Osmotic adjustment in Brevibacterium ammoniagenes: pipecolic acid accumulation at elevated osmolalities

Gouesbet, Gwenola; Blanco, Carlos; Hamelin, J.; Bernard, Théophile

doi:10.1099/00221287-138-5-959

Cited by 36 publications

(35 citation statements)

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“…Transport assays and radioactivity counting were performed as described by Perroud and Le Rudulier (34). 14 C-osmoprotectants were produced as previously described (9,19). DNA manipulations.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA

et al. 1996

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Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA geneencoded osmoprotectant uptake system A from E. chrysanthemi 3937. OusA belongs to the superfamily of solute ion cotransporters. This osmotically inducible system allows the uptake of glycine betaine, proline, ectoine, and pipecolic acid and presents strong similarities in nucleotide sequence and protein function with the proline/ betaine porter of Escherichia coli encoded by proP. The control of ousA expression is clearly different from that of proP. It is induced by osmotic strength and repressed by osmoprotectants. Its expression in E. coli is controlled by H-NS and is rpoS dependent in the exponential phase but unaffected by the stationary phase.Microbial pathogens encounter extremely diverse environments both inside and outside their hosts. In response to these adverse conditions, they undergo striking adaptations in order to survive and retain virulence. The growth of Erwinia chrysanthemi, which is involved in a systemic soft rot disease on a variety of higher plants, is influenced by desiccation (18, 21). We have recently analyzed the influence of osmotic strength on E. chrysanthemi growth and pathogenicity in the absence and presence of osmoprotectants (10). The consequences for pathogenicity were estimated by the effect of osmotic pressure on transcription of pel genes and pectate lyase activity. The transcription of the pelE gene, encoding the major extracellular pectate lyase enzyme, is induced in medium of high osmolarity, whereas the cellular growth rate was reduced. Osmoprotectants such as glycine betaine, proline, ectoine, and pipecolic acid were shown to be accumulated in the cells through an osmoinducible mechanism and stimulated growth. However, pelE transcription was reduced to basal levels.Although uptake and accumulation of osmoprotectants have been observed in many bacteria, transporter structural genes have been characterized in only a few microorganisms. Considering the regulation of their transcription, a knowledge of which is essential to understanding osmoregulation, only the proP and proU operons of Escherichia coli and Salmonella typhimurium have been analyzed in depth (6-8, 13, 14, 20, 27, 28, 31, 32). The identification of structural domains involved in osmosensing and substrate binding could be improved by comparison of ProP and ProU with other osmoprotectant transporters in other bacteria. In this report, we characterize one of the osmoprotectant transporters of E. chrysanthemi, analogous to ProP of E. coli. MATERIALS AND METHODSBacterial strains, plasmids, media, and growth conditions. The E. coli and E. chrysanthemi strains and plasmids used are described in Table 1. Cells were grown in LB or M63 glucose (0.2%) medium (33). E. chrysanthemi cells were usually incubated at 30ЊC, and E. coli cells were incubated at 37ЊC. The osmoprotectants choline, glycine betaine, proline, ectoin...

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Section: Methodsmentioning

confidence: 99%

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA

et al. 1996

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“…Neutral and acidic compounds were discarded by washing the resin with distilled water, and the cationic fraction (containing amino acids and related molecules) was recovered after elution with 2 M NH40H.' After concentration, the solutions were analyzed by paper chromatography and thinlayer chromatography, paper high-voltage electrophoresis, and high-pressure liquid chromatography as described by Gouesbet et al (16). Prior to quantitative analysis, the neutral fraction (containing sugars) was treated as described by Larsen et al (21) and analyzed for trehalose content.…”

Section: Methodsmentioning

confidence: 99%

Osmoadaptation in rhizobia: ectoine-induced salt tolerance

et al. 1994

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), we have investigated the involvement of this molecule in the osmotic adaptation of Rhizobium meliloti. Ectoine appeared almost as effective as glycine betaine in improving the growth of R. meliboti under adverse osmotic conditions (0.5 M NaCI). Moreover, improvement of growth of rhizobial strains insensitive to glycine betaine was also observed. Ectoine transport proved inducible, periplasmic protein dependent, and, as shown by competition experiments, distinct from the transport of glycine betaine. Medium osmolarity had little effect on the uptake characteristics, since the rate of influx increased from 12 to only 20 nmol min-' mg of proteinwhen NaCI concentrations were raised from 0 to 0.3 or 0.5 M, with a constant of transport of 80 FM. Natural-abundance "C-nuclear magnetic resonance and radiolabelling assays showed that ectoine, unlike glycine betaine, is not intracellularly accumulated and, as a consequence, does not repress the synthesis of endogenous compatible solutes (glutamate, N-acetylglutaminylglutamine amide, and trehalose). Furthermore, the strong rise in glutamate content in cells osmotically stressed in the presence of ectoine suggests that, instead of being involved in osmotic balance restoration, ectoine should play a key role in triggering the synthesis of endogenous osmolytes. Hence, we believe that there are at least two distinct classes of osmoprotectants: those such as glycine betaine or glutamate, which act as genuine osmolytes, and those such as ectoine, which act as chemical mediators.Rhizobia are soil bacteria which display symbiotic interactions with specific legume hosts. Most of these bacteria are very sensitive to a soil water deficit, which adversely affects their dinitrogen fixation capacity and hence the productivity of the whole legume plant (1,8,19,38). Much attention has been focused during the last decade on the mechanisms of osmotic adaptation in several rhizobium species (18,41,42). Rhizobium meliloti, the microsymbiont in alfalfa root nodules, has been extensively investigated (5,6,22,36), and the mechanisms governing turgor restoration of bacterial cells growing under hyperosmotic conditions have been partially elucidated. Specifically, concomitant accumulations of potassium and glutamate ions were pointed out as the primary response in R meliloti (6), and the same mechanism has been found in most of the bacterial species investigated thus far (7, 27). Trehalose and the dipeptide N-acetylglutaminylglutamine amide (NAGGN) (37) are also accumulated, probably to help the bacterial cells in balancing the high osmotic pressure of the medium. Among a series of exogenously supplied osmoprotectants, glycine betaine and proline betaine proved the most effective (3, 14).Their effect is quite similar to that found in members of the family Enterobacteriaceae (23) except that lowering medium osmolality triggers catabolism of betaines in R meliloti (3, 14, 36). A transport system involving a periplasmic glycine betainebinding protein was identified previously (24, 40). Glycine beta...

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“…(1992). The purified molecule was assayed by cochromatography with unlabelled commercial pipecolic acid and HPLC analysis (Gouesbet et al, 1992). The specific activity (1.83 GBq mmol-l) was deduced from the HPLC analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Extraction and identification of solutes. Solutes were identified after extraction and chromatographic analysis as described by Gouesbet et al (1992). Transport assays.…”

Section: Methodsmentioning

confidence: 99%

Pipecolic acid is an osmoprotectant for Escherichia coli taken up by the general osmoporters ProU and ProP

et al. 1994

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Osmotic adjustment in Brevibacterium ammoniagenes: pipecolic acid accumulation at elevated osmolalities

Cited by 36 publications

References 36 publications

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA

Osmoadaptation in rhizobia: ectoine-induced salt tolerance

Pipecolic acid is an osmoprotectant for Escherichia coli taken up by the general osmoporters ProU and ProP

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