RESUMEN
The interruption of the sblA gene of Streptomyces lividans was previously shown to lead to relief of glucose repression of the normally strongly glucose-repressed alpha-amylase gene. In addition to this relief, an early entry into stationary phase was observed when cells were grown in a minimal medium containing glucose as the main carbon source. In this study, we established that this mutant does not resume growth after the transition phase when cultured in the complex glucose-rich liquid medium R2YE and sporulates much earlier than the wild-type strain when plated on solid R2YE. These phenotypic differences, which were abolished when glucose was omitted from the R2YE medium, correlated with a reduced glucose uptake ability of the sblA mutant strain. sblA was shown to encode a bifunctional enzyme possessing phospholipase C-like and phosphoinositide phosphatase activities. The cleavage of phosphoinositides by SblA seems necessary to trigger the glucose-dependent renewed growth that follows the transition phase. The transient expression of sblA that takes place just before the transition phase is consistent with a regulatory role for this gene during the late stages of growth. The tight temporal control of sblA expression was shown to depend on two operator sites. One, located just upstream of the -35 promoter region, likely constitutes a repressor binding site. The other, located 170 bp downstream of the GTG sblA translational start codon, may be involved in the regulation of the degradation of the sblA transcript. This study suggests that phosphoinositides constitute important regulatory molecules in Streptomyces, as they do in eukaryotes.
Asunto(s)
Glucosa/metabolismo , Fosfatidilinositoles/metabolismo , Streptomyces lividans/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Represión Enzimática/genética , Represión Enzimática/fisiología , Regulación Bacteriana de la Expresión Génica , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Streptomyces lividans/genética , Streptomyces lividans/crecimiento & desarrollo , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Open reading frame SCO3571 of Streptomyces coelicolor encodes a protein of the cyclic AMP (cAMP) receptor protein (CRP) superfamily of regulatory proteins. A mutant revealed a dramatic defect in germination, followed by growth delay and earlier sporulation. This phenotype correlates with those of an adenylate cyclase (cya) mutant that cannot synthesize cAMP. This finding suggests that S. coelicolor may use a Cya-cAMP-CRP system to trigger complex physiological processes such as morphogenesis.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Streptomyces/crecimiento & desarrollo , Streptomyces/fisiología , Secuencia de Bases , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/genética , Elementos de Facilitación Genéticos , Datos de Secuencia Molecular , Morfogénesis , Esporas Bacterianas/fisiología , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
An immunodominant envelope glycoprotein is encoded by the human herpesvirus 8 (HHV-8) (also termed Kaposi's sarcoma-associated herpesvirus) K8.1 gene. The functional role of glycoprotein K8.1 is unknown, and recognizable sequence homology to K8.1 is not detectable in the genomes of most other closely related gammaherpesviruses, such as herpesvirus saimiri or Epstein-Barr virus. In search for a possible function for K8.1, we expressed the ectodomain of K8.1 fused to the Fc part of human immunoglobulin G1 (K8.1DeltaTMFc). K8.1DeltaTMFc specifically bound to the surface of cells expressing glycosaminoglycans but not to mutant cell lines negative for the expression of heparan sulfate proteoglycans. Binding of K8.1DeltaTMFc to mammalian cells could be blocked by heparin. Interestingly, the infection of primary human endothelial cells by HHV-8 could also be blocked by similar concentrations of heparin. The specificity and affinity of these interactions were then determined by surface plasmon resonance measurements using immobilized heparin and soluble K8.1. This revealed that K8.1 binds to heparin with an affinity comparable to that of glycoproteins B and C of herpes simplex virus, which are known to be involved in target cell recognition by binding to cell surface proteoglycans, especially heparan sulfate. We conclude that cell surface glycosaminoglycans play a crucial role in HHV-8 target cell recognition and that HHV-8 envelope protein K8.1 is at least one of the proteins involved.
Asunto(s)
Heparitina Sulfato/metabolismo , Herpesvirus Humano 8/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/metabolismo , Cartilla de ADN , Ratones , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
We have surveyed the publicly available genome sequence of Corynebacterium diphtheriae (www.sanger.ac.uk) to identify components of the phosphotransferase system (PTS), which plays a central role in carbon metabolism in many bacteria. Three gene loci were found to contain putative pts genes. These comprise: (i) the genes of the general phosphotransferases enzyme I (ptsI) and HPr (ptsH), a fructose-specific enzyme IIABC permease (fruA), and a fructose 1-phosphate kinase (fruK); (ii) a gene that encodes an enzyme IIAB of the fructose/mannitol family, and a novel HPr-like gene, ptsF, that encodes an HPr domain fused to a domain of unknown function; (iii) and a gene for a glucose-specific enzyme IIBCA (ptsG). A search for genes that may be putative PTS-targets or that may operate in general carbon regulation revealed a possible regulatory gene encoding an antiterminator protein downstream from ptsG. Furthermore, genes were detected encoding glycerol kinase, glucose kinase, and a homologue of the global activator of carbon catabolite repression in Escherichia coli, CAP. The possible significance of these observations in carbon metabolism and the novel features of the detected genes are discussed.
