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L Sequeira

Publications and source records attributed to L Sequeira.

34 records · Page 2Linked to original sources
PubMed1982

Hydroxyproline-rich bacterial agglutinin from potato : extraction, purification, and characterization.

A protein, extracted from Katahdin potato (Solanum tuberosum L. cv ;Katahdin') tubers and purified by ion exchange chromatography and gel filtration, agglutinates avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum, but only weakly agglutinates virulent strains. The agglutinin has very low hemagglutinating activity (in contrast to potato lectin) and is a glycoprotein containing about 61% carbohydrate. The carbohydrate moiety contains 91% (weight%) arabinose, 5% galactose, 3% glucose, and 1% glucosamine. The protein portion is rich in hydroxyproline (42%), lysine (16%), serine (9%), and proline (9%). The entire agglutinin has a molecular weight of 91,000 +/- 5,000 and is very basic (pI > 11). Shape estimations based on the concentration dependence of the sedimentation coefficient, the high viscosity ([eta] = 92.7), the frictional coefficient (f/f(o) = 2.15), and axial ratio (a/b = 25) indicate that the agglutinin is a prolate ellipsoid.

Journal Article↗
PubMed1981

Agglutination of Erwinia stewartii Strains with a Corn Agglutinin: Correlation with Extracellular Polysaccharide Production and Pathogenicity.

A bacterial agglutinin was extracted from ground corn (WI hybrid 64A x W117) seed with phosphate-buffered saline (pH 6.0) and precipitated with (NH(4))(2)SO(4) at 70% saturation. The activities of this agglutinin against 22 strains of Erwinia stewartii (agent of bacterial wilt of corn) that varied in virulence were determined. Specific agglutination (agglutination titer per milligram of protein per milliliter) values were correlated negatively with virulence ratings. Strains with high specific agglutination values (15 or higher) were avirulent or weakly virulent; strains with low specific agglutination values (10 or lower) were highly virulent, with two exceptions. Avirulent strains produced butyrous colonies and released only small amounts of extracellular polysaccharide (EPS) into the medium, and the cells lacked capsules; virulent strains produced fluidal colonies and released large amounts of EPS, and the cells were capsulated. There was a strong correlation between the amount of EPS produced by each strain (as determined by increase in viscosity of the medium) and the specific agglutination value; in contrast, lipopolysaccharide compositions were similar in all strains. When cells of six fluidal strains were washed by repeatedly centrifuging and resuspending them in buffer, they were agglutinated more strongly by corn agglutinin than were unwashed cells. When avirulent cells were washed, their specific agglutination values did not increase significantly. Eight EPS-deficient mutants of E. stewartii, selected for resistance to the capsule-dependent bacteriophage K9, had lower virulence but higher specific agglutination than did their corresponding wild-type parents. Production of EPS appears to be essential for virulence; EPS may prevent agglutination of bacteria in the host, thus allowing their multiplication.

Journal Article↗
PubMed1980

Lipopolysaccharide Composition of the Wilt Pathogen, Pseudomonas solanacearum: CORRELATION WITH THE HYPERSENSITIVE RESPONSE IN TOBACCO.

In the induction of the hypersensitive response (HR) in tobacco by Pseudomonas solanacearum, the recognition between host and pathogen is thought to involve an interaction between plant lectins and bacterial lipopolysaccharide (LPS). The LPS of a series of strains of P. solanacearum were examined to determine if there are structural differences that might account for the ability or inability of these strains to induce the hypersensitive response. Analysis of the components of LPS by gas chromatography indicates a clear difference in sugar composition between the HR-inducing and non-HR-inducing strains, especially in terms of the percentage of glucose, xylose and rhamnose. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows there are two distinct kinds of LPS, differing greatly in size, which correspond to rough and smooth LPS in other systems. In addition, a phage, CH154, was isolated which lyses non-HR-inducing bacteria and which is inactivated by LPS from these bacterial strains. Therefore, differences in LPS structure correlate strongly with host recognition of Pseudomonas solanacearum.

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PubMed1980

A New Bacterial Agglutinin from Soybean: I. ISOLATION, PARTIAL PURIFICATION, AND CHARACTERIZATION.

A new bacterial agglutinin was isolated from seeds of the soybean cultivar Clark. Purification was carried out by ammonium sulfate precipitation and ion-exchange chromatography. The agglutinin is a heat-labile glycoprotein most active at pH 4.0. Addition of Ca(2+), Mn(2+) and Mg(2+) did not enhance the agglutinating activity of this glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate showed that the agglutinin is composed of two subunits of approximately 50,000 daltons each. In the undissociated state, it agglutinates Xanthomonas phaseoli var. sojensis, the causal agent of bacterial pustule disease of soybean, at concentrations as low as 10 micrograms protein per milliliter but has no hemagglutinating activity. The agglutinin could be distinguished from previously reported soybean lectins on the basis of solubility in ammonium sulfate, lack of hemagglutinating activity, molecular weight, hapten specificity, and immunological determinants.

