J Wildl Dis, 1993 Oct, 29(4), 596 - 8 Naturally occurring Brucella suis biovar 4 infection in a moose (Alces alces); Honour S et al.; A debilitated adult female moose (Alces alces) shot east of the MacKenzie River, Northwest Territories, Canada, had large fluctuant masses over both carpi . Only the forelimbs were available for examination . Carpal pathology included bilateral bursitis and osteomyelitis of subjacent bone . In addition severe osteomyelitis with fractures was observed in the left lateral and right medial digits . Brucella suis biovar 4 was isolated from the right medial first phalanx . This is believed to be the first reported case of infection with this organism in a wild moose . The bacterium is common in caribou (Rangifer tarandus) in the region. J Wildl Dis, 1993 Oct, 29(4), 591 - 5 Mixed infection of an acid-fast bacterium and an imperfect fungus in a Napoleon fish (Cheilinus undulatus); Wada S et al.; A Napoleon fish (Cheilinus undulatus) was infected with both an acid-fast bacterium and an imperfect fungus . This is the first report of an acid-fast bacterial infection in Cheilinus undulatus, and the first observation of an imperfect fungus in the swim bladder of a tropical marine fish. J Clin Microbiol, 1993 Oct, 31(10), 2745 - 50 Comparison of polymerase chain reaction, culture, and western immunoblot serology for diagnosis of Bordetella pertussis infection; Grimprel E et al.; Polymerase chain reaction (PCR) amplification of the pertussis toxin promoter region was used to detect Bordetella pertussis infection in nasopharyngeal aspirates collected from 24 infants and children infected with pertussis and 13 adult contacts during an epidemiological study . The sensitivity of this PCR assay was approximately one bacterium, and the assay was specific for B . pertussis in tests with other Bordetella species and other respiratory pathogens . The pertussis case definition required a cough with a duration of more than 21 days for infants and children and laboratory confirmation by serology as the primary detection method for infants, children, and adults . The sensitivity of PCR and culture on Bordet-Gengou agar medium was assessed with regard to the case definitions . In the group of infants and children (index cases), the sensitivities of the culture and the PCR were 54.1% (13 of 24) and 95.8% (23 of 24), respectively . In the adult group (household contacts), the sensitivities of the two methods were 15.4% (2 of 13) and 61.5% (8 of 13), respectively . PCR combined with pertussis-specific serology appears to be a useful tool for diagnosis of pertussis especially in epidemiological studies. Int J Pept Protein Res, 1993 Oct, 42(4), 326 - 41 Prediction of transmembrane helices from hydrophobic characteristics of proteins; Ponnuswamy PK et al.; Membrane proteins, requiring to be embedded into the lipid bilayers, have evolved to have amino acid sequences that will fold with a hydrophobic surface in contact with the alkane chains of the lipids and polar surface in contact with the aqueous phases on both sides of the membrane and the polar head groups of the lipids . It is generally assumed that the characteristics of the aqueous parts of the membrane proteins are similar to those of normal globular proteins, and the embedded parts are highly hydrophobic . In our earlier works, we introduced the concept of 'surrounding hydrophobicity' and developed a hydrophobicity scale for the 20 amino acid residues, and applied it successfully to the study of the family of globular proteins . In this work we use the concept of surrounding hydrophobicity to indicate quantitatively how the aqueous parts of membrane proteins compare with the normal globular proteins, and how rich the embedded parts are in their hydrophobic activity . We then develop a surrounding hydrophobicity scale applicable to membrane proteins, by mixing judicially the surrounding hydrophobicities observed in the crystals of the membrane protein, photosynthetic reaction center from the bacterium Rhodopseudomonas viridis, porin from Rhodobacter capsulatus and a set of 64 globular proteins . A predictive scheme based on this scale predicts from amino acid sequence, transmembrane segments in PRC and randomly selected 26 membrane proteins to 80% level of accuracy . This is a much higher predictive power when compared to the existing popular methods . A new procedure to measure the amphipathicity of sequence segments is proposed, and it is used to characterize the transmembrane parts of the sample membrane proteins. Eur J Biochem, 1993 Oct 1, 217(1), 435 - 9 Stimulatory effect of NH4+ on the transport of leucine and glucose in an anaerobic alkaliphile; Koyama N; An anaerobic alkaliphile, EP01, specifically requires NH4+ for the acceleration of amino acid and glucose transport {Koyama, N . (1988) FEBS Lett . 253, 187-189} . In this paper, we attempted to clarify how NH4+ is involved in the transport system . The bacterium acidifies the cytoplasm, which was suggested to result in NH4+ accumulation when NH4Cl was added to the medium . Increase of the NH4Cl concentration administered to the medium caused the acceleration of leucine and glucose transport, which was accompanied by an increase in the internal pH and the absolute internal concentration of NH4+, whereas a decrease in the concentration ratio of internal NH4+/external NH4+ was observed . The addition of 3 mM NH4Cl, which resulted in significant stimulation of leucine and glucose transport, raised the internal NH4+ concentration by 42 mM, but the internal pH only by 0.1 units . It seems more likely that leucine and glucose transport are accelerated depending on the increase in the internal NH4+ concentration rather than the increase in the internal pH . By the imposition of an inwardly directed Na+ gradient, the K(+)-loaded membrane vesicles accumulated leucine and glucose, indicating that a sodium chemical potential is available for active transport . The membrane of the bacterium exhibited a Na(+)-stimulated ATPase activity which was remarkably enhanced by the addition of NH4Cl, depending on its concentration, and was inhibited by vanadate . Leucine and glucose transport were inhibited by vanadate . Based on these results, we propose a mechanism in which NH4+ contributes internally to leucine and glucose transport, depending on its concentration, by the activation of a Na(+)-translocating ATPase responsible for the generation of a sodium chemical potential. Arch Biochem Biophys, 1993 Oct, 306(1), 215 - 22 Amino acid sequence of a high redox potential ferredoxin (HiPIP) from the purple phototrophic bacterium Rhodopila globiformis, which has the highest known redox potential of its class; Ambler RP et al.; Rhodopila globiformis HiPIP has a redox potential (ca . 450 mV) that is 100 mV higher than any other known iron-sulfur protein . The amino acid sequence contains 57 residues and can be aligned with that of Thiobacillus ferrooxidans without any insertions or deletions and is 51% identical . Rp . globiformis HiPIP is also similar to that of Rhodocyclus tenuis, but six- and two-residue gaps must be postulated and there is only 37% identity . Most of the amino acid residues near the iron-sulfur cluster are similar in these two species based on inspection of the three-dimensional structure of Rc . tenuis HiPIP . The reason for the higher redox potential may be a more hydrophilic environment of the Rp . globiformis HiPIP iron-sulfur cluster due to the above two deletions and to substitution of Ser 32 for Gly . Rp . globiformis is unusual in that it has a cytochrome c2 in addition to the HiPIP, and it too has a very high redox potential . These results suggest that the cytochrome c2 and HiPIP may function interchangeably and that the species normally resides in a very high potential environment, although it is not known to grow aerobically in the dark. Trends Microbiol, 1993 Oct, 1(7), 255 - 60 Helicobacter pylori: microbiology of a 'slow' bacterial infection; Blaser MJ; The bacterium Helicobacter pylori lives in the gastric mucus layer of humans and induces a chronic inflammatory response that can result in both peptic ulceration and gastric neoplasms . Helicobacter pylori infection can be considered as a 'slow', adaptive and autoregulating process . The mechanisms by which this slow bacterial pathogen survives and interacts with the host immune system may provide a model for other persistent mucosal pathogens. J Clin Microbiol, 1993 Oct, 31(10), 2709 - 14 Isolation and characterization of "Flexispira rappini" from laboratory mice; Schauer DB et al.; A bacterium with an unusual ultrastructure and possessing a fusiform protoplasmic cylinder, spiral periplasmic fibers, and bipolar tufts of sheathed flagella was identified in the intestinal mucosae of laboratory mice . The organism was cultured under microaerophilic conditions and was found to rapidly hydrolyze urea . On the basis of 16S rRNA gene sequence analysis, the organism was shown to be "Flexispira rappini." "F . rappini" is closely related to members of the genus Helicobacter and has been reported to be associated with human gastroenteritis and ovine abortion . "F . rappini" has not previously been observed in the gastrointestinal tracts of mice. Gene, 1993 Sep 30, 132(1), 107 - 12 Complete sequence of omp1, the structural gene encoding the 40-kDa outer membrane protein of Fusobacterium nucleatum strain Fev1; Bolstad AI et al.; The sequence of the omp1 gene coding for the 40-kDa outer membrane protein (OMP) of the Gram- oral bacterium, Fusobacterium nucleatum strain Fev1, has been determined . Degenerate oligodeoxyribonucleotide primers were used to prime the amplification of a 120-mer sequence of the gene . This sequence was successively used for constructing new primers applied in asymmetrical, symmetrical, and inverse polymerase chain reaction using as template genomic DNA, self-ligated DNA fragments, or fragments ligated into either pGEM-7Zf+ or pACYC184 . The codon usage of the gene was unusual in that A or T was used as the third base in the codon triplets in all cases, except for those amino acids (aa) which have only one or two possible codon choices . Only 35 of the 61 sense codons were used . The aa sequence of the protein was deduced; it consisted of 348 aa (M(r) 39,954), which is in good agreement with the 40-kDa size estimated from electrophoretic analyses . The mature protein was preceded by a 20-aa signal peptide. Biochim Biophys Acta, 1993 Sep 13, 1144(2), 161 - 9 Picosecond energy transfer and trapping kinetics in living cells of the green bacterium Chloroflexus aurantiacus; Muller MG et al.; The excitation energy transfer and trapping processes in intact cells of Chloroflexus aurantiacus were studied by picosecond time-resolved fluorescence spectroscopy . The fluorescence decay kinetics is investigated over the near infrared emission range between 730 nm and 920 nm using various excitation wavelengths and excitation intensities . The data were analyzed by global decay analysis and are presented as decay-associated spectra (DAS) . The specific dependence of the decay kinetics on the excitation wavelength and on the photochemical redox state of the reaction center (RC) allows the identification of the energy transfer and trapping components . The DAS provide evidence for two chlorosomal energy transfer processes . The first one occurs between the chlorosomal bacteriochlorophyll (BChl)-c and the BChl-a792 complex (B792) in the chlorosomal baseplate with an equilibration time constant of 15-16 ps, while the second one occurs from the B792 pigments to the BChl-a806 pigments in the B806-866 complex with a time constant of 35-40 ps . The overall energy trapping process in whole cells is mainly determined by the kinetics of the primary charge separation process in the RCs . With open RCs (QA oxidized) the trapping time constant is 70-90 ps, while the trapping process with closed RCs (QA reduced) takes as long as 180-200 ps . The results on whole cells reported here are interpreted in conjunction with those reported earlier for the various isolated complexes, i.e., two different chlorosome preparations (Holzwarth, A.R., Muller, M.G . and Griebenow, K . (1990) J . Photochem . Photobiol . B 5, 457-465), the B806-866 complex (Griebenow, K., Muller, M.G . and Holzwarth, A.R . (1991) Biochim . Biophys . Acta 1059, 226-232) and isolated reaction centers (Muller, M.G., Griebenow, K . and Holzwarth, A.R . (1991) Biochim . Biophys . Acta 1098, 1-12) . Based on these data, a unified and self-consistent scheme for the primary processes in the whole photosynthetic system of C . aurantiacus is presented . The BChl antenna pigment groups are arranged to form a linear energy transfer cascade with four energy transfer steps from shorter-wavelength- to longer-wavelength-absorbing antenna pools . The overall fluorescence decay kinetics of the photosynthetic system of C . aurantiacus turns out to be 'trap-limited' by the reaction center rather than 'diffusion-limited' by the energy transfer processes. J Biol Chem, 1993 Sep 5, 268(25), 18987 - 93 Purification and properties of NADPH-dependent tylosin reductase from Streptomyces fradiae; Huang SL et al.; A reductase of Streptomyces fradiae was speculated to catalyze reduction of tylosin to relomycin, an industrially undesirable product . The activity of tylosin reductase was closely related to bacterial