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L Sequeira

Publications and source records attributed to L Sequeira.

At least 19 recordsLinked to original sources
PubMed1992

Tyrosine phosphorylation of a membrane protein from Pseudomonas solanacearum.

We have investigated a tyrosine kinase activity from Pseudomonas solanacearum, an economically important plant pathogen. In vitro incubation of membrane fractions with [gamma-32P]ATP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85-kDa phosphoprotein. Phosphorylation of this protein on tyrosine residues was demonstrated by phosphoamino acid analysis of base hydrolysis products and by immunoanalysis of Western blots (immunoblots) with antiphosphotyrosine monoclonal antibody. In vitro incubation of membranes with ATP was not required for recognition by the antibody, indicating that the 85-kDa protein is phosphorylated in vivo. These results demonstrate that membranes from P. solanacearum exhibit a tyrosine kinase activity toward an endogenous membrane protein. This bacterium provides an opportunity to study the structure and function of a prokaryotic tyrosine kinase.

Adenosine Triphosphate↗
PubMed1992

Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum.

Several Pseudomonas solanacearum strains which produced no detectable extracellular polysaccharide (EPS) in planta had been reported to remain highly virulent when tested at high inoculum concentrations (P. Xu, M. Iwata, S. Leong, and L. Sequeira, J. Bacteriol. 172:3946-3951, 1990; P. Xu, S. Leong, and L. Sequeira, J. Bacteriol. 170:617-622, 1988). Two of these mutants, KD700 and KD710, have now been molecularly and genetically mapped to the EPSI gene cluster described by Denny and Baek (Mol. Plant-Microbe Interact. 4:198-206, 1991). When a range of inoculum concentrations was used, these two mutants and all other EPS-defective mutants tested were found to be reduced in virulence to eggplants and tobacco relative to the wild-type strain. Thus, EPS consistently is required for the wild-type level of virulence in P. solanacearum.

Mutation↗
PubMed1991

The use of subtractive hybridization to obtain a DNA probe specific for Pseudomonas solanacearum race 3.

Pseudomonas solanacearum, the causal agent of bacterial wilt, has been classified into three races based on host range and into five biovars based on physiological properties. Strains of race 3 belong exclusively to biovar 2 and primarily affect potatoes. Although this race is thought to have originated in the Andean highlands, it has unusual physiological properties that make it a potential threat to potatoes grown at the cooler latitudes worldwide. Consequently, there is need for a rapid and sensitive method for detection of race 3 strains. We have used subtractive hybridization to enrich for race 3-specific DNA sequences in total race 3 genomic DNA, and thereby obtained a 2 kb clone homologous to DNA from all 28 race 3 strains tested, but with only five of 90 non-race 3 strains. In addition, two larger regions of the genome, containing a minimum of 23 kb of DNA, are also specific for race 3. Deletion of this DNA did not affect virulence. This race 3-specific DNA is a potentially useful diagnostic tool for the detection of race 3 strains.

Blotting, Southern↗
PubMed1991

A gene cluster required for coordinated biosynthesis of lipopolysaccharide and extracellular polysaccharide also affects virulence of Pseudomonas solanacearum.

Bacterial cell surface components can be important determinants of virulence. At least three gene clusters important for extracellular polysaccharide (EPS) biosynthesis have been previously identified in the plant pathogen Pseudomonas solanacearum. We have found that one of these gene clusters, named ops, is also required for lipopolysaccharide (LPS) biosynthesis. Mutations in any complementation unit of this cluster decreased EPS production, prevented the binding of an LPS-specific phage, and altered the mobility of purified LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, restoration of LPS biosynthesis alone was not sufficient to restore virulence to the wild-type level, suggesting that EPS is important for pathogenesis.

Bacteriophages↗
PubMed1991

Genetic and biochemical characterization of a Pseudomonas solanacearum gene cluster required for extracellular polysaccharide production and for virulence.

