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Hydra.icgeb.trieste.itInternational Scholarly Research NetworkISRN MicrobiologyVolume 2012, Article ID 484176, 6 pagesdoi:10.5402/2012/484176 Research Article
Chromosomal Arrangement of AHL-Driven Quorum Sensing
Circuits in Pseudomonas
Zsolt Gelencs´er,1 Borisz Galb´ats,1, 2 Juan F. Gonzalez,3 K. Sonal Choudhary,4
Sanjarbek Hudaiberdiev,4 Vittorio Venturi,3 and S´andor Pongor4
1 Faculty of Information Technology, P´azm´any P´eter Catholic University, Pr´ater u. 50/a, 1083 Budapest, Hungary2 Bioinformatics Group, Biological Research Center, Temesv´ari krt 62, 6726 Szeged, Hungary3 Microbiology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 32149 Trieste, Italy4 Protein Structure and Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 32149 Trieste, Italy Correspondence should be addressed to Vittorio Venturi, email@example.com and S´andor Pongor, firstname.lastname@example.org Received 26 October 2011; Accepted 16 November 2011 Academic Editors: A. Hamood, S. Heeb, and S. Matthijs Copyright 2012 Zsolt Gelencs´er et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
Pseudomonas spp. are able to colonize a large variety of environments due to their wide adaptability which is also associated with anN-acyl homoserine lactone (AHL) gene regulation mechanism called quorum sensing (QS). In this article we present a systematicoverview of the genomic arrangement patterns of quorum sensing genes found in Pseudomonas and compare the topologies withthose found in other bacterial genomes. We find that the topological arrangement of QS genes is more variable than previouslythought but there are a few unifying features that occur in many of the topological arrangements. We hypothesize that the negativeregulators of QS that are often found between the canonical luxR/ and luxI-family genes may be crucial for stabilizing the output ofQS circuits.
beneficial bacteria either by antagonizing plant deleteriousmicroorganisms or by directly influencing plant disease resis- Fluorescent Pseudomonas spp. are able to colonize highly tance and growth .
dynamic environments such as soil, water, plants, as well as Bacteria often possess a regulatory system, known as quo- animals, including humans. This wide adaptability is asso- rum sensing (QS), to modulate gene expression as a function ciated with their resourceful metabolic potential and their of their cell density (for reviews see [3, 4]). In Gram negative ability to control gene expression via regulatory elements bacteria, the most common QS system is regulated by the N- highly represented in their large genomes. For example, the acyl homoserine lactone signaling molecules (AHLs). Studies opportunistic human pathogen Pseudomonas aeruginosa is a of the mechanisms and role of QS in several Pseudomonas notorious member of this genus and is extensively studied for spp. indicated that the most common signal molecules used its ability to cause chronic human opportunistic infections are in fact AHLs . These signals were first described in in immunocompromised patients . In addition to human the marine bioluminescent bacterium Vibrio fischeri in which pathogens, also important plant pathogens are present in this QS regulates light production (reviewed by ). The model group of bacteria; Pseudomonas syringae is an important N-AHL QS system consists of two proteins belonging to model of plant pathogenic bacteria since its pathovars can the LuxI and LuxR families, respectively, [4, 7]. LuxI- infect many diﬀerent plants (c.f. http://www.pseudomonas- family proteins are cytoplasmic enzymes responsible for AHL syringae.org/). Plant-growth-promoting fluorescent pseu- synthesis . AHLs are synthesized from S-adenosyl meth- domonads are also studied for their ability to colonize ionine, which provides the homoserine lactone moiety, and plant-related niches, like the rhizosphere (e.g., P. fluorescens, acyl carrier protein, which provides the fatty acyl moiety.
