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sucrose utilization by zymomonas mobilis: formation of a levan.1. molar growth-yield coefficients of zymomonas mobilis for glucose, fructose, glucose plus fructose, and sucrose are reported. yield coefficients for sucrose are appreciably lower than those for the equivalent concentrations of glucose plus fructose. 2. only 2.6% of [u-(14)c]glucose supplied in the growth medium is incorporated into cell substance by z. mobilis utilizing glucose as the energy source. 3. during growth on sucrose a levan is formed. it has been characterized and shown to resemble ...19664287843
the vitamin nutrition of zymomonas anaerobia. 19734579123
glucose-6-phosphate dehydrogenase in cell free extracts of zymomonas mobilis. 19684885089
the nutrition of zymomonas anaerobia. 19704922569
energies of activation and uncoupled growth in streptococcus faecalis and zymomonas mobilis.growing cultures of streptococcus faecalis at temperatures above 30 c have activation energies for both rates of growth and glycolysis of 10.3 kcal mole(-1), and a constant growth yield; when growth takes place below this temperature, the growth yield decreases and the activation energy for growth increases to 21.1 kcal mole(-1), but the activation energy for glycolysis is unchanged. the adenosine triphosphate pool in the organisms behaves differently above and below 30 c, suggesting that the en ...19674964479
effect of starvation on the viability and cellular constituents of zymomonas anaerobia and zymomonas mobilis. 19705488464
microcalorimetric study of glucose permeation in microbial cells.a microcalorimetric method for measuring the influence of extracellular glucose concentration on the rate of catabolism is described. this method has been applied to anaerobically growing cultures of zymomonas mobilis and of a respiratory-deficient ("petite") mutant of saccharomyces cerevisiae (strain yfa). the michaelian kinetics recorded with both organisms were apparently related to glucose transport. with z. mobilis, it was found that, in the range of glucose concentrations at which this org ...19685650082
[further observations on the antagonist action of zymomonas mobilis (linder) (1928), kluyver and van niel (1936)]. 19685761009
transformation of zymomonas mobilis by a hybrid plasmid.the transformation of zymomonas mobilis by plasmid dna was achieved using a modification of the cacl2 method for escherichia coli. the highest frequency of transformation obtained was 5 x 10(3) transformants/micrograms dna. the success of the method depended upon the use of a plasmid which is a cointegrate between a z. mobilis cryptic plasmid and an e. coli plasmid carrying two selectable drug resistance markers.19846098907
high-productivity alcohol fermentations using zymomonas mobilis.a process is under development at the university of new south wales to produce fermentation ethanol faster and more efficiently. the process is based on the micro-organism zymomonas mobilis, which has higher specific rates of ethanol production and higher yields when compared with the traditionally used yeasts. by using hollow fibre membranes for cell recycle, high productivity continuous processes have been studied at laboratory-scale. pilot scale evaluations (500 litres) are now in progress th ...19836100832
use of differential dye-ligand chromatography with affinity elution for enzyme purification: 2-keto-3-deoxy-6-phosphogluconate aldolase from zymomonas mobilis.2-keto-3-deoxy-6-phosphogluconate aldolase (ec 4.1.2.14) has been isolated from extracts of zymomonas mobilis using differential dye-ligand chromatography and affinity elution with product/product analog. the one-step procedure gives an enzyme with specific activity 600 units mg-1. only 1 out of 47 dyes, procion yellow mx-gr, bound the enzyme completely in 20 mm phosphate buffer, ph 6.5. a column of navy he-r adsorbent was used first to remove most of the potentially adsorbing proteins.19846326622
use of differential dye-ligand chromatography with affinity elution for enzyme purification: 6-phosphogluconate dehydratase from zymomonas mobilis.using differential dye-ligand chromatography and affinity elution with a substrate analog, 6-phosphogluconate dehydratase (ec 4.2.1.12) has been isolated from extracts of zymomonas mobilis in a one-step procedure with 50% recovery. the specific activity of freshly isolated enzyme was 245 units mg-1. the enzyme contains iron, and it is rapidly inactivated in oxidizing conditions. it is inhibited by glycerophosphates, most strongly by the d-alpha-isomer which structurally corresponds to half of th ...19846326623
an iron-activated alcohol dehydrogenase.an alcohol dehydrogenase isolated from zymomonas mobilis was found to be activated by ferrous ions but not by zinc, after inactivation with metal-complexing agents. cobaltous ions also re-activated to a lesser extent. it is suggested that in this species the alcohol dehydrogenase naturally contains iron. kinetic studies on the iron-treated enzyme indicate an 'alcohol activation' phenomenon, which may have physiological relevance in overcoming product inhibition during fermentation.19836343121
self-transmissible plasmid in zymomonas mobilis carrying antibiotic resistance.the cryptic plasmid prut41 from zymomonas mobilis was examined for its biological properties. this plasmid was found to be conjugally transferred from z. mobilis cp4 to escherichia coli bm21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). covalently closed circular plasmid dna was isolated from eight transconjugants of e. coli bm21. these plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native prut41 pl ...19846364969
antibacterial activity of different zymomonas mobilis strains.twenty different zymomonas mobilis strains were found to produce a substance which inhibited or killed various other zymomonas , escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa strains. this antibacterial activity could be detected in cross-streak tests and as zones of clearing in lawns of the test bacteria. when zymomonas strains are used as recipients in conjugation experiments, their antibacterial activity can be used to advantage for removal of unwanted donor cells from th ...19846427556
zymomonas ethanol fermentations.studies on various industrial raw materials indicate that a zymomonas process has its greatest commercial potential in fermenting starch-based substrates. high yields, productivities and ethanol concentrations can be achieved. genetic manipulation is now being used to extend the substrate range to lactose and other carbohydrates.19846444136
