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Biomedical subjects

V J Morris

Publications and source records attributed to V J Morris.

At least 19 recordsLinked to original sources
PubMed1997

Cloning of the aceF gene encoding the phosphomannose isomerase and GDP-mannose pyrophosphorylase activities involved in acetan biosynthesis in Acetobacter xylinum.

The aceF gene from Acetobacter xylinum was identified and cloned from a genomic DNA library. The complete DNA sequence was determined and computer analysis of the translated gene sequence revealed homology with the deduced amino acid sequence of xanB from Xanthomonas campestris. Therefore aceF is likely to encode a bifunctional enzyme with mannose-6-phosphate isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. PMI and GMP activities were detected in strains of Escherichia coli expressing the cloned aceF gene.

Amino Acid Sequence↗
PubMed1997

Effect of deacetylation on the synergistic interaction of acetan with locust bean gum or konjac mannan.

It has been discovered that deacetylation of the bacterial polysaccharide acetan promotes synergistic interactions with either locust bean gum (LBG) or konjac mannan (KM). Acetan is similar in structure to xanthan, and adopts a similar 5-fold conformation in the solid state. Like xanthan, it shows a thermally reversible order (helix)-disorder (coil) transition in solution. Both polymers have a cellulosic backbone with charged (anionic) sidechains attached at O-3 of alternate glucosyl residues, but the sidechains in acetan are longer (pentasaccharide rather than trisaccharide) and do not contain pyruvic substituents. Acetan has two sites of acetylation, one at O-6 of the inner mannosyl residue of the carbohydrate sidechains (as in xanthan) and the other on the polymer backbone (believed to be at O-6 of the branched glucosyl residues). Solutions of acetan or deacetylated acetan were equilibrated against 10 mM potassium chloride (to stabilise the ordered conformation) and were mixed (at 25 degrees C) with solutions of LBG or KM, also equilibrated against 10 mM potassium chloride. Unlike xanthan, native acetan showed no evidence of synergistic interaction with either LBG or KM. After deacetylation, however, large enhancements were observed in dilute-solution viscosity, and thermoreversible gels were formed at higher concentrations. With KM as co-synergist, gel melting was accompanied by an intense endotherm in differential scanning calorimetry. The magnitude of this endotherm increased with storage time at 25 degrees C, reaching a final value of delta H approximately 15.9 J/g (in comparison with delta H approximately 5.0 J/g for the order-disorder transition of deacetylated acetan alone). It is suggested that interaction occurs by formation of heterotypic junctions between the acetan backbone and unsubstituted regions of the plant polysaccharide, and that the acetate groups on native acetan promote solubility and hence inhibit association.

Acetobacter↗
PubMed1997

Effect of o-acyl substituents on the functional behaviour of Rhizobium meliloti succinoglycan.

The effects of selective removal of acetyl or succinyl substituents on the functionality of succinoglycan polysaccharide have been studied by comparing the behaviour of the polysaccharides isolated from native Rhizobium meliloti strain Rm1021, and genetically modified R. meliloti species. Removal of the succinyl groups was found to dramatically improve pseudoplasticity of the aqueous succinoglycan samples and also increase the cooperativity of the order-disorder transition exhibited by the polysaccharide. Removal of the acetyl substituent led to a decrease in the order-disorder transition temperature, whereas the removal of the succinyl groups led to an increase.

Calorimetry, Differential Scanning↗
PubMed1997

Atomic force microscopy of plant cell walls, plant cell wall polysaccharides and gels.

Methods developed for the routine imaging of polysaccharides by atomic force microscopy (AFM) have been used to image plant polysaccharides from higher plants (pectin) and algae (carrageenan). These methods have been extended to image K-carrageenan association in hydrated films. Finally, AFM has been used to image polysaccharide architecture in moist plant cell walls. Simple experimental and image processing methods have been used to enhance molecular structure in 'rough' cell wall surfaces.

Cell Wall↗
PubMed1996

The cpsABCDE genes involved in polysaccharide production in Streptococcus salivarius ssp. thermophilus strain NCBF 2393.

