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PubMed1980

Anabolic-catabolic glucose utilization and product formation of Streptomyces griseus.

If in stationary growth phases appearing in the submerged cultivation of Streptomyces griseus several times the consumption rates of glucose and oxygen and the rates of streptomycin formation are put in relation, the following results are obtained: The yield coefficient Ysp of glucose was below 0.1. Since from the stoichiometric equation of turnover for the biosynthesis of the streptomycin follows that the substrate and the product are in the same weight relation, it was possible to check whether the quantity of glucose that does not appear in the streptomycin is used for the energy supplying synthesis or conservation reactions. As a characteristic value was built up the quotient qo3/qgluc that in total substrate oxidations takes the numerical value 6. This quotient varied between 2.0 and 6.0 so that anabolic side reactions during the production of secondary substances were concluded from. As possibilities are discussed syntheses of analytically disregarded primary metabolites or preliminary steps of the biosynthesis of streptomycin. Due to the decrease of the enthalpy production by anabolic reaction steps in stationary growth phases follows a physiological-energetical importance of microbial product syntheses with likely evolutive action.

Culture Media↗
PubMed2026

Widespread marine and freshwater distributions of active sulfoquinovose-degrading bacteria.

Sulfoquinovose (SQ), a sulfonated sugar produced on a gigaton scale each year, contributes to global sulfur cycling, yet the microbes and pathways mediating its turnover in the environment have been inferred largely from genomic potential rather than direct activity. Here, we coupled incubations of environmental samples with 13C-labeled SQ to DNA-stable isotope probing to identify active SQ carbon assimilators across estuary, mangrove, and lake ecosystems. In estuarine communities, Vibrio and Cognatishimia incorporated SQ-derived 13C; Novosphingobium dominated in the mangrove, and Agrobacterium in the lake. Pure-culture experiments, coupled with comparative proteomics and gene knockout validation, demonstrated that Vibrio strains degrade SQ via modified sulfoglycolytic Embden-Meyerhof-Parnas and Entner-Doudoroff pathways to produce the environmentally significant organosulfur 2,3-dihydroxypropanesulfonate. Comparative genomic analyses suggested that closely related genome representatives of Novosphingobium, Cognatishimia, and Agrobacterium encode the sulfolytic SQ monooxygenase pathway. A global survey of aquatic microbial genomes indicated that over 9% harbor SQ degradation clusters, supporting a widespread distribution of bacterial SQ catabolic potential in aquatic environments.

Fresh Water↗
PubMed2003

Xylose reductase from the Basidiomycete fungus Cryptococcus flavus: purification, steady-state kinetic characterization, and detailed analysis of the substrate binding pocket using structure-activity relationships.

Xylose reductase has been purified to apparent homogeneity from cell extracts of the fungus Cryptococcus flavus grown on D-xylose as carbon source. The enzyme, the first of its kind from the phylum Basidiomycota, is a functional dimer composed of identical subunits of 35.3 kDa mass and requires NADP(H) for activity. Steady-state kinetic parameters for the reaction, D-xylose + NADPH + H(+)<--> xylitol + NADP(+), have been obtained at pH 7.0 and 25 degrees C. The catalytic efficiency for reduction of D-xylose is 150 times that for oxidation of xylitol. This and the 3-fold tighter binding of NADPH than NADP(+) indicate that the enzyme is primed for unidirectional metabolic function in microbial physiology. Kinetic analysis of enzymic reduction of aldehyde substrates differing in hydrophobic and hydrogen bonding capabilities with binary enzyme-NADPH complex has been used to characterize the substrate-binding pocket of xylose reductase. Total transition state stabilization energy derived from bonding with non-reacting sugar hydroxyls is approximately 15 kJ/mol, with a major contribution of 5-8 kJ/mol made by interactions with the C-2(R) hydroxy group. The aldehyde binding site is approximately 1.2 times more hydrophobic than n-octanol and can accommodate linear alkyl chains of <or=6 carbons. Hydrophobic interactions provide a total binding energy of approximately 10 kJ/mol. Specificity for the aldehyde substrate is achieved through large decreases in apparent K(m) ( approximately 100-fold) and smaller but significant increases in turnover number ( approximately 5-fold). We observed up to 250-fold preference of xylose reductase for reaction with pyridine carbaldehydes, 4-nitro-benzaldehyde, and alpha-oxo-aldehydes over reaction with D-xylose, perhaps reflecting a secondary role of this enzyme in detoxication metabolism of reactive endogenous aldehydes and compounds of xenobiotic origin.

