Molecular Microbiology (2008) 67(2), 241–253 ᭿ doi:10.1111/j.1365-2958.2007.06042.x First published online 6 November 2007 MicroReview Gac/Rsm signal transduction pathway of g-proteobacteria: from RNA recognition to regulation of social behaviour Karine Lapouge,1 Mario Schubert,2 Introduction Frédéric H.-T. Allain2 and Dieter Haas1* 1Département de Microbiologie Fondamentale, Bacteria respond to changing environments by adjusting Université de Lausanne, Bâtiment Biophore, CH-1015 the cellular levels of mRNAs, stable RNAs (that is, rRNAs Lausanne, Switzerland. and tRNAs) and small RNAs (sRNAs). Whereas the regu- 2Institute of Molecular Biology and Biophysics, ETH lation of transcription initiation is crucial in this adaptation, Zürich, CH-8093 Zürich, Switzerland. subsequent control of translation initiation can be just as important. Recent studies have shown that two major classes of sRNAs influence the rate of translation initiation Summary in bacteria (Majdalani et al., 2005; Storz et al., 2005). sRNAs of the first class interact with 5′ leader regions of In many g-proteobacteria, the conserved GacS/GacA target mRNAs by base pairing. Such interactions interfere (BarA/UvrY) two-component system positively con- with ribosome binding when they occur at or near the trols the expression of one to five genes specifying Shine-Dalgarno (SD) sequence of mRNAs. The opposite small RNAs (sRNAs) that are characterized by effect, stimulation of ribosome binding, can also be repeated unpaired GGA motifs but otherwise appear observed in situations where sRNAs change the second- to belong to several independent families. The GGA ary structure of target mRNAs by base pairing with an motifs are essential for binding small, dimeric RNA- upstream region. The RNA chaperone Hfq facilitates binding proteins of a single conserved family desig- these base pairing interactions in Gram-negative bacteria, nated RsmA (CsrA). These proteins, which also occur but seems to be dispensable in Gram-positive bacteria in bacterial species outside the g-proteobacteria, act (Heidrich et al., 2006; Bohn et al., 2007). sRNAs of the as translational repressors of certain mRNAs when second class, which have a high affinity for RNA-binding these contain an RsmA/CsrA binding site at or near proteins of the RsmA/CsrA family, can relieve translational the Shine-Dalgarno sequence plus additional binding repression owing to these proteins by sequestering them sites located in the 5Ј untranslated leader mRNA. (Majdalani et al., 2005; Storz et al., 2005; Babitzke and Recent structural data have established that the Romeo, 2007). RsmA and CsrA are acronyms for regula- RsmA-like protein RsmE of Pseudomonas fluore- tor of secondary metabolism and carbon storage regulator scens makes specific contacts with an RNA consen- respectively. In pseudomonads, sRNAs that bind RsmA/ sus sequence 5Ј-A/ CANGGANGU/ -3Ј (where N is any U A CsrA proteins are typically produced under the positive nucleotide). Interaction with an RsmA/CsrA protein control of a two-component system, termed GacS/GacA promotes the formation of a short stem supporting an (for global activation of antibiotic and cyanide synthesis). ANGGAN loop. This conformation hinders access Other g-proteobacteria also have GacS/GacA homo- of 30S ribosomal subunits and hence translation logues, many of which bear different names (see Table 1). initiation. The output of the Gac/Rsm cascade varies The general characteristics of the Gac/Rsm signal trans- widely in different bacterial species and typically duction pathway are outlined in Fig. 1. The target genes involves management of carbon storage and expres- that are translationally regulated by this regulatory sion of virulence or biocontrol factors. Unidentified cascade, and hence the output, vary considerably among signal molecules co-ordinate the activity of the Gac/ various bacteria (Table 1). However, as we wish to point Rsm cascade in a cell population density-dependent out in this review, two features are conserved: in general, manner. mutants blocked in this regulatory pathway are impaired Accepted 2 November, 2007. *For correspondence. E-mail dieter. in social behaviour and there appears to exist a com- [email protected]; Tel. (+41) 21 692 56 31; Fax (+41) 21 692 56 35. mon molecular basis of the RNA-RsmA/CsrA protein © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 242 K. Lapouge, M. Schubert, F. H.-T. Allain and D. Haas Table 1. Mutants affected in gacS and gacA homologues in g-proteobacteria. GacS/GacA- Species GacS/GacA homologues Major GacS/GacA-controlled phenotypes dependent sRNAs References Acinetobacter baumannii GacS/GacA Citrate utilization RsmX, RsmY, RsmZa Dorsey et al. (2002) Azotobacter vinelandii GacS/GacA Alginate, poly-b-hydroxy-butyrate, encystment ? Castañeda et al. (2001) Erwinia carotovora ssp. carotovora GacS/GacA (ExpS/ExpA) Extracellular pectinases, cellulase, protease, virulence, motility RsmB Cui et al. (2001) Erwinia chrysanthemi ?