Requirement for autoinducer in transcriptional negative autoregulation of the Vibrio fischeri luxR gene in Escherichia coli

Dunlap, Paul V.; Ray, Jasodhara

doi:10.1128/jb.171.6.3549-3552.1989

Cited by 43 publications

(63 citation statements)

References 19 publications

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“…In this regard, LuxR weakly represses expression of its own gene. Moreover, under normal conditions, this autorepression depends on VAI (15,16) suggesting that interaction with the signal is required for DNA binding. Unfortunately, TraR does not repress any known genes, making it difficult to discriminate between functions involved in transcriptional activation and DNA binding.…”

mentioning

confidence: 99%

Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity

Luo

Farrand

1999

Proc. Natl. Acad. Sci. U.S.A.

143

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TraR, a member of the LuxR family of quorum-sensing transcription factors, is responsible for the population density-dependent regulation of Ti plasmid conjugal transfer. The protein requires as coinducer an acylhomoserine lactone signal molecule called AAI (Agrobacterium autoinducer) that is produced by the bacteria themselves. TraR only activates its target genes, making it difficult to determine whether interaction with AAI is required for binding DNA or for initiating transcription. To assess this, we converted TraR into a repressor by placing a copy of the tra box, an 18-bp inverted repeat believed to be the recognition site for this protein, over the ؊10 region of a promoter driving expression of lacZ. Repression of this promoter by TraR depended on AAI or, at higher concentrations, VAI, the closely related signal of Vibrio fischeri. C-terminal deletions as short as 2 aa and N-terminal deletions as short as 4 aa in TraR abolished both repressor and activator functions. The Cterminal mutants were strongly dominant over TraR, suggesting that they can form heteromultimers with the wild-type activator. Mutants of TraR with substitutions at Asp-10 and Gly-123 failed to activate a positively controlled reporter but continued to repress the chimeric promoter in an AAIdependent manner. We conclude that TraR recognizes the tra box as its binding site, that binding of TraR to this site depends on AAI, and that the N-terminal half of the protein contains one or more domains that are required for activation but not for multimerization, for interaction with the acylhomoserine lactone, or for DNA binding.

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mentioning

confidence: 99%

Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity

Luo

Farrand

1999

Proc. Natl. Acad. Sci. U.S.A.

143

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“…Alternatively, supernatants were extracted with acidified ethyl acetate (100 l1 of glacial acetic acid in 1 liter of ethyl acetate) and dried as described previously (10) and then redissolved in assay medium. Next, an exponential-phase culture (A66, = 0.3) of the autoinducer assay strain, E. coli PD100(pPD749, pJR5;51) (9), was added to a final A,,o of 0.01, and subsequent luminescence and growth were monitored. The growth rates of the autoinducer assay strain within experiments were essentially similar.…”

Section: Methodsmentioning

confidence: 99%

“…For the measurement of autoinducer activity, filter-sterilized supernatants of high-density cultures (A6, of 1.5 in LBS for V fischeri and 2.0 for E. coli) (i.e., conditioned media) were mixed 1:1 with autoinducer assay medium (an LB-Tris medium) (9). Alternatively, supernatants were extracted with acidified ethyl acetate (100 l1 of glacial acetic acid in 1 liter of ethyl acetate) and dried as described previously (10) and then redissolved in assay medium.…”

Section: Methodsmentioning

confidence: 99%

“…Consistent with earlier results, luminescence in MJ-211 exhibited substantial lux operon transcriptional activation in the absence of added autoinducer. To determine if the luxI mutant actually produced autoinducer activity, cell-free supernatants of medium conditioned by the growth of MJ-211 were examined by a sensitive assay for autoinducers that activate expression of the V fischeri luminescence system (9). Medium conditioned by MJ-211 activated lux operon expression (Fig.…”

Section: Production Of Autoinducer Activity By Uxi Mutants Of Vmentioning

confidence: 99%

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Mutant analysis of luminescence and autoinduction in a marine bacterium

