Rethinking the roles of CRP, cAMP, and sugar-mediated global regulation in the Vibrionaceae.

Many proteobacteria modulate a suite of catabolic genes using the second messenger cyclic 3', 5'-AMP (cAMP) and the cAMP receptor protein (CRP). Together, the cAMP-CRP complex regulates target promoters, usually by activating transcription. In the canonical model, the phosphotransferase system (PTS), and in particular the EIIA(Glc) component for glucose uptake, provides a mechanistic link that modulates cAMP levels depending on glucose availability, resulting in more cAMP and activation of alternative catabolic pathways when glucose is unavailable. Within the Vibrionaceae, cAMP-CRP appears to play the classical role in modulating metabolic pathways; however, it also controls functions involved in natural competence, bioluminescence, pheromone signaling, and colonization of animal hosts. For this group of marine bacteria, chitin is an ecologically relevant resource, and chitin's monomeric sugar N-acetylglucosamine (NAG) supports robust growth while also triggering regulatory responses. Recent studies with Vibrio fischeri indicate that NAG and glucose uptake share EIIA(Glc), yet the responses of cAMP-CRP to these two carbon sources are starkly different. Moreover, control of cAMP levels appears to be more dominantly controlled by export and degradation. Perhaps more surprisingly, although CRP may require cAMP, its activity can be controlled in response to glucose by a mechanism independent of cAMP levels. Future studies in this area promise to shed new light on the role of cAMP and CRP.

Growth on glucose decreases cAMP-CRP activity while paradoxically increasing intracellular cAMP in the light-organ symbiont Vibrio fischeri.

Proteobacteria often co-ordinate responses to carbon sources using CRP and the second messenger cyclic 3', 5'-AMP (cAMP), which combine to control transcription of genes during growth on non-glucose substrates as part of the catabolite-repression response. Here we show that cAMP-CRP is active and important in Vibrio fischeri during colonization of its host squid Euprymna scolopes. Moreover, consistent with a classical role in catabolite repression, a cAMP-CRP-dependent reporter showed lower activity in cells grown in media amended with glucose rather than glycerol. Surprisingly though, intracellular cAMP levels were higher in glucose-grown cells. Mutant analyses were consistent with predictions that CyaA was responsible for cAMP generation, that the EIIA(Glc) component of glucose transport could enhance cAMP production and that the phophodiesterases CpdA and CpdP consumed intracellular and extracellular cAMP respectively. However, the observation of lower cAMP levels in glycerol-grown cells seemed best explained by changes in cAMP export, via an unknown mechanism. Our data also indicated that cAMP-CRP activity decreased during growth on glucose independently of crp's native transcriptional regulation or cAMP levels. We speculate that some unknown mechanism, perhaps carbon-source-dependent post-translational modulation of CRP, may help control cAMP-CRP activity in V.fischeri.

Modeling Analysis of Signal Sensitivity and Specificity by Vibrio fischeri LuxR Variants.

The LuxR protein of the bacterium Vibrio fischeri belongs to a family of transcriptional activators that underlie pheromone-mediated signaling by responding to acyl-homoserine lactones (-HSLs) or related molecules. V. fischeri produces two acyl-HSLs, N-3-oxo-hexanoyl-HSL (3OC6-HSL) and N-octanoyl-HSL (C8-HSL), each of which interact with LuxR to facilitate its binding to a "lux box" DNA sequence, thereby enabling LuxR to activate transcription of the lux operon responsible for bioluminescence. We have investigated the HSL sensitivity of four different variants of V. fischeri LuxR: two derived from wild-type strains ES114 and MJ1, and two derivatives of LuxRMJ1 generated by directed evolution. For each LuxR variant, we measured the bioluminescence induced by combinations of C8-HSL and 3OC6-HSL. We fit these data to a model in which the two HSLs compete with each other to form multimeric LuxR complexes that directly interact with lux to activate bioluminescence. The model reproduces the observed effects of HSL combinations on the bioluminescence responses directed by LuxR variants, including competition and non-monotonic responses to C8-HSL and 3OC6-HSL. The analysis yields robust estimates for the underlying dissociation constants and cooperativities (Hill coefficients) of the LuxR-HSL complexes and their affinities for the lux box. It also reveals significant differences in the affinities of LuxRMJ1 and LuxRES114 for 3OC6-HSL. Further, LuxRMJ1 and LuxRES114 differed sharply from LuxRs retrieved by directed evolution in the cooperativity of LuxR-HSL complex formation and the affinity of these complexes for lux. These results show how computational modeling of in vivo experimental data can provide insight into the mechanistic consequences of directed evolution.

