RESUMEN
The conditioning of culture medium by the production of growth-regulatory substances is a well-established phenomenon with eukaryotic cells. It has recently been shown that many prokaryotes are also capable of modulating growth, and in some cases sensing cell density, by production of extracellular signaling molecules, thereby allowing single celled prokaryotes to function in some respects as multicellular organisms. As Escherichia coli shifts from exponential growth to stationary growth, many changes occur, including cell division leading to formation of short minicells and expression of numerous genes not expressed in exponential phase. An understanding of the coordination between the morphological changes associated with cell division and the physiological and metabolic changes is of fundamental importance to understanding regulation of the prokaryotic cell cycle. The ftsQA genes, which encode functions required for cell division in E. coli, are regulated by promoters P1 and P2, located upstream of the ftsQ gene. The P1 promoter is rpoS-stimulated and the second, P2, is regulated by a member of the LuxR subfamily of transcriptional activators, SdiA, exhibiting features characteristic of an autoinduction (quorum sensing) mechanism. The activity of SdiA is potentiated by N-acyl-homoserine lactones, which are the autoinducers of luciferase synthesis in luminous marine bacteria as well as of pathogenesis functions in several pathogenic bacteria. A compound(s) produced by E. coli itself during growth in Luria Broth stimulates transcription from P2 in an SdiA-dependent process. Another substance(s) enhances transcription of rpoS and (perhaps indirectly) of ftsQA via promoter P1. It appears that this bimodal control mechanism may comprise a fail-safe system, such that transcription of the ftsQA genes may be properly regulated under a variety of different environmental and physiological conditions.
Asunto(s)
Proteínas Bacterianas/genética , División Celular , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/fisiología , Secuencia de Bases , Medios de Cultivo , Proteínas de Unión al ADN/fisiología , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Lactonas/farmacología , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Secuencias Repetitivas de Ácidos Nucleicos , Factor sigma/genética , Transactivadores/genética , Transcripción Genética , Vibrio/genéticaRESUMEN
The phenomenon of cell-density-dependent control of gene expression, called autoinduction, has long been a subject of interest and investigation in bioluminescent marine bacteria. It is now becoming clear that many other bacteria, including animal and plant pathogens, use an autoinduction mechanism to regulate a variety of functions. Cell-density-dependent gene expression provides an excellent example of multicellular behaviour in the prokaryotic kingdom where a single cell is able to communicate and sense when a minimal population unit, a 'quorum' of bacteria, is achieved in order for certain behaviour of the population to be performed efficiently. Regulation of bacterial bioluminescence has been studied for many years and represents the best model system for understanding the mechanism of cell-density-dependent gene expression. This review will focus on transcriptional regulation of the Vibrio fischeri luminescence genes emphasizing the role of the transcriptional activator LuxR and possible autoinduction mechanisms that occur in E. coli. Alternative views and opinions regarding the molecular details of the autoinduction mechanism will be discussed.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras , Transactivadores , Transcripción Genética , Vibrio/genética , Vibrio/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Genes Bacterianos , Mediciones Luminiscentes , Datos de Secuencia Molecular , Operón , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)], a naturally occurring inhibitor of GS. These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range. At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity. ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins. The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds. Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds. Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT. 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution. The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor.
Asunto(s)
Aminobutiratos/farmacología , Escherichia coli/enzimología , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Unión Competitiva , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática/efectos de los fármacos , Reactivadores Enzimáticos , Glutamato-Amoníaco Ligasa/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Fosforilación , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
The enzymes for luminescence in Vibrio fischeri are induced only when a sufficient concentration of a metabolic product (autoinducer) specifically produced by this species accumulates. It has previously been shown that the autoinducer is 3-oxohexanoyl homoserine lactone and that it enters the cells by simple diffusion. To further study the mechanism of induction, we have synthesized several analogs of the autoinducer. The analogs were tested with V. fischeri for their inducing activity and for their ability to inhibit the action of the natural autoinducer. The compounds were found to display various combinations of inducing and inhibiting abilities. None of the compounds tested appeared to have any effect on cells of V. harveyi strain MAV or Photobacterium leiognathi strain 721, but several of the compounds decreased light output by P. phosphoreum strain 8265. These studies show that 1) the site of action of the autoinducer is not highly sterically constrained 2) the autoinducers of other species of luminous bacteria are likely to be quite different from that of V. fischeri and 3) a simple mode in which one autoinducer molecule binds to a single receptor protein site and thus initiates luciferase synthesis is inadequate. The analogs should prove useful in the study of the binding site and mode of action of the autoinducer.