CHEMBIOCHEM HIGHLIGHTS DOI: 10.1002/cbic.201300758
A New Bacterial Chemical Signal: Mapping the Chemical Space Used for Communication Stefan Schulz*[a]
Extracellular chemical communication is a major channel for the exchange of information and is a prerequisite for life. Its biological and chemical aspects have been investigated in many species, from animals, plants and fungi to bacteria and yeasts. Arthropods, especially insects,[1] are favoured for research in this area, but bacterial communication has been explored by chemists since the end of the last century. In these communication systems the term pheromone (more precisely “semiochemical”) is used to describe a compound that transmits information from one individual to another. In bacteria, typical semiochemicals are autoinducers that convey information in a concentration-dependent manner, a phenomenon called “quorum sensing”; it is regulated by a feedback loop.[2] The identification and functional characterisation of a new type of autoinducer has been reported recently by the Bode and Heermann groups;[3] they showed that pyrones represent a new compound class in bacterial communication systems. The quorum sensing system of proteobacteria usually involves LuxI, an autoinducer synthase that produces N-acylhomoserine lactones (AHLs) with variable, mostly fatty-acidderived acyl chains (e.g., 5 and 8, Scheme 1). These AHLs are sensed by a cognate receptor LuxR, which upregulates luxI expression when a certain threshold concentration is reached. Many bacteria have been found to produce and use AHLs for communication.[4] Nevertheless, other signalling compounds must exist, because in many proteobacteria homologues of LuxR are found, but not the corresponding LuxI homologues.[5] Such receptors have been called “orphan” or “solo” LuxRs. The insect pathogenic proteobacterium Photorhabdus luminescens does not produce AHLs, but does express the solo receptor PluR. By using a comparative proteomoic analysis with a DpluR mutant, it was shown that PluR detects an endogenous signal.[3] The analysis revealed that PluR regulates two proteins from the pcfABCDEF operon that are responsible for cell clumping (pcf: Photorabdus clumping factor; see Figure 1). The mutant did not show cell clumping behaviour. For identification of the respective signal a pcfA fluorescence reporter strain was constructed and used in a cell-clumping assay. The target compounds were isolated by XAD 16 adsorption and their structures were elucidated by NMR and MS methods. These compounds proved to be pyrones (termed “photopyrones” by the authors) carrying two alkyl side chains. Photopyr-
[a] Prof. Dr. S. Schulz Institut fr Organische Chemie, Technische Universitt Braunschweig Hagenring 30, 38106 Braunschweig (Germany) E-mail: [email protected]
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. PpyS–PluR signalling in P. luminescens. The photopyrone synthase PpyS produces photopyrones (PPYs) that are sensed by the orphan LuxRtype receptor PluR. Upon signal detection, PluR activates transcription of the pcfABCDEF operon, which in turn leads to cell clumping. Reprinted by permission from Macmillan Publishers Ltd.[3] Copyright: 2013.
ones with branched and unbranched side chains were found; of these photopyrone D (PPYD, 1) showed the highest activity in the reporter system (at as little as 3.5 pm). In the related species Photorhabdus temperata, variants with shorter side chains were found (e.g., PPYA (3) and PPYB (2)), thus demonstrating clear strain-specific photopyrones. PPYA, PPYB and PPYD proved to be active in the bioassays, whereas various AHLs did not induce any activity. Computer modelling and docking experiments with a crystal structure[3, 6] of QscR from Pseudomonas aeruginosa as a model (32 % sequence identity), suggested that PluR is indeed the receptor of the photopyrones. Only two of six conserved amino acids residues of LuxR homologues are present in PluR, thus indicating a different bonding motif,[7] as found for AHL receptors. The results were obtained by using mutants modified at key amino acid positions. These mutants showed largely reduced or absent activity compared to the wild-type protein. Biosynthesis of photopyrones is likely performed with headto-head fusion of two short-chain fatty acids mediated by PpyS. This was confirmed by heterologous expression of the ppyS gene and branched fatty acid synthase genes in Escherichia coli. In the final part of their work,[3] the authors investigated the entire signalling system. The pcf operon was overexpressed in E. coli under the control of a promoter induced by arabinose. The E. coli cells clumped together when arabinose was added, thus showing the function of the pcf operon. Finally, the whole ChemBioChem 2014, 15, 498 – 500
498
CHEMBIOCHEM HIGHLIGHTS
www.chembiochem.org
Scheme 1. Known bacterial signalling compounds (1–10) and similar ones without known function (11, 12).