Asunto(s)
Proteínas Bacterianas , Corynebacterium diphtheriae/enzimología , Corynebacterium diphtheriae/genética , Genoma Bacteriano , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Secuencia de Bases , Mapeo Cromosómico , Secuencia de Consenso , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismoRESUMEN
The high-GC Gram-positive actinomycete Corynebacterium glutamicum is commercially exploited as a producer of amino acids that are used as animal feed additives and flavor enhancers. Despite its beneficial role, carbon metabolism and its possible influence on amino acid metabolism is poorly understood. We have addressed this issue by analyzing the phosphotransferase system (PTS), which in many bacteria controls the flux of nutrients and therefore regulates carbon metabolism. The general PTS phosphotransferases enzyme I (EI) and HPr were characterized by demonstration of PEP-dependent phosphotransferase activity. An EI mutant exhibited a pleiotropic negative phenotype in carbon utilization. The role of the PTS as a major sugar uptake system was further demonstrated by the finding that glucose and fructose negative mutants were deficient in the respective enzyme II PTS permease activities. These carbon sources also caused repression of glutamate uptake, which suggests an involvement of the PTS in carbon regulation. The observation that no HPr kinase/phosphatase could be detected suggests that the mechanism of carbon regulation in C. glutamicum is different to the one found in low-GC Gram-positive bacteria.
Asunto(s)
Proteínas Bacterianas , Corynebacterium/enzimología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Alimentación Animal , Animales , Corynebacterium/genética , Fructosa/metabolismo , Glucosa/metabolismo , Mutagénesis , FenotipoRESUMEN
The catabolite control protein CcpA is a central regulator in low-G+C-content gram-positive bacteria. It confers carbon catabolite repression to numerous genes required for carbon utilization. It also operates as a transcriptional activator of genes involved in diverse phenomena, such as glycolysis and ammonium fixation. We have cloned the ccpA region of Lactobacillus pentosus. ccpA encodes a protein of 336 amino acids exhibiting similarity to CcpA proteins of other bacteria and to proteins of the LacI/GalR family of transcriptional regulators. Upstream of ccpA was found an open reading frame with similarity to the pepQ gene, encoding a prolidase. Primer extension experiments revealed two start sites of transcription for ccpA. In wild-type cells grown on glucose, mRNA synthesis occurred only from the promoter proximal to ccpA. In a ccpA mutant strain, both promoters were used, with increased transcription from the distant promoter, which overlaps a presumptive CcpA binding site called cre (for catabolite responsive element). This suggests that expression of ccpA is autoregulated. Determination of the expression levels of CcpA in cells grown on repressing and nonrepressing carbon sources revealed that the amounts of CcpA produced did not change significantly, leading to the conclusion that the arrangement of two promoters may ensure constant expression of ccpA under various environmental conditions. A comparison of the genetic structures of ccpA regions revealed that lactic acid bacteria possess the gene order pepQ-ccpA-variable while the genetic structure in bacilli and Staphylococcus xylosus is aroA-ccpA-variable-acuC.
Asunto(s)
Proteínas Bacterianas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Lactobacillus/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Southern Blotting , Western Blotting , Carbono/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/química , Lactobacillus/crecimiento & desarrollo , Lactobacillus/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Proteínas Represoras/química , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
Our research group is studying the phosphotransferase system (PTS) of Streptomyces coelicolor, which, in other bacteria, is centrally involved in carbon source uptake and regulation. We have surveyed the public available S. coelicolor genome sequence produced by the ongoing genome sequencing project for pts gene homologues (http://www.sanger.ac.uk/Projects/S_coelicolor/). Three genes encoding homologues of the general PTS components enzyme I (ptsI), HPr (ptsH), and enzyme IIA(Crr) (crr; IIA(Glc)-homologue) and six genes encoding homologues of sugar-specific PTS components were identified. The deduced primary sequences of the sugar-specific components shared significant similarities to PTS permeases of the mannitol/fructose family and of the glucose/sucrose family. A model is presented, in which possible functions of the novel described PTS homologues are discussed.
