RESUMEN
Rhodobacter sphaeroides has two chemotaxis clusters, an Escherichia coli-like cluster with membrane-spanning chemoreceptors and a less-understood cytoplasmic cluster. The cytoplasmic CheA is split into CheA4, a kinase, and CheA3, a His-domain phosphorylated by CheA4 and a phosphatase domain, which together phosphorylate and dephosphorylate motor-stopping CheY6. In bacterial two-hybrid analysis, one major cytoplasmic chemoreceptor, TlpT, interacted with CheA4, while the other, TlpC, interacted with CheA3. Both clusters have associated adaptation proteins. Deleting their methyltransferases and methylesterases singly and together removed chemotaxis, but with opposite effects. The cytoplasmic cluster signal overrode the membrane cluster signal. Methylation and demethylation of specific chemoreceptor glutamates controls adaptation. Tandem mass spectroscopy and bioinformatics identified four putative sites on TlpT, three glutamates and a glutamine. Mutating each glutamate to alanine resulted in smooth swimming and loss of chemotaxis, unlike similar mutations in E. coli chemoreceptors. Cells with two mutated glutamates were more stoppy than wild-type and responded and adapted to attractant addition, not removal. Mutating all four sites amplified the effect. Cells were non-motile, began smooth swimming on attractant addition, and rapidly adapted back to non-motile before attractant removal. We propose that TlpT responds and adapts to the cell's metabolic state, generating the steady-state concentration of motor-stopping CheY6~P. Membrane-cluster signalling produces a pulse of CheY3/CheY4~P that displaces CheY6~P and allows flagellar rotation and smooth swimming before both clusters adapt.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas Bacterianas/genética , Células Quimiorreceptoras/metabolismo , Rhodobacter sphaeroides/genética , Proteínas Bacterianas/metabolismo , Quimiotaxis/genética , Citoplasma/genética , Citoplasma/fisiología , Citosol/metabolismo , Proteínas de Escherichia coli/genética , Eliminación de Gen , Histidina Quinasa/genética , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Fosforilación/genética , Procesamiento Proteico-Postraduccional/genética , Rhodobacter sphaeroides/fisiología , Transducción de Señal/genética , Espectrometría de Masas en TándemRESUMEN
Rhodobacter sphaeroides is a metabolically diverse photosynthetic alphaproteobacterium found ubiquitously in soil and freshwater habitats. Here we present the annotated genome sequence of R. sphaeroides WS8N.
Asunto(s)
Agua Dulce/microbiología , Genoma Bacteriano , Rhodobacter sphaeroides/genética , Secuencia de Bases , Datos de Secuencia Molecular , Rhodobacter sphaeroides/aislamiento & purificaciónRESUMEN
We have developed a stable isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria Rhodobacter sphaeroides and Paracoccus denitrificans. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the lacI(q) gene from pYanni3 and the lac promoter, P(A1/04/03), from pJBA24.
Asunto(s)
Paracoccus denitrificans/genética , Plásmidos/genética , Rhodobacter sphaeroides/genética , Mapeo Cromosómico , Expresión Génica/efectos de los fármacos , Genes Bacterianos/efectos de los fármacos , Vectores Genéticos , Isopropil Tiogalactósido/farmacología , Resistencia a la Kanamicina/genética , Operón Lac , Datos de Secuencia Molecular , Paracoccus denitrificans/efectos de los fármacos , Regiones Promotoras Genéticas , Replicón , Rhodobacter sphaeroides/efectos de los fármacosRESUMEN
Rhodobacter sphaeroides has a complex chemosensory system, with several loci encoding multiple homologues of the components required for chemosensing in Escherichia coli. The operons cheOp2 and cheOp3 each encode complete pathways, and both are essential for chemosensing. The components of cheOp2 are predominantly localized to the cell pole, whereas those encoded by cheOp3 are predominantly targeted to a discrete cluster in the cytoplasm. Here we show that the expression of the two pathways is regulated independently. Overlapping promoters recognized by sigma(28) and sigma(70) RNAP holoenzyme transcribe cheOp2, whereas cheOp3 is regulated by one of the four sigma(54) homologues, RpoN3. The different regulation of these operons may reflect the need for balancing responses to extra- and intracellular signals under different growth conditions.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Operón/genética , ARN Polimerasa Sigma 54/genética , Rhodobacter sphaeroides/genética , Factor sigma/genética , Secuencia de Bases , Quimiotaxis/genética , ARN Polimerasas Dirigidas por ADN/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Sitio de Iniciación de la TranscripciónRESUMEN
The Escherichia coli two-component chemosensory pathway has been extensively studied, and its response regulator, CheY, has become a paradigm for response regulators. However, unlike E. coli, most chemotactic nonenteric bacteria have multiple CheY homologues. The roles and cellular localization of the CheYs in Rhodobacter sphaeroides were determined. Only two CheYs were required for chemotaxis, CheY(6) and either CheY(3) or CheY(4). These CheYs were partially localized to either of the two chemotaxis signaling clusters, with the remaining protein delocalized. Interestingly, mutation of the CheY(6) phosphorylatable aspartate to asparagine produced a stopped motor, caused by phosphorylation on alternative site Ser-83 by CheA. Extensive mutagenesis of E. coli CheY has identified a number of activating mutations, which have been extrapolated to other response regulators (D13K, Y106W, and I95V). Analogous mutations in R. sphaeroides CheYs did not cause activation. These results suggest that although the R. sphaeroides and E. coli CheYs are similar in that they require phosphorylation for activation, they may differ in both the nature of the phosphorylation-induced conformational change and their subsequent interactions with the flagellar motor. Caution should therefore be used when projecting from E. coli CheY onto novel response regulators.
Asunto(s)
Proteínas Bacterianas/metabolismo , Quimiotaxis/fisiología , Rhodobacter sphaeroides/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Asparagina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Escherichia coli/genética , Escherichia coli/fisiología , Hemaglutininas/química , Técnicas In Vitro , Espectrometría de Masas , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Estructura Terciaria de Proteína , Rhodobacter sphaeroides/genética , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismoRESUMEN
Trypanosoma brucei switches between variant surface glycoproteins (VSGs) allowing immune escape. The active VSG is in one of many telomeric bloodstream form VSG expression sites (BESs), also containing expression site-associated genes (ESAGs) involved in host adaptation. The role of BES sequence diversity in parasite virulence can best be understood through analysis of the full repertoire of BESs from a given T. brucei strain. However, few BESs have been cloned, as telomeres are highly underrepresented in standard libraries. We devised a strategy for isolating the repertoire of T. brucei 427 BES-containing telomeres in Saccaromyces cerevisiae by using transformation-associated recombination (TAR). We isolated 182 T. brucei 427 BES TAR clones, 167 of which could be subdivided into minimally 17 BES groups. This set gives us the first view of the breadth and diversity of BESs from one T. brucei strain. Most BESs ranged between 40 and 70 kb (average, 57 +/- 17 kb) and contained most identified ESAGs. Phylogenetic comparison of the cohort of BES promoter and ESAG6 sequences did not show similar trees, indicating rapid evolution most likely mediated by sequence exchange between BESs. This cloning strategy could be used for any T. brucei strain, facilitating research on the biodiversity of telomeric gene families and host-pathogen interactions.