P. Putida, and P. chlororaphis), where they can act as plant After synthesis, the signal can move freely across the bacterial ISRN Microbiology membranes and accumulates both intra- and extracellularly unannotated genes in the complete genomes. Still we found in proportion to cell density. Above a critical threshold con- a few unannotated genes that were accepted on the condition centration or cell density, AHLs interact directly with the that they were in one of the previously observed topological LuxR-family protein, which in most cases results in the for- arrangements. From a total of over 4.3 million genes ana- mation of homodimers. These complexes can then bind at lyzed, we found 624 R genes (29 unannotated), 269 I genes specific sequences called lux-boxes that are located in the (12 unannotated), 39 L genes (11 unannotated), and 36 M promoter region of target QS-regulated genes, aﬀecting their genes (36 unannotated). Out of the 1346 complete genomes, 143 were found to contain QS genes in the vicinity of other Many important phenotypes are regulated by AHL QS QS genes (i.e., within a distance of 3000 nt). All of these were and QS has been suggested as a possible target to control proteobacterial genomes. We do not consider our analysis as bacterial colonization . QS regulates phenotypes related comprehensive because, among other things, it was based on to both pathogenesis (virulence associated factors like toxins, the reading frames given in the genome annotations, and we motility, secreted enzymes, and biofilm-related genes/pro- left Rhizobia and Agrobacterium species out of the survey teins) and to beneficial eﬀects in plant growth promoting rhi- because the arrangement of their QS genes is diﬀerent from zobacteria (PGPR) (e.g., production of antibiotic and anti- Pseudomonas. We found a few conflicts with respect to the fungal compounds and induction of systemic resistance in gene functions assigned in the genome annotations but not the plant [10–13]).
AHL QS is particularly interesting in the pseudomonads due to the presence, diversity, and complexity of regulatory 3. Genomic Topologies of
circuits present in various species. In the case of P. aeruginosa, AHL-Driven QS Circuits
AHL QS seems conserved and ubiquitous, being composedof a complex hierarchy of two LuxI/R pairs and a series of We found two major types of topological arrangements that regulators [5, 14]. In fact, it has been estimated that quorum we term RI and RXI, respectively, (Tables 1 and 2). In RI, the sensing regulates up to 3% of P. aeruginosa genes. On the two genes are vicinal while in RXI there is at least one addi- other hand, most strains of P. fluorescens and P. putida do tional gene between the two LuxI and LuxR family genes.
not possess an AHL QS system [15, 16]. In this study, we 3.1. The RI Topology. There are 3 possible variations, namely performed an in-depth systematic study on the chromo- tandem (unidirectional), convergent, divergent. All of these somal arrangement and synteny of AHL QS systems in pseu- are found in proteobacteria, Pseudomonas does not seem to domonads in order to determine the commonalities and dif- contain the divergent topology which can, however, be found ferences that may exist between pseudomonads and other in other gamma proteobacteria.
bacteria. Previous studies concentrated either on the pres-ence or absence of AHL QS genes in bacteria , or on the 3.2. The RXI Topology. In these topologies, one or more regulatory design principles of selected QS systems [18, 19].
genes are found between the R and the I genes. All the 8 Here, we present a survey of AHL-driven QS circuits in pseu- possible arrangements are found in proteobacteria, however, domonads and compare the chromosomal arrangements only 3 in Pseudomonas where the X gene is most frequently L with those found in other bacterial genomes.
(RsaL, found in P. aeruginosa, P. putida, and P. fuscovaginaespecies). M is much more frequently found in Burkholderia,the only Pseudomonas to contain M is P. fuscovaginae, which 2. QS Genes in Complete Bacterial Genomes
is at the same time, the only pseudomonad found so far tocontain both L and M genes. Both the L and the M genes We used the sequence data of 1346 full bacterial proteomes have their canonical topologies which are shown in the table found at the NCBI bacterial genome repository as well as separately, denoted as RLI (L1) and RMI (M1), respectively.
published QS operon sequences from NCBI GenBank (data Both of these topologies can be found both in Burkholderia last accessed on June 12, 2011). Draft genome sequences and in Pseudomonas. However, some Burkholderia species were excluded from the analysis because of the uncer- contain an additional copy of M, which is in a non-canonical tain annotations we found in some of them. The search arrangement, either because there are one to five additional included standard bioinformatics methodologies and man- genes betweem R and M (M2 topology, found in B. pseu- ual curation (see supplementary materials available online domallei 1106a, B. pseudomallei 1710b, B. pseudomallei 668, at doi: 10.5402/2012/484176). We started our search for B. pseudomallei K96243, B. thailandensis E264), or because luxR sensor/regulators, luxI AHL synthases, rsaL, and rsaM the otherwise constitutive R gene is missing in the immediate repressor homologues in complete bacterial genomes and vicinity of the MI tandem (M3 topology, B. ambipharia included only a set of selected examples of Pseudomonas data AMMD, and MC40-6). On the other hand, P. fluorescens from incomplete genomes (Tables 1 and 2). For luxR, luxI, NCIMB 10586 contains a gene coding for an enzyme, mupX rsaL, and rsaM we use the symbols R, I, L, and M, res- in the X position .