Cyclic AMP receptor protein regulates pheromone-mediated bioluminescence at multiple levels in Vibrio fischeri ES114.

Bioluminescence in Vibrio fischeri ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also modulated in response to environmental stimuli. Previous studies suggested that expression of the pheromone-dependent bioluminescence activator LuxR is regulated in response to glucose by cyclic AMP (cAMP) receptor protein (CRP) (P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 164:45-50, 1985; P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 170:4040-4046, 1988; P. V. Dunlap, J. Bacteriol. 171:1199-1202, 1989; and W. F. Friedrich and E. P. Greenberg, Arch. Microbiol. 134:87-91, 1983). Consistent with this model, we found that bioluminescence in V. fischeri ES114 is modulated by glucose and stimulated by cAMP. In addition, a Δcrp mutant was ∼100-fold dimmer than ES114 and did not increase luminescence in response to added cAMP, even though cells lacking crp were still metabolically capable of producing luminescence. We further discovered that CRP regulates not only luxR but also the alternative pheromone synthase gene ainS. We found that His-tagged V. fischeri CRP could bind sequences upstream of both luxR and ainS, supporting bioinformatic predictions of direct regulation at both promoters. Luminescence increased in response to cAMP if either the ainS or luxR system was under native regulation, suggesting cAMP-CRP significantly increases luminescence through both systems. Finally, using transcriptional reporters in transgenic Escherichia coli, we elucidated two additional regulatory connections. First, LuxR-independent basal transcription of the luxI promoter was enhanced by CRP. Second, the effect of CRP on the ainS promoter depended on whether the V. fischeri regulatory gene litR was also introduced. These results suggest an integral role for CRP in pheromone signaling that goes beyond sensing cell density.

Contribution of rapid evolution of the luxR-luxI intergenic region to the diverse bioluminescence outputs of Vibrio fischeri strains isolated from different environments.

Vibrio fischeri serves as a valuable model of bacterial bioluminescence, its regulation, and its functional significance. Light output varies more than 10,000-fold in wild-type isolates from different environments, yet dim and bright strains have similar organization of the light-producing lux genes, with the activator-encoding luxR divergently transcribed from luxICDABEG. By comparing the genomes of bright strain MJ11 and the dimmer ES114, we found that the lux region has diverged more than most shared orthologs, including those flanking lux. Divergence was particularly high in the intergenic sequence between luxR and luxI. Analysis of the intergenic lux region from 18 V. fischeri strains revealed that, with one exception, sequence divergence essentially mirrored strain phylogeny but with relatively high substitution rates. The bases conserved among intergenic luxR-luxI sequences included binding sites for known regulators, such as LuxR and ArcA, and bases of unknown significance, including a striking palindromic repeat. By using this collection of diverse luxR-luxI regions, we found that expression of P(luxI)-lacZ but not P(luxR)-lacZ transcriptional reporters correlated with the luminescence output of the strains from which the promoters originated. We also found that exchange of a small stretch of the luxI-luxR intergenic region between two strains largely reversed their relative brightness. Our results show that the luxR-luxI intergenic region contributes significantly to the variable luminescence output among V. fischeri strains isolated from different environments, although other elements of strain backgrounds also contribute. Moreover, the lux system appears to have evolved relatively rapidly, suggesting unknown environment-specific selective pressures.