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Flavin-linked dehydrogenation of ether glycols by cell-free extracts of a soil bacterium.

Payne, W. J. (University of Georgia, Athens), and R. L. Todd. Flavin-linked dehydrogenation of ether glycols by cell-free extracts of a soil bacterium. J. Bacteriol. 91:1533-1536. 1966.-Cell-free extracts of bacterium TEG-5 grown on tetraethylene glycol dehydrogenated a variety of ether glycols and nonylphenoxy and secondary alcohol ethoxy derivatives. Nicotinamide nucleotides did not serve as electron acceptors, but ferricyanide was effective. Dialysis of crude extract depressed activity with tetraethylene glycol, which was restored then by flavine adenine dinucleotide (FAD) or boiled extract supernatant fluid (BES) but not by other flavins. Precipitatation of extract protein at pH 4.0 at 80% ammonium sulfate saturation dissociated FAD and yielded an inactive fraction. Activity was restorable by FAD and BES but not by other flavins. Ethylene glycol was not dehydrogenated by the acid ammonium sulfate fraction with FAD. Atabrine inhibited tetraethylene glycol oxidation, and the inhibition was relieved by FAD but not by other flavins. Tergitols which have sulfated ethoxy side chains on secondary alcohols were not dehydrogenated, but those with free ethoxy side chains on identical alcohols were.

Alcohols↗

Antagonistic effect of monovalent cations in maintenance of cellular integrity of a marine bacterium.

The susceptibility of a marine bacterium, designated isolate c-A1, to lysis in distilled water and in salt solutions has been found to be a function of Na(+) concentration. Optical densities of cells pre-exposed to 0.05 m MgCl(2) were maintained in 1.0 m KCl, whereas those of cells pre-exposed to 1.0 m NaCl were not maintained at any KCl concentration tested. Cells transferred from MgCl(2) to low concentrations of NaCl underwent more extensive lysis than did those transferred to distilled water. The degree of disruption of cells transferred to distilled water from mixtures of 0.05 m MgCl(2) and NaCl (0 to 1.0 m) was dependent on the concentration of NaCl; similar results were obtained with LiCl, but not with KCl. In electron micrographs of thin sections, c-A1 cell envelopes consisted of two double-track layers which fractured and peeled apart on lysis after pre-exposure to NaCl-MgCl(2) mixtures. Envelope eruptions or "hernias" occurred only in lysed cells pre-exposed to NaCl alone. No evidence for a functional lytic enzyme was found. Comparative studies on a terrestrial pseudomonad with a multilayered envelope indicated that preexposure to NaCl did not enhance the susceptibility of this cell to lysis in distilled water. The lytic susceptibility of the marine bacterium is considered to be the consequence of competition between specific monovalent cations and Mg(++) for electrostatic interactions with components of the cell envelope of this organism.

Bacteria↗

Identification of diaminopimelic acid in the Legionnaires disease bacterium.

Diaminopimelic acid was found to be a component of the cell wall of the Legionnaires disease bacterium, thus providing additional evidence that the organism is a bacterium. The presence of this amino acid was determined by gas-liquid chromatography and confirmed by gas chromatography-mass spectrometry.

Amino Acids↗

Stability of viable-bacterium counts in liquid media used for preparation of inocula and subsequent impact on antimicrobial susceptibility test results.

We evaluated the consequences of prolonging the time between initial bacterial inoculum suspension preparation and susceptibility test inoculation. Extending the current National Committee for Clinical Laboratory Standards-recommended time of 15 min between suspension preparation and test inoculation should allow laboratories more flexibility to optimize efficiency from the standpoint of workflow. We assessed the length of time for which viable-bacterium counts remain stable in three liquid media at room temperature. Fifty isolates were examined in water, saline, and cation-supplemented Mueller-Hinton broth (CSMHB). Disk diffusion and microdilution MIC tests were performed on nine of these. Our results suggest that directly prepared inoculum suspensions of members of the family Enterobacteriaceae, Pseudomonas aeruginosa, staphylococci, and enterococci can be held for up to 6 h in water or saline prior to inoculation of disk diffusion and MIC tests without compromising test accuracy. The same organisms can be held for at least 1 h in CSMHB. Viridans group streptococci can be held for up to 6 h in saline and CSMHB and for up to 3 h in water. Similarly, Haemophilus influenzae and Streptococcus pneumoniae isolates may be held in CSMHB for up to 3 h. Because of an early decrease in viable-bacterium counts in water and saline with some H. influenzae and S. pneumoniae isolates, we recommend that National Committee for Clinical Laboratory Standards recommendations be followed for these species.

Anti-Bacterial Agents↗

Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice.