pectively, and refer to them as "QS genes". Solo R genes as well as other lonely occurrences of QS genes were 4. The X Genes
not considered. This cautious approach of manual curationwas adopted because we were primarily interested in the In Pseudomonas, the genes in the X position are predomi- genomic arrangements and not so much in finding hitherto nantly negative regulators of the QS response. RsaL (L)  ISRN Microbiology Table 1: Typical chromosomal arrangements of AHL-driven quorum sensing circuits in Pseudomonas.
Occurrence in other Proteobacteria P. aeruginosa LESB58P. aeruginosa PA7 P. aeruginosa PAO1P. aeruginosa UCBPP-PA14P. syringae pv. phaseolicola1448AP. syringae pv. syringaeB728a P. syringae pv. tomato str.
DC3000P. chlororaphis PCL1391P. fluorescens 2–79∗ P. aeruginosa LESB58P. aeruginosa PA7P. aeruginosa PAO1 P. aeruginosa UCBPP-PA14 P. putida WCS358P. putida IsoF∗, PCL1445∗P. fuscovaginae UPB0736 P. fuscovaginae UPB0736 P. fluorescens NCIMB 10586 ∗ Pattern does not contain the overlap indicated.
∗∗RI patterns = two genes are in vicinity and RXI patterns = one additional gene is between luxR and LuxI.
was shown to belong to the tetrahelical superclass of H-T-H I genes (2-to 68 nt) while P. fluorescens genomes do not.
proteins . Members of this family are widespread repres- In the L1 type QS circuits of P. aeruginosa, the overlaps are sors in bacteria and bind to DNA as dimers. We found that 10 nt while in P. fuscovaginae the overlap is 20 nt. On the homologues of RsaL frequently occur outside QS circuits in contrary, the L1 circuits of P. putida are not overlapping, various bacterial genomes (data not shown). In P. fuscovagi- though the open reading frames of R and L are only 4 nt nae, RsaL binds to DNA next to the lux box and prevents apart. Tsai and Winans noted that the overlapping R2-like ar- expression of the R gene . In contrast, RsaM (M) is a rangement is common to QS circuits in which R proteins are protein of unknown structure that seems to occur only in the able to fold, dimerize, bind DNA, and regulate transcription context of QS circuits. M was found to negatively regulate in the absence of AHLs; moreover, these proteins are antag- QS in P. fuscovaginae . Finally, mupX of P. fluorescens onized by their cognate AHLs . The same authors also NCIMB10586 is an amidase-hydrolase that was shown to argued that the expression of one member of a convergent degrade the AHL signal produced by the same species thereby and overlapping gene pair might be antagonized by the exp- decreasing the QS response .
ression of the other member, either via RNA polymerasecollisions or by hybridization of the two complementarymRNAs .
5. Overlapping Genes
Two topologies, R2 and L1, contain overlaps at the proximal 6. Regulatory Implications
ends of convergent genes. Such overlaps are not uncommonin tightly coregulated gene circuits of bacteria , for ins- The most conspicuous feature of the various circuit topolo- tance restriction modification systems . The R2 type gies is the potential negative regulatory eﬀect of R on I which, of arrangements in P. syringae contain overlapping R and as mentioned above, goes in parallel with the well known ISRN Microbiology Table 2: Examples of Pseudomonas species with of AHL-driven Finally, we mention that the chromosomal arrangements quorum sensing networks.
found in QS genes seem more varied than expected so thesearch for common regulatory principles remains an impor- tant task for future research.
P. aeruginosa LESB58 Z. Gelencs´er and B. Galb´ats are Ph.D. and undergraduate stu- P. aeruginosa PA7  dents, respectively, at the Faculty of Information Technology, P´azm´any P´eter Catholic University, Budapest. J. F. Gonzalez, S. Hudaiberdiev, and K. S. Choudhary are Ph.D. students at P. aeruginosa PAO1  ICGEB, Trieste. Work at the Szeged Biological Center wassupported by OTKA Grant K. 84335.
