Identification of para-Cresol as a Growth Factor for Methanoplanus endosymbiosus

Growth of the methanogenic bacterium Methanoplanus endosymbiosus is dependent on the presence of ruminal fluid. Ruminal fluid could be replaced by the eluate of a rumen-derived anaerobic digester. From the eluate of the digester, a growth-stimulatory component was purified and identified as p-cresol. Authentic p-cresol supported a half-maximal growth rate of the organism at 50 nM concentration.     

Characterization and mode of action of an exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritima

An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105 degrees C and a temperature optimum of 80 degrees C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 U.mg(-1)). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed.     

The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.     

ATP synthesis from 2,3-diphosphoglycerate by cell-free extract of Methanobacterium thermoautotrophicum (strain ΔH)

Cell-free extracts of Methanobacterium thermoautotrophicum were found to catalyze ATP synthesis from an endogeneous substrate. Synthesis was stimulated under hydrogen atmosphere and inhibited by KCL (K _i =150 mM). Comparison of the properties of a number of cell constituents showed the endogeneous substrate to be 2,3-diphosphoglycerate. The compound is converted into 3-phosphoglycerate, and via 2-phosphoglycerate and phosphoenolpyruvate into pyruvate, at which the latter reaction is linked with ATP synthesis.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme m, 2-(methylthio)ethanesulfonate - HS-HTP 7-mercaptoheptanoyl-l-threonine phosphate - CoM-SS-HTP the heterodisulfide of HS-CoM and HS-HTP - BCFE bolled cell-free extract - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - PEP phosphoenolpyruvate - 2,3-DPG 2,3-diphosphoglycerate - cDPG cyclic 2,3-diphosphoglycerate - 3-PG 3-phosphoglycerate - 2-PG 2-phosphoglycerate     

Analysis of multiparasitism by Eupelmus vuilleti (Craw) (Eupelmidae) and Dinarmus basalis (Rond) (Pteromalidae) in the presence of one of their common hosts,Bruchidius atrolineatus (Pic) (Coleoptera Bruchidae)

Bruchidius atrolineatus (Pic) is a tropical beetle (Coleoptera Bruchidae) that develops during the larval and pupal stages in the seeds of a legume Vigna unguiculata (Walp). Two species of Hymenoptera, Dinarmus basalis (Rond) and Eupelmus vuilleti (Craw), solitary ectoparasitoids of the larvae and pupae of B. atrolineatus, were introduced successively in the presence of their hosts, varying the interval between the two introductions. When D. basalis females were introduced 24 h, 3 days or 7 days after E. vuilleti, multiparasitism was low. The females had low fecundity, and their eggs were not distributed randomly over the different available hosts. When E. vuilleti females were introduced second, they oviposited on the different hosts availabe and did not avoid multiparasitism. The presence of hosts already parasitised by D. basalis increased the reproduction of E. vuilleti, and the fecundity of the females was higher than in control batches with E. vuilleti alone. E. vuilleti seems capable of detecting the ovipositor shafts drilled by the D. basalis females, and by introducing its own ovipositors killing the D. basalis eggs or larvae. When interspecific competition was occurring the number of E. vuilleti adults emerging from the seeds was no different from that observed in control batches with E. vuilleti alone, and there were always fewer D. basalis adults than in control batches (D. basalis alone). This interspecific competition reduces the influence of the two parasitoids in the biological control of bruchid populations.     

