Morphological differentiation observed in manganese oxidizing bacterial colonies

Bacteria play a vital role in bringing about Mn(II) oxidation in the natural environment. A study was conducted to identify the potential threat offered by these bacteria in bringing about biomineralisation of manganese dioxide on titanium surfaces exposed to seawater. During the study it was observed that the bacteria such as Pseudomonas and Bacillus formed brown colonies on agar plates amended with Mn2+ indicating their ability to oxidize Mn(II). These colonies showed distinct morphologies when grown on plates containing Mn(II) while they formed normal colonies in the absence of Mn.(II).Hence it is possible that these morphologically distinct structures produced by the bacterial colonies assist these bacteria to perform this function of Mn-oxidation.

Localized agglutinin staining in muscle capillaries from normal and very old atrophic human muscle using winged bean ( Psophocarpus tetragonolobus ) lectin

The winged bean (Psophocarpus tetragonolobus) agglutinin (total lectin) and its basic (WBA I) and acidic isoform (WBA II) were used to analyze capillaries in sections from human muscle. The microvessels were clearly labeled after incubation with the lectins in both normal muscle and in old muscles with age-related type II atrophy or muscle fiber grouping. Muscle fibers, nerves, and connective tissue remained unstained. The total lectin detected muscle capillaries from all blood group AB0 individuals. The isoform WBA I reacted only with blood vessels in blood group A and B individuals, while the blood vessels in blood group 0 individuals were demonstrated with WBA II. WBA I staining was inhibited by p-nitrophenyl α-galactopyranoside and N-acetylgalactosamine, whereas 2′-fucosyllactose and preincubation with an antibody against type-1 chain H abolished capillary staining with WBA II. The study demonstrates the usefulness of WBA as a marker of capillaries in human muscle.

Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl galactosides to the basic agglutinin from winged bean (Psophocarpus tetragonolobus)

The binding of winged bean basic agglutinin (WBA I) to 4-methylumbelliferyl (MeUmb) galactosides was examined by extrinsic fluorescence titration and stopped-flow spectrofluorimetry. Upon binding to WBA I, MeUmb alpha-galactosides show quenching in fluorescence intensity, decrease in UV absorbance with a concomitant blue shift, and decrease in fluorescence excited-state lifetimes. However, their beta-analogues show enhancement in fluorescence intensity, increase in UV absorbance with a red shift, and an increase in fluorescence excited-state lifetimes. This implies that the umbelliferyl groups of alpha- and beta-galactosides experience non-polar and polar microenvironments, respectively, upon binding to WBA I. Replacement of the anomeric hydroxyl group of galactose by 4-methylumbelliferyl moiety increases the affinity of resulting saccharides. Substitution of C-2 hydroxyl of galactose by an acetamido group leads to increased affinity due to a favorable entropy change. This suggests that acetamido group of MeUmb-alpha/beta-GalNAc binds to a relatively non-polar subsite of WBA I. Most interestingly, this substitution also reduces the association rate constants dramatically. Inspection of the activation parameters reveals that the enthalpy of activation is the limiting factor for the differences in the forward rate constants for these saccharides and the entropic contribution to the activation energy is small

Crystallization and Preliminary X-ray Studies of the Basic Lectin from Winged Bean (Psophocarpus tetragonolobus)

The basic lectin from winged bean (Psophocarpus tetragonolobus) could be crystallized using polyethyleneglycol (PEG) 4000 (I), PEG 8000 (II) and 2-methylpentane-2,4-diol (MPD) (III) as precipitants. Crystal forms I and II grew in the presence of methyl-α-Image -galactopyranoside or N -acetylgalactosamine while III grew in the absence of sugar. The three forms have the same space group (P21212) and similar unit cell dimensions with two dimeric molecules in the asymmetric unit. The unit cell dimensions are a = 156·8 Å, b = 89·0 Å, c = 73·3 Å for I, a = 155·5 Å, b = 92·3 Å, c = 72·5 Å for II and a = 148·3 Å, b = 90·7 Å, c = 73·8 Å for III. The crystals, particularly those grown using PEG 8000, are suitable for high resolution X-ray analysis, which is in progress.