the structure of a novel polysaccharide isolated from zymomonas mobilis determined by nuclear magnetic resonance spectroscopy.a novel polysaccharide has been observed in vivo by 13c nuclear magnetic resonance (nmr) spectroscopy of the gram-negative bacterium, zymomonas mobilis. the polysaccharide, which was not removed from the z. mobilis cell by washing with 0.86% saline, was extracted by mild acid treatment. the structure was elucidated by a combination of nmr techniques, including proton and proton-carbon two-dimensional methods. the structure was determined to be -alpha-fructofuranosyl-(2-1)-beta-fructofuranosyl-(2 ...19846489351
31p nuclear magnetic resonance studies of the fermentation of glucose to ethanol by zymomonas mobilis.high resolution 31p nuclear magnetic resonance spectroscopy has been employed to study the fermentation of glucose to ethanol by zymomonas mobilis, strain zm4, a bacterium which uses the entner-doudoroff pathway. the levels of nucleoside triphosphates, sugar phosphates, udp-sugars and pi in intact fermenting cells have been studied with a time resolution of 1 min. it is suggested that a ph gradient is established across the cell membrane during fermentation and that the intracellular ph does not ...19846715367
lipid composition of zymomonas mobilis: effects of ethanol and glucose.zymomonas mobilis is an alcohol-tolerant microorganism which is potentially useful for the commercial production of ethanol. this organism was found to contain cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine as major phospholipids. vaccenic acid was the most abundant fatty acid, with lesser amounts of myristic, palmitic, and palmitoleic acids. no branched-chain or cyclopropane fatty acids were found. previous studies in our laboratory have shown that ethanol ...19836853446
comparison of plasmids in strains of zymomonas mobilis.four strains of zymomonas mobilis were examined for their resistance to antimicrobial agents and found to have similar resistance profiles. plasmid dna was extracted and purified by cscl dye-buoyant density centrifugation; molecular weights were determined by agarose gel electrophoresis and electron microscopy. all four strains harbored a large plasmid (46 x 10(6) da) and a smaller plasmid (16-21 x 10(6) da) whose molecular weight was strain dependent. two strains, ag11 and atcc 10988, had small ...19836856691
characterization of the two alcohol dehydrogenases of zymomonas mobilis. 19817030207
genetic alteration of zymomonas mobilis for ethanol production. 19827066009
minimal medium for isolation of auxotrophic zymomonas mutants.a minimal medium which allowed the sustained, rapid growth of zymomonas mobilis and the isolation of a range of auxotrophic mutants was developed.19827125659
ethanol production by zymomonas mobilis and saccharomyces uvarum on aflatoxin-contaminated and ammonia-detoxified corn.zymomonas mobilis demonstrated greater fermentative activity than saccharomyces uvarum during the 1st day in the fermentation of two lots of aflatoxin-contaminated corn and two corresponding lots of ammonia-detoxified corn. final ethanol yields and conversion efficiencies were generally highest in zymomonas fermentations of ammonia-detoxified corn. aflatoxin levels in postfermentation solids from ammonia-detoxified corn all ranged below the food and drug administration feedstuff guideline of < 2 ...19817214236
useful mutants of zymomonas mobilis alcohol dehydrogenase-2 obtained by the use of polymerase chain reaction random mutagenesis. 19957484407
isolation and sequence analysis of rpoh genes encoding sigma 32 homologs from gram negative bacteria: conserved mrna and protein segments for heat shock regulation.the rpoh genes encoding homologs of escherichia coli sigma 32 (heat shock sigma factor) were isolated and sequenced from five gram negative proteobacteria (gamma or alpha subgroup): enterobacter cloacae (gamma), serratia marcescens (gamma), proteus mirabilis (gamma), agrobacterium tumefaciens (alpha) and zymomonas mobilis (alpha). comparison of these and three known genes from e.coli (gamma), citrobacter freundii (gamma) and pseudomonas aeruginosa (gamma) revealed marked similarities that should ...19957501460
zymomonas mobilis cell viability: measurement method comparison.comparison of three different cell viability methods: slide count, plate count and methylene blue staining techniques, applied on zymomonas mobilis cultures, was performed. the slide technique proved to be faster and more accurate than the plate count method, and both of them far more reliable than the standard methylene blue method which constantly overestimated the zymomonas cell viability. the slide technique is advantageous also because it gives information on the cell morphology changes, no ...19937506015
construction of an integrative shuttle vector for zymomonas mobilis.an integrative shuttle vector, pzmocp1, was constructed by ligating ecorv digests of the plasmid cloning vector pbluescript and pzmp1, a cryptic plasmid of zymomonas mobilis proimi a1. the 7.2-kb plasmid pzmocp1 replicated in escherichia coli and could also be transferred from this host by electroporation to z. mobilis atcc 29191. the transformants were selected by ampicillin resistance. the integrative characteristic was detected by hybridization in situ. the vector was stably maintained in z. ...19957590162
characterization of the zymomonas mobilis glucose facilitator gene product (glf) in recombinant escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport.zymomonas mobilis is known to transport glucose by a facilitated diffusion process. a putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. the z. mobilis glf gene is able to complement glucose transport in an escherichia coli strain that is defective in native glucose transport and glucokinase. in this study, the recombinant e. coli was shown to be capable of influx counte ...19957596282
kinetic analysis of the activation of zymomonas mobilis glucokinase by phosphate.a detailed kinetic analysis of glucokinase ec 2.7.1.2 from zymomonas mobilis has been carried out. this enzyme has an absolute requirement for inorganic phosphate as activator, and the kinetic behaviour can be interpreted as a steady-state ordered mechanism in which glucose is the first substrate. values for each of the kinetic constants have been obtained for the conditions i = 0.12, 30 degrees c, and ph 7.0. direct binding studies have confirmed that atp does not bind to the enzyme without glu ...19957599171
