An aryl-homoserine lactone quorum-sensing signal produced by a dimorphic prosthecate bacterium Lisheng Liaoa,b, Amy L. Schaeferb, Bruna G. Coutinhob, Pamela J. B. Brownc, and E. Peter Greenberga,b,1 aIntegrative Microbiology Research Centre, South China Agricultural University, 510642 Guangzhou, People’s Republic of China; bDepartment of Microbiology, University of Washington, Seattle, WA 98195; and cDivision of Biological Sciences, University of Missouri, Columbia, MO 65211 Contributed by E. Peter Greenberg, June 12, 2018 (sent for review May 15, 2018; reviewed by Helen E. Blackwell and Clay Fuqua) Many species of Proteobacteria produce acyl-homoserine lactone predict whether a LuxI homolog is in the ACP- or CoA-dependent (AHL) compounds as quorum-sensing (QS) signals for cell density- family (11–13). We became interested in the α-Proteobacterium dependent gene regulation. Most known AHL synthases, LuxI-type Prosthecomicrobium hirschii when its genome sequence was pub- enzymes, produce fatty AHLs, and the fatty acid moiety is derived lished (14). This dimorphic prosthecate bacterium has genes coding from an acyl-acyl carrier protein (ACP) intermediate in fatty acid for an AHL QS system, and we predicted the luxI homolog codes for biosynthesis. Recently, a class of LuxI homologs has been shown to a member of the CoA-utilizing family. P. hirschii is a saprophyte that use CoA-linked aromatic or amino acid substrates for AHL synthe- can be isolated from freshwater lakes (15) and exhibits two different sis. By using an informatics approach, we found the CoA class of cell morphologies. Some cells have multiple long prostheca, and LuxI homologs exists primarily in α-Proteobacteria. The genome of other cells have many short prostheca. Although there is very little Prosthecomicrobium hirschii, a dimorphic prosthecate bacterium, known about the genus Prosthecomicrobium, it has been reported that possesses a luxI-like AHL synthase gene that we predicted to en- the short prostheca cell type increases in relative abundance in the late code a CoA-utilizing enzyme. We show the P. hirschii LuxI homolog logarithmic phase of growth (16). This further piqued our interest catalyzes synthesis of phenylacetyl-homoserine lactone (PA-HSL). because it raises the possibility that cell type is influenced by QS. P. hirschii Our experiments show P. hirschii obtains phenylacetate from its We report here that produces phenylacetyl-homoserine environment and uses a CoA ligase to produce the phenylacetyl- lactone (PA-HSL) and uses exogenous phenylacetate for this purpose. CoA substrate for the LuxI homolog. By using an AHL degrading We present evidence that the gene linked to the PA-HSL synthase gene enzyme, we showed that PA-HSL controls aggregation, biofilm codes for the PA-HSL receptor. This QS circuit not only positively formation, and pigment production in P. hirschii. These findings autoregulates PA-HSL synthesis, it also controls aggregation and pig- ment production. We find that putative CoA-utilizing AHL synthases advance a limited understanding of the CoA-dependent AHL syn- α thases. We describe how to identify putative members of the class, are common in -Proteobacteria and rare in other Proteobacteria. we describe a signal synthesized by using an environmental aro- Results matic acid, and we identify phenotypes controlled by the aryl-HSL. The P. hirschii LuxI Homolog Clusters with Members of the CoA- bacterial communication | Prosthecomicrobium | α-Proteobacteria | Utilizing Family of AHL Synthases. We developed a method involv- phenylacetate | sociomicrobiology ing the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO) database and an enzyme nomenclature (EC) populated database (13) to search for likely acyl-CoA–utilizing any bacterial species use quorum-sensing (QS) signals as a LuxI homologs. The KO associated with acyl-CoA–utilizing LuxI Mproxy for cell density. Diffusible acyl-homoserine lactones homologs (K18096) was encoded almost exclusively in genomes of (AHLs) are common QS signals in Proteobacteria. The AHL α-Proteobacteria. We found 974 acyl-CoA-type luxI homologs signals accumulate during growth and at sufficient concentra- tions bind cognate transcription factors, QS signal receptors, Significance which affect transcription of genes in QS regulons (1–3). Generally, AHL production is catalyzed by signal synthases, members of the LuxI protein family. Most known AHLs have Acyl-homoserine lactone (AHL) quorum sensing (QS) has received straight-chain fatty acyl side groups of 4–18 carbons, with varying interest as a possible target for controlling microbial activities and degrees of oxidation at the 3-C position (1). The substrates for as a model for communication involved in coordinating activities characterized fatty acyl AHLs are S-adenosylmethionine (SAM) of bacteria. Recent advances show there are two related families and an acylated-acyl carrier protein (acyl-ACP) (4, 5) from the of AHL QS signal synthesis enzymes, which differ in whether they fatty acid biosynthesis pathway. acquire the organic acid reaction substrate from biosynthetic or Recently, LuxI homologs have been identified that produce AHLs primarily catabolic pathways. Most known AHL synthases utilize signalswitharomaticacidandbranchedaminoacidsidechainsin fatty acids activated with an acyl carrier protein from fatty acid three species of α-Proteobacteria: RpaI from Rhodopseudomonas biosynthesis. Some AHL synthases use coenzyme-A–activated or- palustris catalyzes production of p-coumaroyl-HSL (pC-HSL) (6); ganic acids. We describe a coenzyme-A–dependent AHL synthase CinI from a photosynthetic Bradyrhizobium catalyzes production QS system. We also set forth an experimental approach for dis- of cinnamoyl-HSL (cinn-HSL) (7); and BjaI from Bradyrhizobium covering new signals that will allow further expansion of our japonicum catalyzes production of isovaleryl-HSL (IV-HSL) (8). understanding of the diversity of AHL signaling. These three enzymes use coenzyme-A (CoA)–activated acids rather than ACP-activated acids as substrates (8, 9). Presumably, the CoA Author contributions: L.L., A.L.S., and E.P.G. designed research; L.L., A.L.S., B.G.C., and substrates are derived from the activity of cellular acyl-CoA ligases P.J.B.B. performed research; L.L., A.L.S., B.G.C., and E.P.G. analyzed data; and L.L., A.L.S., P.J.B.B., and E.P.G. wrote the paper. involved in organic acid catabolism. In fact, R. palustris requires ex- – ogenous p-coumarate to produce pC-HSL (6). Phylogenomic analyses Reviewers: H.E.B., University of Wisconsin Madison; and C.F., Indiana University. led to the conclusion that the CoA-utilizing family of AHL synthases The authors declare no conflict of interest. resulted from an evolutionary exaptation event (10). Published under the PNAS license. Relatively little is known about the acyl-CoA utilizing AHL syn- 1To whom correspondence should be addressed. Email: [email protected]. thases and the activities they control.Basedonthesequencesofthe This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. three known acyl-CoA utilizing LuxI homologs and phylogenomic 1073/pnas.1808351115/-/DCSupplemental. MICROBIOLOGY analyses, there are now informatics tools, which can be used to Published online July 2, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1808351115 PNAS | July 17, 2018 | vol. 115 | no. 29 | 7587–7592 Downloaded by guest on September 28, 2021 (K018096) in genomes submitted to the Joint Genome Institute genetic system for P. hirschii has not been established, we Integrated Microbial Genomes (JGI IMG) repository and 957 were addressed the question by using recombinant Pseudomonas putida in α-proteobacterial genomes. This represents about 30% of the F1. The genome of this bacterium has a high GC content similar to 3,010 luxI homologs we found in sequenced α-proteobacterial ge- that of P. hirschii, it can use phenylacetic acid as a carbon and luxI nomes. The single homolog in the recently sequenced genome energy source and the first step in phenylacetate catabolism of P. hirschii (14) was found within the family of genes encoding putative acyl-CoA–dependent AHL synthases by KO classification and in phylogenetic analyses (Fig. 1A and SI Appendix,TableS1). The P. hirschii luxI homolog is linked to a gene coding for a LuxR- A +,5,+,5, like AHL-responsive transcription factor. We named this pair of 41&41&=B5+23$= B5+23$ genes hirI and hirR (Fig. 1B). P. hirschii is a dimorphic appendeged $</$</7B%5$627B%5$62 α &%B0(7($&%B0(7($ -proteobacterial species, which exhibits two distinct cell morphol- 4343B5+23$B5+23$ ogies (Fig. 1C). Because P. hirschii is a Rhizobiales as are the 49,49,B%5$'8B%5$'8 Rhodopseudomonas Bradyrhizobium 5+/,B36($( and species, which produce $98B36($ aryl- or amino AHLs, we sought to identify the HirI-produced AHL. Q939D0_PSECL PHZI _PSEFL P. hirschii Produces Phenylacetyl-HSL. As a first step toward identi- A0A0H3HXG1_BURPE Q4VSJ8 _BURGL fication of the AHL produced by HirI, we grew this bacterium in the A0A0H2WCK4 _BMAI1 14 presence of L-[1- C]-methionine. To synthesize AHLs, bacteria use A0ZSH0_SHEHA the common metabolic intermediate SAM derived from methio- /8;,B$/,)6 14 A0A0Q2RV31_VIBR nine, and when grown in the presence of L-[1- C]-methionine, the 14 A6CUQ4 9_VIBR AHL ring is labeled with a C atom (17, 18). Because AHLs are %-4/B3$53 diffusible and solvent extractable, we extracted the culture super- '6*B*$/&6 natant fluid with ethyl acetate and fractionated the extract by /$6,B36($( 4,78B36()/ C18-reverse phase HPLC. Nearly all of the radiolabel was found (9/*B5+23; in a single HPLC fraction (Fig. 2A). As expected if the radiola- *'B%5$-3 48:B5+,5' beled compound was an AHL, the radiolabeled HPLC fraction was eliminated by pretreatment with purified AiiA AHL lactonase );=B5+,)5 A p 75$,B$*5)& (Fig. 2 ). None of the synthetic AHLs we tested, including C- +++B5+,5' HSL, cinn-HSL, and IV-HSL, coeluted with the radiolabeled P. ($*,B(17$* hirschii compound.