Mechanism of lectin-cell interactions: thermodynamic and kinetic analysis of the binding of winged bean acidic lectin (WBA II) to human erythrocytes

In order to identify the forces involved in the binding and to understand the mechanism involved, equilibrium and kinetic studies were performed on the binding of the winged bean acidic lectin to human erythrocytes. The magnitudes of delta S and delta H were positive and negative respectively, an observation differing markedly from the lectin-simple sugar interactions where delta S and delta H are generally negative. Analysis of the sign and magnitudes of these values indicate that ionic and hydrogen bonded interactions prevail over hydrophobic interactions resulting in net -ve delta H (-37.12 kJ.mol-1) and +ve delta S (14.4 J.mole-1 K-1 at 20 degrees C), thereby suggesting that this entropy driven reaction also reflects conformational changes in the lectin and/or the receptor. Presence of two kinds of receptors for WBA II on erythrocytes, as observed by equilibrium studies, is consistent with the biexponential dissociation rate constants (at 20 degrees C K1 = 1.67 x 10(-3) M-1 sec-1 and K2 = 11.1 x 10(-3) M-1 sec-1). These two rate constants differed by an order of magnitude accounting for the difference in the association constants of the two receptors of WBA II. However, the association process remains monoexponential suggesting no observable difference in the association rates of the lectin molecule with both the receptors, under the experimental conditions studied. The thermodynamic parameters calculated from kinetic data correlate well with those observed by equilibrium. A two-step binding mechanism is proposed based on the kinetic parameters for WBA II-receptor interaction

Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus)

The thermodynamics of the binding of D-galactopyranoside (Gal), 2-acetamido-2-deoxygalactopyranoside (GalNAc), methyl-alpha-D-galactopyranoside, and methyl-beta-D-galactopyranoside to the basic agglutinin from winged bean (WBAI) in 0.02 M sodium phosphate and 0.15 M sodium chloride buffer have been investigated from 298.15 to 333.15 K by titration calorimetry and at the denaturation temperature by differential scanning calorimetry (DSC). WBAI is a dimer with two binding sites. The titration calorimetry yielded single-site binding constants ranging from 0.56 +/- 0.14 x 10(3) M-1 for Gal at 323.15 K to 7.2 +/- 0.5 x 10(3) M-1 for GalNAc at 298.15 K and binding enthalpies ranging from -28.0 +/- 2.0 kJ mol-1 for GalNAc at 298.15 K to -14.3 +/- 0.1 kJ mol-1 for methyl-beta-D-galactopyranoside at 322.65 K. The denaturation transition consisted of two overlapping peaks over the pH range 5.6-7.4. Fits of the differential scanning calorimetry data to a two-state transition model showed that the low temperature transition (341.6 +/- 0.4 K at pH 7.4) consisted of two domains unfolding as a single entity while the higher temperature transition (347.8 +/- 0.6 K at pH 7.4) is of the remaining WBAI dimer unfolding into two monomers. Both transitions shift to higher temperatures and higher calorimetric enthalpies with increase in added ligand concentration at pH 7.4. Analysis of the temperature increase as a function of added ligand concentration suggests that one ligand binds to the two domains unfolding at 341.6 +/- 0.6 K and one ligand binds to the domain unfolding at 347.8 +/- 0.6 K.

Carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus)

The carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus) was investigated by quantitative precipitin analysis using blood group A, B, H, Le and I substances and by precipitation inhibition with various mono- and oligosaccharides. The lectin precipitated best with A1 substances and moderately with B and A2 substances, but not with H or Le substances. Inhibition assays of lectin-blood group A1 precipitation demonstration that A substance-derived oligosaccharides having the common structure: d-Ga1NAcα(1 → 3)d-Gal-(β1 → Image ) to a d-Glc, were the best inhibitors and about 8 and 4 times more active than d-Ga1NAc and d-Ga1NAcα(1 → 3)d-Ga1, respectively. A difucosyl A-specific oligosaccharide (A-penta), a monofucosyl (A-tetra) and a non-fucosyl containing (A5 II) oligosaccharide, d-Ga1NAcα(1 → 3)d-Ga1β(1 → 3)d-G1cNAc, had almost the same reactivity, suggesting that the fucose linked to the sub-terminal d-Ga1 or to the third sugar, d-GlcNAc, from the non-reducing end made no contribution to the carbohydrate binding. Although a terminal non-reducing d-Ga1NAc or d-Ga1 residue was indispensible for binding, the lectin bound not only to these terminal non-reducing galactopyranosyl residues, but also showed increased binding to oligosaccharides in which it was bonded to a sub-terminal d-Ga1 joined to a d-GlcNAc residue, as in blood group A or B substances. This defines the site, thus far, as complementary to a disaccharide plus the β linkage to the third sugar (d-Glc or d-GlcNAc) from the non-reducing end. The role of the β(1 → 3) or β(1 → 4) linkage of the sub-terminal non-reducing d-Gal to the d-GlcNAc requires further study.

