, 1994 Aug 15, 224(1), 63 - 70 Chemical structure of the lipid A component of lipopolysaccharides of the genus Pectinatus; Helander IM et al.; The chemical structure of the lipid A components of smooth-type lipopolysaccharides isolated from the type strains of strictly anaerobic beer-spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were analyzed . The hydrophilic backbone of lipid A was shown, by controlled degradation of lipopolysaccharide combined with chemical assays and 31P-NMR spectroscopy, to consist of the common beta 1-6-linked disaccharide of pyranosidic 2-deoxy-glucosamine (GlcN), phosphorylated at the glycosidic position and at position 4' . In de-O-acylated lipopolysaccharide, the latter phosphate was shown to be quantitatively substituted with 4-amino-4-deoxyarabinose, whereas the glycosidically linked phosphate was present as a monoester . Laser-desorption mass spectrometry of free dephosphorylated lipid A revealed that the distal (non-reducing) GlcN was substituted at positions 2' and 3' with (R)-3-(undecanoyloxy)tridecanoic acid, whereas the reducing GlcN carried two unsubstituted (R)-3-hydroxytetradecanoic acids at positions 2 and 3 . The lipid A of both Pectinatus species were thus of the asymmetric hexaacyl type . The linkage of lipid A to polysaccharide in the lipopolysaccharide was relatively resistant to acid-catalyzed hydrolysis, enabling the preparation of a dephosphorylated and deacylated saccharide backbone . Methylation analysis of the backbone revealed that position 6' of the distal GlcN of lipid A was the attachment site of the polysaccharide . Despite the quantitative substitution of the lipid A 4'-phosphate by 4-amino-4-deoxyarabinose, which theoretically should render the bacteria resistant to polymyxin, P . cerevisiiphilus was shown to be susceptible to this antibiotic . P . cerevisiiphilus was, however, also susceptibile to vancomycin and bacitracin, indicating that the outer membrane of this bacterium does not act as an effective permeability barrier. Biochemistry, 1994 Aug 9, 33(31), 9237 - 44 Stabilization of reduced primary quinone by proton uptake in reaction centers of Rhodobacter sphaeroides; Kalman L et al.; Proton binding stoichiometry and kinetics of charge recombination were measured after single flash excitation in reaction centers from the purple photosynthetic bacterium, Rhodobacter sphaeroides strain R-26, where the native ubiquinone in the primary quinone acceptor site QA was removed and replaced by (benzo-, naphtho-, and anthra-) quinones of various structures and redox midpoint potentials . The observed proton binding stoichiometry was small (0.2-0.4 H+/QA-) and not specific to quinones in the acidic and neutral pH ranges . Above pH 9, however, significant differences were detected; reaction centers reconstituted by menadione failed to take up protons above pH 9.5 . The pH dependence of the free energy change (stabilization) of the semiquinone was determined by integration of the proton uptake stoichiometry as a function of pH . Ubiquinone had the largest (100 meV at pH 5) and menadione the smallest (49 meV at pH 5) stabilization energy compared to those at very high (> 11) pH . In the case of the anthraquinone-reconstituted reaction center, acceptable agreement was obtained above pH 9 for the stabilization energies derived from energetic parameters of the thermally activated electron transfer (back reaction) and from proton binding stoichiometries . The stabilization at high pH could be attributed to a single protonatable amino acid, which might be either in the QB (secondary quinone) pocket (Glu L212) or in the vicinity of the QA binding domain (Tyr H40) . It was shown that this residue had a negligible energy of interaction with bacteriopheophytin and that its coupling to the semiquinone was sensitive to the structure and physicochemical properties of QA.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Aug 9, 33(31), 9220 - 8 Respiratory enzymes of Thiobacillus ferrooxidans . Kinetic properties of an acid-stable iron:rusticyanin oxidoreductase; Blake RC 2nd et al.; Rusticyanin is an acid-stable, soluble blue copper protein found in abundance in the periplasmic space of Thiobacillus ferrooxidans, an acidophilic bacterium capable of growing autotrophically on soluble ferrous sulfate . An acid-stable iron:rusticyanin oxidoreductase activity was partially purified from cell-free extracts of T . ferrooxidans . The enzyme-catalyzed, iron-dependent reduction of the rusticyanin exhibited three kinetic properties characteristic of aerobic iron oxidation by whole cells . (i) A