system (PluR, the pcfABCDEF operon and PpyS) was reconstituted in E. coli. Experiments with these constructs showed that photopyrones are indeed used for cell–cell communication. The compounds triggered secretion of proteins that induce cell clumping. As found in other quorum sensing systems, PPYD production increases during the exponential phase and is highest in the stationary phase; however positive feedback on ppyS expression was not observed. Although these compounds are also produced by the bacterium in the host insect larvae, a direct effect on insect mortality was not observed. However, E. coli expressing the constructs proved highly toxic to the insect larvae. The reason for the clumping behaviour and its benefit for Photorabdus spp. is not known. Closely related to the photopyrones are pseudopyronines (e.g., 4), as have been isolated from Pseudomonas spp.;[8] these differ from photopyrones only in side-chain length and methylation. It seems likely that these compounds function as signals, too. This finding might indicate a more widespread occurrence and importance, similar to that of AHLs like 5 and 8, the most common class of bacterial signalling compounds.[4] Whether the predominance of AHLs in proteobacteria is actually true or is only an artefact of the wealth of information on the genetics of their production, processing and function (allowing ever more studies to be produced) is open to discussion. The number of structural classes of compounds used for communication within bacteria is still small. Given the chemical diversity of bacterial metabolites and their different occurrences in so many ecological niches, there should be a wealth of different signalling compounds waiting to be unravelled. Within the structural features of known bacterial signalling compounds, a certain motif seems to show up. Like the AHLs, photopyrones exhibit side chains (derived from fatty acid me 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
tabolism) attached to a polar group. Bacterial signalling compounds with this feature include the autoinducer-1 of Vibrio cholerae ((S)-3-hydroxytridecan-4-one (6)),[9] the hydroxyalkylquinolone signal family (e.g., 9 from P. aeruginosa),[10] the butyrolactone family in streptomycetes[11] (e.g., A-factor (7))[12] and methylenomycin furans (MMFs, e.g. 10), which are responsible for methylenomycin production in streptomycetes.[13] Several other compounds that have been discussed as potential signalling compounds also fit this scheme, such as the longchain N-acylated amino acids arginine, tryptophane and tyrosine (12) found frequently in hitherto uncultured microorganisms[14] or N-acylated alanine methyl esters (11) produced by marine Roseovarius bacteria.[15] The lipophilic side chain probably helps entering the target cell and reaching the receptor. In summary, the results of the described in-depth study, covering all aspects from identification, function and biosynthesis, are fascinating. They clearly show the function of the photopyrones as autoinducers and broaden our knowledge on the chemistry and function of autoinducers. To elucidate new bacterial signalling compounds (called either pheromones, quorum sensing compounds, autoinducers or semiochemicals), these studies explore an extremely promising avenue. The results of such studies will be certainly of great importance for many fields, from medicine to agriculture and basic research. To understand what is really going on in the invisible world, communication is key. Keywords: LuxR · pheromones · photopyrones Photorhabdus · quorum sensing · signal transduction
·
[1] W. Francke, S. Schulz in Comprehensive Natural Products II (Eds.: L. N. Mander, H.-W. Liu), Elsevier, Oxford, 2010.
