RESUMO

Siderophore nutrition tests with Caulobacter crescentus strain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin. C. crescentus did not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [59Fe]apoferrichrome and 59Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [Kd ], 1 nM; Michaelis-Menten constant [KM ], 0.1 nM; Vmax, 19 pMol/109 cells/min) were similar to those of Escherichia coli FhuA. Transport properties for 59Fe-citrate were similar to those of E. coli FecA (KM , 5.3 nM; Vmax, 29 pMol/109 cells/min). Bioinformatic analyses implicated Fur-regulated loci 00028, 00138, 02277, and 03023 as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 to Escherichia coli FepA and BtuB. A Δ02277 mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the 02277 structural gene as hutA (for heme/hemoglobin utilization).IMPORTANCE The physiological roles of the 62 putative TBDT of C. crescentus are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of C. crescentus, identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition by C. crescentus, its heme/hemoglobin transporter (HutA).

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RESUMO

Enteroinvasive "Escherichia coli" strains (EIEC) of different serotypes isolated from patients with acute diarrhea were examined for the ability to produce siderophores and iron-regulated outer membrane proteins (IROMP). For iron starvation cultures were grown at 37ºC in LB supplied with 200(micro)M of (alpha)-(alpha)'dypirydil. All strains produced enterobactin and twelve (40(per cent)) produced aerobactin. The strains showed IROMP varying from 67-82 kDa. Proteins were either induced or stimulated by the iron starvation. Differences were observed in the electrophoretic profile among the serotypes, originating 5 electrophoretic profiles. All serotypes expressed proteins of 82kDa (FepA) and 76 kDa (IutA) (except serotype O28ac:H(-) that did not produce the 76 kDa protein). Several strains (O29:H(-), O144:H(-), 0152:H(-), and O167:H(-)) expressed IutA in the outer membrane, in the abscence of aerobactin production. Additionally to well characterized proteins (FepA and IutA), we found two IROMP of unknown function in some serotypes: a 71 kDa protein was detected in three profiles and a 67 kDa protein was present in serotype O152:H(-). Moreover, two bands (39 and 43 kDa) which were not iron-regulated bound specifically to human lactoferrin

Assuntos

RESUMO

Enteroinvasive Escherichia coli strains (EIEC) of different serotypes isolated from patients with acute diarrhea were examined for the ability to produce siderophores and iron-regulated outer membrane proteins (IROMP). For iron starvation cultures were grown at 37°C in LB supplied with 200 muM of FONT FACE="Symbol">a /font>-alphadypirydil. All strains produced enterobactin and twelve (40%) produced aerobactin. The strains showed IROMP varying from 67-82 kDa. Proteins were either induced or stimulated by the iron starvation. Differences were observed in the electrophoretic profile among the serotypes, originating 5 electrophoretic profiles. All serotypes expressed proteins of 82 kDa (FepA) and 76 kDa (IutA) (except serotype O28ac:H- that did not produce the 76 kDa protein). Several strains (O29:H-, O144:H-, O152:H-, and O167:H-) expressed IutA in the outer membrane, in the absence of aerobactin production. Additionally to well characterized proteins (FepA and IutA), we found two IROMP of unknown function in some serotypes: a 71 kDa protein was detected in three profiles and a 67 kDa protein was present in serotype O152:H-. Moreover, two bands (39 and 43 kDa) which were not iron-regulated bound specifically to human lactoferrin.

Cepas de Escherichia coli enteroinvasora de diferentes sorotipos isoladas de pacientes com diarréia aguda foram examinadas quanto a capacidade de produzir sideróforos e proteínas de membrana externa reguladas pelo ferro (IROMP). O crescimento bacteriano em meio com deficiência em Fe foi obtido em caldo Lúria acrescido de 200 mM de alfa- FONT FACE="Symbol">a /font>dipiridil. Todas as cepas produziram enterobactina e 40% produziram aerobactina. As cepas produziram IROMPs com MM variando de 82-67 kDa. As proteínas foram induzidas ou estimuladas pela deficiência de ferro. Diferenças foram observadas no perfil eletroforético entre os sorotipos, originando 5 perfis eletroforéticos. Todos os sorotipos, com exceção do sorotipo O28ac:H- (onde a proteína de 76 kDa não foi produzida), expressaram proteínas de 82 kDa (FepA) e 76 kDa (IutA). Várias cepas (O29:H-, O144:H-, O152:H- e O167:H-) expressaram IutA na membrana externa, na ausência da produção de aerobactina. Além das proteínas (FepA e IutA), foram encontradas em alguns sorotipos duas IROMPs de função desconhecida: uma proteína de 71 kDa foi detectada em 3 perfis e uma de 67 kDa presente no sorotipo O152:H-. Além disso, 2 bandas (39 e 43 kDa), as quais não foram reguladas pelo ferro, mostraram afinidade à lactoferrina humana.

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We report a novel phenomenon of high genetic instability, related to auxotrophy, in strains of Proteus mirabilis. Among P. mirabilis strains harboring the R plasmid Kept in our laboratory collection, and some freshly isolated strians from clinical material, 54 per cent of the samples presented auxotrophy at frequencies higher than 10(-3). Prototrophic closes gave rise to auxotrophic ones at frequencies not explainable by the usual mutation mechanisms. The instability mainly affected the carbamoyl phosphate synthetase gene (car), which leads to a double requirement for arginine and uracil for growth in minimal medium. Other genes were also affected, at a lower frequency. The car mutation does not revert to prototrophy. A similar phenomenon of instability was induced in Escherichia coli strain HB 101 upon introduction of a drug-resistance plasmid from P. mirabilis. We have ruled out the hypothesis of a transposon in the generation of auxotrophy.

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RESUMO

A novel antigen expression system has recently been developed by means of bacterial flagella, which are potent immunogens (Newton et al., Science 244:70-72,1989). Here we show the insertion of two epitopes from the cholera toxin B-subunit in Salmonella flagelin, CTP-1 (residues 8-20) and CTP-3 (residues 50-64) (Jacob et al., Proc. Natl Acad. Sci. USA 81: 7893-7896, 1984). First, we inserted CTP-1 for expression as a flagellar fusion; a hybrid flagellin expressing CTP-1 and CTP-3 in the hypervariable region of the flagellin gene was also constructed in an attempt to increase the cholera toxin neutralization potential, as well as to address some practical questions concerning the flagellar antigen expression system. The resulting constructs were non-motile although expression of chimeric flagellin was detected by immunoblotting and electron microscopy. One of the constructs (CTP-l + CTP-3) severely affected flagellin expression.

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