survey of 14 different anions indicated that catalysis by the oxidoreductase occurred only in the presence of sulfate or selenate, an anion specificity identical to that of whole cells . (ii) Saturation with both sulfatoiron(II) and the catalyst produced a concentration-independent rate constant of 3 s-1 for the reduction of the rusticyanin, which is an electron transfer reaction sufficiently rapid to account for the flux of electrons through the iron respiratory chain . (iii) Values for the enzyme-catalyzed pseudo-first-order rate constants for the reduction of the rusticyanin showed a hyperbolic dependence on the concentration of sulfatoiron(II) with a half-maximal effect at 300 microM, a value similar to the apparent KM for iron shown by whole cells . On the basis of these favorable comparisons between the behavior patterns of isolated biomolecules and those of whole cells, this iron:rusticyanin oxidoreductase is postulated to be the primary cellular oxidant of ferrous ions in the iron respiratory electron transport chain of T . ferrooxidans. Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7543 - 7 Reduced Rho-dependent transcription termination permits NusA-independent growth of Escherichia coli; Zheng C et al.; NusA and Rho are essential Escherichia coli proteins that influence transcription elongation and termination . We show that an E . coli derivative unable to express NusA, because its sole nusA gene contains a large deletion/substitution, is viable providing that the bacterium also carries a rho mutation that reduces transcription termination . This Rho-mediated suppression is not allele specific, since either a mutation changing amino acid 134 {rho(E134D)} or a mutation changing amino acid 352 (rho1) allows growth of a nusA-deleted E . coli . However, both rho mutations similarly decrease transcription termination 8- to 9-fold . We propose that the essential role of NusA is to enhance pausing of RNA polymerase at certain sites, permitting tight coupling of transcription and translation . This coupling interferes with Rho access to and/or movement on the nascent RNA and blocks premature termination of transcription . Thus, NusA-dependent coupling should be less important in a mutant with low Rho activity . The fact that E . coli grows without NusA argues that NusA should be considered an accessory factor rather than a subunit of RNA polymerase. Appl Environ Microbiol, 1994 Aug, 60(8), 3006 - 10 Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium Shewanella colwelliana D; Coon SL et al.; Shewanella colwelliana D is a marine procaryote which produces a diffusible brown pigment that correlates with melA gene expression . Previously, melA had been cloned, sequenced, and expressed in Escherichia coli; however, the reaction product of MelA had not been identified . This report identifies that product as homogentisic acid, provides evidence that the pigment is homogentisic acid-melanin (pyomelanin), and suggests that MelA is p-hydroxyphenylpyruvate hydroxylase . This is the first report of pyomelanin in an obligate marine bacterium. J Bacteriol, 1994 Aug, 176(15), 4726 - 33 Augmented rates of respiration and efficient nitrogen fixation at nanomolar concentrations of dissolved O2 in hyperinduced Azoarcus sp . strain BH72; Hurek T et al.; Azoarcus sp . strain BH72 is an aerobic diazotrophic bacterium that was originally found as an endophyte in Kallar grass . Anticipating that these bacteria are exposed to dissolved O2 concentrations (DOCs) in the nanomolar range during their life cycle, we studied the impact of increasing O2 deprivation on N2 fixation and respiration . Bacteria were grown in batch cultures, where they shifted into conditions of low pO2 upon depletion of O2 by respiration . During incubation, specific rates of respiration (qO2) and efficiencies of carbon source utilization for N2 reduction increased greatly, while the growth rate did not change significantly, a phenomenon that we called "hyperinduction." To evaluate this transition from high- to low-cost N2 fixation in terms of respiratory kinetics and nitrogenase activities at nanomolar DOC, bacteria which had shifted to different gas-phase pO2s in batch cultures were subjected to assays using leghemoglobin as the O2 carrier . As O2 deprivation in batch cultures proceeded, respiratory Km (O2) decreased and Vmax increased . Nitrogenase activity at nanomolar DOC increased to a specific rate of 180 nmol of C2H4 min-1 mg of protein-1 at 32 nM O2 . Nitrogenase activity was proportional to respiration but not to DOC in the range of 12 to 86 nM O2 . Respiration