ChemBioChem 2014, 15, 498 – 500
499
CHEMBIOCHEM HIGHLIGHTS [2] a) Chemical Communication among Bacteria (Eds.: S. C. Winans, B. L. Bassler), ASM Press, Washington, DC, 2008; b) C. M. Waters, B. L. Bassler, Annu. Rev. Cell Dev. Biol. 2005, 21, 319 – 346. [3] A. O. Brachmann, S. Brameyer, D. Kresovic, I. Hitkova, Y. Kopp, C. Manske, K. Schubert, H. B. Bode, R. Heermann, Nat. Chem. Biol. 2013, 9, 573 – 578. [4] J. S. Dickschat, Nat. Prod. Rep. 2010, 27, 343 – 369. [5] R. J. Case, M. Labbate, S. Kjelleberg, ISME J. 2008, 2, 345 – 349. [6] M. J. Lintz, K.-I. Oinuma, C. L. Wysoczynski, E. P. Greenberg, M. E. A. Churchill, Proc. Natl. Acad. Sci. USA 2011, 108, 15763 – 15768. [7] A. V. Patankar, J. E. Gonzlez, FEMS Microbiol. Rev. 2009, 33, 739 – 756. [8] a) F. Kong, M. P. Singh, G. T. Carter, J. Nat. Prod. 2005, 68, 920 – 923; b) M. Chu, R. Mierzwa, L. Xu, L. He, J. Terracciano, M. Patel, W. Zhao, T. A. Black, T. M. Chan, J. Antibiot. 2002, 55, 215 – 218.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org [9] D. A. Higgins, M. E. Pomianek, C. M. Kraml, R. K. Taylor, M. F. Semmelhack, B. L. Bassler, Nature 2007, 450, 883 – 886. [10] E. Dziel, F. Lpine, S. Milot, J. He, M. N. Mindrinos, R. G. Tompkins, L. G. Rahme, Proc. Natl. Acad. Sci. USA 2004, 101, 1339 – 1344. [11] J. M. Willey, A. A. Gaskell, Chem. Rev. 2011, 111, 174 – 187. [12] S. Horinouchi, Biosci. Biotechnol. Biochem. 2007, 71, 283 – 299. [13] C. Corre, L. Song, S. O’Rourke, K. F. Chater, G. L. Challis, Proc. Natl. Acad. Sci. USA 2008, 105, 17510 – 17515. [14] S. F. Brady, J. Clardy, Org. Lett. 2005, 7, 3613 – 3616. [15] H. Bruns, V. Thiel, S. Voget, D. Patzelt, R. Daniel, I. Wagner-Dçbler, S. Schulz, Chem. Biodiversity 2013, 10, 1559 – 1573. Received: December 2, 2013 Published online on January 16, 2014
ChemBioChem 2014, 15, 498 – 500
500
A New Bacterial Chemical Signal: Mapping the Chemical Space Used for Communication Stefan Schulz*[a]
Extracellular chemical communication is a major channel for the exchange of information and is a prerequisite for life. Its biological and chemical aspects have been investigated in many species, from animals, plants and fungi to bacteria and yeasts. Arthropods, especially insects,[1] are favoured for research in this area, but bacterial communication has been explored by chemists since the end of the last century. In these communication systems the term pheromone (more precisely “semiochemical”) is used to describe a compound that transmits information from one individual to another. In bacteria, typical semiochemicals are autoinducers that convey information in a concentration-dependent manner, a phenomenon called “quorum sensing”; it is regulated by a feedback loop.[2] The identification and functional characterisation of a new type of autoinducer has been reported recently by the Bode and Heermann groups;[3] they showed that pyrones represent a new compound class in bacterial communication systems. The quorum sensing system of proteobacteria usually involves LuxI, an autoinducer synthase that produces N-acylhomoserine lactones (AHLs) with variable, mostly fatty-acidderived acyl chains (e.g., 5 and 8, Scheme 1). These AHLs are sensed by a cognate receptor LuxR, which upregulates luxI expression when a certain threshold concentration is reached. Many bacteria have been found to produce and use AHLs for communication.[4] Nevertheless, other signalling compounds must exist, because in many proteobacteria homologues of LuxR are found, but not the corresponding LuxI homologues.[5] Such receptors have been called “orphan” or “solo” LuxRs. The insect pathogenic proteobacterium Photorhabdus luminescens does not produce AHLs, but does express the solo receptor PluR. By using a comparative proteomoic analysis with a DpluR mutant, it was shown that PluR detects an endogenous signal.[3] The analysis revealed that PluR regulates two proteins from the pcfABCDEF operon that are responsible for cell clumping (pcf: Photorabdus clumping factor; see Figure 1). The mutant did not show cell clumping behaviour. For identification of the respective signal a pcfA fluorescence reporter strain was constructed and used in a cell-clumping assay. The target compounds were isolated by XAD 16 adsorption and their structures were elucidated by NMR and MS methods. These compounds proved to be pyrones (termed “photopyrones” by the authors) carrying two alkyl side chains. Photopyr-
[a] Prof. Dr. S. Schulz Institut fr Organische Chemie, Technische Universitt Braunschweig Hagenring 30, 38106 Braunschweig (Germany) E-mail: [email protected]
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. PpyS–PluR signalling in P. luminescens. The photopyrone synthase PpyS produces photopyrones (PPYs) that are sensed by the orphan LuxRtype receptor PluR. Upon signal detection, PluR activates transcription of the pcfABCDEF operon, which in turn leads to cell clumping. Reprinted by permission from Macmillan Publishers Ltd.[3] Copyright: 2013.