A bacterium with a spiral shape and bipolar, single, sheathed flagella was isolated from the livers of mice with active, chronic hepatitis. The bacteria also colonized the cecal and colonic mucosae of mice. The bacterium grew at 37 degrees C under microaerophilic and anaerobic conditions, rapidly hydrolyzed urea, was catalase and oxidase positive, reduced nitrate to nitrite, and was resistant to cephalothin metronidazole. On the basis of 16S rRNA gene sequence analysis, the organism was classified as a novel helicobacter, Helicobacter hepaticus. This new helicobacter, like two other murine Helicobacter species, H. muridarum and "H. rappini," is an efficient colonizer of the gastrointestinal tract, but in addition, it has the pathogenic potential to elicit persistent hepatitis in mice.

Animals↗

Direct quantification of the enteric bacterium Oxalobacter formigenes in human fecal samples by quantitative competitive-template PCR.

Homeostasis of oxalic acid appears to be regulated, in part, by the gut-associated bacterium Oxalobacter formigenes. The loss of this bacterium from the gut flora is associated with an increased susceptibility to hyperoxaluria, a condition which can lead to the formation of calcium oxalate crystalluria and kidney stones. In order to identify and quantify the presence of O. formigenes in clinical specimens, a quantitative-PCR-based assay system utilizing a competitive DNA template as an internal standard was developed. This quantitative competitive-template PCR test allows for the rapid, highly specific, and reproducible quantification of O. formigenes in fecal samples and provides a prototype for development of DNA-based quantitative assays for enteric bacteria.

Bacteria↗

Leptotrichia amnionii sp. nov., a novel bacterium isolated from the amniotic fluid of a woman after intrauterine fetal demise.

A novel bacterium was isolated and characterized from the amniotic fluid of a woman who experienced intrauterine fetal demise in the second trimester of pregnancy. The bacterium was a slow-growing, gram-negative anaerobic coccobacillus belonging to the genus LEPTOTRICHIA: Unlike Leptotrichia sanguinegens, the isolate did not grow in chopped-meat glucose broth or on sheep blood agar upon subculturing. The isolate was characterized by sequencing and analyzing its 16S rRNA gene. The 1,493-bp 16S ribosomal DNA sequence had only 96% homology with L. sanguinegens. Several phylogenetic analyses indicated that L. amnionii is a distinct species and most closely related to L. sanguiegens.

Adult↗

Corynebacterium resistens sp. nov., a new multidrug-resistant coryneform bacterium isolated from human infections.

Five strains of an unknown, multidrug-resistant coryneform, gram-positive rod were isolated from blood, bronchial aspirate, and abscess specimens. Four of the five strains isolated were highly resistant to antimicrobial agents, including beta-lactams, aminoglycosides, macrolides, quinolones, and tetracyclines, except for glycopeptides. In immunocompromised patients, bacteremia associated with this organism was rapidly fatal. This coryneform bacterium was nonmotile, lipophilic, and nonsaccharolytic. Lack of pyrazinamidase activity differentiated this organism from other lipophilic corynebacteria. Chemotaxonomic studies indicated that this multidrug-resistant coryneform bacterium belongs to the genus Corynebacterium. Comparative 16S rRNA gene sequencing and DNA-DNA hybridization analyses revealed that the five isolates were genetically identical and that they represent a new subline within the genus Corynebacterium, for which we propose the designation Corynebacterium resistens sp. nov. The type strain of Corynebacterium resistens is GTC 2026T (SICGH 158T, JCM 12819T, CCUG 50093T).

Corynebacterium↗

Draft genome sequence of a novel hydrogenogenic CO-oxidizing bacterium that is phylogenetically closely related to the family Thermicanaceae.

An anaerobic, thermophilic hydrogenogenic carbon monoxide (CO)-oxidizing Bacillota bacterium, 3446A, was isolated from freshwater pond sediments in Kyoto, Japan. The 3.11-Mbp draft genome sequence (guanine-cytosine content, 50.4%) encodes CODH-ECH genes, consistent with hydrogenogenic CO oxidation. Phylogenomic analysis suggests that Bacillota bacterium 3446A represents a novel genus-level lineage within the family Thermicanaceae.

Thermicanaceae↗

New spiral bacterium in gastric mucosa.

A new spiral bacterium, distinct from Campylobacter pylori, was found in the gastric mucosa of six patients with gastrointestinal symptoms. All patients had chronic active type B gastritis and four had oesophagitis. Culture and microscopy for C pylori infection was negative. These unculturable spiral organisms were probably an incidental finding in patients presenting for upper gastrointestinal endoscopy, but it is not possible to say from this small series whether these organisms cause chronic active gastritis. The organism is helical, 3.5-7.5 microns long and 0.9 micron in diameter with truncated ends flattened at the tips, and up to 12 sheathed flagella 28 nm in diameter at each pole. It is proposed that this spiral bacterium should be called "Gastrospirillum hominis Gen.nov., Sp.nov."