growth, suggesting involvement of the enzyme in a primary metabolism . The reductase activity was improved significantly in vivo and in vitro . The enzyme was also partially stabilized in vitro . Using a simple five-step chromatographic procedure, the reductase was purified 480-fold to apparent homogeneity . The purified reductase had a molecular mass of 270 kDa and consisted of two different subunits of 26 and 7 kDa at 1:1 ratio . The enzyme exhibited an absorption maximum at 405 nm and was inhibited by exogenous FAD or FMN, indicating a flavin as its prosthetic group . Tylosin reductase was optimally active at pH 7.0-7.2 and 40 degrees C with NADPH as a preferred electron donor . The Km of the enzyme for tylosin was 1.4 mM and that for NADPH was 0.15 mM . The Vmax for the enzymatic reaction was 917 mumol of tylosin formed/min/mg protein . The enzymatic conversion of tylosin to relomycin was coupled to that of NADPH to NADP+ at a stoichiometric ratio of 1:1 . Tylosin reductase showed a broad substrate specificity toward all macrolide aldehydes (as normal and shunt metabolites of tylosin biosynthesis) tested . Thus, the enzyme may have a physiological role of macrolide detoxification for the bacterium. Arch Biochem Biophys, 1993 Sep, 305(2), 252 - 60 Fluorinated probes to measure carboxylesterase activities using 19F NMR spectroscopy: application to erythrocytes and Helicobacter pylori; Mendz GL et al.; Eight fluorinated compounds were tested as putative probes to measure carboxylesterase activity employing 19F nuclear magnetic resonance spectroscopy . The method takes advantage of the sensitivity of fluorine resonances to the changes in the chemical bonding in the covalent structures where they are located . Determination of the kinetic parameters of ethyl fluoroacetate and diethyl fluoromalonate in hemolysates showed that these probes were well suited to study carboxylesterase activities in complex systems the potential of these probes for noninvasive applications was demonstrated in measurements of carboxylesterase kinetic parameters in intact erythrocytes . The presence of carboxylesterases was established in several strains of the bacterium Helicobacter pylori employing 19F nuclear magnetic resonance spectroscopy, and the kinetic parameters of these enzymes for ethyl fluoroacetate and diethyl fluoromalonate were measured. Infect Immun, 1993 Sep, 61(9), 3892 - 900 Specific adherence of Borrelia burgdorferi extracellular vesicles to human endothelial cells in culture; Shoberg RJ et al.; Borrelia burgdorferi produces extracellular vesicles which contain some of the outer surface proteins of the bacterium (e.g., OspA and OspB) . Borrelial vesicles, isolated by differential centrifugation and filtration, were tested for the ability to bind to cultured human umbilical vein endothelial (HUVE) cells in culture . The recently described lipoprotein OspD was expressed on vesicles . Vesicles exhibited differential expression of OspB and OspD in a relationship with passage number and medium serum supplement type, respectively . Qualitative immunoblotting analyses demonstrated dose-dependent, passage number-dependent adsorption of vesicles by HUVE cells . This adsorption was demonstrated to be dependent upon a borrelial component of the vesicle and not due to the presence of minor contamination with intact spirochetes . Quantitative experiments examining inhibition of B . burgdorferi-HUVE association as a function of prior vesicle-HUVE association demonstrated dependence upon (i) a borrelial component(s) in the vesicle, (ii) low passage number, and (iii) vesicle protein concentration . However, vesicle pretreatment of the HUVE cell monolayer was not requisite for this inhibition . Vesicles from highly passaged borrelias were noninhibitory for B . burgdorferi-HUVE cell association, regardless of the serum used to supplement the medium . The use of vesicles as a tool for studying B . burgdorferi pathogenesis and/or physiology is proposed. Zentralbl Bakteriol, 1993 Sep, 280(1-2), 38 - 50 Long term infection of the gastric mucosa with Helicobacter species does induce atrophic gastritis in an animal model of Helicobacter pylori infection; Lee A et al.; Gastric atrophy is a precursor lesion in the development of gastric cancer . It has been proposed that atrophy is part of a natural progression of inflammatory changes that result from long term infection with the bacterium Helicobacter pylori . The aim of this study was to test this hypothesis using an animal model of human Helicobacter infection . Conventional mice were infected with either a cat isolate of Helicobacter felis or a human isolate of "Gastrospirillum hominis" . All infected mice showed a slowly progressive chronic gastritis with increasing numbers of infiltrating mononuclear cells and polymorphonuclear leucocytes . After a year and a half, the inflammatory reaction was so severe that atrophic changes were seen in both the antral and fundic mucosa . Control animals initially showed no inflammatory changes however as the animals aged, the gastric mucosa of some animals became infected with a bacterium Helicobacter muridarum that normally inhabits the small and large bowel of the rodent . The presence of this bacterium was also associated with gastritis and atrophic changes . This is the first report of experimentally induced atrophic changes induced by a gastric bacterium and opens the way for important experiments that will help better understand the induction of gastric cancer. Microb Pathog, 1993 Sep, 15(3), 177 - 85 Lack of pathotype specific gene in human Coxiella burnetii isolates; Stein A et al.; Human Q fever, due to the obligate intracellular bacterium Coxiella burnetii may be acute or chronic . The acute form is rarely fatal, although the chronic form, mostly represented by chronic Q fever endocarditis, is severe and usually fatal in lack of appropriate treatment . A correlation between the human disease state and plasmid types has been described, and specific DNA sequences unique to each plasmid type and which would code for specific pathotypes have been characterized . Because this gene specificity of the pathogenesis was hypothesized only on a small number of isolates, we evaluated seven reference isolates and 30 recent C . burnetii isolates from France for which all clinical data were available using the polymerase chain reaction by employing two specific primer sets . Our studies indicate that the previously described CbhE' plasmid-gene is not unique to C . burnetii isolates associated with acute disease, which would mean that the hypothesis of the correlation between gene specificity and pathotype has to be revised. Appl Environ Microbiol, 1993 Sep, 59(9), 2844 - 50 Glucose toxicity in Prevotella ruminicola: methylglyoxal accumulation and its effect on membrane physiology; Russell JB; When the ruminal bacterium prevotella ruminicola B(1)4-M was grown in a defined medium with an excess of glucose (3.6 mM ammonia and 50 mM glucose), the cells accumulated large amounts of cellular polysaccharide and the viable cell number decreased at least 1,000-fold . This decrease in viability was correlated with an accumulation of methylglyoxal in the supernatant (3 to 4 mM) . Other genetically distinct strains of P . ruminicola produced methylglyoxal, but methylglyoxal production was not ubiquitous among the strains . When P . ruminicola B(1)4-M was grown in continuous culture (dilution rate, 0.1 h-1) with an excess of glucose, there was an oscillating pattern of growth and cell death which was correlated with the accumulation and washout of methylglyoxal from the culture vessel . Mutants which resisted an excess of glucose took up glucose at a slower rate and produced less methylglyoxal than the wild type . These mutants were, however, not stable . There was always a long lag time, and the mutants could only be maintained with a daily transfer schedule . When the mutants were transferred less frequently, methylglyoxal eventually accumulated and the cultures died . The mutants transported glucose at a threefold-slower rate than the wild type, and in each case the carrier had more than one binding site for glucose . Because glucose transport could not be driven by phosphoenolpyruvate or ATP, the glucose carrier of P . ruminicola is probably a proton symport system . When P . ruminicola B(1)4-M cultures were treated with 4 mM methylglyoxal, the delta psi decreased even though intracellular ATP concentrations were high.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1993 Sep, 175(17), 5725 - 7 Identification of tlc and gltA mRNAs and determination of in situ RNA half-life in Rickettsia prowazekii; Cai J et al.; RNAs of Rickettsia prowazekii, an obligate intracytoplasmic bacterium, have been identified and analyzed by an RNase protection assay . Total RNA, a mixture of host cell RNA and rickettsial RNA, was isolated from rickettsia-infected mouse L929 cells by the hot-phenol method . After hybridization with specific antisense RNA probes and digestion with RNase, the protected products were analyzed by electrophoresis and autoradiography . The results show that there is only one mRNA species for the ATP/ADP translocase gene (tlc) but two mRNA species for the citrate synthase gene (gltA) . RNA half-lives were determined by measuring the RNA remaining after addition of rifampin . The half-lives of tlc mRNA, gltA mRNA I, and gltA mRNA II in R . prowazekii are 8.4 +/- 0.6, 12.3 +/- 1.3, and 20.5 +/- 1.8 min, respectively . However, the half-lives of tlc mRNA and gltA mRNA I in recombinant Escherichia coli strains are 2.9 +/- 0.1 and 1.4 +/- 0.1 min, respectively . The 16S rRNA in R . prowazekii was also examined and shown to be stable. Biochemistry, 1993 Aug 31, 32(34), 8871 - 9 The reaction center associated tetraheme cytochrome subunit from Chromatium vinosum revisited: a reexamination of its EPR properties; Nitschke W et al.; The heme components of chromatophore membranes from the purple bacterium Chromatium vinosum have been studied by EPR . Five different heme species could be distinguished on the basis of their g values, redox midpoint potentials, and orientations of heme planes with respect to the membrane plane: gz = 2.94, Em = +10 mV, 40 degrees-50 degrees; gz = 2.94, Em = +10 mV, 0 degree; gz = 3.1, Em = +330 mV, 90 degrees; gz = 3.3, Em = 360 mV, 30 degrees; gz = 3.4, Em = 0 mV, no detectable orientation . Four of these five hemes (gz = 3.3, gz = 3.1, and 2x gz = 2.94) were ascribed to the tetraheme cytochrome subunit associated with the photosynthetic reaction center of this bacterium . Some of the results obtained have already been reported previously {Tiede, D.M., Leigh, J.S., & Dutton, P.L . (1978) Biochim . Biophys . Acta 503, 524-544} and have led to a model for the tetraheme cytochrome subunit in Chromatium which is significantly different from the three-dimensional structure of the reaction center associated subunit in the purple bacterium Rhodopseudomonas viridis . The additional data obtained in our work, however, require a reinterpretation of the previously published results . The model arrived at is in general agreement with the X-ray structure from Rhodopseudomonas viridis . A model rationalizing the detailed differences between the structure of the Rhodopseudomonas viridis cytochrome subunit and the data obtained on tetraheme subunits from other photosynthetic bacteria is presented. FEBS Lett, 1993 Aug 30, 329(3), 319 - 23 Energy transfer processes in Rhodopseudomonas palustris grown under low-light conditions . Heterogeneous composition of LH 2 complexes and parallel energy flow pathways; Nishimura Y et al.; Excitation energy flow in the purple photosynthetic bacterium Rhodopseudomonas palustris grown under a low-light intensity was studied by time-resolved fluorescence spectroscopy in the ps time range . This bacterium synthesized the B824 component under this light condition . Time-resolved spectra at 20 degrees C indicated the sequential energy flow in the order of B803, B856, B882 and B900, long wavelength antenna . An emission from B803 was not observed . A remarkable feature was the emission from B824 throughout the measuring time . After the excitation pulse of 100 ps, the spectra did not change any further, indicating the establishment of an equilibrium among components . Based on the energy distribution after equilibrium, parallel energy transfer pathways to LH 1 were suggested; one including B824 integrated in the B803-824-856 complex, and the other, from the B803-856 complex to B882 . The latter was the dominant energy flow pathway in this bacterium. FEBS Lett, 1993 Aug 23, 329(1-2), 35 - 9 Two-step epoxidation of hyoscyamine to scopolamine is catalyzed by bifunctional hyoscyamine 6 beta-hydroxylase; Hashimoto T et al.; In several solanaceous plants, hyoscyamine is first hydroxylated at the 6 beta-position, and then epoxidized to scopolamine . We expressed hyoscyamine 6 beta-hydroxylase (H6H) in Escherichia coli as a fusion protein with maltose-binding protein . The crude cell extract from the bacterium that expressed the soluble fusion protein showed a strong hydroxylase activity and a weak epoxidase activity . When 100 microM of hyoscyamine was fed to the recombinant bacterium, the alkaloid was first converted to 6 beta-hydroxy hyoscyamine, and then to scopolamine, which was almost the only alkaloid found in the culture after