Infection of host plants by Pseudomonas solanacerum results in wilting, which is thought to be due largely to the occlusion of xylem vessels by the P. solanacearum extracellular polysaccharide (EPS) that primarily consists of N-acetylgalactosamine (GalNAc). By means of Tn3 mutagenesis, we identified a 6.5-kb gene cluster that contains five complementation units required for EPS production and virulence in this bacterium. There was positive correlation between the amount of EPS produced in culture and (i) in planta growth and (ii) virulence. Based on analysis of beta-glucuronidase-gene fusions, these genes are expressed both in broth cultures and in planta and may be constitutive. Both wild-type and mutant strains contained similar amounts of UDP-GalNAc, the predicted primary substrate for EPS synthesis. Thus, the EPS mutants we obtained should be useful in the analysis of steps in the assembly of the polysaccharide and how this process is related to virulence.

Cloning, Molecular↗
PubMed1990

Highly virulent strains of Pseudomonas solanacearum that are defective in extracellular-polysaccharide production.

Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum. By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants. One such mutant, KD700, was studied in detail. By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment. This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60. The Tn5-containing fragment was introduced into other P. solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700. N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P. solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700. In contrast, GalNac was readily detected in similar fluids of plants infected by K60. Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60. No differences in growth rates in culture or in planta between the mutant and the parental strains were observed. Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P. solanacearum.

Carbohydrates↗
PubMed1990

Identification of a locus that regulates multiple functions in Pseudomonas solanacearum.

When Pseudomonas solanacearum K60 carries a multicopy plasmid containing cosmid clone pE6C (from the wild-type strain K60) or pBE6 (from the nonpathogenic strain B1), several phenotypic changes are observed, including the following: loss of virulence, reduced extracellular polysaccharide production, and increased polygalacturonase activity. Both cosmids contain a common 8-kilobase DNA region that is required for the phenotype shift. Saturation mutagenesis of pBE6 with Tn3-gus suggested that a single transcriptional unit of at least 1 kilobase is responsible for the phenotype shift. In maxicell assays, subclones containing this transcriptional unit expressed a single protein of about 25 kilodaltons.

Cosmids↗
PubMed1988

Molecular cloning of genes that specify virulence in Pseudomonas solanacearum.

The suicide plasmid pSUP2021 was used to introduce Tn5 into the Pseudomonas solanacearum wild-type strain K60. We isolated eight avirulent mutants after screening 6,000 kanamycin-resistant transconjugants by inoculating eggplant (Solanum melongena L. cv. Black Beauty) and tobacco (Nicotiana tabacum L. cv. Bottom Special) seedlings. The Tn5-containing EcoRI fragments from the eight mutants were unique, suggesting that numerous genes specify virulence in this species. These EcoRI fragments were cloned into pBR322 or pUC12, and one of the clones, pKD810, was transformed into K60. All of the kanamycin-resistant, ampicillin-sensitive transformants were avirulent. Three randomly selected avirulent transformants were shown to carry the Tn5-containing fragment in place of the wild-type fragment and to exhibit the same hybridization pattern as the original KD810 mutant did. With pKD810 as a probe, we identified cosmids carrying the wild-type virulence genes by using a genomic library of K60 prepared in pLAFR3. Two of the homologous cosmids, pL810A and pL810C, when introduced into KD810 by transformation, restored virulence and normal growth of this mutant in tobacco. Altogether, these data indicate that the gene(s) interrupted by Tn5 insertion in KD810 is essential for the virulence of P. solanacearum. Further characterization of this gene is now being completed by subcloning, transposon mutagenesis, and complementation analysis.

