RESUMEN
A broadly conserved membrane-associated protein required for the functional interaction of kinesin-I with axonal cargo was identified. Mutations in sunday driver (syd) and the axonal transport motor kinesin-I cause similar phenotypes in Drosophila, including aberrant accumulations of axonal cargoes. GFP-tagged mammalian SYD localizes to tubulovesicular structures that costain for kinesin-I and a marker of the secretory pathway. Coimmunoprecipitation analysis indicates that mouse SYD forms a complex with kinesin-I in vivo. Yeast two-hybrid analysis and in vitro interaction studies reveal that SYD directly binds kinesin-I via the tetratricopeptide repeat (TPR) domain of kinesin light chain (KLC) with K(d) congruent with 200 nM. We propose that SYD mediates the axonal transport of at least one class of vesicles by interacting directly with KLC.
Asunto(s)
Transporte Axonal , Proteínas Portadoras/metabolismo , Proteínas de Drosophila , Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Secuencia de Aminoácidos , Animales , Conducta Animal , Transporte Biológico , Proteínas Portadoras/genética , Compartimento Celular , Clonación Molecular , Drosophila/genética , Proteínas de Insectos/metabolismo , Larva/genética , Proteínas de la Membrana/genética , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Unión Proteica , Subunidades de Proteína , Técnicas del Sistema de Dos Híbridos , Red trans-Golgi/químicaRESUMEN
Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomonas aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and the lysogenic (c) repressor. The c open reading frame (ORF), located at the leftmost end of the phage genome and transcribed from right to left, has four possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiation codon. Plasmids were constructed expressing either the wild-type repressor ORF or the ORFs containing the mutated GTA codons. When introduced into Pseudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrated that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension analyses to bp 972-940 from the left end of the phage genome. When the D3112 c repressor was overexpressed and purified as a fusion protein with a C-terminal six-histidine extension (cts15-His6), it showed high affinity for a 261-bp PvuII fragment localized directly upstream of the c repressor ORF. Our results indicate that although D3112 c shows higher amino acid similarity to the lambda family of repressors than it does to those of Mu and D108, it appears that its structure and function more accurately reflect an evolutionary ancestry with those from transposable coliphages Mu and D108.
Asunto(s)
Elementos Transponibles de ADN/genética , Genes Virales/genética , Lisogenia/genética , Fagos Pseudomonas/genética , Proteínas Represoras/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón Iniciador/genética , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Genes Reporteros/genética , Lisogenia/inmunología , Datos de Secuencia Molecular , Mutación/genética , Sistemas de Lectura Abierta/genética , Regiones Operadoras Genéticas/genética , Regiones Promotoras Genéticas/genética , Fagos Pseudomonas/fisiología , Pseudomonas aeruginosa/virología , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Viral/análisis , ARN Viral/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/aislamiento & purificación , Proteínas Represoras/metabolismo , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.
Asunto(s)
Transporte Axonal , Proteínas de Unión al Calcio/metabolismo , Colina O-Acetiltransferasa/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Proteínas Musculares/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Clonación Molecular , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Drosophila melanogaster/enzimología , Genes Letales/genética , Prueba de Complementación Genética , Cinesinas/química , Cinesinas/genética , Cinesinas/metabolismo , Larva/citología , Larva/enzimología , Larva/metabolismo , Ratones , Datos de Secuencia Molecular , Movimiento , Proteínas Musculares/química , Proteínas Musculares/genética , Mutación/genética , Sistema Nervioso/citología , Sistema Nervioso/embriología , Neuronas/citología , Neuronas/enzimología , Neuronas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
Mucin-specific adhesion of Pseudomonas aeruginosa plays an important role in the initial colonization of this organism in the airways of cystic fibrosis patients. We report here that the flagellar cap protein, FliD, participates in this adhesion process. A polar chromosomal insertional mutation in the P. aeruginosa fliD gene made this organism nonadhesive to mucin in an in vitro mucin adhesion assay. The adhesive phenotype was restored by providing the fliD gene alone on a multicopy plasmid, suggesting involvement of this gene in mucin adhesion of P. aeruginosa. Further supporting this observation, the in vitro competition experiments demonstrated that purified FliD protein inhibited the mucin adhesion of nonpiliated P. aeruginosa PAK-NP, while the same concentrations of PilA and FlaG proteins of P. aeruginosa were ineffective in this function. The regulation of the fliD gene was studied and was found to be unique in that the transcription of the fliD gene was independent of the flagellar sigma factor sigma28. Consistent with this finding, no sigma28 binding sequence could be identified in the fliD promoter region. The results of the beta-galactosidase assays suggest that the fliD gene in P. aeruginosa is regulated by the newly described transcriptional regulator FleQ and the alternate sigma factor sigma54 (RpoN).