one week . Therefore, H6H catalyzes two consecutive reactions that oxidize hyoscyamine to scopolamine. Biochem J, 1993 Aug 15, 294 ( Pt 1), 69 - 77 High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme; Wang C et al.; We have cloned the cDNA of rat hsc70 (clathrin-uncoating ATPase) into a T7 expression system and have expressed this enzyme in Escherichia coli . The recombinant clathrin-uncoating ATPase is in the cytosolic fraction of the bacterium and is soluble . It was purified to homogeneity by DEAE-cellulose and ATP-agarose column chromatography . From 1 litre of bacterial culture (0.3-0.4 g of proteins), 5-20 mg of pure recombinant clathrin-uncoating ATPase was routinely obtained . The cloned enzyme is capable of dissociating clathrin from bovine coated vesicle . Furthermore, it is not methylated on basic amino acid residues and is not blocked at the N-terminus, indicating that these modifications on hsc70 are not essential for uncoating of clathrin . Binding of {alpha-32P}ATP by purified recombinant hsc70 was analysed by Scatchard plot . The results indicate that there one high-affinity binding component with a Kd (dissociation constant) of 0.2-0.3 microM . The peptide-stimulated ATPase activities of recombinant hsc70 at 37 degrees C with respect to S-peptide peptides P3a and GT4 at a concentration of 1.2 mM are 142 +/- 6, 214 +/- 8 and 362 +/- 5 pmol/h per micrograms of hsc70 protein respectively . The EC50 values of hsc70 ATPase for S-peptide, peptides P3a and GT4 are 2, 0.67 and 0.17 mM respectively . On the other hand, the dissociation constants of S-peptide, peptides P3a and GT4 for recombinant hsc70 are 7.6, 13 and 100 microM respectively . Thus peptide GT4 is the only peptide examined for which the binding constant is comparable with the EC50 for stimulation ATPase activity, albeit it has the lowest affinity for hsc70. Biochem J, 1993 Aug 15, 294 ( Pt 1), 293 - 9 Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria; Fiermonte G et al.; The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been expressed at an abundant level in Escherichia coli . It accumulates in the bacterium as inclusion bodies, and none of the protein was detected in the bacterial inner membrane . The mitochondrial ADP/ATP carrier, a member of the same super-family of transport proteins as the oxoglutarate carrier, has also been expressed in E . coli . However, the expression of the ADP/ATP carrier in bacteria retards their growth, and so the levels of expression that were attained were lower than those of the oxoglutarate carrier . The oxoglutarate carrier inclusion bodies have been disaggregated with the detergent N-dodecanoyl-sarcosine, and the protein has been incorporated into liposomes . In its ability to transport oxoglutarate and malate and other known substrates of the carrier in mitochondria, and in its inhibition characteristics by a wide range of non-competitive and competitive inhibitors, this reconstituted oxoglutarate carrier is similar to the natural protein in the inner membranes of mitochondria, and to the carrier that has been purified from mitochondria and reconstituted in liposomes . These experiments remove significant obstacles to crystallization trials and to site-directed mutagenesis of the oxoglutarate carrier. Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7824 - 8 Reversible opening of the blood-brain barrier by anti-bacterial antibodies; Tuomanen EI et al.; The leukocyte adhesion molecule CR3 (CD11b/CD18, Mac-1) promotes leukocyte transmigration into tissues by engaging an unknown cognate ligand on the surface of vascular endothelial cells . Filamentous hemagglutinin (FHA), an adhesin of the bacterium Bordetella pertussis, binds to CR3 . We hypothesized that FHA mimics the native ligand for the CR3 integrin on endothelial cells and predicted that anti-FHA antibodies should bind to endothelial cells, interfere with leukocyte recruitment, and induce endothelial permeability . Anti-FHA monoclonal antibodies bound to cerebral microvessels in sections from human brain and upon intravenous injection into rabbits . Antibody binding correlated with the ability to recognize two polypeptides in extracts of human cerebral vessels that were also bound by CD18 . In vivo, antibody binding not only interfered with transmigration of leukocytes into cerebrospinal fluid but also induced a dose-dependent reversible increase in blood-brain barrier permeability sufficient to improve delivery of intravenously administered therapeutic agents to brain parenchyma. Immunol Today, 1993 Aug, 14(8), 387 - 91 Current perspectives in reactive arthritis; Kingsley G et al.; Reactive arthritis (ReA) is an inflammatory arthritis triggered by infection, usually urethritis or gastroenteritis, and is strongly associated with the MHC class I antigen HLA-B27 . Two recent observations have excited interest: first, antigen and DNA from the triggering bacteria have been identified in the joint and, second, ReA synovial T cells have been found to respond specifically to the bacterium that caused the initiating infection . Because the trigger of ReA, its onset and the MHC association are all clearly defined, we can investigate hypotheses that are impossible to study in other forms of human arthritis . Here, Gabrielle Kingsley and Jochen Sieper review the topic in the light of a recent workshop. Infect Immun, 1993 Aug, 61(8), 3570 - 3 Expression of a functional Porphyromonas gingivalis fimbrillin polypeptide in Escherichia coli: purification, physicochemical and immunochemical characterization, and binding characteristics; Sharma A et al.; Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses . A structural subunit of the fimbriae, fimbrillin, has been shown to be important in binding of the bacterium to saliva-coated oral surfaces . In the present study, a coding region of the fimbrillin gene from P . gingivalis 2561 was amplified by the polymerase chain reaction and cloned into the pET-11d vector . The recombinant plasmid was transformed into Escherichia coli BL21, and protein expression was induced with isopropyl-beta-D-thiogalactopyranoside . The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and gel filtration chromatography . The purified recombinant fimbrillin polypeptide, r-fim 10-337, corresponded to amino acid residues 10 to 337 of the deduced amino acid sequence of fimbrillin . In immunoblot analysis, r-fim 10-337 reacted with antibodies to fimbrillin purified from P . gingivalis, as well as with antibodies to synthetic peptides corresponding to the amino acid sequence of fimbrillin . The apparent molecular mass of r-fim 10-337 was estimated to be 41 kDa on sodium dodecyl sulfate-polyacrylamide gels . The r-fim 10-337 polypeptide was capable of inhibiting the binding of P . gingivalis 2561 to saliva-coated hydroxyapatite beads . These results suggest that the fimbrillin subunit polypeptide plays an important role in binding of P . gingivalis cells to saliva-coated surfaces . We describe here the successful expression and purification of a functionally and immunologically reactive recombinant P . gingivalis fimbrillin subunit from E . coli. Appl Environ Microbiol, 1993 Aug, 59(8), 2631 - 7 Cellobiose versus glucose utilization by the ruminal bacterium Ruminococcus albus; Thurston B et al.; Cellulose degradation and metabolism in the rumen can be adversely affected by the presence of soluble sugars, but relatively little information is available on substrate preferences of cellulolytic bacteria . When the ruminal bacterium Ruminococcus albus was incubated with a combination of cellobiose and glucose, the organism preferentially utilized the disaccharide . This preference appeared to be related to repression of glucose uptake systems in cellobiose-grown cells . Glucose transport kinetics exhibited low- and high-affinity uptake, and high-affinity transport was apparently driven by ATP hydrolysis . Bacterial yield in continuous culture was as much as 38% greater when the organism was grown on cellobiose versus glucose, and the increased yield could be partially attributed to constitutive cellobiose phosphorylase activity . The maintenance coefficient of glucose-grown cells was significantly greater than that of cells provided with cellobiose (0.225 g of glucose per g of protein per h versus 0.042 g of cellobiose per g of protein per h), and this result suggested that more energy was devoted to glucose uptake . Substrate affinities were examined in carbon-excess continuous culture, and affinities for glucose and cellobiose were relatively low (0.97 and 3.16 mM, respectively) . Although R . albus maintained a proton motive force of approximately 60 mV from pH 6.7 to 5.5, growth ceased below pH 6.0, and this inhibition of growth may have been caused by a depletion of ATP at low pH. Eur J Biochem, 1993 Aug 1, 215(3), 633 - 43 Enzymes of a novel autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus, the 3-hydroxypropionate cycle; Strauss G et al.; The phototrophic bacterium Chloroflexus aurantiacus can grow autotrophically but seems not to assimilate CO2 via any of the known autotrophic pathways . Holo {Holo, H . (1989) Arch . Microbiol . 151, 252-256} proposed a new pathway in which 3-hydroxypropionate is formed from acetyl-CoA . Previous studies excluded the operation of known CO2 fixation pathways and provided indirect evidence for the suggested pathway based on 13C-labelling experiments . Here all enzyme activities of the postulated cyclic CO2 fixation mechanism are demonstrated in vitro . In essence, acetyl-CoA is carboxylated and reductively converted via 3-hydroxypropionate to propionyl-CoA . Propionyl-CoA is carboxylated and converted via succinyl-CoA and CoA transfer to malyl-CoA . Malyl-CoA is cleaved to acetyl-CoA and glyoxylate . Thereby, the first CO2 acceptor molecule acetyl-CoA is regenerated, completing the cycle and the net CO2 fixation product glyoxylate is released . This cycle represents the fourth autotrophic pathway in nature and is designated the 3-hydroxypropionate cycle. Biochem J, 1993 Aug 1, 293 ( Pt 3), 683 - 9 A glycosulphatase that removes sulphate from mucus glycoprotein; Roberton AM et al.; A novel glycosulphatase has been purified from a mucus glycopeptide-degrading Prevotella from the colon . The purified enzyme removed inorganic {35S}sulphate from 35S-labelled native rat gastric mucus glycoprotein . Desulphation of mucus glycoprotein was initially rapid (19% complete after 10 min) but then plateaued, reaching only 33% after 3 h . Crude periplasmic extracts could remove 79% of the radioactivity as inorganic sulphate . These results suggest that steric hindrance may limit the access of the purified glycosulphatase to the mucus glycoprotein oligosaccharide chains in the absence of glycosidases, and/or that the enzyme may have the wrong specificity for some of the remaining sulphated sugars in the chains . The apparent molecular mass of the enzyme was 111 kDa as judged from gel exclusion chromatography, and it appeared to be composed of two identical subunits . The enzyme was localized in the periplasm of the bacterium, and using pig gastric mucus glycopeptide as a growth substrate markedly increased enzyme levels . Enzymic activity increased at the end of the growth phase . The substrate specificity of the enzyme was tested against low-molecular-mass sulphated molecules . The monosaccharides glucose 6-sulphate and N-acetylglucosamine 6-sulphate were rapidly desulphated, the latter being the major sulphated sugar in some mucus glycoproteins . Lactose 6-sulphate, galactose 6-sulphate, sulphated steroids and unsaturated disaccharide sulphate breakdown products from chondroitin sulphate were not desulphated . Glycosulphatases which can remove sulphate from mucus glycoproteins may play an important role in the degradation of highly sulphated mucus glycoproteins in the digestive tract, and could modify the effectiveness of mucus glycoproteins in mucosal protection. Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7124 - 8 Single core polypeptide in the reaction center of the photosynthetic bacterium Heliobacillus mobilis: structural implications and relations to other photosystems; Liebl U et al.; The gene for a reaction center core polypeptide from the anoxygenic photosynthetic bacterium Heliobacillus mobilis was cloned and sequenced . The deduced amino acid sequence consists of 609 residues with a molecular mass of 68 kDa . An adjacent open reading frame is not transcribed under our experimental conditions . No evidence for a second related reaction center core gene was found . The primary sequence of the reaction center protein (P800 protein) shows a high percentage of sequence identity to photosystem I in a cysteine-containing loop, which is the putative binding site of the iron-sulfur center FX and in the preceding hydrophobic region . Our data imply a homodimeric organization of the reaction center . This is fundamentally different from photosystem I and most other photosynthetic reaction centers, where the reaction center core is composed of two similar but nonidentical subunits. J Infect Dis, 1993 Aug, 168(2), 379 - 85 An uncultured gastric spiral organism is a newly identified Helicobacter in humans; Solnick JV et al.; "Gastrospirillum hominis" is an uncultivated spiral bacterium in human gastric mucosa that is larger and more tightly coiled than Helicobacter pylori . In an attempt to determine if this organism is a new species of Helicobacter, its 16S rRNA gene was cloned and sequenced . Gastric mucosa from 2 patients infected with "Gastrospirillum hominis" was fed to specific pathogen-free mice . Electron microscopy of gastric tissue confirmed that the mice became colonized with "Gastrospirillum hominis." The 16S rRNA gene from bacterial target sequences was amplified directly from mouse stomach tissue by the polymerase chain reaction (PCR), cloned into Escherichia coli, and sequenced . Both fragments were 16S rRNA genes from the Helicobacter genus that were most closely related to Helicobacter felis . "Gastrospirillum hominis" is probably a newly recognized Helicobacter infection in humans . Because this is the only Helicobacter organism that infects both humans and small animals, it may be particularly suited for studies of pathogenesis. J Bacteriol, 1993 Aug, 175(15), 4662 - 9 Purine metabolism by intracellular Chlamydia psittaci; McClarty G et al.; Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism . C . psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines . C . psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell . Exogenous hypoxanthine and inosine were not utilized by the parasite . In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C . psittaci AA Mp . Crude extract prepared from highly purified C . psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity . Adenosine kinase activity was detected, but guanosine kinase activity was not . There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine . Thus, there is no interconversion of nucleotides . Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract. J Bacteriol, 1993 Aug, 175(15), 4652 - 61 Pyrimidine metabolism by intracellular Chlamydia psittaci; McClarty G et al.; Pyrimidine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined mutations affecting pyrimidine metabolism . C . psittaci AA Mp cannot synthesize pyrimidines de novo, as assessed by its inability to incorporate aspartic acid into nucleic acid pyrimidines . In addition, the parasite cannot take UTP, CTP, or dCTP from the host cell, nor can it salvage exogenously supplied uridine, cytidine, or deoxycytidine . The primary source of pyrimidine nucleotides is via the salvage of uracil by a uracil phosphoribosyltransferase . Uracil phosphoribosyltransferase activity was detected in crude extracts prepared from highly purified C . psittaci AA Mp reticulate bodies . The presence of CTP synthetase and ribonucleotide reductase is implicated from the incorporation of uracil into nucleic acid cytosine and deoxycytidine . Deoxyuridine was used by the parasite only after cleavage to uracil . C . psittaci AA Mp grew poorly in mutant host cell lines auxotrophic for thymidine . Furthermore, the parasite could not synthesize thymidine nucleotides de novo . C . psittaci AA Mp could take TTP directly from the host cell . In addition, the parasite could incorporate exogenous thymidine and thymine into DNA . Thymidine kinase activity and thymidine-cleaving activity were detected in C . psittaci AA Mp reticulate body extract . Thus, thymidine salvage was totally independent of other pyrimidine salvage. Infect Immun, 1993 Aug, 61(8), 3503 - 10 Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila; Barker J et al.; The surface properties of Legionella pneumophila were examined by analyzing outer membrane (OM) proteins, lipopolysaccharides (LPS), and cellular fatty acids after growth within Acanthamoeba polyphaga and in vitro under various nutrient-depleted conditions . Intra-amoeba-grown legionellae were found to differ in several respects from cells grown in vitro; most notably, they contained a 15-kDa OM protein and a monounsaturated straight-chain fatty acid (18:1(9)) . These compounds were also found in abundant quantities in the host amoeba . Immunoblot analysis of intra-amoeba-grown legionellae with antiacanthamoebic serum revealed that both the bacterial whole cells and Sarkosyl-extracted OMs contained amoebic antigens . The findings suggest that the 15-kDa OM protein is likely to be of amoebic origin and associates with the OM of the bacterium . It is proposed that disruption of amoebic membranes, as a result of intra-amoebic infection, may liberate macromolecules, including a 15-kDa polypeptide, a major constituent of the amoebic membrane, which adhere to the surface of the legionellae . Growth under specific nutrient depletions also had a significant effect on the surface composition of L . pneumophila . Cells grown under phosphate depletion were markedly sensitive to protease K digestion and contained lower levels of LPS, as observed by silver staining of the digests on polyacrylamide gels . Intra-amoeba-grown cells contained more bands than the in vitro-grown organisms, reflecting further differences in the nature of the LPS . The whole-cell fatty acids of the phosphate-depleted cells were appreciably different from those of cells grown under other nutritional conditions . We found no evidence for expression of iron-regulated OM proteins under iron depletion. Biophys J, 1993 Aug, 65(2), 652 - 60 Study of wild type and genetically modified reaction centers from Rhodobacter capsulatus: structural comparison with Rhodopseudomonas viridis and Rhodobacter sphaeroides; Baciou L et al.; Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy . In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type . In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type . In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed . In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala . We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor . The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type . At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants . In contrast to Rb . sphaeroides, in the wild type Rb . capsulatus, kBP substantially increases in the pH range 7-10.(ABSTRACT TRUNCATED AT 250 WORDS) Genet Res, 1993 Aug, 62(1), 23 - 9 Evidence for a Wolbachia symbiont in Drosophila melanogaster; Holden PR et al.; The bacterial cell division gene, ftsZ, was used as a specific probe to show the presence of a symbiotic bacterium in two wild type strains of Drosophila melanogaster . Under stringent hybridization conditions we have shown that the bacterium is transferred to the progeny of these strains from infected mothers and can be eradicated by treatment with the antibiotic tetracycline . We have characterized this bacterium, by amplifying and sequencing its 16S rRNA gene, as being a member of the genus Wolbachia, an organism that is known to parasitize a range of insects including Drosophila simulans . In a series of reciprocal crosses no evidence was found that the symbiont causes cytoplasmic incompatibility (CI) which is known to occur in infected strains of D . simulans . The implications of these findings are discussed. Biochem Biophys Res Commun, 1993 Jul 30, 194(2), 733 - 40 Cloning of gene responsible for tributyltin chloride (TBTC1) resistance in TBTC1-resistant marine bacterium, Alteromonas sp . M-1; Fukagawa T et al.; A genetic library of tributyltin chloride (TBTC1)-resistant marine bacterium, Alteromonas sp . M-1, was constructed using plasmid vector pUC 19 . Three positive clones were obtained from E . coli JM 109 transformed with the plasmids by the method of replica plating to LB medium containing 1 mM TBTC1 . These clones could grow in LB liquid medium containing 100 microM TBTC1 . Plasmids harbouring genes of Alteromonas sp . M-1 were designated pTBT1, pTBT2 and pTBT3 which contain 1.8 kb Hind III-fragment, 4.8 kb Pst I-fragment and 7.8 kb Pst I-fragment, respectively . Nucleotide sequence of the shortest fragment, 1.8 kb Hind III-fragment was determined, revealing an open reading frame (ORF) was contained in the fragment . The ORF was 324 bp (108 amino acids) . The 48.5% of the amino acids encoded was hydrophobic, suggesting that the product relating to TBTC1 resistance might be membrane related protein . Homology search in amino acids alignment indicated that the product has homology with transport proteins. FEBS Lett, 1993 Jul 26, 327(2), 199 - 202 Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme; Baykov AA et al.; 1,1-Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H(+)-translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata . The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 microM . Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate . By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase . It is concluded that the mitochondrial pyrophosphatase and the H(+)-pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme. FEBS Lett, 1993 Jul 19, 327(1), 68 - 70 Picosecond dynamics of excitations in light-harvesting complex B800-850 from Chromatium minutissimum studied using fluorescence spectrochronography; Godik V et al.; The picosecond dynamics of excitations in the isolated B800-850 light-harvesting complex of the purple sulfur bacterium Chromatium minutissimum has been studied using picosecond fluorescence spectrochronography . A short-lived component of about 20 ps lifetime has been found at 77K at the short wavelength part of the B850 fluorescence spectrum similar to that previously described for the core antenna bacteriochlorophyll band B880 of Rhodospirillum rubrum . Evidence has been presented indicating that this component is likely to reflect excitation energy relaxation step(s) involving both photoexcited bacteriochlorophyll and the protein environment . A new kinetic scheme of excitation transfer from the peripheral antenna to the photoreceptor units in purple bacteria is suggested which takes into account these findings. Gene, 1993 Jul 15, 129(1), 33 - 40 Use of a promoter-probe vector system in the cloning of a new NifA-dependent promoter (ndp) from Bradyrhizobium japonicum; Weidenhaupt M et al.; Many of the symbiotic nitrogen-fixation genes in the soybean root nodule bacterium, Bradyrhizobium japonicum, are transcribed from -24/-12 promoters that are recognized by the sigma 54-RNA polymerase and activated by the transcriptional regulator protein, NifA . Several lines of evidence suggest that the B . japonicum genome has more than those seven NifA-regulated promoters which were characterized previously . Here, we present a strategy aimed at the cloning of new NifA-activated promoters . It makes use of (i) a promoter-probe vector into which random B . japonicum genomic fragments were cloned in front of a promoterless reporter gene and (ii) a screening procedure that allowed us to distinguish constitutive promoters from promoters that were specifically activated by NifA under microaerobic or anaerobic conditions . With certain modifications, the system may be generally applicable to clone positively regulated, anaerobically induced genes . A novel NifA-dependent promoter region (ndp) of B . japonicum was found by these means . The transcription start point was mapped, and its 5'-flanking DNA carried a -24/-12-type promoter sequence plus potential binding sites for NifA and integration host factor . Further transcript analyses confirmed that maximal transcription from this promoter occurred only in the presence of NifA and sigma 54 during anaerobic growth of B . japonicum . In Escherichia coli, expression of beta-galactosidase derived from a transcriptional ndp::lacZ fusion was activated 11-fold by B . japonicum NifA, and this activation also required sigma 54 but was independent of NtrC . The DNA around ndp shared no similarity with known sequences in databases.