Asunto(s)
Proteínas Bacterianas , Evolución Molecular , Genoma Bacteriano , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Filogenia , Streptomyces/genética , Streptomyces/metabolismo , Secuencia de Aminoácidos , Bacterias/clasificación , Bacterias/genética , Transporte Biológico , Secuencia de Consenso , Proteínas de Escherichia coli , Prueba de Complementación Genética , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Programas Informáticos , Streptomyces/enzimologíaRESUMEN
Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.
Asunto(s)
Glucoquinasa/genética , Glucoquinasa/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Glucoquinasa/química , Cinética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not well-conserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein. Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose. HPr-P could also phosphorylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.
Asunto(s)
Proteínas Bacterianas , Genes Bacterianos , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Streptomyces/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/genética , Clonación Molecular , Secuencia de Consenso , Escherichia coli , Proteínas de Escherichia coli , Evolución Molecular , Prueba de Complementación Genética , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos , Fragmentos de Péptidos/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/clasificación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosforilación , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/metabolismo , Filogenia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Staphylococcus aureus/genéticaRESUMEN
HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in gram-positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6-bisphosphate and inhibited by inorganic phosphate. In contrast to wild-type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation-dependent carbon control mechanisms in Gram-positive bacteria.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas , Adenosina Trifosfato/farmacología , Regulación Alostérica/fisiología , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Activación Enzimática/fisiología , Inhibidores Enzimáticos , Escherichia coli/genética , Fructosadifosfatos/farmacología , Genoma Bacteriano , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas , Proteínas Recombinantes/química , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
The Klebsiella pneumoniae genes scrA and scrB are indispensable for sucrose (Scr) utilisation. Gene scrA codes for an Enzyme IIScr (IIScr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), while scrB encodes a sucrose 6-phosphate specific invertase. A 3.7 kbscr AB DNA fragment has been cloned from K. pneumoniae and expressed in Escherichia coli. Its nucleotide sequence was determined and the coding regions for scrA (1371 bp) and scrB (1401 bp) were identified by genetic complementation, enzyme activity test and radiolabelling of the gene products. In addition, the nucleotide sequence of the scrB gene from conjugative plasmid pUR400 isolated from Salmonella typhimurium was also determined and errors in the previously published sequence of the scrA gene of pUR400 were corrected. Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes. Based on the analysis of seven IIScr proteins, a hypothetical model of the secondary structure of IIScr is proposed.
Asunto(s)
Glicósido Hidrolasas/genética , Klebsiella pneumoniae/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Mapeo Cromosómico , Clonación Molecular , ARN Polimerasas Dirigidas por ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica/genética , Genes Bacterianos , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Klebsiella pneumoniae/enzimología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Plásmidos , Regiones Promotoras Genéticas/genética , Estructura Secundaria de Proteína , Ribosomas/metabolismo , Salmonella typhimurium/genética , Análisis de Secuencia , Homología de Secuencia de Ácido Nucleico , Sacarosa/metabolismo , Proteínas Virales , beta-FructofuranosidasaRESUMEN
The complete sequence of the Mycoplasma genitalium chromosome has recently been determined. We here report analyses of the genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system, PTS. These genes encode (1) Enzyme I, (2) HPr, (3) a glucose-specific Enzyme IICBA, (4) an inactive glucose-specific Enzyme IIB, lacking the active site cysteyl residue, and (5) a fructose-specific Enzyme IIABC. Some of the unique features of these genes and their enzyme products are as follows. (1) Each of the genes is encoded within a distinct operon. (2) Both Enzyme I and HPr have basic isoelectric points. (3) The glucose-specific Enzyme IIC bears a centrally located, hydrophilic, 200 amino acyl residue insert that lacks sequence similarity with any protein in the current database. (4) The fructose-specific Enzyme II has a domain order (IIABC), different from those of previously characterized fructose permeases, and its IIA domain more closely resembles the IIANtr protein of Escherichia coli than other fructose-specific IIA domains. The potential significance of these novel features is discussed.