the effect of temperature on enzymes used in diagnostics.a number of enzymes that are used in clinical analysis have been studied in relation to the effect of temperature on their activity. both vmax and km were determined over a temperature range from 13 to 55 degrees c. whereas vmax values increased steadily until denaturation point with all enzymes, the effect of temperature on km was more variable. with most enzymes there was a gradual increase in km, often with a sharp rise close to the denaturation temperature. in most cases, km did not increase ...19957664474
the glutamate uptake regulatory protein (grp) of zymomonas mobilis and its relation to the global regulator lrp of escherichia coli.after being expressed in escherichia coli jc5412, which is defective in glutamate transport, a zymomonas mobilis gene which enabled this strain to grow on glutamate was cloned. this gene encodes a protein with 33% amino acid identity to the leucine-responsive regulatory protein (lrp) of e. coli. although overall glutamate uptake in e. coli was increased, the protein encoded by the cloned fragment repressed the secondary h+/glutamate transport system gltp by interaction with the promoter region o ...19957665494
a novel aerobic respiratory chain-linked nadh oxidase system in zymomonas mobilis.membrane vesicles prepared from zymomonas mobilis oxidized nadh exclusively, whereas deamino-nadh was little oxidized. in addition, the respiratory chain-linked nadh oxidase system exhibited only a single apparent km value of approximately 66 microm for nadh. the nadh oxidase was highly sensitive to the respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-n-oxide. however, the nadh:quinone oxidoreductase was not sensitive to 2-heptyl-4-hydroxyquinoline-n-oxide and was highly resistant to anot ...19957665502
sequence analysis and overexpression of the zymomonas mobilis tgt gene encoding trna-guanine transglycosylase: purification and biochemical characterization of the enzyme.trna-guanine transglycosylase (tgt) is involved in the biosynthesis of the hypermodified trna nucleoside queuosine (q). it catalyzes the posttranscriptional base exchange of the q precursor 7-aminomethyl-7-deazaguanine (preq1) with the genetically encoded guanine in the anticodon of trna(asp), trna(asn), trna(his), and trna(tyr). a partially sequenced gene upstream of the dna ligase (lig) gene of the zymomonas mobilis chromosome shows strong homology to the tgt gene of escherichia coli (k.b. sha ...19957665516
comparative energetics of glucose and xylose metabolism in ethanologenic recombinant escherichia coli b.this study compared the anaerobic catabolism of glucose and xylose by a patented, recombinant ethanologenic escherichia coli b 11303:ploi297 in terms of overall yields of cell mass (growth), energy (atp), and end product (ethanol). batch cultivations were conducted with ph-controlled stirred-tank bioreactors using both a nutritionally rich, complex medium (luria broth) and a defined salts minimal medium and growth-limiting concentrations of glucose or xylose. the value of gamma atp was determine ...19957668846
analysis of intact hopanoids and other lipids from the bacterium zymomonas mobilis by high-performance liquid chromatography.hopanoids and other lipids were extracted from zymomonas mobilis and quantitatively analyzed by high-performance liquid chromatography. previous methods for hopanoid analysis required derivatization of the hopanoids via periodate oxidation or acetylation. the current method employs a normal-phase silica gel column, a ternary gradient of hexane-isopropanol-water-triethylamine, and detection with a flame ionization detector. three major hopanoid classes were separated and quantified by this new me ...19957710085
semipreparative separation of intact hopanoids from zymomonas mobilis.hopanoids are an important class of molecules that play a structural and physiological role in the membrane processes of prokaryotic and plant cells. studies on the function of hopanoids require milligram quantities but have been limited by current procedures for isolation and characterization: most separations have isolated only derivatized compounds of hopane in microgram quantities. our method employs aminopropyl bonded-phase solid-phase extraction columns with sequential elution and silica s ...19957710086
purification and characterization of an extracellular levansucrase from pseudomonas syringae pv. phaseolicola.levansucrase (ec 2.4.1.10), an exoenzyme of pseudomonas syringae pv. phaseolicola, was purified to homogeneity from the cell supernatant by chromatography on tmae-fraktogel and butyl-fraktogel. the enzyme has molecular masses of 45 kda under denaturing conditions and 68 kda during gel filtration of the native form. in isoelectric focusing, active bands appeared at ph 3.55 and 3.6. maximum sucrose cleaving activities were measured at ph 5.8 to 6.6 and 60 degrees c. the enzyme was highly tolerant ...19957751294
crystallization and preliminary x-ray analysis of glucose-fructose oxidoreductase from zymomonas mobilis.glucose-fructose oxidoreductase (e.c. 1.1.99.-) from the ethanol-producing gram-negative bacterium zymomonas mobilis is a periplasmic, soluble enzyme that forms a homotetramer of 160 kda with one nadp(h) cofactor per subunit that is tightly, but noncovalently, bound. the enzyme was crystallized by the hanging drop vapor diffusion method using sodium citrate as precipitant. the obtained crystals belong to the space group p2(1)2(1)2, with unit cell constants of 84.6 a, 94.1 a, and 117.0 a, consist ...19947756998
zymomonas mobilis research in the pernambuco federal university. 19937764199
cloning and characterization of a pair of genes that stimulate the production and secretion of zymomonas mobilis extracellular levansucrase and invertase.a 1.7-kb dna fragment cloned from zymomonas mobilis genomic dna complemented the inability to grow on sucrose of a suc- mutant of z. mobilis that was deficient in the production of both extracellular levansucrase and invertase. analysis of the nucleotide sequence of the fragment found two open reading frames (orfs), both of which did not correspond to the structural gene for the levansucrase or the invertase. by subcloning each orf into two different suc- mutants of z. mobilis, it has been found ...19947764692
strain improvement of zymomonas mobilis for ethanol production. 19947764783
cloning of the acetobacter xylinum cellulase gene and its expression in escherichia coli and zymomonas mobilis.a dna fragment corresponding to carboxymethylcellulase activity of acetobacter xylinum ifo 3288 was isolated and cloned in escherichia coli, and the dna sequence was determined. the dna fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 da. the protein encoded in the dna fragment expressed in e. coli hydrolyzed a carboxymethylcellulose. this gene was subcloned into the shuttle vector [pza22; mis ...19947765731