Erythrocyte-binding studies on an acidic lectin from winged bean (Psophocarpus tetragonolobus)

n acidic lectin (WBA II) was isolated to homogeneity from the crude seed extract of the winged bean (Psophocarpus tetragonolobus) by affinity chromatography on lactosylaminoethyl-Bio-Gel. Binding of WBA II to human erythrocytes of type-A, -B and -O blood groups showed the presence of 10(5) receptors/cell, with high association constants (10(6)-10(8) M-1). Competitive binding studies with blood-group-specific lectins reveal that WBA II binds to H- and T-antigenic determinants on human erythrocytes. Affinity-chromatographic studies using A-, B-, H- and T-antigenic determinants coupled to an insoluble matrix confirm the specificity of WBA II towards H- and T-antigenic determinants. Inhibition of the binding of WBA II by various sugars show that N-acetylgalactosamine and T-antigenic disaccharide (Thomsen-Friedenreich antigen, Gal beta 1-3GalNAc) are the most potent mono- and di-saccharide inhibitors respectively. In addition, inhibition of the binding of WBA II to erythrocytes by dog intestine H-fucolipid prove that the lectin binds to H-antigenic determinant.

Purification and properties of uridine hydrolase from mung-bean (Phaseolus radiatus) seedlings

The occurrence in plants of an enzyme system catalyzing the cleavage of uridine has been demonstrated. The enzyme from Phaseolus radiatus was purified about 132-fold with 24% recovery by a combination of procedures involving mild acid treatment, ammonium sulphate fractionation, negative adsorption on calcium phosphate gel and DEAE-cellulose chromatography. The enzyme cleaves uridine to uracil and ribose in the absence of phosphate indicating that the mechanism of cleavage was hydrolytic rather than phosphorolytic. The enzyme is specific to uridine and does not act on other purine and pyrimidine compounds. The enzyme shows maximum activity at pH 7.4 and has a temperature optimum of 45 °. It does not require metal ions for activity. Inhibition of the enzyme by p-chloromercuribenzoate as well as N-ethylmaleimide and the reversal of p-chloromercuribenzoate inhibition by sulfhydryl agents indicate the probable involvement of readily oxidizable sulfhydryl groups in enzyme activity.

Nucleotidases in plants : I. Partial purification and properties of the enzyme hydrolyzing flavine adenine dinucleotide from mung bean seedlings (Phaseolus radiatus)

The occurrence of an enzyme hydrolyzing flavine adenine dinucleotide (FAD) was demonstrated in a number of seed extracts. The enzyme from Phaseolus radiatus was purified 104-fold by fractionation with ammonium sulfate and ethanol and by negative adsorption on alumina Cγ gel. The enzyme cleaves the POP bond of FAD to yield flavine mononucleotide and adenosine monophosphate. When reduced glutathione is added to the enzyme, it cleaves FAD at the COP bond to yield riboflavine, adenosine, and pyrophosphate, Both the activities are optimal at a pH of 7.2 and at a temperature of 37 . The Km for both the activities is 1.65 × 10−5 M. The stoichiometry and the identity of the products of both the treated and untreated enzyme were established. The untreated enzyme was not inhibited by pCMB or arsenite, but the treated enzyme was sensitive to both these inhibitors. The inhibition by pCMB could be reversed by monothiols and the inhibition by arsenite by dithiols.

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins - Implication of the C-H center dot center dot center dot O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of C-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha-and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H ... O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.

