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International 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, and S´andor Pongor, 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 [2].
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 [5]. These signals were first described in in immunocompromised patients [1]. 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 [6]). 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 Plant-growth-promoting fluorescent pseu- synthesis [8]. 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 [9]. 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 [17], 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 [21].
pectively, and refer to them as "QS genes". Solo R genes[20] 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) [22] 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 [23]. 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 [24]. 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 [24]. Finally, mupX of P. fluorescens onized by their cognate AHLs [27]. 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 [21].
ression of the other member, either via RNA polymerasecollisions or by hybridization of the two complementarymRNAs [27].
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 [25], for ins- The most conspicuous feature of the various circuit topolo- tance restriction modification systems [26]. 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 [29] 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 [30] ICGEB, Trieste. Work at the Szeged Biological Center wassupported by OTKA Grant K. 84335.
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