purification and characterization of cycloinulooligosaccharide fructanotransferase (cftase) from bacillus circulans mci-2554.cycloinulooligosaccharide fructanotransferase (cftase) that produces cyclofructan from inulin was purified about 69-fold from a culture broth of bacillus circulans mci-2554 by column chromatographies on deae-toyopearl, qae-toyopearl, hydroxyapatite, and phenyl-sepharose. the molecular mass of the enzyme was estimated to be 115 kda by sds-polyacrylamide gel electrophoresis and gel filtration, indicating a monomer structure. maximal activity was observed at ph 7.5 and 45 degrees c. the enzyme was ...19957765973
cloning and characterization of zymomonas mobilis genes encoding extracellular levansucrase and invertase.the genes encoding the extracellular levansucrase and invertase of zymomonas mobilis have been cloned and sequenced. the levansucrase gene, sucze2, spans 1269 bp and encodes an m(r) 46,790 polypeptide, and the invertase gene, sucze3, is of 1239 bp and encodes an m(r) 46,110 polypeptide. the 5'-terminal sequences of both genes corresponded to the n-terminal amino acid sequences of the secreted levansucrase and invertase, implying that the secretion of both enzymes does not involve proteolytic pro ...19957766026
improved strains of recombinant escherichia coli for ethanol production from sugar mixtures.hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. ethanologenic escherichia coli that have been previously investigated preferentially ferment hexose sugars. in some cases, xylose fermentation was slow or incomplete. the purpose of this study was to develop improved ethanologenic e. coli strains for the fermentation of pentoses in sugar mixtures. using fosfomycin as a selective agent, glucose-negative mutants of e. coli ko11 (containing chro ...19957766137
functional expression of the glucose transporter of zymomonas mobilis leads to restoration of glucose and fructose uptake in escherichia coli mutants and provides evidence for its facilitator action.the zymomonas mobilis genes encoding the glucose facilitator (glf), glucokinase (glk), or fructokinase (frk) were cloned and expressed in a laciq-ptac system using escherichia coli k-12 mutants deficient in uptake and phosphorylation of glucose and fructose. growth on glucose or fructose was restored when the respective genes (glf-glk or glf-frk) were expressed. in e. coli glf+ strains, both glucose and fructose were taken up via facilitated diffusion (km, 4.1 mm for glucose and 39 mm for fructo ...19957768841
evaluation of image analysis and laser granulometry for microbial cell sizing.a direct cell size measurement technique and an image analysis based sizing method were developed. the former consisted of a manual size measurement of the two-dimensional cell images on a video screen, with automatic data recording. this method was chosen as the reference. the latter, a semiautomatic method took advantage of a commercial computer program designed for image processing and particle morphology analysis. it gave average and median size values which were compatible with the manual m ...19957771762
molecular cloning and characterization of the extracellular sucrase gene (sacc) of zymomonas mobilis.the zymomonas mobilis gene sacc that encodes the extracellular sucrase (protein b46) was cloned and expressed in escherichia coli. the gene was found to be present downstream to the already described levansucrase gene sacb in the cloned chromosomal fragment of z. mobilis. the expression product was different from sacb and exhibited sucrase but not levansucrase activity; therefore, sacc behaves like a true sucrase. expression of sacc in e. coli jm109 and xl1 was very low; overexpression was obser ...19957778976
enzymes from zymomonas mobilis and their application to glucose determination. 19957785863
cloning, sequencing and characterization of the alkaline phosphatase gene (phod) from zymomonas mobilis.the phod gene encoding the membrane-bound alkaline phosphatase (alpi) from zymomonas mobilis cp4 was cloned and sequenced. both the translated sequence and the properties of the recombinant enzyme were unusual. z. mobilis alpi was monomeric (m(r) 62,926) and hydrolysed nucleotides more effectively than sugar phosphates. the translated sequence contained a single hydrophobic segment near the n-terminus which may serve as a membrane-anchor in z. mobilis, although the recombinant enzyme was recover ...19957875572
imidazole acetol phosphate aminotransferase in zymomonas mobilis: molecular genetic, biochemical, and evolutionary analyses.hish encodes imidazole acetol phosphate (iap) aminotransferase in zymomonas mobilis and is located immediately upstream of tyrc, a gene which codes for cyclohexadienyl dehydrogenase. a plasmid containing hish was able to complement an escherichia coli histidine auxotroph which lacked the homologous aminotransferase. dna sequencing of hish revealed an open reading frame of 1,110 bp, encoding a protein of 40,631 da. the cloned hish product was purified from e. coli and estimated by sodium dodecyl ...19957883715
zymomonas mobilis squalene-hopene cyclase gene (shc): cloning, dna sequence analysis, and expression in escherichia coli.using a dna probe from the gene encoding squalene-hopene cyclase (shc, ec 5.4.99.-) from the gram-positive bacterium alicyclobacillus acidocaldarius, we have cloned a 4.3 kb hindiii fragment of chromosomal dna from zymomonas mobilis. an open reading frame of 1977 bp was detected that could encode a protein of 658 amino acids with a calculated molecular mass of 74077 da. under the control of lac or tac promoters, this gene, shc, was expressed in escherichia coli k12 strains and its product had sq ...19957894707
comparative fermentation behaviour and chemical characteristics of saccharomyces and zymomonas fermented culled apple juice.ethanol production from culled apple juice showed that fermentability of the juice could be enhanced by addition of dahp or ammonium sulphate in saccharomyces and dahp in zymomonas fermentation. addition of trace elements inhibited both the fermentations and ethanol, consequently. with respect to by-products of fermentation, no clear advantage of zymomnas fermentation of culled apple juice could be observed. differences in physico-chemical characteristics of the fermented apple juice were also n ...19947896319