Adult↗

Presence of Mycobacterium avium subspecies paratuberculosis in suspensions of ovine trichostrongylid larvae produced in faecal cultures artificially contaminated with the bacterium.

A reference strain of Mycobacterium avium subspecies paratuberculosis was added to faecal larval cultures of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis. Samples of the larvae produced were cultured for the presence of the bacterium in modified BACTEC 12B medium, both before and after exposure to gamma irradiation. The water used to wash the larvae off the faecal cultures was also tested for the presence of the bacterium. Positive growth was confirmed as M. avium subspecies paratuberculosis by IS900 polymerase chain reaction and restriction endonuclease analysis of the product. M. avium subspecies paratuberculosis was detected in the unirradiated larval suspensions and wash waters of all three nematode species, and in the irradiated H. contortus larval suspension.

Animals↗

Isolation of a methanogenic bacterium, Methanosarcina sp. strain FR, for its ability to degrade high concentration of perchloroethylene.

Tetrachloroethylene (PCE) is a toxic compound essentially used as a degreasing and dry-cleaning solvent. A methanogenic and sulfate-reducing consortium that dechlorinates and mineralizes high concentrations of PCE was derived from anaerobically digested sludge obtained from a waste water treatment plant (Bourg-en-Bresse, France). A methanogenic bacterium, strain FR, was isolated from this acclimated consortium. On the basis of morphological and physiological characteristics, strain FR was classified in the genus of Methanosarcina. Phylogeny analysis with the 16S rRNA gene sequence revealed that strain FR is highly related to Methanosarcina mazei and Methanosarcina frisia (99.6 and 99.5% identity, respectively). High concentrations (50-87 microM) of PCE were completely dechlorinated by strain FR cultures at the rate of 76 nM-mg protein(-1).day(-1). PCE dechlorination produced a nonidentified compound. The tracer experiments with [13C]PCE revealed that the product was nonchlorinated. Dechlorination of PCE to trichloroethylene was still active in the presence of boiled cell extract of the strain FR. However, no further dechlorination was observed. This result suggests that a cofactor rather than an enzymatic system is responsible for the first dechlorination of PCE. Dechlorination-active fractions purified from cell extracts on a XAD-4 column revealed the presence of F(420), F(430), and cobamides cofactors. This is the first report of the isolation of a methanogenic bacterium with the ability to dechlorinate high concentrations of PCE to a nonchlorinated product.

Cell-Free System↗

Effects of NaCl on the uptake of alpha-[14C]aminoisobutyric acid by the halotolerant bacterium Halomonas elongata.

The alpha-aminoisobutyric acid (AIB) transport system of the halotolerant bacterium, Halomonas elongata, was examined. Cells were grown in L-alanine defined medium with 0.05, 0.375, 1.37, 2.5, or 3.4 M NaCl. Each group of cells was resuspended in buffered salts with different NaCl concentrations (0.05, 0.375, 1.37, 2.5, and 3.4 M) and the uptake of alpha-[14C]AIB was measured. Optimum AIB uptake occurred in the 0.375 M NaCl solution for the lower salt grown cells and the 1.37 M NaCl solution for the higher salt grown cells. When cells were grown in the higher salt media and suspended in hypoosmotic solutions, appreciable AIB uptake occurred; but for cells grown in lower salt media and suspended in hyperosmotic solutions, the uptake was dramatically reduced. This effect was mainly attributed to cell plasmolysis which in turn resulted in some cell death. The AIB uptake was Na+ specific and this analogue was not metabolized after being transported into the cells. An amino acid competition study gave a pattern similar to that of a marine bacterium.

Amino Acids↗

Cold shock proteins and cold acclimation proteins in the psychrotrophic bacterium Pseudomonas putida Q5 and its transconjugant.

The production of cold shock proteins (csps) and cold acclimation proteins (caps) was characterized in the psychrotrophic bacterium Pseudomonas putida Q5 and its transconjugant P. putida Q5T which contains the toluene-degradative TOL (pWWO) plasmid, using two-dimensional gel electrophoresis and computing scanning laser densitometry. Similar growth rates for the psychrotrophic bacterium P. putida Q5 and the transconjugant were found at temperatures ranging from 30 to 0 degree C. Sixteen proteins were quantified and compared in P. putida Q5 and P. putida Q5T following a 25 to 5 degrees C cold shock or constant growth at 5 degrees C. During constant growth at 25 degrees C, a decrease in the synthesis of various proteins occurred in the transconjugant. Following cold shock to 5 degrees C or constant growth at 5 degrees C, csps and caps were produced with a greater number occurring in the transconjugant. This may suggest an additional stress response in the transconjugant owing to metabolic load exerted by the TOL plasmid. Growth of P. putida Q5T with toluate produced seven proteins that appeared to be TOL-plasmid mediated and of which some were also designated as caps.