A 4074-bp EcoRI fragment of Streptococcus salivarius ssp. thermophilus (S. thermophilus) chromosomal DNA containing genes involved in exocellular polysaccharide (EPS) was identified and cloned. The nucleotide sequence of this fragment was determined and found to contain one partial and four complete open reading frames. These were designated cpsA, cpsB, cpsC, cpsD and cpsE and encoded proteins of > 130, 243, 230, 246 and 455 amino acids, respectively, that showed homology with the genes of the cps cluster, involved in polysaccharide biosynthesis, in Streptococcus pneumoniae Type 19F. The cpsA gene is predicted to encode a transcriptional regulator, while cpsC and cpsD are predicted to encode proteins involved in polysaccharide polymerization and export. The cpsE gene is likely to encode the phosphate-prenyl glycosyl-1-phosphate transferase catalyzing the first step in polysaccharide biosynthesis in S. thermophilus. Southern blot analysis revealed that cpsE is found only in polysaccharide producing strains of S. thermophilus.

Amino Acid Sequence↗
PubMed1996

Identification, cloning and sequencing the aceA gene involved in acetan biosynthesis in Acetobacter xylinum.

The aceA gene from Acetobacter xylinum was identified and cloned from a genomic DNA library. The complete DNA sequence was determined and computer analysis of the translated gene sequence revealed homology with the deduced amino acid sequence of gumD from Xanthomonas campestris. Therefore aceA is likely to encode the phosphate-prenyl glucose l-phosphate transferase catalyzing the first step in acetan biosynthesis in A. xylinum.

Amino Acid Sequence↗
PubMed1996

Imaging polysaccharides by atomic force microscopy.

Techniques have been developed for the routine reliable imaging of polysaccharides by atomic force microscopy (AFM). The polysaccharides are deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged under alcohols. The rationale behind the development of the methodology is described and data is presented for the bacterial polysaccharides xanthan, acetan, and the plant polysaccharides l-carrageenan and pectin. Studies on uncoated polysaccharides have demonstrated the improved resolution achievable when compared to more traditional metal-coated samples or replicas. For acetan the present methodology has permitted imaging of the helical structure. Finally, in addition to data obtained on individual polysaccharides, AFM images have also been obtained of the network structures formed by kappa-carrageenan and gellan gum.

Image Processing, Computer-Assisted↗
PubMed1996

Mixed iota-kappa carrageenan gels.

Studies have been made of the gelation of mixtures of iota and kappa carrageenan. Differential scanning calorimetry (DSC) studies suggest that the iota and kappa components gel independently of each other. The DSC data conflict with evidence presented for the currently accepted Domain model for carrageenan gelation (Morris, E.R., Rees, D.A. and Robinson, G.J. J. Mol. Biol. 1980; 138:349). In particular, the present data suggest that iota carrageenan gels show thermoreversible setting and melting behaviour associated with the coil-helix transition. Analysis of rheological data favours an interpenetrating network (IPN) model for gelation of the mixtures.

Calorimetry, Differential Scanning↗
PubMed1996

Structure and conformation of acetan polysaccharide.

Acetan is an anionic bacterial polysaccharide. The chemical repeat unit consists of a cellobiose unit solubilised by attachment of a charged pentasaccharide sidechain to one of the glucose residues. The repeat unit contains two sites of acetylation. 1H and 13C NMR studies, coupled with both basic-methylation and mild-methylation studies, have shown that acetylation occurs at C6 on the (1,2)D-Man and the (1,34)D-Glc residues. A variety of techniques including NMR, optical rotation, circular dichroism and DSC show evidence for a thermoreversible conformational order (helix)-disorder (coil) transition for acetan in aqueous solution. The studies suggest that acetylation of the backbone does not prevent helix formation.

Acetylation↗
PubMed1996

Visualization of plant cell walls by atomic force microscopy.

Atomic force microscopy has been used to visualize the ultrastructure of hydrated plant cell wall material from prepared apple (Malus pumila MILL; Cox orange pippin), water chestnut (Eleocharis dulcis L.), potato (Solanum tuberosum L.; Bintje), and carrot (Daucus carota L.; Amsterdamse bak) parenchyma. Samples of cell wall material in aqueous suspension were deposited onto freshly cleaved mica. Excess water was blotted away and the moist samples were imaged in air at ambient temperature and humidity. The three-dimensional images obtained highlighted the layered structure of the plant cell walls and revealed features interpreted as individual cellulose microfibrils and plasmodesmata.

Biophysical Phenomena↗
PubMed1996

Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum: nucleotide sequence analysis of the aceB, aceC, aceD and aceE genes.