an allosterically insensitive class of cyclohexadienyl dehydrogenase from zymomonas mobilis.the key enzyme of tyrosine biosynthesis in many gram-negative prokaryotes is cyclohexadienyl dehydrogenase. the zymomonas mobilis gene (tyrc) coding for this enzyme was cloned in escherichia coli by complementation of a tyrosine auxotroph. the tyrc gene was 882 bp long, encoding a protein with a calculated molecular mass of 32086 da. the z. mobilis cyclohexadienyl dehydrogenase expressed in e. coli was purified to electrophoretic homogeneity. the subunit molecular mass of the purified enzyme was ...19937916685
isolation of intergeneric hybrids between bacillus subtilis and zymomonas mobilis and the production of thermostable amylase by hybrids.stable hybrids were obtained by protoplast fusion between bacillus subtilis and zymomonas mobilis. all the hybrids were able to hydrolyse starch and possessed ampicillin- and tetracycline-resistant phenotypes. two of the hybrids, bz-1 and bz-2, were hyperproducers of alpha-amylase. the enzyme produced by these hybrids exhibited increased thermostability. the results show that stable intergeneric gene transfer can be achieved through poly(ethylene glycol)-mediated protoplast fusion between two in ...19947917061
reversible dissociation and unfolding of pyruvate decarboxylase from zymomonas mobilis.the denaturation and renaturation process of pyruvate decarboxylase (pdc) from zymomonas mobilis (atcc 29191) has been investigated using guanidine hydrochloride and urea as denaturing agents. the quarternary structure of the homotetramer is strongly stabilized by the cofactors mg2+ and thiamine diphosphate (tdp). the structural transitions were monitored by activity measurements, fluorescence spectroscopy, circular dichroism and gel-filtration chromatography. a three-step denaturation process, ...19947925382
cloning, sequencing and expression of stress genes from the ethanol-producing bacterium zymomonas mobilis: the groesl operon.zymomonas mobilis is unique among bacteria in its ability to produce high levels of ethanol (etoh) during fermentation. elevated etoh concentration, like elevated temperature, is a microbial stress and a universal inducer of stress proteins. for z. mobilis, exposure to high levels of etoh represents a natural stress. by using a simple strategy which combines the genetic tools of escherichia coli and bacillus subtilis, we have cloned genes encoding two of the most abundant stress proteins in z. m ...19947926837
polymerase chain reaction-based random mutagenesis: production and characterization of thermostable mutants of zymomonas mobilis alcohol dehydrogenase-2.the adhb gene encoding alcohol dehydrogenase-2 from zymomonas mobilis has been subjected to random mutagenesis to obtain more thermostable variants of the enzyme. random mutagenesis was accomplished using the polymerase chain reaction in mutagenic conditions. the optimum conditions involved restricting the concentration of one nucleotide to approximately one-tenth the normal amount. this introduced mutations at an average rate of 1 base in 600 in a 30-cycle pcr, sufficient to ensure that the maj ...19947950371
mechanism of alanine excretion in recombinant strains of zymomonas mobilis.a thiamine-auxotrophic strain of zymomonas mobilis (cp4thi/pzy73), in which the alad gene of bacillus sphaericus coding for the alanine dehydrogenase was expressed, synthesizes and excretes alanine at high rates after thiamine starvation and in the presence of high external ammonium concentrations. the mechanism of alanine excretion was studied in this recombinant zymomonas mobilis strain. under production conditions the internal alanine concentration reached values of up to 280 mm and excretion ...19947986805
sorbitol promotes growth of zymomonas mobilis in environments with high concentrations of sugar: evidence for a physiological function of glucose-fructose oxidoreductase in osmoprotection.the gram-negative ethanologenic bacterium zymomonas mobilis is able to grow in media containing high concentrations of glucose or other sugars. a novel compatible solute for bacteria, sorbitol, which enhances growth of z. mobilis at glucose concentrations exceeding 0.83 m (15%), is described. added sorbitol was accumulated intracellularly up to 1 m to counteract high external glucose concentrations (up to 1.66 m or 30%). accumulation of sorbitol was triggered by a glucose upshift (e.g., from 0.3 ...19948002594
ethanolic fermentation in transgenic tobacco expressing zymomonas mobilis pyruvate decarboxylase.during oxygen limitation in higher plants, energy metabolism switches from respiration to fermentation. as part of this anaerobic response the expression of genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) is strongly induced. in addition there is ample evidence for post-translational regulation. in order to understand this multi-level regulation of the anaerobic response, we provided tobacco with the constitutive capacity of ethanolic fermentation by expressing a pdc ...19948026460
analysis of the escherichia coli genome. v. dna sequence of the region from 76.0 to 81.5 minutes.the dna sequence of a 225.4 kilobase segment of the escherichia coli k-12 genome is described here, from 76.0 to 81.5 minutes on the genetic map. this brings the total of contiguous sequence from the e.coli genome project to 725.1 kb (76.0 to 92.8 minutes). we found 191 putative coding genes (orfs) of which 72 genes were previously known, and 110 of which remain unidentified despite literature and similarity searches. seven new genes--arse, arsf, arsg, tref, xylr, xylg, and xylh--were identified ...19948041620
two genes for carbohydrate catabolism are divergently transcribed from a region of dna containing the hexc locus in pseudomonas aeruginosa pao1.the hexc locus of pseudomonas aeruginosa pao1 was localized to a 247-bp segment of chromosomal dna on the multicopy broad-host-range vector pro1614. the presence of this plasmid (ppz196) in strain pao1 produced the so-called "hexc effect," a two- to ninefold increase in the activities of four carbohydrate catabolism enzymes, glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. the extent of the hexc effect was restricted, ...19948045900
glucose repression in streptomyces coelicolor a3(2): a likely regulatory role for glucose kinase.the glucose kinase gene (glka-orf3) of streptomyces coelicolor a3(2) plays an essential role in glucose utilisation and in glucose repression of a variety of genes involved in the utilisation of alternative carbon sources. these genes include daga, which encodes an extracellular agarase that permits agar utilisation. suppressor mutants of glka-orf3 deletion strains capable of utilising glucose (glc+) arise at a frequency of about 10(-5) on prolonged incubation. the glc+ phenotype of the mutants ...19948052232