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/fisiología , Mucinas/fisiología , Pseudomonas aeruginosa/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Pseudomonas aeruginosa/genética , Factor sigma/fisiologíaRESUMEN
Previous work has demonstrated that fleR, the gene for a transcriptional activator belonging to the NtrC subfamily of response regulators, is involved in the regulation of mucin adhesion and flagellar expression by Pseudomonas aeruginosa. This report describes the identification and characterization of fleQ, the gene for another transcriptional regulator which also regulates mucin adhesion and motility in this organism. The complete nucleotide sequence of the fleQ gene was determined on both DNA strands, and an open reading frame (ORF) consisting of 1,493 nucleotides was identified. This ORF coded for a gene product of predicted molecular weight, as confirmed by the overexpression of the fleQ gene as a fusion protein under an inducible promoter. The fleQ gene is flanked by a flagellar operon, fliDSorf126, at the 5' end and the fleSR operon on the 3' end. FleQ also had striking homology to a number of proteins belonging to the NtrC subfamily of response regulators, which work in concert with the alternate sigma factor RpoN (sigma54) to activate transcription. However, FleQ lacks the residues corresponding to Asp-54 and Lys-104 of the NtrC protein which are conserved in most of the members belonging to this subfamily of regulators. In addition, unlike some of the other transcriptional activators of this group, FleQ does not appear to have a cognate sensor kinase. A chromosomal insertional mutation in the fleQ gene abolished mucin adhesion and motility of P. aeruginosa PAK and PAK-NP. Both of these functions were regained by providing the complete fleQ gene on a multicopy plasmid. The location of fleQ immediately upstream of the fleSR operon, which is also necessary for the same process, suggested that these regulators may interact in some way. We therefore examined the regulation of the fleSR operon by fleQ and vice versa. Promoter fusion experiments showed that the fleSR operon was regulated by RpoN and FleQ. On the other hand, the fleQ promoter was independent of RpoN and FleR. FleQ, thus, adds another level of regulation to motility and adhesion in P. aeruginosa, above that of fleSR. We therefore propose the existence of a regulatory cascade which consists of at least two transcriptional regulators, FleQ and FleR, in the control of motility and adhesion in P. aeruginosa.