Cloning, Molecular↗
PubMed1985

Reassembly of a fimbrial hemagglutinin from Pseudomonas solanacearum after purification of the subunit by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Distilled water homogenates of Pseudomonas solanacearum B1, a highly fimbriated strain, strongly agglutinated human group A erythrocytes. The fimbriae and hemagglutinating activity were precipitated from the crude extract with 1% acetic acid, redissolved at pH 10, and precipitated again with 20 mM CaCl2 at pH 6.9. Ca2+, Mg2+, and Zn2+ had similar ability to precipitate the fimbrial hemagglutinin, but Na+ and K+ were much less effective. The fimbrial protein in the precipitate was purified to homogeneity by preparative gel electrophoresis in sodium dodecyl sulfate. The major protein band was eluted, and sodium dodecyl sulfate was removed by chromatography on ion retardation resin (AG 11A8) in 6 M urea. After dialysis against 10 mM sodium acetate (pH 4.5) to remove the urea, the protein reassembled to yield long fibers. These fibers were identical to fimbriae in the crude extract in diameter (6 nm) and in their ability to cause hemagglutination. The purified fimbriae contained no carbohydrates and wee similar to other bacterial fimbriae in amino acid composition, with hydrophobic amino acids comprising 41.8% of the total.

Amino Acids↗
PubMed1985

Surface components involved in bacterial pathogen-plant host recognition.

During their initial association with plant hosts, pathogenic bacteria interact with plant cell walls. The results of this interaction appear to determine whether bacterial multiplication will take place. With one group of bacterial plant pathogens (e.g. Agrobacterium tumefaciens), attachment to the host surface appears essential for pathogenesis. With another group (e.g. Pseudomonas solanacearum), only those strains that do not attach to the host cell wall are able to multiply in the intercellular spaces. Attachment of many incompatible strains to tobacco mesophyll cell walls leads to a rapid hypersensitive response (HR) and a drastic reduction in bacterial multiplication. Our working hypothesis is that these differences in host response to strains of P. solanacearum are the result of a recognition response in which surface components of both host and pathogen play important roles. Our approach is based on the use of spontaneous or transposon (Tn5)-generated mutants of strains K60 (virulent) and B1 (avirulent) that differ in surface components and in their ability to attach to host cells and to induce the HR. A study of the surface components of bacterial and tobacco cell walls has led to the tentative conclusion that bacterial lipopolysaccharide (LPS) and plant hydroxyproline-rich glycoproteins mediate initial attachment, apparently as a result of charge-charge interaction. This initial attachment is reversed by high salt concentrations during the first 15 min, but not thereafter. Firm attachment appears to depend on hydrophobic interactions mediated by bacterial pili. At the normal ionic strength of intercellular fluids, extracellular polysaccharide (EPS) appears to inhibit only the pili-mediated attachment. Several HR- mutants of strain B1 have been obtained by Tn5 insertion, but they remain avirulent on tobacco. We are examining the EPS, LPS and pili production, and the attachment characteristics of these strains.

Cell Adhesion↗
PubMed1984

Chemical Characterization of the Lipopolysaccharide of Pseudomonas solanacearum.

The carbohydrates present in lipopolysaccharide (LPS) from Pseudomonas solanacearum are rhamnose, xylose, 2-amino-2-deoxyglucose, glucose, heptose, and 2-keto-3-deoxyoctonate. LPS extracted from cultures grown on either glycerol or glucose (as the major source of carbon) and extracted after various incubation periods had similar compositions. The LPS from several strains of the bacterium contained the same component sugars, but the amounts of each sugar varied considerably. It was observed, however, that xylose and 2-amino-2-deoxyglucose increased proportionately with rhamnose, the major component. Phenol-water-extracted LPS contained measurable amounts of nucleic acid, protein, and arabinan, but none of these polymers were detected in LPS extracted with phenol-chloroform-petroleum ether. Polysaccharides liberated from LPS by mild acid hydrolysis were purified by gel filtration. Carbohydrate analysis of the LPS from a virulent, fluidal strain (K60) showed that the O-specific antigen consisted of rhamnose, xylose, and 2-amino-2-deoxyglucose in the proportions 4:1:1. The LPS of an avirulent, afluidal strain (B1) lacked the O-specific antigen; the R-core region consisted of rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate. Methylation analysis indicated that the K60 O-specific antigen was composed of a hexasaccharide repeating unit containing 3-, 2-, and 3,4-substituted rhamnopyranosyl residues, 3-substituted 2-amino-2-deoxyglucose, and terminal xylopyranose in the molar ratios 2:1:1:1:1.