Kuo¹

1994

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The marine symbiotic bacterium Vibriofischeri is striking for its ability both to emit light and to dramatically regulate light emission using a cell-to-cell signalling mechanism called autoinduction.The latter is mediated by a signal molecule called the "autoinducer". The mechanistic bases of both luminescence and autoinduction are well known in V. fischeri, but this knowledge is mostly derived from studies of the cloned luminescence and autoinduction genes expressed in Escherichia coli. In this study, luminescence and autoinduction mutations were systematically generated in V. fischeri to explore aspects of luminescence and autoinduction not addressable in E. coli, such as the adaptive significance of luminescence. Most dramatically, the mutants revealed the presence of multiple autoinducers and autoinducer synthases in V. fischeri. One of the autoinducers (autoinducer-2, or AI-2) was chemically purified and shown to be Noctanoyl-L-homoserine lactone. The genetic locus encoding the AI-2 synthase was cloned and designated ain (autoinducer). Manipulation of ain and AI-2 in V. fischeri demonstrated that the function of AI-2 appears to be to inhibit rather than to promote autoinduction. 2 ACKNOWLEDGMENTS

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“…The type of carbon source provided for growth also influences luminescence (Ruby & Nealson, 1976;Friedrich & Greenberg, 1983;Dunlap & Greenberg, 1985, 1988, 1991Dunlap & Ray, 1989;Dunlap, 1989). Therefore, to examine the luminescence behaviour of V. Jischeri MJ-1 growing on CAMP, cells were inoculated into minimal medium broth containing CAMP, glucose, or ribose (10 mM each), and growth and luminescence were monitored.…”

Section: Growth and Luminescence Of Vjischeri On Camp As A Sole Carbmentioning

confidence: 99%

Growth of the marine luminous bacterium Vibrio fischeri on 3':5'-cyclic AMP: correlation with a periplasmic 3':5'-cyclic AMP phosphodiesterase

Dunlap¹,

Muêller²,

Lisa³

et al. 1992

Journal of General Microbiology

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The marine luminous bacterium Vibrio jischeri, the species-specific light-organ symbiont of monocentrid (pinecone) fish, grew on 3':5'-cyclic adenosine monophosphate (CAMP) as a sole source of carbon and energy, a capability not previously described in bacteria. In a minimal, chemically defined medium containing cAMP as the sole source of carbon and energy, V.jischeri cells grew more slowly than on glucose or ribose, but as quickly as on 5'-AMP. Expression of luminescence, which is dependent on cAMP in V.jischeri, was stimulated in cells grown on cAMP compared to cells grown on glucose or ribose. All strains of Kjischeri tested (MJ-I, B-61, ATCC 7744, MJ-A1, CG-A1) grew on CAMP, as did strains of two other marine luminous bacteria, V. fogei and Photobacterium phosphoreum, and strains of the terrestrial enteric bacterium Serratia marcescescens. Other tested species of marine luminous bacteria (P. feiognathi, Shewaneffa [ Afteromonas] hamhi, V. hmeyi, V. orientalis, V. splendidus) and terrestrial enteric bacteria (Escherichia cofi, Salmonella typhimurium), which grew on 5'-AMP or ribose, did not grow on CAMP. Assays on intact cells and periplasmic extracts revealed that V.jischeri MJ-1 produced a periplasmic 3': 5'-CAMP phosphodiesterase (CAMP phosphodiesterase) of very high specific activity 19.0 p o l phosphate released (mg protein)-' min-l ] and narrow substrate specificity (3': 5'-CAMP and 3': 5'-cGMP were attacked). The novel periplasmic location and unusually high activity of cAMP phosphodiesterase appear to account for the ability of this species to grow on CAMP. The periplasmic cAMP phosphodiesterase of Kjischeri might play a role in degrading free cAMP in seawater or in a CAMP-mediated aspect of the light organ symbiosis with monocentrid fish.

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Requirement for autoinducer in transcriptional negative autoregulation of the Vibrio fischeri luxR gene in Escherichia coli

Cited by 43 publications

References 19 publications

Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity

Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity

Mutant analysis of luminescence and autoinduction in a marine bacterium

Growth of the marine luminous bacterium Vibrio fischeri on 3':5'-cyclic AMP: correlation with a periplasmic 3':5'-cyclic AMP phosphodiesterase

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