Aldehyde Reductase↗
PubMed2003

Emergence of protease inhibitor resistance-associated mutations in plasma HIV-1 precedes that in proviruses of peripheral blood mononuclear cells by more than a year.

HIV-1 genotype assay using plasma viruses has been widely applied for detection of resistant viruses in infected individuals, whereas there are only a few reports about proviral genotype in peripheral blood mononuclear cells (PBMCs). To determine which sample, plasma or PBMC, should be used for early detection of drug-resistant viruses during antiretroviral treatment, we analyzed 275 plasma-derived and 211 PBMC-derived HIV-1 protease sequences obtained from HIV-1-infected patients during protease inhibitor (PI) therapy. In 70 of 107 pairs (65.4%) of plasma and PBMC samples taken from the same blood draws, the numbers of PI resistance-associated mutations in the plasma-derived genotype were different from those in the PBMC-derived genotype. Plasma viruses had more PI resistance-associated mutations than PBMC proviruses (P = 0.0004). Analysis of serial samples showed that plasma-derived genotype assay could detect primary mutations about 425 days earlier than PBMC-derived genotype when the plasma viral load was less than 10(4 )copies/mL. Our data suggest that genetic turnover of PBMC proviruses is slower than that of plasma viruses and that time lag between emergence of mutations in plasma-derived and PBMC-derived genotypes correlates inversely with viral load. Plasma viruses should be the material of choice for early detection of drug resistance during antiretroviral treatment.

Antiretroviral Therapy, Highly Active↗
PubMed2003

Purification and properties of 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase involved in microbial degradation of carbazole.

Hydrolysis following meta-ring cleavage by a dioxygenase is a well-known step in aromatic compound metabolism. The 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase from Pseudomonas LD2 is a new member of the small group of characterized aromatic hydrolases that catalyze the cleavage of C-C bonds. In this study, the His(6)-tagged 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid (HOPDA) hydrolase was purified from a recombinant Escherichia coli strain utilizing immobilized metal affinity chromatography. 2-Hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase is a colorless homodimer with no cofactor requirement. The enzyme actively converted HOPDA into benzoic acid and 2-hydroxypenta-2,4-dienoic acid. The enzyme exhibited activity between pH 6.5 and 10.5 with a maximum activity at pH 7.0. The optimum temperature at pH 7.0 was 60 degrees C. The calculated K'(m) for HOPDA was 4.6 microM, the V(max) was 3.3 micromol min(-1), and the K(s) was 70.0 microM. This corresponds to a maximum specific turnover rate of 1300 HOPDAs(-1)dimer(-1). The deduced amino acid sequence of CarC showed 30.3, 31.3, and 31.8% identity with TodF (P. putida F1), XylF (P. putida), and DmpD (Pseudomonas sp. CF600), respectively, which are meta-cleavage compound hydrolases from other Pseudomonads. The amino acid sequence Gly-X-Ser-X-Gly, which is highly conserved in these hydrolases, is also found in CarC. Lysates from a strain expressing enzyme in which the putative active site serine is mutated to alanine showed a significant reduction in activity.

Carbazoles↗
PubMed1976

Nature of Escherichia coli mutants deficient in detergent-resistant and/or detergent-sensitive phospholipase A.

1. Escherichia coli K-12 mutants deficient in detergent-resistant (DR) and detergent-sensitive (DS) phospholipases A and deficient in DS phospholipase A were isolated. 2. The growth, compositions of phospholipids and fatty acids and turnover of phospholipids of three mutants (DR-, DS-, DR-DS-) were compared with those of their parent (DR- was isolated in a previous study). 3. Autodegradations of membrane phospholipids of 18,000 X g supernatants and precipitates of the homogenates of these three mutants were also compared with those of the parent, and the effects of various detergents and organic solvents on these activities were examined. 4. We could not identify any significant physiological role for DR or DS phospholipase A.

Detergents↗
PubMed1985

Characterization of limestones and their effects in vitro and in vivo in dairy cattle.

Capacities of limestones of differing particle size to neutralize acid in vitro and to modify pH and utilization of feedstuffs in vivo were compared. Acid neutralization during pH-stat titrations was faster for fine than for coarse limestone, and mixed microbial cultures were more resistant to pH change when they contained fine calcium carbonate. Diets containing 25:75 corn silage to concentrates and .95% calcium from either coarse or fine limestone were fed to rumen-fistulated heifers. Total ruminal volatile fatty acid concentrations were higher for the fine limestone treatment. Ruminal volumes, dry matter disappearance, and ruminal fluid pH and dilution rate did not differ between fine and coarse limestone treatments. Ruminal fluid volume, osmolality, ratios of acetate to propionate, and concentrations of total volatile fatty acids were unaffected in rumen-fistulated Holstein cows fed 60:40 corn silage to concentrates and either .5% calcium (control) or 1.0% calcium from either coarse or fine limestone. Ruminal pH increased .07 to .10 units with limestone supplementation. Ruminal fluid dilution and particulate turnover rates were slower for the coarse limestone than the control treatment. Differences between coarse and fine limestones in vitro were observed under some conditions in vivo, but they were not consistent.