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Jul 6, 32(26), 6688 - 95 Role of anionic phospholipids in the interaction of doxorubicin and plasma membrane vesicles: drug binding and structural consequences in bacterial systems; de Wolf FA et al.; Anthracycline-membrane interactions play a role in the transport, the cytoplasmic distribution, and possibly also the activity of anthracyclines . Previous work on model membranes has shown that the widely-applied anticancer drug doxorubicin interacts specifically with anionic phospholipids {de Wolf, F . A., et al . (1991) Biochim . Biophys . Acta 106, 67-80} . We have now been able to investigate these interactions, and their selectivity for anionic phospholipids, directly in plasma membranes . Because of the recent availability of Escherichia coli mutants in which the anionic phospholipid content ranges from only 10% to as much as 100% of the total phospholipid content, we used this bacterium as a source of plasma membranes . We compared the interactions of the cationic anthracycline doxorubicin with (1) plasma membranes of different mutant strains, (2) total lipid extracts of these membranes, and (3) synthetic phospholipid mixtures in which a comparable fraction of the phospholipids was negatively charged . The results show that anionic phospholipids are important determinants of doxorubicin binding, not only in model membranes but also in plasma membrane systems . Only in plasma membranes with a very low anionic lipid content was the binding to the anionic phospholipid masked by other factors . Using an unsaturated fatty acid auxotroph grown on {11,11-2H2}oleic acid, it appeared from 2H-NMR data that doxorubicin induces a disordering of acyl chains in bacterial plasma membranes and their total lipid extracts . This indicates that the binding is not purely electrostatic but involves the insertion of drug molecules into the lipid matrix, probably due to hydrophobic interactions. J Biol Chem, 1993 Jul 5, 268(19), 14426 - 31 Nucleotide sequence of the heme subunit of flavocytochrome c from the purple phototrophic bacterium, Chromatium vinosum . A 2.6-kilobase pair DNA fragment contains two multiheme cytochromes, a flavoprotein, and a homolog of human ankyrin; Dolata MM et al.; The gene for the cytochrome subunit of Chromatium vinosum flavocytochrome c (sulfide dehydrogenase) was cloned from an EcoRI digest of chromosomal DNA . The mature cytochrome subunit contains 175 amino acid residues and two heme binding sites in agreement with the previously reported amino acid sequence . There is also a signal peptide of 25 residues, which apparently directs the protein to the periplasmic space . There are two open reading frames upstream of the heme subunit gene, which encode a tetraheme cytochrome c and a homolog of human ankyrin . The gene for the flavoprotein subunit of flavocytochrome c is in frame 15 nucleotides downstream of the stop codon for the cytochrome gene . Messenger RNA was isolated from malate grown cells . The transcript is approximately 3 kilobases in size and does not hybridize with a probe containing the tetraheme cytochrome gene and part of the ankyrin homolog gene . The heme subunit and flavoprotein subunit genes thus appear to form an operon . The flavoprotein subunit has a 30-residue signal peptide . The clone ends 95 amino acids into the N-terminal sequence of the mature flavoprotein subunit (which should contain about 400 residues) . The apparently periplasmic location of flavocytochrome c has important consequences for the presumed function as a sulfide dehydrogenase, because sulfur, which is the product of oxidation, is stored in the cytoplasm . Our results on the location of the enzyme are incompatible with this function. Proc Soc Exp Biol Med, 1993 Jul, 203(3), 323 - 7 Differential effects of ethanol on permissive versus nonpermissive macrophages infected with Legionella pneumophila; Yamamoto Y et al.; The effect of ethanol treatment was studied in terms of effect on permissive versus nonpermissive macrophages for growth of Legionella pneumophila, which is an intracellular bacterium causing pneumonia in immunocompromised patients . It was found that ethanol treatment of permissive macrophages from L . pneumophila-susceptible A/J mice evinced a decrease in replication of the bacteria compared with nontreated infected macrophages . Whereas there was more than a 100-fold increase in Legionella growth over a 48-hr culture period in infected A/J mouse macrophages, treatment of the macrophages with 0.5% ethanol depressed the ability of the macrophages to be infected by Legionella approximately 45% . A lower concentration of ethanol had a lesser effect but still resulted in inhibition of the ability of the cells to replicate Legionella . In contrast to ethanol-induced inhibition of the A/J mouse macrophages to replicate Legionella, macrophages from Legionella-resistant BALB/c mice, which only minimally replicated Legionella (i.e., only a 2-fold increase or less over a 48-hr replicated Legionella (i.e., only a 2-fold increase or less over a 48-hr period), treatment with ethanol resulted in their greater replication of the Legionella . This effect was most marked with the 1.0% concentration of ethanol after 7 days of pretreatment, while the 0.5% and 0.1% concentrations of alcohol caused less enhancement of bacterial growth in the cells, but these concentrations still had a significant enhancement effect . Thus, ethanol had differing effects on growth of the opportunistic intracellular bacterium Legionella in macrophages from permissive versus nonpermissive mice . Studies on the mechanisms involved are in progress. Eur J Biochem, 1993 Jul 1, 215(1), 25 - 35 Detection of the in vivo incorporation of a metal cluster into a protein . The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus; Gollan U et al.; The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate . The MoO4(2-) concentration causing 50% repression of the alternative nitrogenase in nifHDK- cells was 6 nM . If MoO4(2-) was added to a growing nifHDK- culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically . In spite of the fact that C2H4 formation decreased continuously during the duration of the experiment (3 days), the total C2H6 production increased about twofold within the first 24 h, whereas the relative yield of C2H6 increased from 2% (C2H6/C2H4 x 100) in the absence of MoO4(2-), to a maximal value of 69% in the presence of MoO4(2-) (1 mM) after 72 h incubation . This 'Mo effect' appeared to be stronger the higher the MoO4(2-) concentration in the medium and the longer the incubation time . In the presence of ReO4-, WO4(2-) or VO4(3-), a similar effect did not occur . The 'Mo effect' was not observed in a nifHDK- nifE- double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK- nifQ- mutant . Crude extracts from nifHDK- cells cultivated in the presence of MoO4(2-), also showed enhanced production of ethane . Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for the FeMoco . These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase . The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures . The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo protein synthesis . The analysis of nifE-lacZ and nifN-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO4(2-) deficiency . The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed. Aust Vet J, 1993 Jul, 70(7), 241 - 3 Sampling ryegrass to assess the risk of annual ryegrass toxicity; McKay AC et al.; Most stock losses caused by annual ryegrass toxicity occur because stockowners unknowingly allow their stock to graze annual ryegrass (Lolium rigidum) infected with the bacterium Clavibacter toxicus . To help stockowners avoid losses we have developed criteria for a testing service to determine the risk of poisoning before the pasture is grazed . Low, medium and high risk categories were selected using samples of dry, mature ryegrass seedheads collected by stockowners from untreated, infected pastures in South Australia . The proportion of toxic paddocks in each risk category over all the seasons tested was 11%, 32% and 76%, respectively, and these accounted for 7%, 14% and 79% of total stock losses . The proportion of paddocks in which stock were poisoned did not vary significantly between years, was not affected by variation in sample weight, and did not vary between South Australia and Western Australia. Antimicrob Agents Chemother, 1993 Jul, 37(7), 1410 - 3 Antibiotic susceptibilities of Afipia felis in axenic medium and in cells; Maurin M et al.; Afipia felis, one of the putative agents of cat scratch disease (CSD), is a facultative intracellular bacterium . Although CSD is considered not to be susceptible to antibiotic therapy, sporadic case reports indicated that aminoglycosides may be effective . We determined the in vitro antibiotic susceptibilities of three A . felis strains in axenic medium and in a cell model . In axenic medium, A . felis was susceptible to imipenem, aminoglycosides, and rifampin when using either the broth dilution technique or the agar technique . When grown in HeLa cells, A . felis was susceptible to amikacin and tobramycin but was resistant to the other compounds tested . Despite its intracellular location, A . felis can apparently be reached by aminoglycosides . Thus, the in vitro data presented here are in accord with the clinical data obtained in patients suffering CSD. Arch Biochem Biophys, 1993 Jul, 304(1), 117 - 22 Purification and properties of an unusual membrane-derived cytochrome b-561 from the purple phototrophic bacterium Rhodobacter capsulatus, which is structurally related to the bacteriochlorophyll-binding protein, LHII beta; Bartsch RG et al.; An abundant cytochrome b-561 was solubilized from Rhodobacter capsulatus membranes by successive treatments with perchlorate and butanol/water . Neither procedure was effective alone although they could be combined into a single step . Once solubilized, cytochrome b-561 was purified by standard chromatographic procedures used for water-soluble proteins without addition of butanol or detergents . Cytochrome b-561 appears to be highly acidic, it has a size greater than about 1000 kDa as isolated, and the subunit size measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is less than 8 kDa . The redox potential measured by cyclic voltammetry is -65 mV at pH 7 . The N-terminal amino acid sequence is identical to that of the Rb . capsulatus LHII beta light-harvesting bacteriochlorophyll binding protein subunit which has only 48 amino acid residues, and the mass, determined by mass spectroscopy, is identical to that of LHII beta . There is but one heme per two to three peptide chains of 5 kDa, which suggests that the two extraplanar ligands to the heme are on separate subunits . There is strong exciton splitting in the circular dichroism spectrum in the Soret region indicative of heme-heme interaction . The helix content based on far-uv CD is 41% . Together, these properties of cytochrome b-561 are very similar to those of isolated LHII alpha beta bacteriochlorophyll-protein complexes. J Exp Med, 1993 Jul 1, 178(1), 197 - 209 Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine; Stover CK et al.; The current vaccine against tuberculosis, Mycobacterium bovis strain bacille Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for the expression and delivery of protective recombinant antigens (Stover, C.K., V.F . de la Cruz, T.R . Fuerst, J.E . Burlein, L.A . Benson, L.T . Bennett, G.P . Bansal, J.F . Young, M.H . Lee, G.F . Hatfull et al . 1991 . Nature {Lond} . 351:456; Jacobs, W.R., Jr., S.B . Snapper, L . Lugosi and B.R . Bloom . 1990 . Curr . Top . Microbiol . Immunol . 155:153; Jacobs, W.R., M . Tuckman, and B.R . Bloom . 1987 . Nature {Lond.} . 327:532); but as an attenuated intracellular bacterium residing in macrophages, BCG would seem to be best suited for eliciting cellular responses and not humoral responses . Since bacterial lipoproteins are often among the most immunogenic of bacterial antigens, we tested whether BCG expression of a target antigen as a membrane-associated lipoprotein could enhance the potential for a recombinant BCG vaccine to elicit high-titered protective antibody responses to target antigens . Immunization of mice with recombinant BCG vaccines expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi as a membrane-associated lipoprotein resulted in protective antibody responses that were 100-1,000-fold higher than responses elicited by immunization with recombinant BCG expressing OspA cytoplasmically or as a secreted fusion protein . Furthermore, these improved antibody responses were observed in heterogeneous mouse strains that vary in their immune responsiveness to OspA and sensitivity to BCG growth . Thus, expression of protective antigens as chimeric membrane-associated lipoproteins on recombinant BCG may result in the generation of new candidate vaccines against Lyme borreliosis and other human or veterinary diseases where humoral immunity is the protective response. Avian Dis, 1993 Jul-Sep, 37(3), 706 - 14 Inhibition of Mycoplasma gallisepticum growth and attachment to chick tracheal rings by antibodies to a 64-kilodalton membrane protein of M . gallisepticum; Avakian AP et al.; A Mycoplasma gallisepticum (MG) strain R protein of 64 kilodaltons (p64) was partially digested from the surface of the bacterium by trypsin . Monospecific polyclonal anti-p64 IgG inhibited attachment of MG to chick tracheal rings by as much as 69% . However, trypsin treatment of viable MG cells did not reduce attachment to tracheal rings or hemagglutination titer . Anti-p64 IgG inhibited growth of MG strain R in broth and on solid media, inhibited the uptake of radiolabeled thymidine, but did not inhibit hemagglutination . Anti-p64 IgG inhibited growth of eight MG strains on solid medium . The degree of growth inhibition varied widely depending on the strain and correlated positively with the reported virulence of the MG strains with one exception (A5969) . An IgG monoclonal antibody to p64 (MyG 001) inhibited growth of MG strain R on solid and in broth media . The strong attachment-inhibition activity of anti-p64 IgG may result from its growth-inhibiting activity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MG strains suggested that p64 is expressed in higher amounts in vitro in virulent strains (R, S6) than in strains of low virulence (F, M876, K503, K703, K730) . P64 should be used to immunize chickens to determine if it can stimulate a growth and attachment-inhibiting response in the respiratory tract. J Biol Chem, 1993 Jun 25, 268(18), 13364 - 71 Methyl esterification of C-terminal leucine residues in cytosolic 36-kDa polypeptides of bovine brain . A novel eucaryotic protein carboxyl methylation reaction; Xie H et al.; Incubation of cytosolic extracts of bovine brain with S-adenosyl{methyl-3H}methionine results in the predominant {3H}methyl esterification of a 36-kDa polypeptide . This reaction appears to be distinct from any of the three known types of protein carboxyl methylation reactions previously established . We show here that the methylated 36-kDa polypeptide is a component of a cytosolic protein with a native molecular mass estimated at 178 kDa by gel filtration chromatography . The methyl group is not stable on the protein and is lost as {3H}methanol with a half-life of about 180 min at pH 7.0, 37 degrees C . The methyltransferase responsible for this reaction is a cytosolic protein with a native molecular mass of about 40 kDa that is readily separated from the well described protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) . The methyl ester linkage is cleaved by carboxypeptidase Y, suggesting that the 36-kDa polypeptide is methylated on its C-terminal carboxyl group . Extensive digestion of gel-purified 3H-methylated 36-kDa polypeptide with trypsin and leucine aminopeptidase results in a radioactive product that co-chromatographs with authentic L-leucine methyl ester in reverse phase high performance liquid chromatography (HPLC), thin layer chromatography, thin layer electrophoresis, and high resolution-sulfonated polystyrene cation-exchange chromatography . Additionally, the o-phthalaldehyde/beta-mercaptoethanol-derived isoindole derivative of the 3H digestion product co-migrates on HPLC with the corresponding isoindole for L-leucine methyl ester . We demonstrate that a similar methylation system is present in yeast Saccharomyces cerevisiae but not in the bacterium Escherichia coli . These results provide evidence for a new type of reversible posttranslational modification reaction that may function to modulate the activities of its methyl-accepting substrates. Eur J Biochem, 1993 Jun 1, 214(2), 599 - 607 Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb . nov., strain GJ1B; Potin P et al.; The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar {Young, K . S . Bhattacharjee, S . S . & Yaphe, W . (1978) Carbohydr . Res . 