Isolation and characterization of an alginate lyase from Klebsiella aerogenes

The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37°C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acidrich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.Abbreviations poly(ManA) (1–4)--D-mannuronan - poly(GulA) (1–4)--L-guluronan - TBA 2-thiobarbituric acid     

Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide

Escherichia coli K5 polysaccharide has structural analogies with N-acetylheparosan, a non-sulphated precursor of heparin and, for this reason, can be considered an attractive precursor for the production of semi-synthesis heparin analogues. This polysaccharide has two components: a high molecular weight (HMW) one and a low molecular weight (LMW) one, whose ratio varies depending on the action of a lyase enzyme synthesized by the same K5 producer strain. The present paper reports the production of the K5 polysaccharide by a spontaneous E. coli mutant strain lacking the lyase activity. Similar K5 polysaccharide yields, 180 mg l(-1) after 16 h fermentation, were obtained by both the wild and mutant strains, though K5 lyase activity was only observed in the culture filtrates from the wild strain. The time course of the specific filtrate volume (1 m(-2)) and of the specific filtrate flux rate (1 m(-2) h(-1)) during ultrafiltration (UF) of culture filtrates where the lyase enzyme acted on the K5 chain, showed a decrease of UF performance, probably because of membrane fouling by the LMW K5 fraction. In particular, the specific filtrate volume and specific filtrate flux rate of wild strain samples reached respectively 13 l m(-2) and 4 l m(-2) h(-1), compared to 25 l m(-2) and 15 l m(-2) h(-1) obtained from the mutant strain samples. PCR molecular analysis of the DNA region encoding for the lyase enzyme showed that, in the mutant strain, molecular rearrangements occurred in both regulatory and structural regions.     

Regulation of the central metabolism in relation to citric acid production in Saccharomycopsis lipolytica

The mechanism of the massive extracellular production of citric and isocitric acids by Saccharomycopsis lipolytica grown on n-paraffins has been studied. When growth stops, because of nitrogen limitation, the intracellular concentration of ATP sharply rises whereas that of AMP and ADP decreases to a low level. At the same time production of acids begins. The activity of the NAD-dependent isocitrate dehydrogenase which requires AMP for activity becomes very low and prevents the oxidative function of the citric acid cycle whereas isocitrate lyase is not inhibited. As citrate synthase inhibition by ATP appears to be insufficient to stop n-paraffin degradation, citric and isocitric acids accumulation can take place. Massive excretion of these acids, however, probably still involves other physiological changes brought about by nitrogen limitation, possibly some permeabilization of the cell to these acids.This work is a part of a Doctorat de Spécialité Thesis submitted by R. Marchal to the University of Nancy (1975)     

ATP-linked citrate lyase activity in the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum

Cell-free extracts of the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum strains 1C and L have been shown to cleave citrate with the formation of oxaloacetate and acetyl-CoA. This capacity was found in autotrophically grown cells as well as in the cells grown on media with acetate or L-glutamate. Citrate lyase activity in cell-free extracts is only measurable in the presence of citrate, adenosine-5-triphosphate, coenzyme A and Mg²⁺ or Mn²⁺. It is concluded on the basis of the obtained data that C. limicola f. thiosulfatophilum contains adenosine-5-triphosphate-linked citrate lyase (E.C.4.1.3.8). In contrast to green bacteria in the purple bacteria Ectothiorhodospira shaposhnikovii, Rhodospirillum rubrum and Thiocapsa roseopersicina citrate lyase activity was not found.     

Extracellular K5 polysaccharidic antigen recovery performed by different cell separation technologies

The K5 polysaccharidic antigen obtainable from a strain of Escherichia coli is the non-sulphated precursor in heparin biosynthesis; K5 is composed by two components, 16000 and 1500 Da, their ratio depending on the fermentation conditions. In this study an assessment was made of how various cell-culture filtrate separation technologies affected the components themselves and their ratio. A dynamic membrane filtration technology, a tubular ceramic module for tangential flow microfiltration and the standard discontinuous centrifugation were comparatively employed to perform the separation. Experiments carried out on E. coli cultures containing the two components in different ratios showed that the DMF system is a suitable technology for K5 isolation.     