Animal Feed↗
PubMed1995

AIDS pathogenesis: from models to viral dynamics in patients.

The course of HIV infection usually follows a pattern in which the immune system is initially able to limit viral replication, but ultimately fails. It is proposed that disease progression is a consequence of viral replication and evolution within the host, the immune response providing the selection pressure for increasing diversity. When the number of mutants rises above a certain threshold level, the immune system can no longer downregulate all the viral variants simultaneously and symptomatic disease results. Recent studies of the dynamics and kinetics of HIV infection have indicated that 30% of the free virus population in the plasma is replenished each day, a very high turnover rate. After intervention with nevirapine, resistant virus was observed to have replaced wild-type virus in the plasma within 2-4 weeks. Even in late-stage patients, the immune system has the capacity to regenerate large numbers of CD4 cells, but this ability does not continue indefinitely. This implies that to reverse immunodeficiency the principal objective of treatment in the management of HIV infection should be inhibition of viral replication and thus prevention of lymphocyte destruction.

Acquired Immunodeficiency Syndrome↗
PubMed1982

Field observations of methane concentrations and oxidation rates in the southeastern bering sea.

Measurements of methane oxidation rates were made in southeastern Bering Sea water samples with [C]methane. The rate at which CO(2) evolved from samples exposed to one methane concentration was defined as the relative methane oxidation rate. Rate determinations at three methane concentrations were used to estimate methane oxidation kinetics. The rate constant calculated from the kinetics and the observed methane concentration in the same water sample were used to calculate an in situ methane oxidation rate and the turnover time. First-order kinetics were observed in essentially all experiments in which methane oxidation kinetics were measured. Relative methane oxidation rates were greater in waters collected at inshore stations than at the offshore stations and were greater in bottom samples than in surface samples. In most water samples analyzed, there was essentially no radioactivity associated with the cells. The resulting respiration percentages were therefore very high with a mean of >98%. These data suggest that most of the methane was used by the microflora as an energy source and that very little of it was used in biosynthesis. The relative methane oxidation rates were not closely correlated with methane concentrations and did not appear to be linked to either oxygen or dissolved inorganic nitrogen concentrations. However, there was a significant correlation with relative microbial activity. Our data suggest that the methane oxidizers were associated with the general microbial heterotrophic community. Since these organisms did not appear to be using methane as a carbon source, it is unlikely that they have been isolated and identified as methane oxidizers in the past.

Journal Article↗
PubMed1981

Characteristics of the norepinephrine-stimulated phosphatidylinositol turnover in rat pineal cell dispersions.

Dispersed rat pineal cells can be used for the study of the phosphatidylinositol effect. The response to ( - )-norepinephrine of the incorporation of 32Pi into phospholipids is linear with time and cell concentration, stereospecific, and mediated through alpha-1-adrenergic receptors. Na+ in the incubation medium is obligatory for labeling of phosphatidylinositol and phosphatidylcholine by 32P. In the absence of K+, incorporation of 32P is drastically lowered and no stimulation by norepinephrine occurs. Rb+ can replace K+. Omission of Ca2+ or substitution with Sr2+ preferentially lowers incorporation of radioactivity into phosphatidylcholine. Mg2+ is not required for basal or stimulated labeling.

Animals↗
PubMed2003

Characterization of small bowel resection and intestinal adaptation in germ-free rats.

BACKGROUND: After massive small bowel resection (SBR), the remnant bowel adapts by increasing enterocyte proliferation and apoptosis. The purpose of this study was to investigate the relevance of luminal bacteria on postresection intestinal cell turnover. METHODS: Male germ-free (GF) and normally colonized control rats underwent either a 75% mid-SBR or sham operation. In other experiments, normally colonized control rats were given antibiotics in the drinking water. After 7 days, the remnant ileum was harvested and adaptation verified by alterations in wet weight, crypt depth, and villus height. Proliferation and apoptosis were measured in crypts as the percent of crypt cells staining for Ki-67 or the number of apoptotic bodies per crypt. RESULTS: Both GF and control rats demonstrated significant increases in all adaptive parameters. Proliferation was increased after SBR in both groups, but significantly greater in the GF animals over control. This response could not be recapitulated after antibiotic treatment. Apoptosis increased equally after SBR in all groups. CONCLUSION: Resection-induced intestinal adaptation occurs normally in GF animals. Epithelial-microbial interactions are probably not involved in the activation of enterocyte apoptosis. The germ-free studies offer the possibility that luminal bacteria may attenuate the proliferative response of the enterocyte to massive small bowel resection.

