RESUMEN
DNA uptake by naturally competent bacteria provides cells with both genetic information and nucleotides. In Haemophilus influenzae, competence development requires both cAMP and an unidentified signal arising under starvation conditions. To investigate this signal, competence induction was examined in media supplemented with nucleic acid precursors. The addition of physiological levels of AMP and GMP reduced competence 200-fold and prevented the normal competence-induced transcription of the essential competence genes comA and rec-2. The rich medium normally used for growth allows only limited competence. Capillary electrophoresis revealed only a subinhibitory amount of AMP and no detectable GMP, and the addition of AMP or GMP to this medium also reduced competence 20- to 100-fold. Neither a functional stringent response system nor a functional phosphoenolpyruvate:glycose phosphotransferase system (PTS) was found to be required for purine-mediated repression. Added cAMP partially restored both transcription of competence genes and competence development, suggesting that purines may reduce the response to cAMP. Potential binding sites for the PurR repressor were identified in several competence genes, suggesting that competence is part of the PUR regulon. These observations are consistent with models of competence regulation, in which depleted purine pools signal the need for nucleotides, and support the hypothesis that competence evolved primarily for nucleotide acquisition.
Asunto(s)
ADN Bacteriano , Haemophilus influenzae/genética , Precursores de Ácido Nucleico , Nucleósidos de Purina , Adenosina Monofosfato , AMP Cíclico , Genes Bacterianos , Guanosina Monofosfato , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato , Transcripción GenéticaRESUMEN
Development of competence for DNA uptake by the bacterium Haemophilus influenzae is tightly regulated, and expression of the cell's complement of competence genes is absolutely dependent on the cAMP-CRP complex. A second regulator of competence may maximize competence under starvation conditions. Several investigators have recently identified a consensus sequence (competence regulatory element, CRE) in the promoter regions of some competence genes and have proposed that this may be a binding site for Sxy (TfoX), a putative positive regulator of competence. However, a scoring method that reliably ranks candidate binding sites according to affinity for the cognate binding protein predicts that the cAMP-CRP complex will bind CRE sequences with high affinity. Moreover, the predicted Sxy protein lacks recognizable DNA-binding motifs and has not been shown to bind DNA. No other consensus sequences (putative binding sites) were identified in the promoter regions of competence genes. These observations suggest that the proposed competence-specific regulatory elements are in fact CRP-binding sites, and highlight the central role of cAMP-an established bacterial mediator of the response to nutritional stress-in competence regulation. Minor sequence elements uniquely conserved in the set of CRE sequences are predicted to reduce CRP affinity, and a model is suggested in which a secondary regulator of competence genes may interact with CRP under certain conditions to stabilize the initiation complex.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Haemophilus influenzae/metabolismo , Transactivadores , Animales , Proteínas Bacterianas/metabolismo , Sitios de Unión , AMP Cíclico/metabolismo , Escherichia coli/metabolismo , Regiones Promotoras GenéticasRESUMEN
Changes in intracellular 3',5' cyclic AMP (cAMP) concentration regulate the development of natural competence in Haemophilus influenzae. In Escherichia coli, cAMP levels are modulated by a cAMP phosphodiesterase encoded by the cpdA gene. We have used several approaches to demonstrate that the homologous icc gene of H. influenzae encodes a functional cAMP phosphodiesterase and that this gene limits intracellular cAMP and thereby influences competence and other cAMP-dependent processes. In E. coli, expression of cloned icc reduced both cAMP-dependent sugar fermentation and beta-galactosidase expression, as has been shown for cpdA. In H. influenzae, an icc null mutation increased cAMP-dependent sugar fermentation and competence development in strains where these processes are limited by mutations reducing cAMP synthesis. When endogenous production of cAMP was eliminated by a cya mutation, an icc strain was 10,000-fold more sensitive to exogenous cAMP than an icc+ strain. The icc strain showed moderately elevated competence under noninducing conditions, as expected, but had subnormal competence increases at onset of stationary phase in rich medium, and on transfer to a nutrient-limited medium, suggesting that excessive cAMP may interfere with induction. Consistent with this finding, a cya strain cultured in 1 mM cAMP failed to develop maximal competence on transfer to inducing conditions. Thus, by limiting cAMP levels, the H. influenzae cAMP phosphodiesterase may coordinate its responses to nutritional stress, ensuring optimal competence development.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , AMP Cíclico/metabolismo , Haemophilus influenzae/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Secuencia de Bases , Clonación Molecular , AMP Cíclico/biosíntesis , AMP Cíclico/farmacología , Cartilla de ADN , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/genética , Mutagénesis Sitio-DirigidaRESUMEN
Changes in intracellular cAMP concentration play important roles in Haemophilus influenzae, regulating both sugar utilization and competence for natural transformation. In enteric bacteria, cAMP levels are controlled by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in response to changes in availability of the preferred sugars it transports. We have demonstrated the existence of a simple PTS in H. influenzae by several methods. We have cloned the H. influenzae ptsI gene, encoding PTS Enzyme I; genome analysis locates it in a pts operon structurally homologous to those of enteric bacteria. In vitro phosphorylation assays confirmed the presence of functional PTS components. A ptsI null mutation reduced fructose uptake to 1% of the wild-type rate, and abolished fructose fermentation even when exogenous cAMP was provided. The ptsI mutation also prevented fermentation of ribose and galactose, but utilization of these cAMP-dependent sugars was restored by addition of cAMP. In wild-type cells the non-metabolizable fructose analogue xylitol prevented fermentation of these sugars, confirming that the fructose PTS regulates cAMP levels. Development of competence under standard inducing conditions was reduced 250-fold by the ptsI mutation, unless cells were provided with exogenous cAMP. Competence is thus shown to be under direct nutritional control by a fructose-specific PTS.