Asunto(s)
Proteínas Bacterianas , Genes Bacterianos , Genoma Bacteriano , Mycoplasma/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Análisis de Secuencia de ADN/métodos , Evolución Molecular , Familia de Multigenes , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/clasificación , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/genética , FilogeniaRESUMEN
A 4471 bp region between crr and cysM on the Salmonella typhimurium chromosome (49.5 min) has been sequenced. Five ORFs were found within this region, one of which is likely to be the putative regulatory gene, ptsJ, that corresponds in map position to a gene which when mutated allows expression of a cryptic Enzyme I of the phosphotransferase system. The deduced amino acid sequence of the encoded protein is similar to those of several open reading frames (ORFs) including ORFT2 of Rhodobacter spheroides with which it is 28% identical throughout most of its length (comparison score of 21 S.D.). PtsJ exhibits a putative, N-terminal, helix-turn-helix, DNA binding domain that is similar in sequence to those in members of the GntR family of transcriptional regulators. Analyses of the sequences of the ORFs encoded within this region are presented.
Asunto(s)
Proteínas de Escherichia coli , Genes Bacterianos , Genes Reguladores , Sistemas de Lectura Abierta , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/genética , Salmonella typhimurium/genética , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas de Unión al ADN/genética , Secuencias Hélice-Asa-Hélice , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Proteínas Represoras/genética , Mapeo Restrictivo , Rhodobacter sphaeroides/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Streptomyces lividans, S. coelicolor and S. griseofuscus were examined for the presence of the enzymes of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). All three species were shown to possess Enzyme I, HPr and fructose-specific Enzyme II (IIFru) activities. In S. lividans and S. coelicolor, all three PTS enzymes were fructose-inducible, but in S. griseofuscus the system was expressed constitutively. These organisms apparently lack the HPr(Ser) kinase and HPr(Ser-P) phosphatase that characterize low-GC Gram-positive bacteria.
Asunto(s)
Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Streptomyces/enzimología , Bacillus subtilis/enzimología , Fructosa/metabolismo , Prueba de Complementación Genética , Concentración de Iones de Hidrógeno , Cinética , Fosforilación , Especificidad de la Especie , Streptomyces/genéticaRESUMEN
We have characterized a new family of proteins (the ROK family) which includes six transcriptional repressors for sugar catabolic operons, three sugar kinases, and three unidentified open reading frames. Analysis of the aligned sequences and phylogenetic tree construction allow predictions regarding the functional nature of conserved domains and residues within these proteins as well as the pathway of evolutionary divergence that gave rise to the family.
Asunto(s)
Bacterias/genética , Evolución Biológica , Fosfotransferasas/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Bacterias/enzimología , Proteínas Bacterianas/genética , Sitios de Unión/genética , Carbohidratos , Proteínas de Unión al ADN/genética , Genes Bacterianos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
In enteric bacteria, chromosomally encoded permeases specific for lactose, maltose, and melibiose are allosterically regulated by the glucose-specific enzyme IIA of the phosphotransferase system. We here demonstrate that the plasmid-encoded raffinose permease of enteric bacteria is similarly subject to this type of inhibition.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/farmacología , Plásmidos/genética , Rafinosa/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Transporte Biológico , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Gram-negative bacteria are able to respond chemotactically to carbohydrates which are substrates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The mechanism of signal transduction in PTS-mediated chemotaxis is different from the well-studied mechanism involving methyl-accepting chemotaxis proteins (MCPs). In PTS-mediated chemotaxis, carbohydrate transport is required, and phosphorylation seems to be involved in both excitation and adaptation. In this review the roles of the components of the PTS in chemotactic signal transduction are discussed.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Quimiotaxis , Bacterias Gramnegativas/metabolismo , Proteínas de la Membrana/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Transducción de Señal , Proteínas Bacterianas/metabolismo , Transporte Biológico , Proteínas Quimiotácticas Aceptoras de Metilo , Modelos Biológicos , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
During the molecular analysis of a plasmid-coded sucrose metabolic pathway of enteric bacteria, a gene, scrY, was found whose product, ScrY, had all the properties of a bacterial porin (Schmid et al., 1988). Loss of this protein (Mr 58 kDa), localized in the outer membrane, led, as shown here, to an increase in the apparent Km for sucrose transport in whole cells from 10 microM in wild-type cells to 300 microM in mutant cells. This contrasts with the Km for sucrose phosphorylation as measured in membrane vesicles from mutant and wild-type cells, which remained unchanged at about 10 microM, and reflects the activity of the sucrose-specific Enzymell of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) responsible for uptake through the inner membrane. Furthermore, the presence of ScrY restored growth on maltodextrins in cells devoid of LamB, thus complementing the lack of this maltoporin. The amino acid sequence deduced from the DNA sequence was determined for the plasmid-coded and the ScrY porin coded in the chromosome of Klebsiella pneumoniae. Both show high identity (86%) to each other, and to the channel domain of LamB, further corroborating the conclusion that they constitute porins.