characterization and sequence of phoc, the principal phosphate-irrepressible acid phosphatase of morganella morganii.phosphatase activities were investigated in morganella morganii, which is one of the few enterobacterial species producing high-level phosphate-irrepressible acid phosphatase activity (hpap phenotype), and the gene encoding the major phosphate-irrepressible acid phosphatase was cloned, sequenced, and its product characterized. using p-nitrophenyl phosphate as substrate, morganella produced a major phosphate-irrepressible acid phosphatase (named phoc) which is associated with the hpap phenotype, ...19948081499
nucleotide and derived amino acid sequences of an extracellular sucrase gene (invb) of zymomonas mobilis zm1 (atcc10988).dna sequence analysis of a previously cloned 4.5 kb dna fragment showed that the extracellular sucrase gene (invb) of zymomonas mobilis was located in the 155 bp downstream of levansucrase gene (levu). the invb gene had an open reading frame of 1242 bp and the deduced amino acid sequence was 413 residues with a molecular weight of 46,107. the translated sequence of z. mobilis invb was in good agreement with the 10 n-terminal amino acid residues determined by amino acid sequencing. the amino acid ...19948086457
metal-catalyzed oxidation of fe2+ dehydrogenases. consensus target sequence between propanediol oxidoreductase of escherichia coli and alcohol dehydrogenase ii of zymomonas mobilis.we have studied two enzymes of a newly described family of dehydrogenases with high sequence homology, 1,2-propanediol oxidoreductase of escherichia coli and alcohol dehydrogenase ii of zymomonas mobilis. these enzymes perform their metabolic role under anaerobic conditions; in the presence of oxygen, they show a very similar inactivation pattern by a metal-catalyzed oxidation system. titration of histidine residues with diethyl pyrocarbonate showed one histidine residue less in the oxidized enz ...19948120011
reconstruction of glucose uptake and phosphorylation in a glucose-negative mutant of escherichia coli by using zymomonas mobilis genes encoding the glucose facilitator protein and glucokinase.expression of the zymomonas mobilis glf (glucose facilitator protein) and glk (glucokinase) genes in escherichia coli zsc113 (glucose negative) provided a new functional pathway for glucose uptake and phosphorylation. both genes were essential for the restoration of growth in glucose minimal medium and for acid production on glucose-macconkey agar plates.19948144485
molecular analysis of the erwinia chrysanthemi region containing the kdga and zwf genes.the pathways of pectin and galacturonate catabolism in erwinia chrysanthemi converge to form a common intermediate, 2-keto-3-deoxygluconate, which is phosphorylated to form 2-keto-3-deoxy-6-phosphogluconate (kdgp) and then cleaved by the aldolase encoded by the kdga gene. we cloned the kdga gene of the e. chrysanthemi strain 3937 by complementing an escherichia coli kdga mutation, using an rp4-derivative plasmid. restriction mapping of the kdga region and isolation of kdga-lac fusions allowed th ...19948145647
[design of recombinant plasmids for effective zymomonas mobilis pyruvate decarboxylase (pdk) gene expression in bacillus subtilis cells].the pdk gene from z. mobilis localized on the 4.7-kb sphi dna fragment in plasmid pb201 was subcloned using drai restriction endonuclease into the smai site of the phage cloning vector m13mp19. the derivatives of m13mp19 obtained, containing 1.8-kb inserts of the pdk gene in two opposite orientations, were used for dna sequencing and site-directed mutagenesis. the latter was performed using polymerase chain reaction (pcr) and synthetic deoxyribonucleotides of appropriate structure as primers. in ...19948145744
molecular characterization of microbial alcohol dehydrogenases.there is an astonishing array of microbial alcohol oxidoreductases. they display a wide variety of substrate specificities and they fulfill several vital but quite different physiological functions. some of these enzymes are involved in the production of alcoholic beverages and of industrial solvents, others are important in the production of vinegar, and still others participate in the degradation of naturally occurring and xenobiotic aromatic compounds as well as in the growth of bacteria and ...19948185833
investigation of the cofactor-binding site of zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis.several enzymes require thiamin diphosphate (thdp) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (pdc; ec 4.1.1.1) from zymomonas mobilis, as a model for this group of enzymes. it is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. a sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-gdg-) triplet and ending with a double asparagine (-nn-) sequence, has been identified ...19948198554
molecular cloning and sequence analysis of an azospirillum brasilense indole-3-pyruvate decarboxylase gene.azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (iaa) into the culture media. cosmid p0.2, isolated from an a. brasilense sp245 genome library in plafr1, complements the tn5-induced mutant spm7918 of a. brasilense sp6 which excretes reduced amounts of iaa. restriction mapping and gene expression studies identified a bglii-ecori 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of iaa production in mutan ...19948202090
isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate.incorporation of 13c-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (zymomonas mobilis, methylobacterium fujisawaense, escherichia coli and alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13c-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route f ...19938240251
zymobacter palmae gen. nov., sp. nov., a new ethanol-fermenting peritrichous bacterium isolated from palm sap.zymobacter palmae gen. nov., sp. nov. was proposed for a new ethanol-fermenting bacterium that was isolated from palm sap in okinawa prefecture, japan. the bacterium is gram-negative, facultatively anaerobic, catalase-positive, oxidase-negative, nonsporeforming and peritrichously flagellated. it requires nicotinic acid for growth. it ferments hexoses, alpha-linked di- and tri-saccharides, and sugar alcohols (fructose, galactose, glucose, mannose, maltose, melibiose, saccharose, raffinose, mannit ...19938257279