Asunto(s)
Adhesión Bacteriana/genética , Proteínas de Unión al ADN , Flagelos/genética , Regulación Bacteriana de la Expresión Génica/genética , Pseudomonas aeruginosa/genética , Transactivadores/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/fisiología , Flagelina/análisis , Genes Bacterianos/genética , Datos de Secuencia Molecular , Mucinas/metabolismo , Operón/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa Sigma 54 , Proteínas Recombinantes de Fusión/aislamiento & purificación , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factor sigma/fisiología , Transactivadores/aislamiento & purificación , Transactivadores/fisiología , Factores de Transcripción/genéticaRESUMEN
Because actin can form a complex in vitro containing both gelsolin and DNase I, gelsolin and DNase I have been assumed to bind independently to actin. Although this assumption is consistent with the known crystalline structures of gelsolin with one actin and of actin with DNase I, which suggest that the binding sites on actin for both gelsolin and DNase I are distinct and separate, we propose that a second actin binding site on gelsolin competes with DNase I for actin. Since actin is an inhibitor of DNase I, competition at the second binding site results in activation of DNase I by gelsolin. Covalent cross-linking experiments confirm that DNase I prevents dimerization of actin by gelsolin, consistent with displacement of one actin from gelsolin by DNase I. Activation of DNase I by gelsolin is a novel function for a cytoskeletal protein and could have broad implications for biology, such as a role in initiating apoptosis. These results also may explain why both gelsolin and DNase I decrease sputum viscosity in cystic fibrosis (CF). While the activity of DNase I had originally been attributed to fragmentation of DNA, subsequent data suggested that both gelsolin and DNase I may affect viscosity by depolymerizing filamentous actin. The current results alternatively suggest aht dissociation of the actin-DNase I complex by gelsolin in CF sputum results in activation of the nuclease activity of constitutive DNase I. The nuclease activity of DNase I alone is therefore sufficient to explain the effects of both gelsolin and DNase I on CF sputum.
Asunto(s)
Fibrosis Quística/enzimología , Desoxirribonucleasa I/efectos de los fármacos , Desoxirribonucleasa I/metabolismo , Gelsolina/farmacología , Esputo/enzimología , Actinas/química , Actinas/metabolismo , Animales , Bovinos , Reactivos de Enlaces Cruzados , Fibrosis Quística/metabolismo , Desoxirribonucleasa I/química , Electroforesis en Gel de Agar , Activación Enzimática/efectos de los fármacos , Etidio , Fluorescencia , Humanos , Conejos , Esputo/efectos de los fármacos , Esputo/metabolismoRESUMEN
Pseudomonas aeruginosa colonizes the mucus of patients with chronic lung diseases by a specific mechanism involving an adhesin-receptor system. Several adhesins have been implicated in the adhesion of P. aeruginosa to cells, but the identity of the principal adhesin(s) involved in adhesion to mucin is unknown. Mutagenesis studies have indicated that P. aeruginosa adhesion is under the control of the rpoN gene, which also regulates pilin synthesis, flagellum formation, and other functions. Mutagenesis of certain flagellar genes that are not controlled by RpoN, e.g., flif, also indicates a close relationship between adhesion and flagellar genes and not necessarily an independent effect of rpoN on adhesion. Mutants of certain early flagellar genes lead to the loss of both adhesion and motility, whereas mutants of certain late genes, e.g., fliC, the gene for flagellin, lose motility but retain adhesion. Recent studies indicate that both motility and adhesion are regulated by a two-component regulatory system called fleS-R, which in turn is controlled by another regulator in a cascade that involves rpoN. A fleR mutant possessing pili adheres poorly to mucins, definitively showing that pili do not play a major role in adhesion to mucin. It is unclear whether the adhesin is a flagellar protein or another protein that uses the flagellar export apparatus for localization or both. Finding the gene under control of rpoN may provide answers to these questions.