supported N2 fixation more efficiently at high than at low respiratory rates, the respiratory efficiency increasing from 0.14 to 0.47 mol of C2H4 mol of O2 consumed-1 . We conclude that (i) during hyperinduction, strain BH72 used an increasing amount of energy generated by respiration for N2 fixation, and (ii) these bacteria have a high respiratory capacity, enabling them to develop ecological niches at very low pO2, in which they may respire actively and fix nitrogen efficiently at comparatively high rates. Am J Med Sci, 1994 Aug, 308(2), 83 - 7 Passive protection of mice against lethal Francisella tularensis (live tularemia vaccine strain) infection by the sera of human recipients of the live tularemia vaccine; Drabick JJ et al.; The relative role that humoral immunity plays in protection against infection with the intracellular bacterium, Francisella tularensis, remains controversial . Cellular immunity is thought to play the major and perhaps only role . The authors, in this article, investigate the immunologic and protective properties of immune serum collected from human recipients of the live tularemia vaccine (LVS) . Sera of recipients of the vaccine demonstrated reactivity with the vaccine strain by enzyme-linked immunosorbent assay and Western blot analysis . This reactivity appeared to be directed primarily against the lipopolysaccharide of LVS and demonstrated complete cross-reactivity with fully virulent F . tularensis (Schu4) . Pooled immune sera protected mice fully against a 10,000 LD50 challenge with the LVS strain relative to non-immune sera . The protection was abrogated by dilution or preadsorption with the LVS strain but not by preadsorption with Escherichia coli, which suggests specificity of protection . The authors conclude that antibodies to the LVS strain of F . tularensis are generated by live vaccination in humans and play a significant role in protection of mice against lethal challenge with the same organism . These antibodies crossreact completely with fully virulent F . tularensis, but whether they play a role in protection against fully virulent human tularemia strains requires further experimentation. Infect Immun, 1994 Aug, 62(8), 3472 - 8 Defective antigen presentation by Mycobacterium tuberculosis-infected monocytes; Gercken J et al.; In this study we investigated the effect of an in vitro infection with Mycobacterium tuberculosis on the ability of human monocytes to present the soluble antigen tetanus toxoid to T cells . We observed that tetanus toxoid-specific T-cell proliferation was markedly reduced when monocytes were infected with large numbers (bacterium-to-monocyte ratio, 50:1) of both viable and heat-killed mycobacteria . The level of antigen-induced gamma interferon release also was decreased when M . tuberculosis-containing monocytes were used as antigen-presenting cells . However, mycobacterium-infected monocytes did not show or trigger suppressive activity, because the presence of mycobacterium-infected monocytes did not affect the T-cell response induced by tetanus toxoid-pulsed control monocytes . When M . tuberculosis-infected monocytes were fixed with paraformaldehyde, they were not able to serve as antigen-presenting cells even in the presence of untreated accessory monocytes . Moreover, the uptake of both viable and heat-killed M . tuberculosis cells reduced the expression of human leukocyte antigen DR on monocytes . With regard to accessory function, monocytes infected with large numbers of mycobacteria were less efficient as accessory cells than were control monocytes in cultures of T cells activated with pokeweed mitogen . These results indicate that infection with large numbers of M . tuberculosis cells impairs the ability of monocytes to process and/or present soluble antigen and to serve as accessory cells in T-cell activation. Arch Biochem Biophys, 1994 Aug 1, 312(2), 349 - 56 The Entner-Doudoroff pathway in Helicobacter pylori; Mendz GL et al.; Evidence for the presence of the enzymes of the Entner-Doudoroff pathway in Helicobacter pylori was obtained using 1H and 31P nuclear magnetic resonance spectroscopy . Bacterial lysates generated 6-phosphogluconate and NADH or NADPH in incubations with glucose-6-phosphate and NAD+ or NADP+, indicating the presence of glucose-6-phosphate dehydrogenase activities . Formation of pyruvate was observed in time courses of incubations of bacterial lysates with 6-phosphogluconate as the only substrate, suggesting the presence of 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase activities . The existence of these enzymes and of