/GacA Extracellular pectinases, cellulase, protease, TTSS, virulence ? Lebeau et al. (2008) Escherichia coli BarA/UvrY Central carbon metabolism, biofilm, virulenceb, motility CsrB, CsrC Suzuki et al. (2002); Weilbacher et al. (2003); Tomenius et al. (2006) Legionella pneumophila LetS/LetA Cytotoxicity, virulence, motility RsmY, RsmZa Hammer et al. (2002); Molofsky and Swanson (2003) ᭿ Journal compilation © 2007 Blackwell Publishing Ltd, Pseudomonas aeruginosa GacS/GacA AHL, HCN, pyocyanin, lipase, elastase, biofilm, virulence, RsmY, RsmZ Rahme et al. (1995); motility Reimmann et al. (1997); Parkins et al. (2001); Kay et al. (2006) Pseudomonas chlororaphis GacS/GacA AHL, phenazines, HCN, surfactants, 2,3-butanediol, ? Chancey et al. (1999); (aureofaciens) protease, biocontrol Schmidt-Eisenlohr et al. (2003); Han et al. (2006); Girard et al. (2006) Pseudomonas entomophila GacS/GacA Protease, hemolysin, virulence RsmY, RsmZc Vodovar et al. (2006) Pseudomonas fluorescens GacS/GacA DAPG, HCN, pyoluteorin, pyrrolnitrin, protease, RsmX, RsmY, RsmZ Haas and Défago (2005); phospholipase, biocontrol, H2O2 resistance, motility Kay et al. (2005); Heeb et al. (2005); Dubuis et al. (2007) Pseudomonas marginalis LemA/GacA Pectinases, virulence ? Liao et al. (1997) Pseudomonas syringae pv. syringae GacS(LemA)/ GacA Syringomycin, syringolin, AHL, alginate, protease, virulence RsmX, RsmY, RsmZc Willis et al. (2001); Quinones et al. (2004) Pseudomonas syringae pv. tomato GacS/GacA Coronatine, AHL, TTSS, virulence RsmXa, RsmY, RsmZ Chatterjee et al. (2003) Pseudomonas tolaasii PheN(RtpA)/? Tolaasin, protease, virulence, motility ? Grewal et al. (1995); Murata et al. (1998) Pseudomonas viridiflava RepA/RepB Extracellular pectinase, protease, alginate, virulence ? Liao et al. (1996) Salmonella enterica BarA/SirA TTSS, invasion, motility CsrB, CsrC Altier et al. (2000); ssp. Typhimurium Fortune et al. (2006) Molecular Microbiology Serratia marcescens PigW/PigQ Prodigiosin ? Williamson et al. (2006) Serratia plymuthica GrrS/GrrA Extracellular protease, pyrrolnitrin, biocontrol ? Ovadis et al. (2004) Vibrio cholerae VarS/VarA HapR-dependent virulence factors CsrB1a, Lenz et al. (2005) CsrB2a (= CsrC), CsrB3a (= CsrD) Vibrio fischeri GacS/GacA Bioluminescence, squid colonization CsrB1a, CsrB2a Whistler and Ruby (2003) © 2007 The Authors a. Predicted by Kulkarni et al. (2006). b. In uropathogenic strains. , 67 c. Predicted by BLASTN. , 241–253 AHL, N-acyl-homoserine lactone; DAPG, 2,4-diacetylphloroglucinol; TTSS, type III secretion system; ? indicates that GacS/GacA-dependent sRNAs or regulators have not yet been identified. Regulation of RsmA/CsrA binding to RNA 243 Fig. 1. General characteristics of the Gac/Rsm signal transduction pathway in g-proteobacteria. The highest number of sRNA genes (five) is predicted in Photobacterium profundum (Kulkarni et al., 2006). The highest number of genes for small RNA-binding proteins (four) appears to occur in P. syringae pv. tomato (Rife et al., 2005). ↓, positive effect; ^, negative effect; dotted line, positive feedback loop; X, unknown hypothetical component. interaction, which ultimately determines the output of the compromised envZ mutant of Escherichia coli (Nagasawa regulatory pathway. The emphasis of this review will be on et al., 1992). The response regulator GacA was discov- the mechanisms that regulate target gene expression in ered as a master regulator of antifungal metabolites in the the Gac/Rsm cascade, on common features of target biocontrol bacterium Pseudomonas fluorescens CHA0 genes and on recent insight gained by structural analysis (Laville et al., 1992). Evidence that GacS and GacA form of a complex formed between an RsmA/CsrA-type protein a two-component system came from genetic studies in and a target RNA. P. syringae (Rich et al., 1994) and later from in vitro phos- photransfer experiments with the GacS/GacA homo- Putting the pieces of a jigsaw together logues BarA/UvrY in E. coli (Pernestig et al. 2001). At the hierarchical level below GacS/GacA, two genes that When the components of the Gac/Rsm pathway were first encode GacA-controlled sRNAs were initially found as a described in various bacteria, the researchers had widely multicopy suppressor (TRR) of a phaseolotoxin-negative, different objectives. The sensor kinase GacS (originally presumably GacA-defective mutant of P. syringae pv. designated LemA) was found in the plant pathogen phaseolicola (Rowley et al., 1993) and as an activator Pseudomonas syringae pv. syringae as a key regulator of of extracellular virulence