P. aeruginosaUCBPP-PA14  P. fuscovaginae UPB0736  B. W. Holloway and A. F. Morgan, "Genome organization in Pseudomonas," Annual Review of Microbiology, vol. 40, pp. 79– P. syringae pv.
phaseolicola 1448A   D. J. O'Sullivan and F. O'Gara, "Traits of fluorescent Pseudo- P. syringae pv. syringae Psyr 1622/Psyr 1621 monas spp. involved in suppression of plant root pathogens," Microbiological Reviews, vol. 56, no. 4, pp. 662–676, 1992.
P. syringae pv. tomato str.
 A. Camilli and B. L. Bassler, "Bacterial small-molecule sig- naling pathways," Science, vol. 311, no. 5764, pp. 1113–1116, P. chlororaphis PCL1391  C. Fuqua, M. R. Parsek, and E. P. Greenberg, "Regulation of P. fluorescens 2–79  gene expression by cell-to-cell communication: acyl-homo-serine lactone quorum sensing," Annual Review of Genetics, P. fluorescens NCIMB vol. 35, pp. 439–468, 2001.
 V. Venturi, "Regulation of quorum sensing in Pseudomonas," P. putida WCS358  FEMS Microbiology Reviews, vol. 30, no. 2, pp. 274–291, P. putida IsoF  P. putida PCL1445   E. G. Ruby, "Lessons from a cooperative, bacterial-animal association: the Vibrio fischeri-Euprymna scolopes light organsymbiosis," Annual Review of Microbiology, vol. 50, pp. 591–624, 1996.
positive regulatory eﬀect. In other words, R seems both to  N. A. Whitehead, A. M. L. Barnard, H. Slater, N. J. L. Simpson, activate and to inhibit the I genes in a number of cases. In and G. P. C. Salmond, "Quorum-sensing in Gram-negative RXI circuits, R activates an X gene that decreases the eﬀect bacteria," FEMS Microbiology Reviews, vol. 25, no. 4, pp. 365– of I. In the R2 circuits, the negative eﬀect follows from the overlap between the convergently transcribed R and I genes  K. M. Pappas, C. L. Weingart, and S. C. Winans, "Chemical . Regulatory circuits in which an element can both acti- communication in proteobacteria: biochemical and structural vate and inhibit another element are termed incoherent feed studies of signal synthases and receptors required for inter- forward loops or IFFLs [41, 42]. In contrast to simple feed cellular signalling," Molecular Microbiology, vol. 53, no. 3, pp.
forward arrangements, IFFLs can exhibit a number of com- 755–769, 2004.
plex behavior patterns (for a review see ). Perhaps the  T. B. Rasmussen, M. E. Skindersoe, T. Bjarnsholt et al., "Iden- most important of these is the stabilization of the output tity and eﬀects of quorum-sensing inhibitors produced by signals: while simple feed forward circuits have no inherent Penicillium species," Microbiology, vol. 151, no. 5, pp. 1325– limits on their output, IFFL networks have bounded output which ensures robustness against fluctuations in the input  T. F. C. Chin-A-Woeng, D. Van Den Broek, B. J. J. Lugten- signal levels. Most often, QS regulatory circuits are simply berg, and G. V. Bloemberg, "The Pseudomonas chlororaphisPCL1391 sigma regulator psrA represses the production of referred to as autoinduction loops which, at least in theory, the antifungal metabolite phenazine-1-carboxamide," Molec- should increase their output without limits. The examples ular Plant-Microbe Interactions, vol. 18, no. 3, pp. 244–253, shown in this survey suggest that a stabilizing, negative regu- latory pathway is present in many QS systems. It was found  J. F. Dubern, B. J. J. Lugtenberg, and G. V. Bloemberg, "The experimentally that deletion of RsaL or RsaM leads to a dra- ppuI-rsaL-ppuR quorum-sensing system regulates biofilm matic increase in AHL production, but the resulting mutants formation of Pseudomonas putida PCL1445 by controlling are less virulent than the wild type , which shows, on the biosynthesis of the cyclic lipopeptides putisolvins I and other hand, that the negative regulatory path may in fact be II," Journal of Bacteriology, vol. 188, no. 8, pp. 2898–2906, a crucial stabilizing element within the QS circuits.