66, 207-212}, were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products . The bacterium was assigned to the genus Alteromonas and the new combination A . agarlyticus (Cataldi) is proposed . An alpha-agarase, i.e . specific for the alpha(1-->3) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CL-6B) and by anion-exchange chromatography (Mono Q column) . The major end product of agarose hydrolysis using the purified enzyme was agarotetraose . Using SDS/PAGE, the purified alpha-agarase was detected as a single band with a molecular mass of 180 kDa . After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography . The isolectric point was estimated to be 5.3 . Enzyme activity was observed using agar as the substrate over the pH range 6.0-9.0 with a maximum value at pH 7.2 in Mops or Tris buffer . The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45 degrees C or by removing calcium . In addition, a beta-galactosidase specific for the end products of the alpha-agarase was present in the alpha-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme . The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues. J Bacteriol, 1993 Jun, 175(12), 3893 - 6 Copy number of the 16S rRNA gene in Rickettsia prowazekii; Pang H et al.; The obligate intracellular parasite, Rickettsia prowazekii, is a slowly growing bacterium with a doubling time of 8 to 12 h . The copy number of the 16S rRNA gene in the rickettsial chromosome was determined to be one . Genomic DNA from R . prowazekii was digested either by a variety of restriction enzymes known not to cut at any site in the rickettsial 16S rRNA gene or by a combination of these noncutting enzymes and SmaI, which cuts the gene only once . Only one DNA fragment in these digests hybridized to a biotinylated probe containing a portion of the rickettsial 16S rRNA gene . Moreover, the density of the rickettsial 16S rRNA gene fragment after hybridization was equal to the density of each of the seven 16S rRNA gene fragments in Escherichia coli. J Bacteriol, 1993 Jun, 175(12), 3776 - 83 Genetics of serine pathway enzymes in Methylobacterium extorquens AM1: phosphoenolpyruvate carboxylase and malyl coenzyme A lyase; Arps PJ et al.; Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that uses the serine pathway for formaldehyde incorporation as its assimilation pathway during growth on one-carbon compounds . A DNA region from M . extorquens AM1 previously shown to contain genes for the serine pathway enzymes malyl coenzyme A (CoA) lyase and hydroxypyruvate reductase has been characterized in more detail . Insertion mutagenesis revealed an additional region required for growth on one-carbon compounds, and all of the insertion mutants in this region lacked activity for another serine pathway enzyme, the acetyl-CoA-independent phosphoenolpyruvate (PEP) carboxylase . Expression analysis with Escherichia coli of DNA fragments that included the malyl-CoA lyase and PEP carboxylase regions identified five polypeptides, all transcribed in the same direction . Three of these polypeptides were expressed from the region necessary for the acetyl-CoA-independent PEP carboxylase, one was expressed from the region containing the malyl-CoA lyase gene, and the fifth was expressed from a region immediately downstream from the gene encoding hydroxypyruvate reductase . All six genes are transcribed in the same direction, but the transposon insertion data suggest that they are not all cotranscribed. Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 4897 - 901 Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium; Langin D et al.; The human hormone-sensitive lipase (HSL) gene encodes a 786-aa polypeptide (85.5 kDa) . It is composed of nine exons spanning approximately 11 kb, with exons 2-5 clustered in a 1.1-kb region . The putative catalytic site (Ser423) and a possible lipid-binding region in the C-terminal part are encoded by exons 6 and 9, respectively . Exon 8 encodes the phosphorylation site (Ser551) that controls cAMP-mediated activity and a second site (Ser553) that is phosphorylated by 5'-AMP-activated protein kinase . Human HSL showed 83% identity with the rat enzyme and contained a 12-aa deletion immediately upstream of the phosphorylation sites with an unknown effect on the activity control . Besides the catalytic site motif (Gly-Xaa-Ser-Xaa-Gly) found in most lipases, HSL shows no homology with other known lipases or proteins, except for a recently reported unexpected homology between the region surrounding its catalytic site and that of the lipase 2 of Moraxella TA144, an antarctic psychrotrophic bacterium . The gene of lipase 2, which catalyses lipolysis below 4 degrees C, was absent in the genomic DNA of five other Moraxella strains living at 37 degrees C . The lipase 2-like sequence in HSL may reflect an evolutionarily conserved cold adaptability that might be of critical survival value when low-temperature-mobilized endogenous lipids are the primary energy source (e.g., in poikilotherms or hibernators) . The finding that HSL at 10 degrees C retained 3- to 5-fold more of its 37 degrees C catalytic activity than lipoprotein lipase or carboxyl ester lipase is consistent with this hypothesis. J Bacteriol, 1993 Jun, 175(11), 3278 - 88 Cloning and genetic characterization of the Helicobacter pylori and Helicobacter mustelae flaB flagellin genes and construction of H . pylori flaA- and flaB-negative mutants by electroporation-mediated allelic exchange; Suerbaum S et al.; Helicobacter pylori is one of the most common human pathogens . It causes chronic gastritis and is involved in the pathogenesis of gastroduodenal ulcer disease and possibly gastric carcinoma . Helicobacter mustelae is a bacterium closely related to H . pylori that causes gastritis and ulcer disease in ferrets and is therefore considered an important animal model of gastric Helicobacter infections . Motility, even in a viscous environment, is conferred to the bacteria by several sheathed flagella and is regarded as one of their principal virulence factors . The flagellar filament of H . pylori consists of two different flagellin species expressed in different amounts . The gene (flaA) encoding the major flagellin has recently been cloned and sequenced . Here we report the cloning and sequencing of two highly homologous new flagellin genes from H . pylori 85P and H . mustelae NCTC 12032 . The nucleotide sequence of the H . pylori gene proved that it encoded the second flagellin molecule found in H . pylori flagellar filaments . The genes were named flaB . The H . mustelae and H . pylori flaB genes both coded for proteins with 514 amino acids and molecular masses of 54.0 and 53.9 kDa, respectively . The proteins shared 81.7% identical amino acids . The degree of conservation between H . pylori FlaB and the H . pylori FlaA major flagellin was much lower (58%) . Both flaB genes were preceded by sigma 54-like promoter sequences . Mapping of the transcription start site for the H . pylori flaB gene by a primer extension experiment confirmed the functional activity of the sigma 54 promoter . To evaluate the importance of both genes for motility, flaA- and flaB-disrupted mutants of H . pylori N6 were constructed by electroporation-mediated allelic exchange and characterized by Western blot (immunoblot) analysis and motility testing . Both mutations selectively abolished the expression of the targeted gene without affecting the synthesis of the other flagellin molecule . Whereas flaA mutants were completely nonmotile, flaB mutants retained motility. Infect Immun, 1993 Jun, 61(6), 2369 - 76 Purification and characterization of a protease from Porphyromonas gingivalis capable of degrading salt-solubilized collagen; Sojar HT et al.; An enzyme capable of hydrolyzing the substrate 4-phenylazobenzyloxycarbonyl-L-prolyl-leucyl-glycyl-prolyl-D-ar gin ine (pZ-peptide), pZ-peptidase, was purified from the oral bacterium Porphyromonas gingivalis . pZ-peptidase hydrolyzed salt-solubilized type I collagen from rat skin, rat plasma low-molecular-weight kininogen, and transferrin at room temperature in the presence of calcium and dithiothreitol . pZ-peptidase did not cleave acid-soluble type I calf skin collagen, type V placental collagen, lysozyme, albumin, or human plasma fibrinogen . Furthermore, the purified enzyme did not hydrolyze N-alpha-benzoyl-DL-Arg-p-nitroanilide, Gly-Pro-p-nitroanilide, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, N-p-tosyl-Gly-Pro-Lys-p-nitroanilide, azoalbumin, or azocasein . Under reducing conditions, the native enzyme migrated as a single band at 120 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . However, when heated to 100 degrees C for 10 min in SDS under reducing conditions, the enzyme migrated as a major band at 50 kDa and a minor band at 60 kDa on SDS-polyacrylamide gel electrophoresis . Zymography using calf skin gelatin revealed the gelatin-cleaving activity of the enzyme as evidenced by a diffuse band in the range of 120 to 300 kDa under reducing conditions at room temperature, suggesting that this is the native form of the enzyme . However, incubation at 50 degrees C for 10 min under reducing conditions showed gelatin-cleaving activity at a distinct band of 60 kDa . A minimum temperature of 50 degrees C was required to dissociate the 60-kDa chain from the native complex in active form on gelatin zymography . The ability of the enzyme to cleave other proteins, including kininogen and transferrin, suggests that it has specificity for the Pro-X-Gly sequence found in several proteins, including collagen. Mol Microbiol, 1993 Jun, 8(6), 1105 - 14 The obligate intracellular bacterium Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside triphosphates; Tipples G et al.; Using well-characterized mutant host cell lines, deficient in specific enzymes of energy and nucleotide metabolism, we addressed numerous questions regarding nucleotide metabolism in the obligate intracellular bacterium Chlamydia trachomatis . The results presented indicate that C . trachomatis: (i) does not absolutely depend on mitochondrial generated ATP for survival; (ii) does have a significant draw on host-cell NTP pools but does not have a detrimental effect on the ability of the host cell to maintain its energy charge; (iii) lacks the ability to synthesize purine and pyrimidine nucleotides de novo; (iv) is not capable of interconverting purine nucleotides; and (v) possesses the pyrimidine metabolic-pathway enzymes CTP synthetase and deoxycytidine nucleotide deaminase . In total our results indicate that C . trachomatis is auxotrophic for host-cell ATP, GTP and UTP . In contrast, CTP can be obtained from the host cell or it can be synthesized from UTP by the parasite. Microbiol Rev, 1993 Jun, 57(2), 293 - 319 Biology of Frankia strains, actinomycete symbionts of actinorhizal plants; Benson DR et al.; Frankia strains are N2-fixing actinomycetes whose isolation and cultivation were first reported in 1978 . They induce N2-fixing root nodules on diverse nonleguminous (actinorhizal) plants that are important in ecological successions and in land reclamation and remediation . The genus Frankia encompasses a diverse group of soil actinomycetes that have in common the formation of multilocular sporangia, filamentous growth, and nitrogenase-containing vesicles enveloped in multilaminated lipid envelopes . The relatively constant morphology of vesicles in culture is modified by plant interactions in symbiosis to give a diverse array of vesicles shapes . Recent studies of the genetics and molecular genetics of these organisms have begun to provide new insights into higher-plant-bacterium interactions that lead to productive N2-fixing symbioses . Sufficient information about the relationship of Frankia strains to other bacteria, and to each other, is now available to warrant the creation of some species based on phenotypic and genetic criteria. Appl Environ Microbiol, 1993 Jun, 59(6), 1774 - 8 Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1; Blasco R et al.; The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells . The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates . The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method . The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups . Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor. Radiat Res, 1993 Jun, 134(3), 261 - 4 Experimental and theoretical cross sections for Escherichia coli mutants B, B/r, and Bs-1 after heavy-ion irradiation; Katz R et al.; Data for the inactivation of three Escherichia coli mutants by energetic heavy ions are fitted by the track theory of a one-hit detector in an extended target mode . The respective E0's are 46, 36.5, and 12.6 Gy for E . coli B, B/r, and Bs-1 and a0, the assumed target radius, is 0.5 microns for all three . The parameter E0, the D37 with gamma rays, is measured directly, while a0 is fitted to the data . It is significant that neither a point target model nor calculations with a0 = 0.2 and 1.0 micron fit the data . Our fitted target radius, a0, approximates the size of the bacterium . These results raise questions as to why the E . coli mutants are one-hit detectors, and concerning the differences in the E0's in relation to a mechanistic interpretation of cell killing. Microb Releases, 1993 Jun, 2(1), 53 - 9 Glutamate uptake and synthesis by Escherichia coli cells in seawater: effects on culturability loss and glycinebetaine transport; Gauthier MJ et al.; In filtered natural seawater supplemented with potassium glutamate, the ability of Escherichia coli MC4100 cells to grow on a complex medium was enhanced as a logarithmic function of the external glutamate concentration . By comparison, a glutamate-respiring strain of E . coli exhibited a greater decline in culturability in seawater, suggesting a protective influence of the accumulated amino acid . Potassium glutamate increased the uptake of 14C-glycinebetaine by E . coli MC4100 cells in seawater and enhanced the protective effects of the betaine against culturability loss, possibly by increasing the expression of the ProU transport system . This bacterium apparently was able to synthesize glutamate because a protective effect (i.e . a lower culturability loss) was observed in seawater when supplemented with precursor compounds (2-oxoglutarate and glutamine) . The combination of 2-oxoglutarate and glutamine resulted in the greatest protection of cells, possibly due to the synthesis of glutamate through glutamine 2-oxoglutarate amino transferase activity . The possible influence of glutamate and its precursors on survival of E . coli cells in the natural marine environment is considered, since glutamate, glutamine and betaines have been found in marine coastal waters and sediments. Trends Microbiol, 1993 Jun, 1(3), 99 - 104 Macrophage control of Brucella abortus: influence of cytokines and iron; Baldwin CL et al.; Brucella abortus is a facultative intracellular bacterium that can infect and replicate in mononuclear phagocytes . Recent work has elucidated the role of cytokines in activating macrophages to inhibit the intracellular replication of brucellae, and in recruiting macrophages to the site of infection in vivo . There is also evidence that iron increases the ability of cytokine-activated macrophages to control intracellular brucellae by mechanisms involving reactive oxygen intermediates. J Hosp Infect, 1993 Jun, 24(2), 95 - 108 A multidisciplinary investigation of a cluster of deaths on a paediatric intensive care unit; Maguire H et al.; During late December 1989 and early January 1990, a cluster of six unexplained deaths occurred on a paediatric intensive care unit (PICU) among children with congenital heart disease who had undergone cardiac surgical procedures . The children were all aged three years or less . In each case death was preceded by an unexpected increase in ventilatory pressure requirement followed by the development of a similar pulmonary shadowing on chest radiography . The radiological abnormality was felt to be consistent with a pneumonitis associated with some small airway disease . The clustering of these deaths, occurring in a similar unusual manner, was felt to constitute an outbreak warranting investigation . An Incident Committee was established to plan and manage a large multidisciplinary investigation during which the unit was temporarily closed . Following extensive investigation no bacterium, virus, fungus or other pathogen, toxic agent, or any other explanation for the cluster of deaths could be found . The possibility that the cluster occurred by chance remains although this was felt to be unlikely. Mol Microbiol, 1993 Jun, 8(6), 1017 - 24 Tn5-mediated bleomycin resistance in Escherichia coli requires the expression of host genes; Blot M et al.; The transposon Tn5 expresses a gene, ble, whose product increases the viability of Escherichia coli and also confers resistance to the DNA-cleaving antibiotic bleomycin and the DNA-alkylating agent ethylmethanesulphonate . We find that the Ble protein induces expression of an alkylation inducible gene, aidC, and that both the AidC gene product and DNA polymerase I are required for Ble to confer bleomycin resistance . These findings support models in which Ble enhances DNA repair and suggest that Tn5 confers a fitness advantage to the host bacterium by increasing the repair of spontaneous DNA lesions . Such co-operation between a transposon and its host suggests that Tn5 is a symbiotic rather than a selfish DNA element. FEBS Lett, 1993 May 24, 323(1-2), 159 - 62 Spectral hole burning study of intact cells of green bacterium Chlorobium limicola; Fetisova ZG et al.; Spectral hole burning studies of intact cells of the green bacterium, Chlorobium limicola, have proven that the Qy-absorption system of antenna bacteriochlorophyll c (BChl c) should be interpreted in terms of the delocalized exciton level structure of an oligomer . For the first time the 0-0 band of the lowest exciton state of BChl c oligomers has been directly detected as the lowest energy inhomogeneously broadened band (FWHM approximately 100 cm-1; position of maximum, at approximately 774 nm) of the near-infrared BChl c band of 1.8K excitation spectrum (FWHM = 830 cm-1; position of maximum, at 751 nm). J Mol Biol, 1993 May 20, 231(2), 501 - 4 Two-dimensional crystallization of the light-harvesting complex from Rhodospirillum rubrum; Ghosh R et al.; Homogeneous detergent-solubilized B873 light-harvesting complexes from a carotenoid-less mutant of the purple non-sulfur bacterium, Rhodospirillum rubrum G9, were reassembled spontaneously into two-dimensional (2D) hexagonal arrays during extensive and controlled dialysis . As the complexes contain only 1 to 2 mol phospholipid per mol alpha beta dimer, the arrays formed by a self assembly process are primary due to protein-protein interactions . The hexagonal lattices were analyzed by negative stain electron microscopy and digital image processing . They exhibited a unit cell size of 12.3 nm, in close agreement with the particle diameter of the active photo-unit in native chromatophore membranes . The unit cell contains a central 5 nm stain-filled depression, embraced by a ring with an outer diameter of 10 nm. Arch Biochem Biophys, 1993 May 15, 303(1), 44 - 50 Isolation and characterization of flavoredoxin, a new flavoprotein that permits in vitro reconstitution of an electron transfer chain from molecular hydrogen to sulfite reduction in the bacterium Desulfovibrio gigas; Chen L et al.; A new FMN-containing flavoprotein isolated from Desulfovibrio gigas provided maximum coupling efficiency for the reduction of bisulfite from molecular H2 . This protein, which is distinct from flavodoxin and for which the name flavoredoxin is proposed, is required for reconstitution of an electron transfer chain between hydrogenase and bisulfite reductase . A Ca(2+)-binding protein functions as a modulator in the presence of Ca2+ in the process . The finding of a membrane-bound cytochrome c with a molecular weight of 104,000 Da that is also active in this electron transfer chain provides an explanation for the energetic linkage between periplasmic and cytoplasmic proteins in this sulfate-reducing bacterium. J Biol Chem, 1993 May 15, 268(14), 10636 - 44 Purification and characterization of a novel dimeric ferredoxin (FdIII) from Rhodobacter capsulatus; Jouanneau Y et al.; A new ferredoxin, called FdIII, has been isolated and purified from the photosynthetic bacterium Rhodobacter capsulatus . Its complete amino acid sequence has been determined . The FdIII polypeptide consists of 100 residues, including 9 cysteines and has a calculated molecular mass of 10,688 Da, which was confirmed by electrospray mass spectrometry . In its native form, FdIII is a homodimer as deduced from molecular sieve chromatography and non-denaturing polyacrylamide gel electrophoresis, as well as cross-linking experiments . The dimeric ferredoxin was found to contain 15.2 +/- 0.6 iron atoms and 13 +/- 1 inorganic sulfur atoms, consistent with the presence of four {4Fe-4S} clusters/molecule . The UV visible absorption spectrum of oxidized FdIII exhibited maxima at 282 and at 386 nm and a shoulder near 314 nm . FdIII was fully reduced by excess dithionite at pH 8.0 or photochemically using 5-deazaflavin . Anaerobic oxidative titration of reduced FdIII with thionin indicated that each FdIII monomer exchanges two electrons . Exposure of FdIII to air resulted in a rapid and irreversible oxidative denaturation of the Fe-S clusters . The EPR spectrum of fully reduced FdIII showed a broad signal with an average g value of 1.94 that integrated to about two spins/monomer . EPR analysis of partially reduced FdIII (approximately 20% reduction) revealed a complex set of signals which was interpreted as being the resulting sum of the contribution of two distinct paramagnetic centres . Based on its biochemical and spectroscopic properties, it is concluded that FdIII is a dimeric ferredoxin containing four {4Fe-4S} clusters . The synthesis of FdIII occurs only under growth conditions allowing the derepression of nif genes . Results of in vitro electron transfer assays indicate that FdIII cannot serve as an electron donor to nitrogenase. Biochemistry, 1993 May 11, 32(18), 4719 - 26 Kinetics of photooxidation of soluble cytochromes, HiPIP, and azurin by the photosynthetic reaction center of the purple phototrophic bacterium Rhodopseudomonas viridis; Meyer TE et al.; The photosynthetic reaction center of Rhodopseudomonas viridis contains a bound tetraheme cytochrome c subunit which is the primary electron donor to the photooxidized special pair bacteriochlorophyll . We have tested a variety of soluble electron-transfer proteins for their ability to serve as secondary electron donors to the bacteriochlorophyll via the bound cytochrome by measuring the kinetics of reaction center heme reduction following photooxidation by a laser flash, as a function of soluble protein concentration and ionic strength . All of the soluble redox proteins utilized appear to interact with a negatively charged region on the reaction center and to transfer electrons to the 300-mV heme c-556 of the bound cytochrome . Rps . viridis cytochrome c2 was the best electron donor among those proteins tested, with a second-order rate constant extrapolated to infinite ionic strength of 1.2 x 10(6) M-1 s-1, which is two orders of magnitude larger than that of horse cytochrome c . Rps . viridis cytochrome c2 apparently binds to the reaction center at low ionic strength, as evidenced by a nonlinear dependence of kobs on protein concentration . The limiting first-order electron-transfer rate constant at 6 mM ionic strength is approximately 1300 s-1 . Horse cytochrome c and the reaction center also form a complex, with a limiting first-order rate constant for electron transfer which is 5 times smaller than for cytochrome c2 . Other cytochromes c2 are intermediate in reactivity . More distantly related cytochromes, HiPIP, and azurin are relatively poor electron donors under the conditions of assay.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1993 May 10, 322(2), 111 - 4 The antigenic domain of flagellin from S . paratyphi shares a structural fold with subtilisin; Grewal N et al.; Bacterial flagellin has two domains: the polymerizing domain consisting of N- and C-terminal regions which are partly disordered in the monomeric state; and the central antigenic domain with compact globular structure . The polymerizing domain is highly conserved in flagellins from different species but the antigenic domain is diverse in sequence and size . Whereas the former has direct functional significance for bacterial motility, the latter has not been identified as having a specific function except for defining the distinct serotype of the bacterium . The sequence alignment of flagellin from S . paratyphi with proteins of known three-dimensional structure reveals significant homology of the central 265 residue stretch with the bacterial serine protease, subtilisin . This homology is evident also in the comparison of the predicted secondary structure of flagellin with the observed secondary structural features in subtilisin . The deletions/insertions arising due to optimal alignment of the two proteins occur on the surface loops in the structure . Thus, a domain of S . paratyphi flagellin and subtilisin appear to have similar structural folds. J Biol Chem, 1993 May 5, 268(13), 9484 - 9 Purification and properties of 2'-hydroxybenzalpyruvate aldolase from a bacterium that degrades naphthalenesulfonates; Kuhm AE et al.; 2'-Hydroxybenzalpyruvate aldolase catalyzes the cleavage of 2'-hydroxybenzalpyruvate to salicylaldehyde and pyruvate . This reaction is part of the degradative pathways for naphthalene and naphthalenesulfonates by bacteria . 