ones with branched and unbranched side chains were found; of these photopyrone D (PPYD, 1) showed the highest activity in the reporter system (at as little as 3.5 pm). In the related species Photorhabdus temperata, variants with shorter side chains were found (e.g., PPYA (3) and PPYB (2)), thus demonstrating clear strain-specific photopyrones. PPYA, PPYB and PPYD proved to be active in the bioassays, whereas various AHLs did not induce any activity. Computer modelling and docking experiments with a crystal structure[3, 6] of QscR from Pseudomonas aeruginosa as a model (32 % sequence identity), suggested that PluR is indeed the receptor of the photopyrones. Only two of six conserved amino acids residues of LuxR homologues are present in PluR, thus indicating a different bonding motif,[7] as found for AHL receptors. The results were obtained by using mutants modified at key amino acid positions. These mutants showed largely reduced or absent activity compared to the wild-type protein. Biosynthesis of photopyrones is likely performed with headto-head fusion of two short-chain fatty acids mediated by PpyS. This was confirmed by heterologous expression of the ppyS gene and branched fatty acid synthase genes in Escherichia coli. In the final part of their work,[3] the authors investigated the entire signalling system. The pcf operon was overexpressed in E. coli under the control of a promoter induced by arabinose. The E. coli cells clumped together when arabinose was added, thus showing the function of the pcf operon. Finally, the whole ChemBioChem 2014, 15, 498 – 500
498
CHEMBIOCHEM HIGHLIGHTS
www.chembiochem.org
Scheme 1. Known bacterial signalling compounds (1–10) and similar ones without known function (11, 12).
system (PluR, the pcfABCDEF operon and PpyS) was reconstituted in E. coli. Experiments with these constructs showed that photopyrones are indeed used for cell–cell communication. The compounds triggered secretion of proteins that induce cell clumping. As found in other quorum sensing systems, PPYD production increases during the exponential phase and is highest in the stationary phase; however positive feedback on ppyS expression was not observed. Although these compounds are also produced by the bacterium in the host insect larvae, a direct effect on insect mortality was not observed. However, E. coli expressing the constructs proved highly toxic to the insect larvae. The reason for the clumping behaviour and its benefit for Photorabdus spp. is not known. Closely related to the photopyrones are pseudopyronines (e.g., 4), as have been isolated from Pseudomonas spp.;[8] these differ from photopyrones only in side-chain length and methylation. It seems likely that these compounds function as signals, too. This finding might indicate a more widespread occurrence and importance, similar to that of AHLs like 5 and 8, the most common class of bacterial signalling compounds.[4] Whether the predominance of AHLs in proteobacteria is actually true or is only an artefact of the wealth of information on the genetics of their production, processing and function (allowing ever more studies to be produced) is open to discussion. The number of structural classes of compounds used for communication within bacteria is still small. Given the chemical diversity of bacterial metabolites and their different occurrences in so many ecological niches, there should be a wealth of different signalling compounds waiting to be unravelled. Within the structural features of known bacterial signalling compounds, a certain motif seems to show up. Like the AHLs, photopyrones exhibit side chains (derived from fatty acid me 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
tabolism) attached to a polar group. Bacterial signalling compounds with this feature include the autoinducer-1 of Vibrio cholerae ((S)-3-hydroxytridecan-4-one (6)),[9] the hydroxyalkylquinolone signal family (e.g., 9 from P. aeruginosa),[10] the butyrolactone family in streptomycetes[11] (e.g., A-factor (7))[12] and methylenomycin furans (MMFs, e.g. 10), which are responsible for methylenomycin production in streptomycetes.[13] Several other compounds that have been discussed as potential signalling compounds also fit this scheme, such as the longchain N-acylated amino acids arginine, tryptophane and tyrosine (12) found frequently in hitherto uncultured microorganisms[14] or N-acylated alanine methyl esters (11) produced by marine Roseovarius bacteria.[15] The lipophilic side chain probably helps entering the target cell and reaching the receptor. In summary, the results of the described in-depth study, covering all aspects from identification, function and biosynthesis, are fascinating. They clearly show the function of the photopyrones as autoinducers and broaden our knowledge on the chemistry and function of autoinducers. To elucidate new bacterial signalling compounds (called either pheromones, quorum sensing compounds, autoinducers or semiochemicals), these studies explore an extremely promising avenue. The results of such studies will be certainly of great importance for many fields, from medicine to agriculture and basic research. To understand what is really going on in the invisible world, communication is key. Keywords: LuxR · pheromones · photopyrones Photorhabdus · quorum sensing · signal transduction
·
[1] W. Francke, S. Schulz in Comprehensive Natural Products II (Eds.: L. N. Mander, H.-W. Liu), Elsevier, Oxford, 2010.
ChemBioChem 2014, 15, 498 – 500
499
CHEMBIOCHEM HIGHLIGHTS [2] a) Chemical Communication among Bacteria (Eds.: S. C. Winans, B. L. Bassler), ASM Press, Washington, DC, 2008; b) C. M. Waters, B. L. Bassler, Annu. Rev. Cell Dev. Biol. 2005, 21, 319 – 346. [3] A. O. Brachmann, S. Brameyer, D. Kresovic, I. Hitkova, Y. Kopp, C. Manske, K. Schubert, H. B. Bode, R. Heermann, Nat. Chem. Biol. 2013, 9, 573 – 578. [4] J. S. Dickschat, Nat. Prod. Rep. 2010, 27, 343 – 369. [5] R. J. Case, M. Labbate, S. Kjelleberg, ISME J. 2008, 2, 345 – 349. [6] M. J. Lintz, K.-I. Oinuma, C. L. Wysoczynski, E. P. Greenberg, M. E. A. Churchill, Proc. Natl. Acad. Sci. USA 2011, 108, 15763 – 15768. [7] A. V. Patankar, J. E. Gonzlez, FEMS Microbiol. Rev. 2009, 33, 739 – 756. [8] a) F. Kong, M. P. Singh, G. T. Carter, J. Nat. Prod. 2005, 68, 920 – 923; b) M. Chu, R. Mierzwa, L. Xu, L. He, J. Terracciano, M. Patel, W. Zhao, T. A. Black, T. M. Chan, J. Antibiot. 2002, 55, 215 – 218.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org [9] D. A. Higgins, M. E. Pomianek, C. M. Kraml, R. K. Taylor, M. F. Semmelhack, B. L. Bassler, Nature 2007, 450, 883 – 886. [10] E. Dziel, F. Lpine, S. Milot, J. He, M. N. Mindrinos, R. G. Tompkins, L. G. Rahme, Proc. Natl. Acad. Sci. USA 2004, 101, 1339 – 1344. [11] J. M. Willey, A. A. Gaskell, Chem. Rev. 2011, 111, 174 – 187. [12] S. Horinouchi, Biosci. Biotechnol. Biochem. 2007, 71, 283 – 299. [13] C. Corre, L. Song, S. O’Rourke, K. F. Chater, G. L. Challis, Proc. Natl. Acad. Sci. USA 2008, 105, 17510 – 17515. [14] S. F. Brady, J. Clardy, Org. Lett. 2005, 7, 3613 – 3616. [15] H. Bruns, V. Thiel, S. Voget, D. Patzelt, R. Daniel, I. Wagner-Dçbler, S. Schulz, Chem. Biodiversity 2013, 10, 1559 – 1573. Received: December 2, 2013 Published online on January 16, 2014
ChemBioChem 2014, 15, 498 – 500
500