Adaptation, Physiological↗
PubMed1999

Mineralization in ferritin: an efficient means of iron storage.

Ferritins are a class of iron storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. Iron is stored within the protein shell of ferritin as a hydrous ferric oxide nanoparticle with a structure similar to that of the mineral "ferrihydrite." The eight hydrophilic channels that traverse the protein shell are thought to be the primary avenues by which iron gains entry to the interior of eukaryotic ferritins. Twenty-four subunits constitute the protein shell and, in mammalian ferritins, are of two types, H and L, which have complementary functions in iron uptake. The H chain contains a dinuclear ferroxidase site that is located within the four-helix bundle of the subunit; it catalyzes the oxidation of ferrous iron by O(2), producing H(2)O(2). The L subunit lacks this site but contains additional glutamate residues on the interior surface of the protein shell which produce a microenvironment that facilitates mineralization and the turnover of iron(III) at the H subunit ferroxidase site. Recent spectroscopic studies have shown that a di-Fe(III) peroxo intermediate is produced at the ferroxidase site followed by formation of a mu-oxobridged dimer, which then fragments and migrates to the nucleation sites to form incipient mineral core species. Once sufficient core has developed, iron oxidation and mineralization occur primarily on the surface of the growing crystallite, thus minimizing the production of potentially harmful H(2)O(2).

Animals↗
PubMed1988

Functional abolition of monocyte HLA-DR by aldehyde treatment. A novel approach to studies of class II restriction elements in antigen presentation.

The effect of low concentration aldehyde treatment (0.0012 to 0.005%) on the expression of HLA-DR Ag by human monocytes was investigated. This treatment was shown to selectively abolish the expression of HLA-DR determinants defined by a monomorphic mAb (YE2.36) in a rosette assay. The expression of class I MHC Ag and Fc gamma R remained unaffected. As a result, the presentation of the recall Ag tetanus toxoid and streptokinase-streptodornase (SK-SD) to freshly isolated autologous T cells and T cell clones was completely inhibited. Increasing the concentration of aldehyde to 0.05% consistently produced partial restoration of Ag-presenting capacity. Low dose aldehyde treatment did not affect monocyte viability or membrane turnover. Thus, aldehyde-treated monocytes produced a second generation of HLA-DR and expression was almost completely restored to normal after 24 h of culture. The presentation of monocyte class II Ag as alloantigens was also inhibited by low dose aldehyde treatment but inhibition was much less marked when monocytes were aldehyde treated at 2 h rather than at 24 h of culture. This is consistent with the reexpression of HLA-DR which occurred readily in the first 24 h of culture and much less readily thereafter. Low dose aldehyde treatment did not affect Ag uptake and processing. However, monocytes which had been pulsed with Ag, aldehyde-treated to abolish HLA-DR, and then cultured to allow regeneration of HLA-DR could present Ag only when given a second Ag pulse, suggesting that once the association between microbial Ag and HLA-DR had formed the Ag was not then free to reassociate with novel HLA-DR. Low dose aldehyde treatment did not affect monocyte IL-1 production, neither did it inhibit the detection of HLA-DR by soluble mAb in FACS analysis. These results are consistent with the view that low dose aldehyde treatment disrupts the tertiary structure of human Ia molecules such that allostimulatory determinants and restriction elements for exogenous Ag are rendered inaccessible to T lymphocyte receptors and to cell-bound anti-DR mAb in the rosette assay, although DR determinants may remain accessible to soluble mAb.

Aldehydes↗
PubMed2026

Life in sediments fosters 'sexual' speciation in the Shewanella baltica complex.