2'-Hydroxybenzalpyruvate aldolase has been purified to homogeneity from a bacterium that degrades naphthalenesulfonates (strain BN6) . The enzyme has a molecular weight of about 120,000 and is composed of identical subunits with a molecular weight of about 38,500 . Thus the enzyme appears to exist as a trimeric oligomer . The NH2-terminal amino acid sequence did not show significant homology to other published amino acid sequences . Extensive loss of enzyme activity occurred when the enzyme was incubated with 2'-hydroxybenzalpyruvate in the presence of sodium borhydride . This suggested the intermediate formation of a stable Schiff base between enzyme and substrate . 2'-Hydroxybenzalpyruvate aldolase was inhibited by p-chloromercuribenzoate and by the reaction product salicylaldehyde . The enzyme converted 2'-hydroxybenzalpyruvate, 2',4'- and 2',6'-dihydroxybenzalpyruvate. Genes Dev, 1993 May, 7(5), 895 - 903 Multiple extracellular signals govern the production of a morphogenetic protein involved in aerial mycelium formation by Streptomyces coelicolor; Willey J et al.; The formation of an aerial mycelium by the filamentous bacterium Streptomyces coelicolor is determined in part by a small morphogenetic protein called SapB . A collection of representative bald (bld) mutants, which are blocked in aerial mycelium formation, are all defective in the production of this protein and regain the capacity to undergo morphological differentiation when SapB is supplied exogenously . We now report that most of the bld mutants are rescued for SapB production and aerial mycelium formation when grown near certain other bld mutants . Extracellular complementation experiments of this kind indicate that morphological differentiation is governed by a hierarchical cascade of at least four kinds of intercellular signals . At least one such signal is present in conditioned medium . It is resistant to boiling and protease treatment, and it remains effective even when diluted up to eightfold in fresh medium. Virology, 1993 May, 194(1), 403 - 7 Selection and characterization of a neuraminidase-minus mutant of influenza virus and its rescue by cloned neuraminidase genes; Liu C et al.; A neuraminidase (NA)-deficient mutant, designated NWS-Mvi, of the reassortant influenza virus A/NWS/33HA-A/tern/Australia/G70c/75NA (H1N9), was selected by passaging virus in MDCK cells in a medium containing neuraminidase from the bacterium Micromonospora viridifaciens and polyclonal antiserum against the influenza NA . Growth of the resulting mutant virus is dependent on the addition of neuraminidase to the medium . Western blot analysis showed that the neuraminidase protein was absent from the mutant virus particles, and Northern hybridization showed that RNA segment 6, which contains the coding information for the NA, had undergone massive deletion . Viral protein synthesis in cells infected with the mutant virus was not dependent on the addition of neuraminidase . In the absence of a functional NA, the NWS-Mvi mutant virus can infect MDCK cells with normal cytopathic effects . This neuraminidase-minus influenza virus serves as an excellent source of parent virus for reverse genetics experiments involving genes that encode a functional neuraminidase. Intern Med, 1993 May, 32(5), 359 - 64 The relationship between Helicobacter pylori and oxygen-derived free radicals in the mechanism of duodenal ulceration; Salim AS; This prospective randomized study investigated the possibility that duodenal ulcer relapse associated with Helicobacter Pylori infection is mediated by oxygen-derived free radicals . To this end, the radical scavengers allopurinol (50 mg 4 times daily) and dimethyl sulphoxide (DMSO, 500 mg 4 times daily) were administered orally . One hundred and forty-six consecutive patients with previous symptomatic endoscopy proven duodenal ulceration, which had been shown endoscopically to have healed in the presence of gastric mucosal infection with Helicobacter Pylori, were randomized to receive for the period of one year either placebo, or cimetidine 400 mg at bedtime, or allopurinol, or DMSO . In one hundred and twenty-six patients evaluable for efficacy, the cumulative relapse at one year was: placebo 47%, cimetidine 24%, allopurinol 6% and DMSO 6% . Cimetidine was significantly effective in preventing the relapse (p < 0.01), however allopurinol and DMSO were superior to cimetidine in this respect (p < 0.05) . In the patients who relapsed, ulcer recurrence tended to occur early in those on placebo and cimetidine and to be evenly distributed over the year in those on free radical scavenging therapy . In all groups, ulcer recurrence throughout the maintenance year was more frequently symptomatic than silent . The incidence of infection with Helicobacter Pylori was not influenced by any of the regimens employed and the bacterium was detected with every relapse noted in this study and during the follow-up endoscopy which was carried out at 6 months and at 12 months during the maintenance year . The results suggest that oxygen-derived free radicals are involved in the relapse of duodenal ulceration in patients infected with Helicobacter Pylori. Mol Gen Genet, 1993 May, 239(1-2), 49 - 57 A plant mitochondrial gene encodes a protein involved in cytochrome c biogenesis; Schuster W et al.; Analysis of a transcribed region in the mitochondrial genome of Oenothera revealed an open reading frame (ORF) of 577 codons (orf577) that is also conserved in carrot, here encoding a protein of 579 amino acids (orf579) . RNA editing alters the mRNA sequence of orf577 in Oenothera with 46 C to U transitions, many of which improve sequence similarity with the homologous Marchantia gene orf509 . The deduced polypeptides show significant similarity with the ccl1-encoded protein involved in cytochrome c biogenesis in the photosynthetic bacterium Rhodobacter capsulatus . A highly conserved domain is also found in plastid ORFs, suggesting that these bacterial, chloroplast and mitochondrial genes encode polypeptides with analogous functions in assembly and maturation of cytochromes c. Med Microbiol Immunol (Berl), 1993 May, 182(2), 63 - 76 Immune suppressive effects of Helicobacter pylori on human peripheral blood mononuclear cells; Knipp U et al.; Helicobacter pylori, the causative agent of type-B gastritis and duodenal ulcer in man is described as a bacterium able to stimulate the human immune system . This study demonstrates that H . pylori besides this property possesses an immune suppressive activity . The in vitro proliferation of human peripheral blood mononuclear cells to purified protein derivative of tuberculin (PPD), phytohemagglutinin, and concanavalin A was reduced in a dose-dependent manner by bacteria which had been inactivated by incubation at 56 degrees C as well as by a soluble cytoplasmic fraction of H . pylori . The immune suppressive effect on the mitogen-induced proliferation could be increased by preincubation of the mononuclear cells with H . pylori . The observed effect does not seem to be a specific phenomenon depending on prior exposure of the blood donors to H . pylori, since suppression occurred with mononuclear cells of H . pylori-infected patients as well as of antibody-negative healthy control individuals . The suppressive activity was non-dialyzable, heat-labile (100 degrees C, 30 min) and sensitive to trypsin . Furthermore, the treatment at 100 degrees C caused an increase in the capability of H . pylori to induce lymphoproliferation . This fact indicates that the suppressive factor is also effective on H . pylori antigens . While exogenous interleukin-2, could to a certain extent, restore the responsiveness of the lymphocytes after PPD-stimulation in the presence of H . pylori, the addition of interleukin-1 had no effect on the suppressed lymphoproliferation . Cell-separation and cell-mixing experiments indicated that an influence on monocytes rather than on T cells is the major cause of the observed suppressive effect . Although the immunological mechanisms involved in H . pylori-associated gastritis are not clearly defined, it is reasonable to presume that suppression of host defense mechanisms may contribute to the pathogenesis of this disease. FEMS Microbiol Lett, 1993 May 1, 109(1), 49 - 53 Purification of form L2 RubisCO from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110; Chung SY et al.; Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) was purified from an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus MH-110 . The protein has a M(r) value of approximately 110,000, and is composed of two identical subunits of 55,000 . To our knowledge, the existence of L2-form RubisCO in a chemolithoautotrophic bacterium is first reported in this paper . The N-terminal amino acid sequence determination of the purified enzyme showed high homology with those of the L2-form RubisCO of Rhodospirillum rubrum and the Lx-form RubisCO from Rhodobacter sphaeroides. Appl Environ Microbiol, 1993 May, 59(5), 1607 - 12 Development of an oligonucleotide probe targeting 16S rRNA and its application for detection and quantitation of the ruminal bacterium Synergistes jonesii in a mixed-population chemostat; McSweeney CS et al.; Radiolabelled and fluorescent-dye-conjugated oligonucleotide probes which targeted rRNA sequences were developed for the enumeration of the ruminal bacterium Synergistes jonesii 78-1 in mixed culture . Two probes were tested, and both were highly specific for the respective complementary sequences of the target organism . Individual cells of S . jonesii in pure and mixed cultures were clearly visualized in situ by hybridization with the fluorescent-dye-conjugated probe but could not be detected in natural samples . Therefore the radiolabelled probe was used to monitor the population of S . jonesii introduced into a chemostat which simulated the rumen ecosystem . The S . jonesii probe did not hybridize to RNA extracted from the culture prior to inoculation with the target organism . After inoculation, S . jonesii rRNA represented 4.5% of the total bacterial rRNA and then rapidly declined to < 0.2% before increasing to about 1% of the total bacterial rRNA during the following 3 weeks . This study demonstrates that rRNA-targeted probes could be used for tracking organisms introduced into the rumen ecosystem. Appl Environ Microbiol, 1993 May, 59(5), 1444 - 51 Complete oxidation of toluene under strictly anoxic conditions by a new sulfate-reducing bacterium; Rabus R et al.; A toluene-degrading sulfate-reducing bacterium, strain Tol2, was isolated from marine sediment under strictly anoxic conditions . Toluene was toxic if applied directly to the medium at concentrations higher than 0.5 mM . To provide toluene continuously at a nontoxic concentration, it was supplied in an inert hydrophobic carrier phase . The isolate had oval, sometimes motile cells (1.2 to 1.4 by 1.2 to 2.0 microns) . The doubling time was 27 h . Toluene was completely oxidized to CO2, as demonstrated by measurement of the degradation balance . The presence of carbon monoxide dehydrogenase and formate dehydrogenase indicated a terminal oxidation of acetyl coenzyme A via the CO dehydrogenase pathway . The use of hypothetical intermediates of toluene degradation was tested in growth experiments and adaptation studies with dense cell suspensions . Results do not support a degradation of toluene via one of the cresols or methylbenzoates, benzyl alcohol, or phenylacetate as free intermediate . Benzyl alcohol did not serve as growth substrate; moreover, it was a strong, specific inhibitor of toluene degradation, whereas benzoate utilization was not affected by benzyl alcohol . Sequencing of 16S rRNA revealed a relationship to the metabolically dissimilar genus Desulfobacter and on a deeper level to the genus Desulfobacterium . The new genus and species Desulfobacula toluolica is proposed. Dig Dis Sci, 1993 May, 38(5), 944 - 9 Helicobacter pylori potentiates histamine release from serosal rat mast cells in vitro; Bechi P et al.; Helicobacter pylori seems to be involved in the etiology of peptic ulcer and chronic gastritis . Histamine is fundamental in gastric secretion modulation, and some features of H . pylori-associated gastritis (edema, vasodilatation, inflammatory cell infiltration) are typical of the histamine-mediated response . This in vitro study has been undertaken as a preliminary step, in order to find a possible link between H . pylori and histamine release . H . pylori isolated from gastric biopsies has been tested as whole washed bacterium, whole formalin-