Journal Article↗
PubMed1984

Interaction of Pseudomonas solanacearum Lipopolysaccharide and Extracellular Polysaccharide with Agglutinin from Potato Tubers.

In vitro binding assays were used to study the possible role of a cell wall agglutinin in the attachment to plant cell walls of avirulent strains of the wilt pathogen, Pseudomonas solanacearum. In a nitrocellulose filter assay, radioactively labeled lipopolysaccharide (LPS) from the virulent strain, K60, and the avirulent strain, B1, and extracellular polysaccharide (EPS) from K60 were bound quantitatively by the agglutinin extracted from Katahdin potato tubers. The LPS from B1 had significantly greater agglutinin-binding affinity than that from K60 but not after treatment with deoxycholate, which improved solubility. Highly purified chitotetraose did not inhibit binding of K60 LPS to agglutinin, but binding was inhibited by EPS as well as by diverse anionic polymers (DNA, dextran sulfate, xanthan). Binding of agglutinin to EPS and LPS was inhibited at ionic strengths greater than 0.03 and 0.15 M, respectively. It was concluded that electrostatic charge-charge interactions could account for binding of LPS and EPS to potato agglutinin.

Journal Article↗
PubMed1984

Interaction of Pseudomonas solanacearum with Suspension-Cultured Tobacco Cells and Tobacco Leaf Cell Walls In Vitro.

Attachment of radiolabeled Pseudomonas solanacearum cells to suspension-cultured tobacco cells and tobacco leaf cell walls was measured in vitro by a filtration technique that allowed separation of attached and unattached bacteria. An avirulent strain (B1) attached more rapidly to suspension-cultured cells than did the virulent parent strain (K60), and B1 attachment was less sensitive to inhibition by high ionic strength than was K60. Attachment of B1 bacteria to suspension-cultured cells and to leaf cell walls was comparable (50 to 70%), but only a small proportion (10 to 20%) of K60 bacteria attached to leaf cell walls under optimal conditions. With high bacterial populations (10 bacteria per ml), attachment of K60 to suspension-cultured cells was greatly reduced. Attachment of both strains was completely inhibited by pretreating bacterial cells with heat (41 degrees C) or azide and was partially inhibited by EDTA and kanamycin. The mechanism of attachment is not known, but ionic forces may be involved.

Journal Article↗
PubMed1984

Lipopolysaccharide-Defective Mutants of the Wilt Pathogen Pseudomonas solanacearum.

Lipopolysaccharide (LPS)-defective mutants of Pseudomonas solanacearum were used to test the hypothesis that differences in LPS structure are associated with the ability or inability of different strains to induce a hypersensitive response (HR) in tobacco. To obtain these mutants, LPS-specific bacteriophage of P. solanacearum were isolated and used to select phage-resistant mutants of the virulent, non-HR-inducing strain K60. The LPS of 24 of these mutants was purified and compared with that of K60 and its HR-inducing variant, B1. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LPS from K60 and other smooth strains separated into many evenly spaced bands that migrated slowly, whereas LPS from B1 and most phage-resistant strains separated into one to three bands that migrated rapidly. Carbohydrate analysis showed that the LPS of the phage-resistant strains lacked O-antigen sugars (rhamnose, xylose, and N-acetylglucosamine) and could be grouped into (i) those that had all core sugars (rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate), (ii) those that had no core rhamnose, and (iii) those that lacked all core sugars except for 2-keto-3-deoxyoctonate. The LPS composition of 10 of the rough, phage-resistant mutants was similar to that of the HR-inducing strain, B1, yet none of them induced the HR. Only 2 of 13 mutant strains tested caused wilting of tobacco, and these had rough LPS but produced large amounts of extracellular polysaccharide, unlike most LPS-defective mutants. The evidence did not support the hypothesis that the initial interaction between rough LPS and tobacco cell walls is the determining factor in HR initiation.

Journal Article↗