Understanding how intra- and interspecific differentiation arises in natural microbial populations is central to explaining the processes that drive bacterial evolution. Motivated by the co-occurrence of multiple putative genospecies closely related to Shewanella baltica in Baltic Sea sediments, we investigated the genomic structure of this species complex across fine spatial scales. We analyzed 112 genome sequences from strains collected across several sediment cores and depths (0-6 cm) at Vax&#xf6;n (Stockholm archipelago, Sweden) as well as earlier isolates from this site and allopatric strains from surrounding locations obtained from both sediments and the water column. Using a reverse-ecology population genomics approach, we found unprecedented genomic diversification among sediment-associated strains, which form a species complex resolving into three cohesive evolutionary groups (G1, G2, and G3) with distinct signatures of metabolic specialization including sulfite respiration. While G1 consists predominantly of a single species (S. baltica) with high gene turnover, G2 and G3 comprise an array of divergent putative genospecies and previously reported species consistently recovered from sediments. Patterns of homologous recombination indicate that diversification of the lineages within G2 and G3 is primarily recombination-driven ('sexual') and is associated with specialization in sulfite reduction and utilization of certain carbon sources. The extent of diversity uncovered here far exceeds that reported for S. baltica from other environments, suggesting that a sediment-associated lifestyle promotes the emergence of novel genotypes. These findings expand the known limits of sympatric speciation in prokaryotes beyond subspecific ecotypes, demonstrating that bacterial species can diverge and persist as distinct lineages in the absence of spatial segregation and at microgeographic scales. Furthermore, our results suggest that collective interactions and ecological differentiation can structure sediment-associated bacterial populations strongly enough to drive divergence at the species level.

Journal Article↗
PubMed1999

OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors.

Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 micrograms ml-1; cephaloridine, 4 vs. 32 micrograms ml-1, respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 micrograms ml-1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs. 50 microM, kcat=345 vs. 234 s-1) and cephaloridine (Km=97 vs. 202 microM, kcat=1023 vs. 202 s-1). Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate. For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.

Drug Resistance↗
PubMed1971

Polysome turnover during amino acid starvation in Escherichia coli.

The experiments presented in this paper support earlier evidence that ribosomes are released from polysomes when they encounter a codon for which no charged transfer ribonucleic acid is available. However, it is further shown that these ribosomes then reinitiate and resume translation. The size and the level of polysomes during deprival of an amino acid is a function of the frequency with which that particular amino acid appears in cellular proteins. Polysomes from starved cells are more stable than those from growing cells, and, moreover, polysomes from starved relaxed strains are more stable than those from starved stringent strains.

Amino Acids↗
PubMed2006

Regulation of rumen fermentation during seasonal fluctuations in food intake of muskoxen.

We studied rumen fermentation of castrated adult muskoxen (Ovibos moschatus; n=4) during periods of low (May) and high (August) food intake. Turnover time (17+/-1.8 h) and volume (26+/-3.9 L) of rumen fluid were consistent between May and August and among days within each season. Rumen temperature did not vary significantly during the day (38.8+/-0.29 degrees C) in either season. Rumen osmolality (271.9+/-16.4 vs. 245.9+/-11.4 mOsm kg(-1)) and pH (6.81+/-0.31 vs. 6.39+/-0.15) were higher in May than in August indicating a shift in the allostatic set point. Rumen fluid pH was more variable in May than in August both before and after a single meal of fermentable substrate even though fermentation acids were lower in May than in August (101.0+/-11.0 vs. 126.0+/-8.74 mM). Changing proportions of minor fermentation acids indicated a shift in metabolic pathways even though bacterial numbers were similar between seasons (6.4+/-5.8x10(9) mL(-1)). Allostatic set points probably alter the homeostatic range of conditions and the microbial diversity of fermentations in herbivores from highly seasonal environments.

Animals↗
PubMed2001

Sulfate Reduction at a Lignite Seam: Microbial Abundance and Activity.

In a combined isotope geochemical and microbiological investigation, a setting of multiple aquifers was characterized. Biologically mediated redox processes were observed in the aquifers situated in marine sands of Tertiary age and overlying Quaternary gravel deposits. Intercalated lignite seams define the aquitards, which separate the aquifers. Bacterial oxidation of organic matter is evident from dissolved inorganic carbon characterized by average carbon isotope values between ?18.4 per thousand and ?15.7 per thousand (PDB). Strongly positive sulfur isotope values of up to +50 per thousand (CTD) for residual sulfate indicate sulfate reduction under closed system conditions with respect to sulfate availability. Both, hydrochemical and isotope data are thus consistent with the recent activity of sulfate-reducing bacteria (SRB). Microbiological investigations revealed the presence of an anaerobic food chain in the aquifers. Most-probable-number (MPN) determinations for SRB and fermenting microorganisms reached highest values at the interface between aquifer and lignite seam (1.5 x 103 cells/g sediment dry mass). Five strains of SRB were isolated from highest MPN dilutions. Spore-forming bacteria appeared to dominate the SRB population. Sulfate reduction rates were determined by the 35S-radiotracer method. A detailed assessment indicates an increase in the reduction rate in proximity to the lignite seam, with a maximum turnover of 8.4 mM sulfate/a, suggesting that lignite-drived compounds represent the substrate for sulfate reduction.

Journal Article↗