triose phosphate isomerase was confirmed by observing the appearance of dihydroxyacetone phosphate in time courses of bacterial lysates incubated with 6-phosphogluconate . Aldolase activity was measured by the production of pyruvate and dihydroxyacetone phosphate in lysates incubated with 2-keto-3-deoxy-6-phosphogluconate as the sole substrate . Dehydrogenase, dehydratase and aldolase activities were observed in several bacterial strains including wild types from fresh isolates . Kinetic parameters were measured for the three activities . The cellular location of the enzymes was investigated by comparing the activities measured in the pellet and supernatant fractions obtained by centrifugation of lysate suspensions . The concentration of compounds causing 50% inhibition of enzyme activity was determined from dose-response curves . The data suggested the presence of two glucose-6-phosphate dehydrogenases linked to NAD+ and NADP+ activities . Using inhibitors differences between the H . pylori and mammalian KDPG aldolases were detected . The presence of these enzyme activities in H . pylori provided evidence for the existence of the Entner-Douderoff pathway in the bacterium. Tierarztl Prax, 1994 Aug, 22(4), 301 - 8 {Lyme borreliosis--an infectious disease with interdisciplinary demands}; Maiwald M; Lyme borreliosis is an infectious disease with various clinical manifestations, caused by the bacterium Borrelia burgdorferi . Although symptoms of the disease have been described since more than 100 years ago, it was not until 1982 that the causative organism was discovered . In Europe, Lyme borreliosis is the most common tick-borne disease, and in endemic regions, a considerable number of clinical cases can be found . The course of the disease is in stages and involves various organ systems, such as skin, nervous system, heart, and joints . Laboratory diagnosis is considered to be difficult and relies mainly on the determination of antibodies . A vaccine for use in humans is not yet available, but experimental data support the feasibility of a vaccine development. Microbiology, 1994 Aug, 140 ( Pt 8), 2077 - 84 Multiple lactate dehydrogenase activities of the rumen bacterium Selenomonas ruminantium; Gilmour M et al.; The lactate utilizing strain of Selenomonas ruminantium 5934e was found to contain three lactate dehydrogenase (LDH) activities in sonicated cell extracts . One activity, an NAD dependent L-LDH (L-nLDH) was measured at 15-fold greater levels in extracts of cells grown to mid-exponential phase on glucose compared to cells grown to the equivalent growth stage on DL-lactate . A second nLDH activity specific for D-lactate (D-nLDH) was detected at similar levels in both lactate-grown cell extracts and glucose-grown cell extracts . The third activity, an NAD independent DLDH (D-iLDH) was very low in cells grown on glucose but was induced more than 10-fold when DL-lactate was used as the carbon source . The three LDH activities could be separated by gel filtration . Recovery of the activities was low due to the apparent instability of the enzymes at 4 degrees C, which was most pronounced in the case of the D-iLDH . A Km for lactate of 0.5 mM was estimated for the D-iLDH and this was considerably lower than the values of 45 mM and 70 mM measured for L-nLDH and D-nLDH respectively . It is proposed that the D-iLDH may be largely responsible for the formation of pyruvate in lactate-grown cells of S . ruminantium strain 5934e . Three other lactate utilizing strains of S . ruminantium, HD4, 5521C1 and JW13 exhibited a similar profile of LDH activities to strain 5934e when grown on glucose and DL-lactate. Infect Immun, 1994 Aug, 62(8), 3058 - 65 Specific genetic variants of Actinobacillus actinomycetemcomitans correlate with disease and health in a regional population of families with localized juvenile periodontitis; DiRienzo JM et al.; A geographically homogeneous population of 83 subjects, from 21 families with localized juvenile periodontitis (LJP), and 35 healthy control subjects was monitored, over a 5-year period, for the presence of the periodontal pathogen Actinobacillus actinomycetemcomitans . Restriction fragment length polymorphism (RFLP) analysis was used to monitor the distribution of genetic variants of this bacterium in LJP-susceptible subjects that converted from a healthy to a diseased periodontal status . A . actinomycetemcomitans was cultured from 57% of the LJP family members accessioned into the study . Nine of 36 LJP-susceptible subjects, in seven families, developed signs of periodontal destruction . All but one of