ISRN Microbiology  G. Girard, E. T. van Rij, B. J. J. Lugtenberg, and G. V.
 C. Winstanley, M. G. I. Langille, J. L. Fothergill et al., "Newly Bloemberg, "Regulatory roles of psrA and rpoS in phena- introduced genomic prophage islands are critical determi- zine-1-carboxamide synthesis by Pseudomonas chlororaphis nants of in vivo competitiveness in the liverpool epidemic PCL1391," Microbiology, vol. 152, no. 1, pp. 43–58, 2006.
strain of Pseudomonas aeruginosa," Genome Research, vol. 19,  R. Schuhegger, A. Ihring, S. Gantner et al., "Induction of sys- no. 1, pp. 12–23, 2009.
temic resistance in tomato by N-acyl-L-homoserine lactone-  P. H. Roy, S. G. Tetu, A. Larouche et al., "Complete genome producing rhizosphere bacteria," Plant, Cell and Environment, sequence of the multiresistant taxonomic outlier Pseudomonas vol. 29, no. 5, pp. 909–918, 2006.
aeruginosa PA7," PLoS ONE, vol. 5, no. 1, Article ID e8842,  M. Schuster and E. P. Greenberg, "A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa,"  C. K. Stover, X. Q. Pham, A. L. Erwin et al., "Complete ge- International Journal of Medical Microbiology, vol. 296, no. 2-3, nome sequence of Pseudomonas aeruginosa PAO1, an oppor- pp. 73–81, 2006.
tunistic pathogen," Nature, vol. 406, no. 6799, pp. 959–964,  M. Elasri, S. Delorme, P. Lemanceau et al., "Acyl-homoserine lactone production is more common among plant-associated  D. G. Lee, J. M. Urbach, G. Wu et al., "Genomic analysis reveals Pseudomonas spp. than among soilborne Pseudomonas spp," that Pseudomonas aeruginosa virulence is combinatorial," Ge- Applied and Environmental Microbiology, vol. 67, no. 3, pp.
nome Biology, vol. 7, no. 10, article R90, 2006.
 V. Joardar, M. Lindeberg, R. W. Jackson et al., "Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola  L. Steindler, I. Bertani, L. De Sordi, J. Bigirimana, and V.
1448A reveals divergence among pathovars in genes involved Venturi, "The presence, type and role of N-acyl homoserine in virulence and transposition," Journal of Bacteriology, vol.
lactone quorum sensing in fluorescent Pseudomonas originally 187, no. 18, pp. 6488–6498, 2005.
isolated from rice rhizospheres are unpredictable," FEMS  H. Feil, W. S. Feil, P. Chain et al., "Comparison of the com- Microbiology Letters, vol. 288, no. 1, pp. 102–111, 2008.
plete genome sequences of Pseudomonas syringae pv. syringae  R. J. Case, M. Labbate, and S. Kjelleberg, "AHL-driven quo- B728a and pv. tomato DC3000," Proceedings of the National rum-sensing circuits: their frequency and function among Academy of Sciences of the United States of America, vol. 102, the Proteobacteria," ISME Journal, vol. 2, no. 4, pp. 345–349, no. 31, pp. 11064–11069, 2005.
 C. R. Buell, V. Joardar, M. Lindeberg et al., "The complete  A. B. Goryachev, "Design principles of the bacterial quorum genome sequence of the arabidopsis and tomato pathogen sensing gene networks," Wiley Interdisciplinary Reviews, vol. 1, Pseudomonas syringae pv. tomato DC3000," Proceedings of the no. 1, pp. 45–60, 2009.
National Academy of Sciences of the United States of America,  A. B. Goryachev, "Understanding bacterial cell-cell communi- vol. 100, no. 18, pp. 10181–10186, 2003.
cation with computational modeling," Chemical Reviews, vol.
 S. R. Khan, J. Herman, J. Krank et al., "N-(3-hydroxyhexa- 111, no. 1, pp. 238–250, 2011.
noyl)-L-homoserine lactone is the biologically relevant quor-  S. Subramoni and V. Venturi, "LuxR-family "solos": bachelor mone that regulates the phz operon of Pseudomonas chloro- sensors/regulators of signalling molecules," Microbiology, vol.
raphis strain 30-84," Applied and Environmental Microbiology, 155, no. 5, pp. 1377–1385, 2009.
vol. 73, no. 22, pp. 7443–7455, 2007.
