When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395␣395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395␣395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps ؉ strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.Bacteria that enter into intimate associations with plants and animals, whether pathogenic or mutualistic, respond to signals that are thought to indicate the presence of the host. In the symbiosis between legumes and the bacteria collectively known as rhizobia, one example is the production of Nod factors (lipochitooligosaccharides) by the bacteria in response to flavonoid compounds exuded by the plant (17, 27). Another potential example is the alteration of the lipopolysaccharide (LPS) of Rhizobium etli when grown in the presence of the most potent nod inducers from Phaseolus vulgaris (17), the anthocyanins exuded by germinating seeds (10,22,23). These induced LPS alterations have been detected by the lack of recognition of the altered LPS by certain monoclonal antibodies (23). Modifications of the LPS structure in strains of this and other rhizobial species also have been reported to occur during nodule development and in culture in response to low pH and low oxygen, as well as nod inducers (1,16,18,19,29,31). However, in no case have precise chemical changes been determined, nor is it known whether these induced LPS modifications are important in the development or functioning of the symbiosis.Although little is known of the biological roles of specific structural features of the rhizobial LPS, the importance of wild-type LPS in symbiosis is well established. Studies with mutants of a wide variety of rhizobial s...

Objective: Asthma and gastric reflux disease are widespread and often coexisting diseases with complex interactions, leading some to suspect that asthma symptoms of patients with reflux may improve with anti-reflux therapy. The objective of this study was to determine whether pepsin in saliva, indicative of airway reflux, could be detected in patients with asthma of varying severity and test the requirement of citric acid as a pepsin preservative. Methods: Saliva samples were collected in the clinic (with/without citric acid) and upon waking the following morning from 25 asthmatic patients. Enzyme-linked immunosorbent assay was performed for pepsin and interleukin-8 (IL-8), an inflammatory cytokine induced by pepsin in other airway epithelia. Pepsin induction of IL-8 was tested in a lung epithelial cell culture model. Results: Pepsin was detected in saliva from 14/25 patients (56%; mean concentration of pepsin in specimens where observed ±SD =80.3±87.5 ng/mL); significant agreement was found between samples collected in the presence/absence of citric acid. No significant associations were found with pepsin and clinical measures of asthma severity. IL-8 was detected in saliva from 22/25 patients (88%; mean IL-8 in all specimens where observed =3.27±3.91 ng/mL). IL-8 was significantly upregulated in human lung epithelial cells exposed to pepsin at pH7 in vitro ( P =0.041). Conclusion: In summary, more than half of the asthma patients in this study were found to have pepsin in their saliva, indicative of airway reflux. These data support the use of salivary pepsin as a noninvasive tool for future investigation of airway reflux in a larger cohort. The data further suggest that collection in citric acid as a sample preservative is not warranted and that pooling of multiple saliva samples collected at various timepoints may improve sensitivity of pepsin detection and reduce costs incurred by multiple sample analysis in future studies.

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