Molecular characterization of sediment bacterial communities affected by fish farming

Fish farming introduces nutrients, microbes and a wide variety of chemicals such as heavy metals, antifoulants and antibiotics to the surrounding environment. Introduction of antibiotics has been linked with the increased incidence of antibiotic resistant pathogenic bacteria in the farm vicinities. In this thesis molecular methods such as quantitative PCR and DNA sequencing were applied to analyze bacterial communities in sediments from fish farms and pristine locations. Altogether four farms and four pristine sites were sampled in the Baltic Sea. Two farm and two pristine locations were sampled over a surveillance period of four years. Furthermore, a new methodology was developed as a part of the study that permits amplifying single microbial genomes and capturing them according to any genetic traits, including antibiotic resistance genes. The study revealed that several resistance genes for tetracycline were found at the sediment underneath the aquaculture farms. The copy number of these genes remained elevated even at a farm that had not used any antibiotics since year 2000, six years before this study started. Similarly, an increase in the amount of mercury resistance gene merA was observed at the aquaculture sediment. The persistence of the resistance genes in absence of any selection pressure from antibiotics or heavy metals suggests that the genes may be introduced to the sediment by the farming process. This is also supported by the diversity pattern of the merA gene between farm and pristine sediments. The bacterial community-level changes in response to fish farming were very complex and no single phylogenetic groups were found that would be typical to fish farm sediments. However, the community structures had some correlation with the exposure to fish farming. Our studies suggest that the established approaches to deal with antibiotic resistance at the aquaculture, such as antibiotic cycling, are fundamentally flawed because they cannot prevent the introduction of the resistance genes and resistant bacteria to the farm area by the farming process. Further studies are required to study the entire fish farming process to identify the sources of the resistance genes and the resistant bacteria. The results also suggest that in order to prevent major microbiological changes in the surrounding aquatic environment, the farms should not be founded in shallow water where currents do not transport sedimenting matter from the farms. Finally, the technique to amplify and select microbial genomes will potentially have a considerable impact in microbial ecology and genomics.

sRNAscanner: A Computational Tool for Intergenic Small RNA Detection in Bacterial Genomes

Background:Bacterial non-coding small RNAs (sRNAs) have attracted considerable attention due to their ubiquitous nature and contribution to numerous cellular processes including survival, adaptation and pathogenesis. Existing computational approaches for identifying bacterial sRNAs demonstrate varying levels of success and there remains considerable room for improvement. Methodology/Principal Findings: Here we have proposed a transcriptional signal-based computational method to identify intergenic sRNA transcriptional units (TUs) in completely sequenced bacterial genomes. Our sRNAscanner tool uses position weight matrices derived from experimentally defined E. coli K-12 MG1655 sRNA promoter and rho-independent terminator signals to identify intergenic sRNA TUs through sliding window based genome scans. Analysis of genomes representative of twelve species suggested that sRNAscanner demonstrated equivalent sensitivity to sRNAPredict2, the best performing bioinformatics tool available presently. However, each algorithm yielded substantial numbers of known and uncharacterized hits that were unique to one or the other tool only. sRNAscanner identified 118 novel putative intergenic sRNA genes in Salmonella enterica Typhimurium LT2, none of which were flagged by sRNAPredict2. Candidate sRNA locations were compared with available deep sequencing libraries derived from Hfq-co-immunoprecipitated RNA purified from a second Typhimurium strain (Sittka et al. (2008) PLoS Genetics 4: e1000163). Sixteen potential novel sRNAs computationally predicted and detected in deep sequencing libraries were selected for experimental validation by Northern analysis using total RNA isolated from bacteria grown under eleven different growth conditions. RNA bands of expected sizes were detected in Northern blots for six of the examined candidates. Furthermore, the 5'-ends of these six Northern-supported sRNA candidates were successfully mapped using 5'-RACE analysis. Conclusions/Significance: We have developed, computationally examined and experimentally validated the sRNAscanner algorithm. Data derived from this study has successfully identified six novel S. Typhimurium sRNA genes. In addition, the computational specificity analysis we have undertaken suggests that similar to 40% of sRNAscanner hits with high cumulative sum of scores represent genuine, undiscovered sRNA genes. Collectively, these data strongly support the utility of sRNAscanner and offer a glimpse of its potential to reveal large numbers of sRNA genes that have to date defied identification. sRNAscanner is available from: http://bicmku.in:8081/sRNAscanner or http://cluster.physics.iisc.ernet.in/sRNAscanner/.

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved