RESUMEN
Sinorhizobium meliloti strains lacking BacA function are impaired in symbiosis with alfalfa host plants and display altered sensitivities to a number of compounds relative to wild-type strains. With the goal of finding clues to the currently unknown biological function(s) of BacA, we carried out a genetic analysis to determine which amino acids are critical for protein function and to attempt to ascertain whether the multiple phenotypes that result from a bacA-null allele were the result of a common cause or whether BacA has multiple functions. We have created a set of 20 site-directed mutants in which selected individual amino acids in bacA were replaced with glycine residues. The resulting mutants were characterized to determine how the various amino acid changes affected a number of phenotypes associated with loss of BacA function. Mutants H165G, W182G, D198G, and R284G had null phenotypes for all functions assayed, while mutants W57G, S83G, S231G, and K350G were indistinguishable from wild-type strains. The remaining 12 site-directed mutants demonstrate mixed phenotypic characteristics and fall into a number of distinctly different groups. These observations may be consistent with a role for BacA in multiple, nonoverlapping functions.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana , Sinorhizobium meliloti/fisiología , Secuencia de Aminoácidos , Antibacterianos/farmacología , Bleomicina/farmacología , Etanol/farmacología , Gentamicinas/farmacología , Medicago sativa/microbiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fenotipo , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/citología , Sinorhizobium meliloti/efectos de los fármacos , Dodecil Sulfato de Sodio/farmacología , SimbiosisRESUMEN
Brucella abortus, a mammalian pathogen, and Rhizobium meliloti, a phylogenetically related plant symbiont, establish chronic infections in their respective hosts. Here a highly conserved B. abortus homolog of the R. meliloti bacA gene, which encodes a putative cytoplasmic membrane transport protein required for symbiosis, was identified. An isogenic B. abortus bacA mutant exhibited decreased survival in macrophages and greatly accelerated clearance from experimentally infected mice compared to the virulent parental strain. Thus, the bacA gene product is critical for the maintenance of two very diverse host-bacterial relationships.
Asunto(s)
Proteínas Bacterianas/fisiología , Brucella abortus/patogenicidad , Brucelosis/microbiología , Macrófagos/microbiología , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana , Sinorhizobium meliloti/fisiología , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/genética , Brucella abortus/genética , Brucella abortus/fisiología , Brucelosis/inmunología , Células Cultivadas , Femenino , Hipersensibilidad Tardía , Hígado/microbiología , Macrófagos/inmunología , Medicago sativa/microbiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis Insercional , Sinorhizobium meliloti/genética , Bazo/microbiología , Simbiosis , VirulenciaRESUMEN
Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies. Analysis of outer membrane proteins (OMPs) from B. japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores. One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment. Sequencing of this fragment revealed a single open reading frame of 750 amino acids. A putative N-terminal signal sequence of 14 amino acids which would result in a mature protein of 736 amino acids with a molecular mass of 80,851 Da was predicted. FegA shares significant amino acid similarity with several Fe(III)-siderophore receptors from gram-negative bacteria and has greater than 50% amino acid similarity and 33% amino acid identity with two [corrected] bacterial receptors for hydroxamate-type Fe(III)-siderophores. A dendrogram describing total inferred sequence similarity among 36 TonB-dependent OMPs was constructed; FegA grouped with Fe(III)-hydroxamate receptors. The transcriptional start site of fegA was mapped by primer extension analysis, and a putative Fur-binding site was found in the promoter. Primer extension and RNA slot blot analysis demonstrated that fegA was expressed only in cells grown under iron-limiting conditions. This is the first report of the cloning of a gene encoding a putative Fe(III)-siderophore receptor from nitrogen-fixing rhizobia.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Compuestos Férricos , Ácidos Hidroxámicos , Hierro/farmacocinética , Receptores de Superficie Celular/genética , Rhizobiaceae/genética , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Clonación Molecular , Medios de Cultivo , ADN Bacteriano , Regulación Bacteriana de la Expresión Génica , Bacterias Gramnegativas/metabolismo , Datos de Secuencia Molecular , Conejos , Receptores de Superficie Celular/metabolismo , Rhizobiaceae/efectos de los fármacos , Rhizobiaceae/metabolismo , Análisis de Secuencia de ADNRESUMEN
To identify possible iron sources for bacteroids in planta, soybean (Glycine max L. Merr.) symbiosomes (consisting of the bacteroid-containing peribacteroid space enclosed by the peribacteroid membrane [PBM]) and bacteroids were assayed for the ability to transport iron supplied as various ferric [Fe(III)]-chelates. Iron presented as a number of Fe(III)-chelates was transported at much higher rates across the PBM than across the bacteroid membranes, suggesting the presence of an iron storage pool in the peribacteroid space. Pulse-chase experiments confirmed the presence of such an iron storage pool. Because the PBM is derived from the plant plasma membrane, we reasoned that it may possess a ferric-chelate reductase activity similar to that present in plant plasma membrane. We detected ferric-chelate reductase activity associated with the PBM and suggest that reduction of Fe(III) to ferrous [Fe(II)] plays a role in the movement of iron into soybean symbiosomes.