Acclimatization↗

A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi.

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.

Carbon-Sulfur Ligases↗

Marinobacter lutaoensis sp. nov., a thermotolerant marine bacterium isolated from a coastal hot spring in Lutao, Taiwan.

A heterotrophic and thermotolerant marine bacterium, designated strain T5054, was isolated from a hot spring on the coast of Lutao, Taiwan. It was a strictly aerobic, Gram-negative rod. Cells grown in broth cultures were non-spore-forming and motile by means of one to several polar flagella. It seems that pilus-like structures were produced from both poles of the cells. Strain T5054 required Na+ for growth and exhibited optimal growth at about 45 degrees C, pH 7, and 3-5% NaCl. It contained iso-C15:0 as the most abundant fatty acid and ubiquinone-8 as the only isoprenoid quinone. Its genomic DNA G+C content was 63.5 mol%. The strain did not require vitamins or other organic growth factors, and it grew on glucose, mannitol, and a variety of organic acids and amino acids as sole carbon sources. Characterization data, together with the results of a 16S rDNA-based phylogenetic analysis, indicated that strain T5054 could be classified as a new species in the genus Marinobacter. The name Marinobacter lutaoensis sp. nov. is proposed for this new bacterium. The type strain is T5054 (CCRC 17087; JCM 11179).

Alteromonadaceae↗

Serogroups of the beer spoilage bacterium Megasphaera cerevisiae correlate with the molecular weight of the major EDTA-extractable surface protein.

Megasphaera cerevisiae is a Gram-negative obligate anaerobe that causes turbidity and off-flavour and aroma in beer. Seven isolates of M. cerevisiae were obtained worldwide, and their extractable surface antigens were focused upon to determine if there is more than one serogroup of this bacterium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of ethylenediaminetetraacetic acid (EDTA) bacterial extracts revealed a predominant protein with apparent molecular weights of 46,000, 45,000, and 43,000 for three, two, and two isolates, respectively. When mouse anti-serum generated against any of the EDTA extracts was reacted with denatured bacterial proteins in immunoblots, all bacterial isolates exhibited extensive cross-reactivity involving three antigens, one being the major EDTA-extractable protein. In contrast, when the sera were tested for surface reactivity with intact bacteria, three cross-reactivity groups were observed, with the groups individually comprised of bacteria having the same size major EDTA-extractable surface protein. When BALB/c mice immunized with a bacterium from each of the three serogroups were used for monoclonal antibody (Mab) hybridoma production, bacterial surface-reactive Mabs were obtained whose reactivities parallel the three polyclonal antibody-defined serogroups. Through combining these surface-reactive Mabs, it will be possible to rapidly detect and identify beer contamination by M. cerevisiae belonging to any serogroup.

Animals↗

Isolation of a human intestinal bacterium that transforms mangiferin to norathyriol and inducibility of the enzyme that cleaves a C-glucosyl bond.

The C-glucosyl bond of C-glucosides generally tolerates acid and enzymatic hydrolysis. Many C-glucosides are cleaved by human intestinal bacteria. We isolated the specific bacterium involved in the metabolism of mangiferin (2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone), C-glucosyl xanthone, from a mixture of human fecal bacteria. The anaerobic Bacteroides species named MANG, transformed mangiferin to the aglycone, norathyriol, suggesting cleavage of a C-glucosyl bond. However, B. sp. MANG cleaved C-glucosyl in a dose- and time-dependent manner only when cultivated in the presence of mangiferin. Cleavage was abolished by inhibitors of RNA and protein syntheses, such as rifampicin and chloramphenicol, respectively, indicating that the enzyme that cleaves C-glucosyl is induced by mangiferin. In contrast, mangiferin did not affect bacterial alpha- and beta-glucosidase activities under any conditions. The C-glucosyl-cleavage in cell-free extracts was not altered by potent glucosidase inhibitors such as 1-deoxynojirimycin and gluconolactone. Therefore, the C-glucosyl-cleaving enzyme substantially differs from known glucosidases that cleave O-glucosides. This is the first description of a specific intestinal bacterium that is involved in the metabolism of mangiferin and which produces a novel and inducible C-glucosyl-cleaving enzyme.

Antiviral Agents↗