nucleotide sequence of levansucrase gene (levu) of zymomonas mobilis zm1 (atcc10988).the extracellular levansucrase gene (levu) was cloned from the genomic dna of zymomonas mobilis zm1 and the nucleotide sequence of the levu structural gene was determined. the 3.1 kb ecorv-polylinker fusion dna fragment containing the levu gene had an open reading frame of 1272 bps and the deduced amino acid sequence was 423 residues long with a molecular weight of 46,725. although this protein exhibited little similarity with other known levansucrases, several well-conserved regions were observ ...19938318541
cloning, sequencing, and expression of the zymomonas mobilis phosphoglycerate mutase gene (pgm) in escherichia coli.phosphoglycerate mutase is an essential glycolytic enzyme for zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. the pgm gene encoding this enzyme was cloned on a 5.2-kbp dna fragment and expressed in escherichia coli. recombinants were identified by using antibodies directed against purified z. mobilis phosphoglycerate mutase. the pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untransl ...19938320209
the aroq-encoded monofunctional chorismate mutase (cm-f) protein is a periplasmic enzyme in erwinia herbicola.enteric bacteria possess two species of chorismate mutase which exist as catalytic domains on the amino termini of the bifunctional phea and tyra proteins. in addition, some of these organisms possess a third chorismate mutase, cm-f, which exists as a small monofunctional protein. the cm-f gene (denoted aroq) from erwinia herbicola was cloned and sequenced for the first time. a strategy for selection by functional complementation in a chorismate mutase-free escherichia coli background was devise ...19938335631
isolation, characterization and sequence analysis of the scrk gene encoding fructokinase of streptococcus mutans.a gene encoding an atp-dependent fructokinase from streptococcus mutans gs-5 was identified within a 2 kb dna fragment immediately downstream from the scra gene. the gene cloned in escherichia coli also expressed mannokinase activity. insertional inactivation of this gene in s. mutans markedly decreased both fructokinase and mannokinase activities. nucleotide sequence analysis of the 2 kb fragment revealed an orf starting 199 bp downstream from the scra gene, preceded by potential ribosome-bindi ...19938336109
isoprenoid biosynthesis in bacteria: two different pathways?the biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. cell-free extracts of myxococcus fulvus, staphylococcus carnosus, lactobacillus plantarum and halobacterium cutirubrum converted [14c]acetyl-coa or [14c]hydroxymethylglutaryl-coa to [14c]mevalonic acid. furthermore, [14c]mevalonic acid, [14c]mevalonate-5-phosphate and [14c]mevalonate-5-pyrophosphate were metabolized to [14c]isopentenylpyrophosphate. ...19938405922
classification of rhizomonas suberifaciens, an unnamed rhizomonas species, and sphingomonas spp. in rrna superfamily iv.thermal melting profiles of hybrids between 3h-labeled rrna of rhizomonas suberifaciens, the causal agent of corky root of lettuce, and chromosomal dnas from 27 species of gram-negative bacteria indicated that the genus rhizomonas belongs to superfamily iv of de ley. on the basis of the melting temperatures of dna hybrids with rrnas from the type strains of r. suberifaciens, sphingomonas paucimobilis, and sphingomonas capsulata, rhizomonas strains constitute a separate branch in superfamily iv, ...19938427800
an ultraviolet-sensitive mutant of zymomonas mobilis affecting the stability of its natural plasmid pzmo2.plasmid pzmo2, a 1.9-kb natural plasmid of zymomonas mobilis strain atcc 10988, was found to be absent from the extract of a mutant isolate sensitive to ultraviolet irradiation, methyl methanesulfonic acid, and mitomycin c. this mutant, named uvs51, also exhibited extremely high segregational instability of the recombinant plasmid pds212, whose maintenance in the parental strain is known to be due to pzmo2 sequences. helped conjugations, employing uvs51 as recipient with escherichia coli donors ...19938441765
mutational analysis of segmental stabilization of transcripts from the zymomonas mobilis gap-pgk operon.in zymomonas mobilis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase are transcribed together from the gap-pgk operon. however, higher levels of the former enzyme are present in the cytoplasm because of increased stability of a 5' segment containing the gap coding region. this segment is bounded by an upstream untranslated region which can be folded into many stem-loop structures and a prominent intercistronic stem-loop. mutations eliminating a proposed s ...19938468293
glucose-fructose oxidoreductase, a periplasmic enzyme of zymomonas mobilis, is active in its precursor form.glucose-fructose oxidoreductase (gfor) is a periplasmic enzyme of the ethanologenic, gram-negative bacterium zymomonas mobilis. it contains tightly bound nadp+ as cofactor. in z. mobilis gfor-recombinant strains, a precursor form of gfor was accumulated. to assay the pregfor for its nadp(h) content and enzymatic activity, it was purified from an overproducing strain. using sds-page, the precursor subunit size was determined to approximately 45 kda (compared with a 40 kda subunit size for the mat ...19938472911
zymomonas mobilis--science and industrial application.zymomonas mobilis is undoubtedly one of the most unique bacterium within the microbial world. known since 1912 under the names termobacterium mobilis, pseudomonas linderi, and zymomonas mobilis, reviews on its uniqueness have been published in 1977 and 1988. the bacterium zymomonas mobilis not only exhibits an extraordinarily uniqueness in its biochemistry, but also in its growth behavior, energy production, and response to culture conditions, as well as cultivation techniques used. this uniquen ...19938477453
electroporation and dna-dependent cell death in murine macrophages.the difficulty of transfecting primary macrophages and macrophage cell lines has meant that relatively few studies on regulation of gene expression have been performed in these cells. this study has optimized an electroporation procedure for the macrophage cell line raw 264, but shows that introduction of dna into the cytoplasm of primary macrophages by electroporation is toxic to the cells. it is proposed that this cell death may have a physiological role in defence against certain viral infect ...19938486399
use of in vivo 13c nuclear magnetic resonance spectroscopy to follow sugar uptake in zymomonas mobilis.a noninvasive, in situ, in vivo, and anomer-specific method for studying membrane transport of sugars in bacteria is presented. high-resolution 13c nmr was used to measure the distribution of alpha- and beta-xylose, maltose, mes buffer, and ethanol in the extracellular and the cytoplasmic compartments in dense cell suspensions of zymomonas mobilis, an aerotolerant bacterium that transports xylose but does not further metabolize it. the method relied on a difference in the magnetic susceptibility ...19938489007