Sequence analysis of a 5.323 kb chromosomal DNA fragment from Acetobacter xylinum involved in the biosynthesis of the exopolysaccharide acetan, revealed the presence of four ace genes designated aceB, aceC, aceD and aceE. Comparison of translated gene sequences to the databanks was used to assign putative gene functions. AceB displayed strong homology to a glucose-diphosphoprenyl beta, D-glucose transferase from Xanthomonas campestris, while AceC was homologous to a cellobiosyl-diphosphoprenyl alpha, D-mannose transferase from the same organism. Thus these genes encode enzymes catalyzing the second and third steps of the acetan biosynthetic pathway. AceD and AceE were homologous to ExoP and ExoT respectively from Rhizobium meliloti and are likely to be involved in acetan polymerization and export.

Amino Acid Sequence↗
PubMed1995

NMR studies of acetan and the related bacterial polysaccharide, CR1/4, produced by a mutant strain of Acetobacter xylinum.

Acetan is a bacterial polysaccharide produced by Acetobacter xylinum NRRL B42. Chemical mutagenesis of A.xylinum allowed selection of a mutant strain which produced a new polysaccharide, CR1/4. 2D NMR methods have been used to assign the 1H and 13C spectra of the two polysaccharides and to determine that CR1/4 has the structure shown below. The total number of O-acetyl groups is slightly less than two per repeating unit. [formula: see text] The pentasaccharide side chain of acetan is truncated to a disaccharide unit in CR1/4, but the structures are otherwise identical. In particular, the degree of acetylation is about the same and the O-acetyl groups are located at the same position in both polysaccharides.

Carbohydrate Sequence↗
PubMed1995

Observation of the helical structure of the bacterial polysaccharide acetan by atomic force microscopy.

A method has been developed that has been found to give reproducible images of uncoated polysaccharides by Atomic Force Microscopy (AFM). Aqueous solutions of the polysaccharide are deposited as drops onto freshly cleaved mica surfaces, air dried, and then imaged under butanol. The method has been used to obtain images of the bacterial polysaccharide acetan. In regions within the deposited sample, where the molecules are aligned side-by-side, it has been possible to observe a periodic structure along the polysaccharide chain, attributable to the helical structure of acetan.

Acetobacter↗
PubMed1994

Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum.

We have identified, cloned and sequenced an 8422 base pair fragment of Acetobacter xylinum genomic DNA containing part of the acetan biosynthetic gene cluster. Computer analysis of the nucleotide sequence data generated revealed the presence of six open reading frames. Comparison of the translated sequences of putative genes to the amino acid sequences of genes from other organisms was used to assign functions to the aceA, aceC and manB genes. These genes were predicted to encode a UDP-glycosyl transferase, a GDP-mannosyl transferase and a phosphomannose isomerase/GDP-mannose pyrophosphorylase, respectively.

Bacterial Proteins↗
PubMed1994

Physicochemical characterization of an acetan variant secreted by Acetobacter xylinum strain CR1/4.

Chemical mutagenesis has been used to produce mutants of Acetobacter xylinum NRRL B42 that are cellulose-negative and that produce variants of the acetan structure deficient in the side-chain sugar residues. The product of A. xylinum strain CR1/4 has been shown to possess a tetrasaccharide repeat unit with the side chain terminating in glucuronic acid. X-ray diffraction studies of oriented fibres suggest that the polysaccharide CR1/4 forms a fivefold helix with a pitch of 4.8 nm. Light-scattering studies on CR1/4 solutions suggest a molecular weight of 1.2 x 10(6) with radii of gyration values of 86 nm (aqueous solution) and 67 nm (0.1 M NaCl solution). The magnitude of the measured radii of gyration and the shape of the Holtzer plots suggest that CR1/4 can be described as a stiff coil. Preliminary differential scanning calorimetry data show melting behaviour consistent with order-disorder transitions of a charged helical structure. Rheological studies have revealed new synergistic interactions of CR1/4 with locust bean gum. Comparative studies of acetan and CR1/4 show that decreasing the length of the side chain enhances the solution viscosity.

Calorimetry, Differential Scanning↗
PubMed1993

Solution conformational analysis of the alpha-zein proteins of maize.

Small angle x-ray scattering and viscometric analyses of the alpha-zeins of maize in solution indicated that the molecules were asymmetric. Structure predictions of consensus sequences for the two classes of alpha-zeins, Z19 and Z22, were in good agreement with the alpha-helical contents determined by circular dichroism. Dimensions determined by small angle x-ray scattering and viscometry indicated a predominantly alpha-helical conformation. The data are discussed in relation to models for the solution conformation and to earlier models for alpha-zeins structure.

Amino Acid Sequence↗