Pyruvate formate lyase inRhodospirillum rubrum Ha adapted to anaerobic dark conditions

The fermatation metabolism ofRhodospirillum rubrum Ha was studied after adaptation of both light-anaerobic and dark aerobic to dark anaerobic conditions.Pyruvate was metabolized to acetate, formate, CO₂ and propionate by suspensions of cells adapted to anaerobiosis. Pyruvate cleavage to formate accounted for about two-thirds of the pyruvate decomposed. This process was catalyzed by a coenzyme A dependent pyruvate formate lyase. In carboxylate- and nucleotide-free extracts, the substrate concentrations for half-maximal velocity [S]_0.5V were found to be 1.5 mM for pyruvate and 75 M for coenzyme A.Pyruvate formate lyase could practically not be demonstrated in light-anaerobic photosynthesizing cells. Lyase activity was low at a basic level in darkaerobic respiring cells. After adaptation of both types of cells under growth conditions to dark anaerobiosis lyase activity increased about 10-fold. Highest levels could be observed in cells grown aerobically in the dark on pyruvate after transition to dark anaerobic conditions. It is concluded that pyruvate formate lyase is the characteristic key enzyme of the dark-anaerobic fermentative metabolism ofR. rubrum Ha.     

The Escherichia coli K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm

The intracellular expression of the K5 lyase enzyme, which degrades the K5 polysaccharide, decreased cell surface expression of the Escherichia coli K5 capsule. This indicates that biosynthesis of K5 polysaccharide in the cytoplasm is accessible to the action of K5 lyase and is not synthesized within a protected cytoplasmic compartment.The polysaccharide capsules of bacteria have been studied in most detail in Escherichia coli (13). E. coli has over 80 chemically and serologically distinct polysaccharide capsules, which are designated K antigens and classified into four groups (13). Group 2 polysaccharide capsules, of which K1 and K5 have been most studied (12, 13), are commonly expressed in pathogenic extraintestinal E. coli (1, 3, 7) and closely resemble the capsules of Neisseria meningitidis and Haemophilus influenzae (13). Group 2 capsule gene clusters have a conserved genetic organization comprising three regions. Regions 1 and 3 are common to all group 2 capsule gene clusters and encode the Kps proteins involved in polysaccharide export across the inner membrane, periplasm, and outer membrane. Region 2, flanked by regions 1 and 3, contains the highly variable serotype-specific genes involved in the biosynthesis of the particular polysaccharide (12, 13). In the case of the K5 capsule gene cluster, this involves the kfiABCD genes (9).The biosynthesis of the K5 polysaccharide occurs through the sequential addition of GlcA and GlcNAc residues to the nonreducing end of the polysaccharide chain catalyzed by two glycosyltransferases, KfiA and KfiC (4, 6). Polysaccharide biosynthesis occurs at the cytoplasmic face of the inner membrane and involves a hetero-oligomeric complex, consisting of proteins involved in both biosynthesis and export, that is localized at the pole of the cell (8). Such a complex would facilitate a linkage between polysaccharide synthesis and export, although at this stage the mechanism by which synthesis and export are linked is unclear. A recent paper in which the K1-specific endosialidase was expressed in the cytoplasm of a K1-expressing strain indicated that K1 polysaccharide synthesis may occur within a protected cytoplasmic compartment that is inaccessible to endosialidase cleavage (10). To test whether this was also true for the synthesis of the K5 polysaccharide, we expressed the K5-specific lyase, an enzyme that specifically degrades K5 and is associated with the tail spike of K5-specific bacteriophage (2, 5), in the cytoplasm of a K5-encoding strain. In contrast to the situation with K1, we found that expression of the K5 lyase in the cytoplasm reduced the cell surface expression of K5 polysaccharide, suggesting that unlike K1 polysaccharide synthesis, K5 polysaccharide is not synthesized within a protected cytoplasmic compartment.     

Characterization of citrate lyase from Clostridium sporosphaeroides

Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of l-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in star form.     

Localization of C-S lyase activity in the root cells ofAlbizzia lophanta

Summary The distribution of C-S lyase activity in root cells ofAlbizzia lophanta Benth. plantlets was investigated histochemically. H₂S formed upon cleavage of exogenously applied L-cysteine was precipitated by Pb⁺⁺ in a capture reaction at the site of its formation. Enzyme activity was found to be localized at the root tip and in a layer of cortex cells adjacent to the endodermis throughout the whole length of the root. Distinct areas within the exodermis, distributed in a regular pattern on the root surface, also exhibited the specific reaction. In vivo roots ofAlbizzia lophanta actively excrete the strongly smelling methylene dithiol, formed by enzymatic cleavage of djenkolic acid, the natural substrate of C-S lyase inAlbizzia. The physiological meaning of this compound, as well as the localization and intracellular distribution of C-S lyase activity are discussed.     

The zygote cell wall of Chlamydomonas reinhardii: a structural,chemical and immunological approach

The zygote cell wall of Chlamydomonas reinhardii has been studied using structural, chemical and immunological methods. Monoclonal antibodies and polyclonal antisera that were originally raised to the major hydroxyproline-rich glycoproteins of the vegetative cell wall were used to probe the zygote wall for common antigenic components. These antibodies cross-reacted strongly and specifically with components of the zygote cell wall, and were used to show the origin, route of transport, and the location of these antigens within the zygote cell wall. The zygote cell wall contained about 10% protein, with hydroxyproline accounting for 22.5 mol % of the total amino acids present. Glucose was the most abundant sugar residue, and accounted for 56% of the total sugar present. Gas liquid chromatography-mass spectrometry showed the presence of a (1-3)-d-glucan as the major structural polysaccharide within the zygote cell wall. The (1-3)-d-glucan was detected and localised within the zygote cell wall by immunogold labelling of thin sections. Using an antiserum directed against (1-3)-d-linked glucose units, this polysaccharide was found to be consistently present within the non-staining layer of both young and mature zygote cell walls. (1-3)-d-Glucan was also detected in other wall layer using higher concentrations of antiserum. No intracellular labelling was found, indicating that the plasmamembrane is the site for the synthesis of this polysaccharide within the Chlamydomonas zygote.Abbreviations DGP antiserum to deglycosylated 2-BII glycoprotein - GLC-MS gas liquid chromatography-mass spectrometry - MAC monoclonal antibody centre     

Production of an extracellular polysaccharide byAgrobacterium sp DS3 NRRL B-14297 isolated from soil

A bacterium isolated from soil and identified asAgrobacterium sp produced a water-soluble extracellular polysaccharide capable of producing highly viscous solutions. Gas chromatographic analysis revealed a sugar composition of glucose, galactose and mannose in the molar ratio of 7.52.41, together with 3.7% (w/w) pyruvic acid. Methylation analyses showed the presence of (13)-, (14)- and (16)-linked glucose, (13)- and (14, 16)-linked galactose and a small portion of (13)-linked mannose residues. Succinic acid was not present. The molecular weight of the polysaccharide was estimated by light scattering to be 2×10⁶ Da. The viscosity of solutions containing the polysaccharide remained constant from pH 3 to 11, and decreased by 50% when heated from 5 to 55°C. Maximum yield of the polysaccharide, 20 g L^–1, was reached in 48 h at 30°C incubation.     

Structural analysis of water-soluble fractions obtained fromAspergillus fumigatus mycelium

Fractions were prepared from the water-soluble components ofAspergillus fumigatus mycelium either by lectin-affinity chromatography or salt precipitation. While they varied considerably in their amino-acid composition, each contained a preponderance of aspartic and glutamic acids.¹³C-NMR spectroscopy of these fractions, compared with that of polysaccharide obtained by alkaline extraction, indicated the presence of glycoproteins, the polysaccharide components of which contained -d-Galf units that are part of structures chemically different from those obtained by alkali treatment. In two of the three fractions examined, gas-liquid chromatography-mass spectrometry showed marked differences in the contents of non-reducing end-units of -d-Manp and -d-Galf. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the preparations revealed an array of components, which stained to differing extents with silver stain and with Coomassie Blue and many of which were bound by lectins with specificity for different sugars.     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

Molecular genetics of carbon-phosphorus bond cleavage in bacteria

Phosphonates (Pn) are a large class of organophosphorus molecules that have direct carbon-phosphorus (C - P) bonds in place of the carbon-oxygen-phosphorus ester bond. In bacteria two pathways exist for Pn breakdown for use as a P source: the phosphonatase and C - P lyase pathways. These pathways differ both in regard to their substrate specificity and their cleavage mechanism. The phosphonatase pathway acts on the natural Pn -aminoethylphosphonate(AEPn). In a two-step process it leads to cleavage of the C - P bond by a hydrolysis reaction requiring an adjacent carbonyl group. In contrast the C - P lyase pathway has a broad substrate specificity. It leads to cleavage of substituted Pn (such as AEPn) as well as unsubstituted Pn by a mechanism involving redox or radical chemistry. Due to its broad substrate specificity, the C - P lyase pathway is generally thought to be responsible for the breakdown of Pn herbicides (such as glyphosate) by bacteria. As a way to gain a more in-depth understanding of these Pn degradative pathways, their respective genes have been isolated and characterized. In the absence of a biochemical assay for the C - P lyase pathway such molecular approaches have been especially valuable. The roles of individual genes have been inferred from DNA sequence analysis and mutational effects. Genes for the C - P lyase pathway exist in a fourteen-gene operon that appears to encode both a binding protein-dependent Pn transporter and a C - P lyase. Genes for the phosphonatase pathway also exist in a gene cluster containing Pn uptake and degradative genes. A combination of biochemistry, molecular biology, and molecular genetics approaches has provided more detailed understanding of the mechanisms of C - P bond cleavage. Such basic information may provide a new handle for improvement of Pn degradation capabilities in bacteria, or in other cells in which the respective genes may be introduced and expressed.Abbreviations AEPn -aminoethylphosphonate - C carbon - kbp kilobase pair - kDa kilodalton - MPn methylphosphonate - P phosphorus - P _i inorganic phosphate - Pn phosphonate - psi phosphate starvation inducible     

Insect Induced Self-Pollination

Significant increase of pod production occurs inLupinus palaestinus Boiss. andL. pilosus Murr. following insect visits. The cause of this increase is investigated through (1) examination of the biology of pollination, (2) examination of pod production under various pollination conditions, (3) examination of cross pollination by genetical markers. All data strongly suggest that Insect Induced Self Pollination is the main factor in the increase of pod production of these species in nature.     

Choline derivatives increase two different acid phosphatases in Rhizobium meliloti and Pseudomonas aeruginosa

In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sole carbon and nitrogen source, increase acid phosphatase activity. The enzyme activity of both bacteria could be released into the surrounding medium by EDTA-lysozyme treatment. The R. meliloti acid phosphatase activity of crude periplasmic extracts measured with p-nitrophenylphosphate was not inhibited by the presence of 5 mM choline, betaine, trimethylammonium or phosphorylcholine. The activity could not be detected using phosphorylethanolamine or phosphorylcholine as substrates. Among several phosphoesters tested only pyridoxal-5-phosphate was hydrolyzed at a considerable rate. In 7.5% polyacrylamide slab gel electrophoresis (non-denaturing conditions) of crude extracts obtained from bacteria grown in the presence of serine, glutamate, aspartate or dimethylglycine a phosphatase activity with identical mobility could be detected when alpha-naphthylphosphate or pyridoxal-5-phosphate were used as substrates. In conclusion, although the coline metabolites are capable of increasing acid phosphatase activities in R. meliloti and in P. aeruginosa, there are two different enzymes involved, apparently in different metabolisms.Abbreviations p-NPP p-nitrophenyl phosphate - PLP pyridoxal-5-phosphate - PMP pyridoxamine-5-phosphate Recipient of a Fellowship from the CONICORMember of the SAPIU-CONICETCareer Member of the CONICET     

Isolation and characterization of the exopolysaccharide produced by Daedalea quercina

The production of exopolysaccharide (EPS) by a strain of the basidiomycete Daedalea quercina was investigated. Of seven different carbon sources, glucose and dextrins gave the highest crude polysaccharide yield (4.7–5 g l^–1, 55–60% carbohydrate content) in shake-flask cultures, at 14 days of fermentation. Experiments carried out in a 10 l fermenter, at two different agitation speeds, gave the best results at 300 rpm, resulting in 12–14 g l^–1 of crude exopolysaccharide in 9–11 days. Fractionation of the EPS samples, carried out by tangential flow ultrafiltration, evidenced a single EPS fraction (MW >30 000 Da) in samples from glucose, while two fractions (MW > 30 000 Da and 30 000 > MW > 10 000 Da) were present in samples from dextrins. Fractions characterization by HPLC and proton NMR spectroscopy revealed diversity in composition and structure in the obtained EPS: from glucose mainly an -linked mannan, and from dextrins mainly an - and -linked glucan.     