purification and characterization of an oxygen-labile, nad-dependent alcohol dehydrogenase from desulfovibrio gigas.a nad-dependent, oxygen-labile alcohol dehydrogenase was purified from desulfovibrio gigas. it was decameric, with subunits of m(r) 43,000. the best substrates were ethanol (km, 0.15 mm) and 1-propanol (km, 0.28 mm). n-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as zymomonas mobilis adh2 and bacillus methanolicus mdh.19938491707
cloning and expression in escherichia coli of the dnak gene of zymomonas mobilis.the dnak protein of zymomonas mobilis (dnakz) was identified and found to be 80% identical to the dnak protein of escherichia coli on the basis of the sequence of the n-terminal 21 amino acids. the dnakz gene was cloned and found to be expressed in a thermosensitive dnak mutant of escherichia coli. expression of the foreign gene restored a thermoresistant phenotype but failed to modulate the heat shock response in e. coli.19938491740
development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of pseudomonas syringae.the expression of the ice nucleation gene inaz of pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. a promoterless version of inaz was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its phe1 ...19958526492
the replacement of trp392 by alanine influences the decarboxylase/carboligase activity and stability of pyruvate decarboxylase from zymomonas mobilis.the bulky tryptophan residue 392 located in the deep cleft leading to the active center of pyruvate decarboxylase (pdc) from zymomonas mobilis was changed to alanine which is found in the equivalent position of pdc from yeast. the mutation reduced the decarboxylase activity towards pyruvate by a factor of two (60-70 u/mg), whereas the km (1.1 mm in mes/koh buffer) remains unchanged compared with the wild-type enzyme. the apparent km for thiamine diphosphate (thiamin-p2) in the presence of 5 mm m ...19958536715
overexpression of extracellular sucrase (sacc) of zymomonas mobilis in escherichia coli.the extracellular sucrase (sacc) gene of zymomonas mobilis was overexpressed in escherichia coli bl21 using the t7 polymerase expression system. a low cell density induction method was designed to have maximum expression, and the conditions (iptg concentration, ampicillin addition) were optimised to overexpress to the level of more than 60% of the total cellular protein representing sacc protein.19958566709
the cyanobacterium synechococcus sp. strain pcc 7942 contains a second alkaline phosphatase encoded by phov.a gene (phov) encoding an alkaline phosphatase from synechococcus sp. strain pcc 7942 was isolated by screening a plasmid gene bank for expression of alkaline phosphatase activity in escherichia coli jm103. two independent clones carrying the same alkaline-phosphatase-encoding gene were isolated. one of these clones (pkw1) was further analysed and the nucleotide sequence of a contiguous 3234 bp dna fragment was determined. two complete open reading frames (orf1 and phov) and an incomplete orf3 w ...19958574398
cloning and expression of the unique ca2+-atpase from flavobacterium odoratum.the 60-kda ca2+-atpase from flavobacterium odoratum is kinetically and mechanistically similar to other p-type atpases, suggesting its use as a model system for structure-function studies of ion transport. a portion of the gene was amplified by polymerase chain reaction of genomic dna with degenerate oligonucleotide primers, one based on the n-terminal amino acid sequence of the purified protein and the other based on a consensus sequence for the phosphorylation site of p-type atpases. this gene ...19968617788
ethanol transport in zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer.for the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. the rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees c and 2.7 +/- 0.3s(-1) at 20 degrees c. they were determined by using 1h selective nuclear magnetic resonance spin magnetization transfer. the measurements were done on l-ml cell suspensions. no additi ...19968626306
the role of residues glutamate-50 and phenylalanine-496 in zymomonas mobilis pyruvate decarboxylase.several enzymes require thiamine diphosphate (thdp) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (pdc; ec 4.1.1.1) from zymomonas mobilis, as a model for this group of enzymes. it is well suited for this purpose because of its stability, ease of purification, homotetrameric subunit structure and simple kinetic properties. crystallographic analyses of three thdp-dependent enzymes [müller, lindqvist, furey, schulz, jordan and schneider (1993) structure 1, 95-103] ...19968645153
continuous ethanol production by zymomonas mobilis and saccharomyces cerevisiae in biofilm reactors.continuous ethanol fermentations were performed in duplicate for 60 days with zymomonas mobilis atcc 331821 or saccharomyces cerevisiae atcc 24859 in packed-bed reactors with polypropylene or plastic composite-supports. the plastic composite-supports used contained polypropylene (75%) with ground soybean-hulls (20%) and zein (5%) for z. mobilis, or with ground soybean-hulls (20%) and soybean flour (5%) for s. cerevisiae. maximum ethanol productivities of 536 g l-1 h-1 (39% yield) and 499 g l-1 h ...19968652117
crystal structure of trna-guanine transglycosylase: rna modification by base exchange.trna-guanine transglycosylases (tgt) are enzymes involved in the modification of the anticodon of trnas specific for asn, asp, his and tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. in prokaryotes tgt catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preq1). the crystal structure of tgt from zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic r-fa ...19968654383
the gluemp operon from zymomonas mobilis encodes a high-affinity glutamate carrier with similarity to binding-protein-dependent transport systems.the nucleotide sequence downstream of the grp gene, encoding the glutamate uptake regulatory protein of zymomonas mobilis, was determined. three clustered genes (glue, glum, and glup) close to ghe grp gene, but on the opposite strand, were identified. these genes encode a high-affinity transport system for glutamate and aspartate. the glup gene product is a polypeptide of 25.4 kda and contains segments with significant similarity to the atp-binding proteins of binding-protein-dependent transport ...19968661924
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