Asunto(s)
Adhesinas Bacterianas/genética , Flagelos/genética , Mucinas/genética , Pseudomonas aeruginosa/genética , Adhesión Bacteriana/fisiología , Fimbrias Bacterianas/genética , Genes Bacterianos/fisiología , Humanos , Mutación , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/ultraestructuraRESUMEN
Pseudomonas aeruginosa adheres to the mucosal surfaces of the lungs. This process appears to be mediated by nonpilus adhesins which bind to mucin. To find this nonpilus adhesin(s), mutagenesis of a nonpiliated mutant of P. aeruginosa with transposon Tn5G, followed by a screen for mucin adhesion, was used to isolate a series of mutants unable to adhere to mucin. All of these mutants were also found to be defective in motility. One such mutant, PAK-RR20, is characterized here. The site of the transposon insertion in PAK-RR20 was localized to a gene which is homologous to the fliF gene of other organisms and was flanked by other motility-related genes, fliE and fliG. Both adhesion and motility defects in PAK-RR20 were complemented by providing the fliF gene in trans. Since complementation could have been due to the presence of an internal promoter in the fliF gene or in the Tn5G transposon, which allowed the transcription of the downstream genes, another chromosomal mutant of the fliF gene was constructed by insertional inactivation with an antibiotic resistance cassette. This mutant was also nonmotile and nonadhesive. However, the two defects in this new mutant could not be complemented by the fliF gene in trans, consistent with the interpretation that there is no internal fliF promoter but possibly a functional promoter in the Tn5G transposon. The complete nucleotide sequences of the fliE and fliF genes and a partial nucleotide sequence of the fliG gene of P. aeruginosa were determined. Control of the promoter upstream of the fliE gene was analyzed by construction of a fliE-lacZ fusion and the introduction of this construct into strains of P. aeruginosa with mutations in several regulatory genes. Beta-Galactosidase expression measurements indicated that the fliE promoter does not utilize RpoF (sigma(28)) or RpoN (sigma(54)) sigma factors. The characterization of this gene as being responsible for the loss of adhesion indicates that basal body structures are probably important for localization of the adhesin.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/genética , Genes Bacterianos , Proteínas de la Membrana , Mucinas/metabolismo , Pseudomonas aeruginosa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factor sigma/genéticaRESUMEN
This work has identified two genes (designated fleS and fleR) in Pseudomonas aeruginosa which are highly homologous to members of the subclass of two-component systems involved in transcriptional regulation of a diverse array of genes from sigma 54 promoters. The genes are located upstream from fliE, a flagellar gene of P. aeruginosa, and they are arranged in a putative fleSR operon. FleS has a predicted molecular mass of 43.87 kDa and shows strong homology to histidine kinases which in other two-component systems have been shown to be sensor proteins. FleR has a predicted molecular mass of 51.26 kDa and is homologous to other regulatory proteins that bind to specific upstream activating elements to enhance transcription of genes with sigma 54 promoters. The fleSR system is believed to control both flagellar synthesis and adhesion to mucin. Several lines of evidence are presented. (i) A nonpiliated mutant of P. aeruginosa PAK containing a gentamicin cassette in fleR is nonmotile and nonadhesive. (ii) The fleR mutant regained motility and adhesion when complemented with a wild-type copy of fleR. (iii) A Western blot (immunoblot) of the fleR mutant showed no synthesis of flagellin, and electron microscopy of the fleR mutant confirmed the lack of flagella. Previous work has shown that flagellar mutants with mutations in fliA (sigma 28) or fliC (the structural gene for flagellin) retain adhesion; therefore, these new observations suggest that FleSR regulates both the expression of flagella and the nonpilus adhesin(s) for mucin or that one of the flagellar proteins (other than flagellin) may be responsible for adhesion to mucins.
Asunto(s)
Adhesión Bacteriana/genética , Genes Bacterianos , Pseudomonas aeruginosa/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Flagelos/metabolismo , Genes Reguladores , Datos de Secuencia Molecular , Mucinas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
A guanosine to cytosine transversion at position 2 of the fifth intron of the mitochondrial gene COB blocks the ligation step of splicing. This mutation prevents the formation of a base pair within the P1 helix of this group I intron--the RNA duplex formed between the 3' end of the upstream exon and the internal guide sequence. The mutation also reduces the rate of the first step of splicing (guanosine addition at the 5' splice junction) while stimulating hydrolysis at the 3' intron-exon boundary. Consequently, the ligation of exons is blocked because the 3' exon is removed prior to cleavage at the 5' splice junction. The lesion can be suppressed by second-site mutations that preserve the potential for base-pairing at this position. Because the P1 duplex and the P10 duplex (between the guide sequence and the 3' exon) overlap at the affected pairings represent alternative structures that do not, indeed cannot, form simultaneously.