Journal Article↗
PubMed1980

A New Bacterial Agglutinin from Soybean: II. EVIDENCE AGAINST A ROLE IN DETERMINING PATHOGEN SPECIFICITY.

The activity of a bacterial agglutinin from soybean seed [Glycine max (L.) Merrill cv. Clark] against two bacterial pathogens, Pseudomonas glycinea (causal agent of bacterial blight) and Xanthomonas phaseoli var. sojensis (causal agent of bacterial pustule) was determined. The agglutinin was active against several strains of X. phaseoli var. sojensis grown on nutrient agar, but there was no correlation between pathogenicity and agglutination. Agglutination was affected by the age of the bacterial cells and the growth medium used. None of seven strains of P. glycinea was agglutinated.Bacterial agglutination was inhibited by both purified lipopolysaccharide and extracellular polysaccharide from five strains of X. phaseoli var. sojensis. The lipopolysaccharides and extracellular polysaccharides from other species of bacteria were ineffective.Ultrastructural studies showed that an avirulent strain of X. phaseoli var. sojensis was attached to leaf mesophyll cell walls of the susceptible cultivar Clark by 34 hours after vacuum infiltration. Cells of this avirulent strain were enveloped by fibrillar and granular material at the mesophyll cell wall. In contrast, cells of a virulent strain were not attached or enveloped, and they remained free to multiply in the intercellular spaces.

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PubMed1978

Modification of disease resistance of tobacco callus tissues by cytokinins.

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a "hypersensitive" reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.

Journal Article↗
PubMed1977

Bacterial lipopolysaccharides as inducers of disease resistance in tobacco.

The cell wall component of Pseudomonas solanacearum that induces disease resistance in tobacco was highly heat stable at neutral or alkaline pH but highly labile at acid pH. Activity was unaffected by nucleases and proteases but destroyed by a mixture of beta-glycosidases. Washing of bacterial cell walls released a lipopolysaccharide (LPS) fraction with high inducer activity. Purified LPS, extracted by a variety of procedures from whole cells, isolated cell walls, and culture filtrates of both smooth and rough forms of P. solanacearum, induced disease resistance in tobacco at concentrations as low as 50 microgram/ml. The LPS from the non-plant pathogens Escherichia coli B, E. coli K, and Serratia marcescens was also active. Cell wall protein, free phospholipid, and nucleic acids were not necessary for activity. Moreover, since LPS from rough forms was active, the O-specific polysaccharide of the LPS was not required for activity. Hydrolysis of the remaining core-lipid A linkage or deacylation of lipid A destroyed inducer activity. When injected into tobacco leaves, purified LPS attached to tobacco mesophyll cell walls and induced ultrastructural changes in the host cell similar to those induced by attachment of whole heat-killed bacteria.

Cell Wall↗
PubMed1975

Technique for the Determination of the Rate of Ethylene Production by Pseudomonas solanacearum.

A tube culture system was designed for measurement of ethylene evolved by the phytopathogenic bacterium, Pseudomonas solanacearum. The system consisted of 10 glass tubes joined together in series and coated on the inside surface with a dextrose-peptone-casamino acids agar medium. The system provided a large surface for bacterial growth in relation to the volume of air. The system was seeded with a bacterial suspension (7 x 10(8) cells/ml) drawn through all the tubes by vacuum applied at one end and was then placed in a water bath at 30 C. Air was pumped through the system at 3 ml/min; the outlet was connected directly to the inlet port of a gas sampling loop and ethylene in the sample was determined by gas chromatography.Maximum rate of ethylene production for a fluidal, virulent isolate of P. solanacearum (K60) was 5.5 x 10(-9) moles/min and occurred at the end of lag phase and beginning of stationary phase. Three other fluidal isolates produced ethylene at relatively low rates (2.4-6.4% that of K60). Avirulent, butyrous variants of these isolates grew as well as the virulent forms in most cases, but ethylene production rates per cell were much lower for the avirulent than for the virulent forms. Loss of virulence appears to be accompanied by lower ethylene production.Peak CO(2) production (14.5 mumoles/min) and O(2) consumption (11.7 mumoles/min) for isolate K60 also occurred at the time when the bacterial culture was entering stationary phase. The concentrations of O(2) (11%) and CO(2) (11%) in air present at this time were thought to be neither limiting nor inhibitory to bacterial growth.

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PubMed1974

Soluble peroxidase in fluid from the intercellular spaces of tobacco leaves.

A high proportion of the soluble peroxidase of tobacco (Nicotiana tabacum L. var. Bottom Special) leaves is found in the fluid obtained by centrifugation of a buffer solution previously infiltrated into the intercellular spaces. Only a very small amount of the cytoplasmic enzyme, glucose 6-phosphate dehydrogenase, is present in this fluid. Specific activity data suggest that an active process is responsible for the transfer of soluble peroxidase to the intercellular space and that the intercellular fluid fraction is not simply composed of material moving out of leaf cells by diffusion. The centrifugation method is a satisfactory means of isolating diluted intercellular fluid for biochemical and physiological investigations.

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PubMed1970

Abscisic Acid in tobacco plants: tentative identification and its relation to stunting induced by pseudomonas solanaccarum.

In tobacco plants inoculated with the wilt-inducing bacterium, Pseudomonas solanacearum, there was a correlation between decreased internode elongation, maximum multiplication of the bacterium, and an increase in the growth inhibitor content of stems 4 to 12 days after inoculation, as determined by a wheat coleoptile assay. Initial wilting of the upper leaves was also correlated with an increase in inhibitor content of these tissues.Application of either the partially purified inhibitor from tobacco or pure (+)-abscisic acid to roots, terminal buds, or petioles of tobacco plants caused a reduction of internode length which lasted from 8 to 10 days following a single treatment. Repeated treatment was necessary to obtain growth retardation over a longer period of time.The tobacco inhibitor was tentatively identified as abscisic acid, based on a comparison with authentic abscisic acid on paper, thin layer, column, and gas-liquid chromatography. On the basis of optical rotatory dispersion, circular dichroism, and ultraviolet spectra, the tobacco inhibitor was indistinguishable from abscisic acid. Increases in the inhibitor content of infected tissues are attributed primarily to abscisic acid although other substances, not separable from abscisic acid by the procedures used, could also play a role. The inhibitor was not found in P. solanacearum culture medium.

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PubMed1966

Partial purification and kinetics of indoleacetic Acid oxidase from tobacco roots.

Extracts from roots of Nicotiana tabacum L var. Bottom Special contain oxidative enzymes capable of rapid degradation of indoleacetic acid (IAA) in the presence of Mn(2+) and 2, 4-dichlorophenol. Purification of IAA oxidase was attempted by means of ammonium sulfate fractionation and elution through a column of SE-Sephadex. Two distinct fractions, both causing rapid oxidation of IAA in the absence of H(2)O(2), were obtained. One fraction exhibited high peroxidase activity when guaiacol was used as the electron donor; the other did not oxidase guaiacol. Both enzyme fractions caused similar changes in the UV spectrum of IAA; absorption at 280 mmu was reduced, while major absorption peaks appeared at 254 and 247 mmu. The kinetics of IAA oxidation by both fractions were followed by measuring the increase in absorption at 247 mmu. The peroxidase-containing fraction showed no lag or a slight lag which could be eliminated by addition of H(2)O(2) (3 mumoles/ml). The peroxidase-free fraction showed a longer lag, but addition of similar amounts of H(2)O(2) inhibited the rate of IAA oxidation and did not remove the lag. With purified preparations, IAA oxidation was stimulated only at low concentrations of H(2)O(2) (0.03 mumole/ml). A comparison of K(m) values for IAA oxidation by the peroxidase-containing and peroxidase-free fractions suggests that tobacco roots contain an IAA oxidase which may have higher affinity for IAA and may be more specific than the general peroxidase system previously described from other plant sources. A similar oxidase is present in commercial preparations of horseradish peroxidase. It is suggested that oxidation of IAA by horseradish peroxidase may be due to a more specific component.

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PubMedDate not supplied

Characterization of a negative regulator of exopolysaccharide production by the plant-pathogenic bacterium Pseudomonas solanacearum.

Wild-type strains of the bacterial wilt pathogen Pseudomonas solanacearum exhibit reduced exopolysaccharide production and virulence when transformed with plasmids carrying the epsR locus. To understand the function of epsR, we used mutagenesis and DNA sequencing to identify the gene responsible for the shutoff of exopolysaccharide production. The epsR gene encodes a 236-amino-acid polypeptide that, based on polypeptide sequence homology, has significant similarity to other proteins of the luxR family of environmentally responsive, two-component regulatory systems. When a mutated copy of the epsR gene was marker-exchanged into the wild-type P. solanacearum chromosome, however, we observed no effect on growth in culture or on exopolysaccharide production. This suggests that the EpsR phenotype becomes apparent only via overproduction of the EpsR protein. By means of an antiserum directed against the EpsR protein, we detected the overproduction of EpsR in cell lysates of a strain of P. solanacearum harboring a multicopy plasmid with an active epsR gene but not in one harboring the same plasmid with a mutated epsR gene.

Amino Acid Sequence↗