Purification and properties of swine kidney phosphoprotein phosphatase

Summary Pig intestinal brush borders (BB) were radiolabeled by iodination using the lactoperoxidase-hydrogen peroxide procedure. The BB were then detergent solubilized, centrifuged to remove particulate material, and chromatographed on Sepharose CL-4B. The fractions were incubated with K88+ E. coli using an in vitro binding assay. Binding of the iodinated membranes to K88+ E. coli occurred throughout a wide range of molecular weight components, in excess of 690K daltons to near 25K daltons. The system utilizing intact K88+ E. coli and solubilized BB was shown to be saturable. Prior contact of K88+ E. coli with nonradiolabeled membranes or specific antibodies to K88+ pili inhibited binding of the radiolabeled BB. Simple sugars were tested for their ability to block binding of the labeled BB; partial inhibition occurred with galactose (17.9%), galactosamine (32%), glucose (10.6%), and N-acetylglucosamine (32%). Calcium enhanced binding with as little as 10 M. A 10 × increase in binding occurred with 500 M calcium. Affinity chromatography using K88+ pili coupled on agarose beads avidly bound the labeled BB. The receptor membranes were eluted with high molar concentrations of salt, however considerable degradation occurred. Despite low yields from the affinity system, receptor membranes with higher binding activities were recovered. Protein: glycoprotein ratios were 1:4. Elution with SDS and electrophoresis on 12.5% polyacrylamide gels in the presence of a reducing agent produced two major subunits 35–32K and 23K daltons. These components were recovered from the gels and retained their binding activity. This information suggests that the intestinal receptor responsible for binding of K88+ E. coli is a glycoprotein, that in the native state exists in multimeric forms.     

Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses

The cell-wall sulphated polysaccharide of the red microalga Porphyridium sp. has impressive antiviral activity against Herpessimplex viruses types 1 and 2 (HSV 1, 2) and Varicella zoster virus(VZV). Treatment of cells with 1 g mL^-1 polysaccharideresulted in 50% inhibition of HSV-infection as measured by the plaqueassay. Inhibition of the production of new virus particles was also shownwhen pre-infected cell cultures were treated with the polysaccharide. Inaddition, there was indirect evidence for a strong interaction between thepolysaccharide and HSV and a weak interaction with the cell surface.Depending on the concentration, the polysaccharide completely inhibitedor slowed down the development of the cytopathic effect in HSV or VZVpreinfected cells, but did not show any cytotoxic effects on Vero cells evenwhen a concentration as high as 250 g mL^-1 was used. Itseems therefore that the polysaccharide is able to inhibit viral infection bypreventing adsorption of virus into the host cells and/or by inhibiting theproduction of new viral particles inside the host cells. Thus, this alga seems tobe a good candidate for the development of an antiviral drug.     

Galactomannan formation and guanosine 5′-diphosphate-mannose: galactomannan mannosyltransferase in developing seeds of fenugreek (Trigonella foenum-graecum L., leguminosae)

The time-course of galactomannan and stachyose (digalactosyl-sucrose) deposition in the fenugreek seed endosperm has been determined, and correlated with standard parameters of seed development. During, and only during, the period of galactomannan deposition, endosperm homogenates are capable of catalysing the transfer of labelled d-mannosyl residues from guanosine 5-diphosphate d-[U-¹⁴C]mannose to a soluble polysaccharide product indistinguishable from galactomannan. The mannosyltransferase activity peaks twice, once at the beginning of galactomannan deposition, and again in the middle of the most rapid phase of galactomannan deposition. The enzyme in the later peak sediments with grossly particulate material (1,000 g pellet), whereas the earlier peak contains a considerable proportion of a particulate enzyme sedimenting at 100,000 g. These observations are discussed in the light of existing information on the ultrastructural aspects of galactomannan deposition. The mannosyltransferase is clearly involved in galactomannan formation in vivo, but the status of an accompanying galactosyltransferase is less clear.Abbreviations GDP guanosine 5-diphosphate - UDP uridine 5-diphosphate