Mutants of Rhizobium meliloti have been isolated which are deficient in exopolysaccharide (EPS) production and effective nodulation of alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned R. meliloti exo loci. We also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-beta-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.
The cell surface polysaccharides of wild-type Bradyrhizobium japonicum USDA 110 and a nonnodulating mutant, strain HS123, were analyzed. The capsular polysaccharide (CPS) unpublished data). We found strain HS123 to be a loughtype mutant differing markedly from the wild type in the composition of the LPS. MATERIALS AND METHODSOrganism and culture media. The wild-type B. japonicum USDA 110 and the mutant strain HS123 were obtatined as described previously (18) and were grown on defined medium (13). Six-day-old bacterial cells were harvested by centrifugation at 16,000 x g for 20 min. The pelleted cells were washed three times with phosphate-buffered saline (0.43 g of NaH2PO4, 1.48 g of Na2HPO4, 7.2 g of NaCl per liter), rinsed with deionized water, and freeze-dried.Isolation and fractionation of polysaccharides. The supernatant fluid obtained after the removal of cells by centrifugation was concentrated by rotary evaporation at 40°C. The concentrated solution was dialyzed extensively against distilled water and freeze-dried to yield the crude EPS traction.For the isolation of CPS, the cells suspended in phosphate-buffered saline were stirred in a Waridg blender for a minute at full speed. After the removal of cells by centrifugation, the crude CPS fraction was recovered from the supernatant fluid by dialysis and lyophilization. The CPS and EPS were purified by chromatography on a DEAE-Bio-Gel column as reported by Mort and Bauer (13).LPS was isolated by phenol-water eXtraction of freezedried cells by the method of Johnson and Perry (9). After extraction, the aqueous phase was dialyzed extensively, and the polysaccharides were recovered by freeze-drying. These crude extracts were dissolved in distilled water and passed through a Dowex-1 anion (acetate form)-exchange column to remove any adhering acidic extracellular polysaccharides. After the column was washed with distilled water until no carbohydrate was detected in the effluent (as determined by the phenol-sulfuric acid method [2]), the effluent and the combined washings were concentrated by rotary evaporation and freeze-dried.
Phenol-water cell extracts of virulent Agrobacterium tumefaciens A348 and several avirulent mutants with a reduced ability to attach to plant surfaces were examined. A low-molecular-weight 2-linked-13-D-glucan was identified in the cell wall extracts of the virulent wild-type strain. Analyses of phenol-water extracts and culture filtrates of four mutant strains showed that the mutants did not produce any 2-linked-4-D-glucan. When these mutants were complemented, the ability to produce the glucan described above was restored. These results suggest that there is a role for 2-linked-o-D-glucans in the attachment of A. tumefaciens to plant cells. One avirulent, attachment-defective mutant retained its ability to produce the low-molecular-weight glucan. This mutation, however, mapped to a different transcriptional unit than the mutants deficient in the glucan described above. Thus, it appears that 2-linked-Ip-D-glucan is only one component that may be necessary for attachment of A. tumefaciens to plant cell surfaces.
The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) applied foliarly as the 2-ethylhexyl ester (EHE) to wheat and potatoes, to the soil as the dimethylamine (DMA) salt under apple tree canopies, and preplant as the free acid for wheat, lettuce, and radish was studied to evaluate metabolic pathways. Crop fractions analyzed for (14)C residues included wheat forage, straw, and grain; potato vine and tubers; and apple fruit. The primary metabolic pathway for foliar application in wheat is ester hydrolysis followed by the formation of base-labile 2,4-D conjugates. A less significant pathway for 2,4-D in wheat was ring hydroxylation to give NIH-shift products 2,5-dichloro-4-hydroxyphenoxyacetic acid (4-OH-2,5-D), 4-OH-2,3-D, and 5-OH-2,4-D both free and as acid-labile conjugates. The primary metabolic pathway in potato was again ester hydrolysis. 2,4-D acid was further transformed to 4-chlorophenoxyacetic acid and 4-OH-2,5-D. For the soil applications, (14)C residues in the crops were low, and characterization of the (14)C residues indicated association with or incorporation into the biochemical matrix of the tissue. The degradative pathways observed in wheat are similar to those characterized in other intact plant studies but differ from those in studies in wheat cell suspension culture in that no amino acid conjugates were observed.
Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii, might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii-host interaction and play an important protective role by creating a permeability barrier around the cyst.
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