these conversion subjects harbored A . actinomycetemcomitans . Bacterial variants representative of a single RFLP group (II) showed the strongest correlation with conversion (P < 0.002) . Six of nine conversion subjects were infected with A . actinomycetemcomitans from this group . RFLP group II variants also prevailed in 8 of 22 probands but were absent in the 35 healthy control subjects . In contrast to the selective distribution of group II variants is diseased individuals, variants belonging to RFLP groups XIII and XIV were found exclusively in the control subjects . Thus, the use of RFLP to type clinical isolates of A . actinomycetemcomitans has resulted in the identification of genetic variants that predominate in LJP and health . These results indicate that studies concerned with the pathogenicity of this bacterium in LJP should be focused on the group II variants. Biol Pharm Bull, 1994 Aug, 17(8), 1018 - 22 Enzymatic sulfation of glycosides and their corresponding aglycones by arylsulfate sulfotransferase from a human intestinal bacterium; Konishi-Imamura L et al.; A novel type of arylsulfate sulfotransferase (ASST) from a predominant human intestinal bacterium catalyzes the stoichiometric transfer of a sulfate group from phenolic sulfate esters to phenols . We clarified that polyphenols were better substrates of this enzyme than the corresponding glycosides . Additionally, a coumarin derivative, esculetin, was sulfated by ASST at the 6-position to give 6-monosulfate . Therefore, ASST is more useful for the preparation of sulfated polyphenols at their specific hydroxyl groups and would play an important role in the metabolism of phenolic compounds in vegetable food and traditional medicines. Proc Natl Acad Sci U S A, 1994 Jul 19, 91(15), 7124 - 8 Modification of a hydrogen bond to a bacteriochlorophyll a molecule in the light-harvesting 1 antenna of Rhodobacter sphaeroides; Olsen JD et al.; Site-directed mutagenesis has been used to examine the function of a highly conserved aromatic residue, alpha Trp43, in the light-harvesting 1 antenna of the photosynthetic bacterium Rhodobacter sphaeroides . In this antenna alpha Trp43 is thought to be located near the putative binding site for bacteriochlorophyll; in this work it was changed to both Tyr and Phe, and in each case the main near-infrared absorbance peak was shifted to the blue, from 876 nm to 865 nm and then to 853 nm, respectively . Resonance Raman spectroscopy of the resulting complexes shows a shift of one component of the 1640-cm-1 peak to 1632 cm-1 for the Tyr mutant and to 1660 cm-1 for the Phe mutant . This demonstrates a strengthening of an existing H bond for the Tyr change and a breakage of this bond for the change to Phe . The 1640-cm-1 peak has been previously assigned to H-bonded C2 acetyl carbonyl groups of both bacteriochlorophylls in the light-harvesting 1 antenna dimer {Robert, B . & Lutz, M . (1985) Biochim . Biophys . Acta 807, 10-21} . These results indicate that one of these H bonds is to alpha Trp43, placing this residue in close proximity to the bacteriochlorophyll a macrocycle with which it interacts . The existence of this bond places constraints on the conformation of the alpha polypeptide, and a model of an alpha beta heterodimer is presented incorporating these data. J Mol Biol, 1994 Jul 15, 240(3), 265 - 6 Crystallization and preliminary crystallographic investigation of electron-transfer flavoprotein from the bacterium Methylophilus W3A1; White SA et al.; Electron-transfer flavoprotein from Methylophilus W3A1 has been crystallized using the sitting drop vapour diffusion technique . Hexagonal crystals, suitable for high-resolution structure determination grow to approximately 0.7 mm x 0.7 mm x 0.5 mm in size and diffract to at least 2.2 A . The space group is either P6(1) or P6(5) with unit cell dimensions a,b = 119.1 A and c = 85.6 A. Am J Med Genet, 1994 Jul 15, 51(4), 522 - 6 Robust amplification and ethidium-visible detection of the fragile X syndrome CGG repeat using Pfu polymerase; Chong SS et al.; We report on a method for ethidium bromide detection of FMR1 normal and premutation-size CGG triplet repeats . A set of PCR conditions has been optimized for the polymerase of the hyperthermophilic bacterium, Pyrococcus furiosus . Using this protocol, normal-size alleles ranging from 5 to 50 repeats, as well as most of premutation-size alleles, varying from > 50 to approximately 200 repeats, could be detected by ethidium bromide staining of agarose gels . Since the protocol requires neither autoradiography nor polyacrylamide gel electrophoresis, i