 J. Hothersall, A. C. Murphy, Z. Iqbal et al., "Manipulation  D. W. Wood and L. S. Pierson, "The phzI gene of Pseudomonas of quorum sensing regulation in Pseudomonas fluorescens aureofaciens 30-84 is responsible for the production of a dif- NCIMB 10586 to increase mupirocin production," Applied fusible signal required for phenazine antibiotic production," Microbiology and Biotechnology, vol. 90, no. 3, pp. 1017–1026, Gene, vol. 168, no. 1, pp. 49–53, 1996.
 D. V. Mavrodi, V. N. Ksenzenko, R. F. Bonsall, R. J. Cook, A.
 T. De Kievit, P. C. Seed, J. Nezezon, L. Passador, and B. H.
M. Boronin, and L. S. Thomashow, "A seven-gene locus for Iglewski, "RsaL, a novel repressor of virulence gene expression synthesis of phenazine-1-carboxylic acid by Pseudomonas flu- in Pseudomonas aeruginosa," Journal of Bacteriology, vol. 181, orescens 2-79," Journal of Bacteriology, vol. 180, no. 9, pp.
no. 7, pp. 2175–2184, 1999.
 A. K. El-Sayed, J. Hothersall, S. M. Cooper, E. Stephens, T. J.
 G. Rampioni, F. Polticelli, I. Bertani et al., "The Pseudomonas Simpson, and C. M. Thomas, "Characterization of the mupi- quorum-sensing regulator RsaL belongs to the tetrahelical rocin biosynthesis gene cluster from Pseudomonas fluorescens superclass of H-T-H proteins," Journal of Bacteriology, vol. 189, NCIMB 10586," Chemistry and Biology, vol. 10, no. 5, pp. 419– no. 5, pp. 1922–1930, 2007.
 M. Mattiuzzo, I. Bertani, S. Ferluga et al., "The plant pathogen  I. Bertani and V. Venturi, "Regulation of the N-acyl homoser- Pseudomonas fuscovaginae contains two conserved quorum ine lactone-dependent quorum-sensing system in rhizosphere sensing systems involved in virulence and negatively regulated Pseudomonas putida WCS358 and cross-talk with the statio- by RsaL and the novel regulator RsaM," Environmental nary-phase RpoS sigma factor and the global regulator GacA," Microbiology, vol. 13, no. 1, pp. 145–162, 2011.
Applied and Environmental Microbiology, vol. 70, no. 9, pp.
 D. C. Krakauer, "Stability and evolution of overlapping genes," 5493–5502, 2004.
Evolution, vol. 54, no. 3, pp. 731–739, 2000.
 A. Steidle, M. Allesen-Holm, K. Riedel et al., "Identification  M. K. Kaw and R. M. Blumenthal, "Translational indepen- and characterization of an N-acylhomoserine lactone-depen- dence between overlapping genes for a restriction endonucle- dent quorum-sensing system in Pseudomonas putida strain ase and its transcriptional regulator," BMC Molecular Biology, IsoF," Applied and Environmental Microbiology, vol. 68, no. 12, vol. 11, article 87, 2010.
pp. 6371–6382, 2002.
 C. S. Tsai and S. C. Winans, "LuxR-type quorum-sensing reg-  U. Alon, "Network motifs: theory and experimental appro- ulators that are detached from common scents," Molecular aches," Nature Reviews Genetics, vol. 8, no. 6, pp. 450–461, Microbiology, vol. 77, no. 5, pp. 1072–1082, 2010.
ISRN Microbiology  R. Milo, S. Shen-Orr, S. Itzkovitz, N. Kashtan, D. Chklovskii, and U. Alon, "Network motifs: simple building blocks of com-plex networks," Science, vol. 298, no. 5594, pp. 824–827,2002.
 D. Kim, Y. K. Kwon, and K. H. Cho, "The biphasic behavior of incoherent feed-forward loops in biomolecular regulatorynetworks," BioEssays, vol. 30, no. 11-12, pp. 1204–1211, 2008.
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