RESUMEN
The csbX gene of Azotobacter vinelandii was regulated in an iron-repressible manner from a divergent promoter upstream of the catecholate siderophore biosynthesis (csb) operon and was predicted to encode an efflux pump of the major facilitator superfamily. Other proteins that were most similar to CsbX were encoded by genes found in the catecholate siderophore biosynthesis operons of Aeromonas hydrophila and Stigmatella aurantiaca. Inactivation of csbX resulted in 57-100% decrease in the amount of catecholates released when compared to the wild-type in iron-limited medium. CsbX was most important for the export of the high affinity chelator protochelin with the majority of the catecholates released by csbX mutants being the protochelin intermediates azotochelin and aminochelin.
Asunto(s)
Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Catecoles/metabolismo , Genes Bacterianos , Sideróforos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Transporte Biológico , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Orden Génico , Hierro/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Mutagénesis Insercional , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
In the intestine, epithelial cells continually produce and secrete low levels of nitric oxide (NO). Salmonella sp. invade epithelium by responding to environmental stimuli. The aims of this study were to determine the effect of reactive nitrogen intermediates (RNIs) on S. dublin and S. typhimurium growth and invasion of T84 epithelial monolayers. Intracellular NO formation was inhibited by 7-nitroindazole (7-NI) or N(G)-monomethyl-L-arginine, monoacetate (L-NMMA); extracellular NO and peroxynitrite were scavenged with ferro-hemoglobin or urate. The effect of authentic peroxynitrite (ONOO-); 3-morpholino-sydnonimine (SIN-1), which releases ONOO- via NO and superoxide; spermine NONOate, which releases only NO; or superoxide generated by xanthine oxidase and pterin on S. dublin and S. typhimurium growth and invasion were examined. Inhibition of NO synthesis and scavenging of extracellular NO or peroxynitrite reduced S. dublin invasion into T84 monolayers and enhanced bacterial growth. Pre-exposure of S. dublin to ONOO- and SIN-1 increased subsequent bacterial invasion into T84 monolayers. Conversely, exposure of bacteria to spermine NONOate or superoxide did not affect S. dublin invasion. In contrast, S. typhimurium invasion was not affected by pre-treatment with NO donors. In conclusion, exposure of S. dublin to ONOO- enhances the ability of the bacteria to invade epithelial cells. These results suggest that luminal ONOO- may have a novel role as an extracellular signal between invasive bacteria and epithelial cells.
Asunto(s)
Intestinos/microbiología , Molsidomina/análogos & derivados , Ácido Peroxinitroso/farmacología , Salmonella/efectos de los fármacos , Salmonella/patogenicidad , Espermina/análogos & derivados , División Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Humanos , Intestinos/citología , Intestinos/efectos de los fármacos , Molsidomina/farmacología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitrógeno/metabolismo , Óxidos de Nitrógeno , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/patogenicidad , Espermina/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
In iron-limited medium, Azotobacter vinelandii strain UW produces three catecholate siderophores: the tricatecholate protochelin, the dicatecholate azotochelin and the monocatecholate aminochelin. Each siderophore was found to bind Fe3+ preferentially to Fe2+, in a ligand:Fe ratio of 1:1, 3:2 and 3:1, respectively. Protochelin had the highest affinity for Fe3+, with a calculated proton-independent solubility coefficient of 10439, comparable to ferrioxamine B. Iron-limited wild-type strain UW grown under N2-fixing or nitrogen-sufficient conditions hyper-produced catecholate siderophores in response to oxidative stress caused by high aeration. In addition, superoxide dismutase activity was greatly diminished in iron-limited cells, whereas catalase activity was maintained. The ferredoxin I (Fdl)-negative A. vinelandii strain LM100 also hyper-produced catecholates, especially protochelin, under oxidative stress conditions, but had decreased activities of both superoxide dismutase and catalase, and was about 10 times more sensitive to paraquat than strain UW. Protochelin and azotochelin held Fe3+ firmly enough to prevent its reduction by.O- 2 and did not promote the generation of hydroxyl radical by the Fenton reaction. Ferric-aminochelin was unable to resist reduction by O- 2 and was a Fenton catalyst. These data suggest that under iron-limited conditions, A. vinelandii suffers oxidative stress caused by.O- 2. The catecholate siderophores azotochelin, and especially protochelin, are hyper-produced to offer chemical protection from oxidative damage catalysed by.O- 2 and Fe3+. The results are also consistent with Fdl being required for oxidative stress management in A. vinelandii.