Mode of action of pectin lyase A of Aspergillus niger on differently C(6)-substituted oligogalacturonides

A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C(6)-substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of ester (ethyl esterification) or transamidation resulted in (almost) complete loss of conversion. The PLA activity increased with increasing length of the substrate up to a degree of polymerization (DP) of 8 indicating the presence of at least eight subsites on the enzyme. Product analysis demonstrated the formation of several Delta 4,5 unsaturated products and their saturated counterparts. The Delta 4,5 unsaturated trimer was the main product up to DP 8. For DP 9 and 10 Delta 4,5 unsaturated tetramer was the major product. Based upon the bond cleavage frequencies, a provisional subsite map was calculated, which supports the presence of eight subsites. By limited alkaline de-esterification of fully methyl-esterified pentamer and hexamer two sets of partially methyl-esterified pentamers (x and y methyl groups) and hexamers (a and b methyl groups) were prepared. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) analysis demonstrated that the methyl-ester distribution was fully random. Using these partially methyl-esterified oligogalacturonides as substrates for PLA a 10-fold decrease in reaction rate was recorded compared with the fully methyl-esterified counterparts. Analysis of the methyl-ester distribution of the products showed that PLA tolerates carboxyl groups in the substrate binding cleft. At either subsite +2, +4, or -1 to -4 a free carboxyl group could be tolerated, whereas methyl-esters were obligatory at subsite +1 and +3. So PLA is capable to cleave the bond between a methyl-esterified and a non-esterified galacturonic acid residue, where the newly formed Delta 4,5 unsaturated non-reducing end residue always contains a methyl-ester.     

Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening

The Cnr ( C olourless n on- r ipening) tomato ( Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic polysaccharides to the non-softening and altered cell adhesion phenotype. Cell wall material (CWM) and solubilised fractions of mature green and red ripe fruit were analysed by chemical, enzymatic and immunochemical techniques. No major differences in CWM sugar composition were detected although differences were found in the solubility and composition of the pectic polysaccharides extracted from the CWM at both stages of development. In comparison with the wild type, the ripening-associated solubilisation of homogalacturonan-rich pectic polysaccharides was reduced in Cnr. The proportion of carbohydrate that was chelator-soluble was 50% less in Cnr cell walls at both the mature green and red ripe stages. Chelator-soluble material from ripe-stage Cnr was more susceptible to endo-polygalacturonase degradation than the corresponding material from wild-type fruit. In addition, cell walls from Cnr fruit contained larger amounts of galactosyl- and arabinosyl-containing polysaccharides that were tightly bound in the cell wall and could only be extracted with 4 M KOH, or remained in the insoluble residue. The complexity of the cell wall alterations that occur during fruit ripening and the significance of different extractable polymer pools from cell walls are discussed in relation to the Cnr phenotype.     

Methanobacterium thermoautotrophicum (strain ΔH) contains a membrane-bound cyclic 2,3-diphosphoglycerate hydrolase

Cyclic 2,3-diphosphoglycerate (cDPG) hydrolase activity was demonstrated in cofactor-free extract of Methanobacterium thermoautotrophicum (strain H), but not in crude extract. Only after ultrafiltration or dialysis of crude extract cDPG hydrolase activity could be shown. cCPG hydrolysis was optimal at pH 6.0 and 60°C. Hydrolysis of cDPG occurred under nitrogen or hydrogen atmosphere and was completely inhibited by oxygen. Phosphate and potassium chloride were also strong inhibitors: 50% inhibition occurred at 0.6–0.7 mM phosphate or 0.2 M KCl. The enzyme was localized in the membrane fraction and could be solubilized for approximately 60% by treatment with 25 mM of the detergent CHAPS. The K _m and the V _max for cDPG were determined at 60°C and were 59 mM and 216 mU/mg, respectively. Furthermore, cDPG hydrolase was dependent on the presence of Co²⁺. The role of cDPG and cDPG hydrolase is discussed.Abbreviations cDPG cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-diphosphoglycerate - 2-PG 2-phosphoglycerate - 3-PG 3-phosphoglycerate - PG phosphoglycerate - PEP phosphoenolpyruvate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - TRIS tris(hydroxymethyl)-aminomethane - DTT dithiothreitol - CHAPS 3-([3-cholamidopropyl]-dimethylammonio)-1-propanesulfonate - MOPS 3-(N-morpholino) propanesulfonic acid     

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.     

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.