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The role of pheromones in bacterial interactions
R E V I E W S
The role of pheromones in bacterial interactions Reinhard Wirth, Albrecht Muscholl and Gerhard Wanner
p
heromones were defined (Agrobacterium tumefaciens), An increasing number of bacterial species in 1959 by Karlson and LasR (Pseudomonas aerugiare found to communicate via excreted Liischer ~ as substances pheromones. These signals trigger various nosa) and YenR (Yersinia enterocolitica), to define a LuxR which are secreted to the outresponses, including luminescence, side by an individual and resuperfamily of response reguproduction of virulence factors, lators. A recent comparison 6 ceived by a second individual development of fruiting bodies, lists ten regulators known to be of the same species, in which competence and sporulation, secondary they release a specific action, for induced by AHLs. In addition, metabolism and plasmid transfer. the transcriptional activators example, a definite behaviour R. Wirth * and A. Muscholl are in or a developmental process. The FixJ (Rhizobium meliloti), the Universitat Regensburg, Lehrstuhl fiir principle of minute amounts NarL, MalT, RpoD (EscherMikrobiologie-Archaebakterienzentrum, ichia coli) and GerE (Bacillus being effective holds...', to difUniversit#tstrafle 31, D- 93053 Regensburg, subtilis) belong to this superferentiate these extracellular Germany,; G. Wanner is in the Universit~it M~inchen, Institut f~ir Botanik, Menzinger Strafle 67, signals from intracellularly actfamilyL Likewise, a LuxI superD- 80638 Miinchen, Germany. family of signal-generating (i.e. ing hormones. Pheromones are '=tel: +49 941 943 3182, fax: +49 941 943 2403, AHL-synthesizing) proteins can one of the few means by which [email protected] be defined and recently ten of a single bacterial cell can obtain these have been documented 7. information from others of the Why bacteria like P. fischeri should emit light when same species (the extracellular fibrils of Myxococcus xanthus constitute another mechanism; see below). their critical cell density is above 107ml 1 (indicated by AHL concentrations > ] 0 - 9 M ) remains unknown. Communication via such signals is important for the survival of bacteria in nature, not only as defense Some bioluminescent bacteria live free in the ocean at against predators and competitors but also to supply concentrations of about 10 z cells m1-1, but in the symbiotic organs of luminous fish and squids they exceed information about members of the same species. Pherothe critical cell density, reaching 1011cells ml-'. It could mones belonging to several chemical classes have been be speculated that pheromone-induced enzymes conidentified for a great variety of bacteria. This review tribute to bacterial nutrition in the symbiotic light ordiscusses the biology of some of these systems with the gans. In these organs, doubling times of about 20 h are emphasis on N-acyl-L-homoserine lactones (AHLs), observed, compared with <1 h for growth in nutrientfound in a great variety of Gram-negative bacteria, and rich medium. Interestingly, it was shown recently s that the sex pheromone system of Enterococcus faecalis, a pheromone of Vibrio harveyi not only regulates biowhich is very probably restricted to that Gram-positive luminescence, but also induces the formation of polyspecies (Table t). 3-hydroxybutyrate, which might act as an energy source for maintaining cell viability in the stationary stage of AHL-regulatedsystems AHLs were first identified in luminous bacteria. If growth in this species. For P. fischeri, regulation is more complex than prepresent above a certain concentration, these excreted viously thought 9, as two pheromones are produced substances induce an operon of genes including those from luxI and a third from ain. Similarly, more than for their own production (AHLs are also called autoone pheromone seems to be produced by V. harvey? °. inducers). For Vibrio and Photobacterium species the induced proteins enable the bacteria to emit light. AHLs LuxR of P. fischeri is composed of two domains, of also act in systems totally unrelated to bioluminescence which the amino-terminal domain of 160 amino acids inhibits the DNA-binding activity of the carboxy-ter(see Table 1). AHLs are produced by LuxI-type prominal domain (amino acids 160-250) ~1. Binding of the teins, diffuse freely over the cell membrane and accumulate in the medium. If sufficiently large amounts of autoinducer relieves the inhibition. In contrast, LuxR AHLs are accumulated extracellularly these signals acti- of V. harveyi does not bind an autoinducer 12 and has its own distinct DNA-binding sites. vate an intracellular response regulator of the LuxR The nutritional needs of Erwinia carotovora (a plant type to induce transcription of at least one operon, including a luxI-type gene. Excellent recent reviews 2 pathogen) seem to be a plausible explanation for the pheromone-stimulated expression of enzymes that lyse cover this field. Sufficient sequence similarities exist between differ- plant cell walls 13. The effectiveness of these enzymes is increased if many bacterial cells of the same species ent transcriptional activators, including LuxR (Photobacterium fischeri; also known as Vibrio fischeri), TraR attack susceptible plants. Copyright © 1996 Elsevier Science Ltd. All rights reserved. 0966 842X/96t$15.00 -|'I
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Table 1. Characterization of bacterial pheromone systems" Signal(s)
Structure/minimal active concentration
Bacterial species
Comments
AHLs AHLs
OHHL/IO 9M; HHL/? (prob. 10-8M); OOHL/? (prob. 10 7M) HBHL/IO-6M; ?/?
Photobacterium fischeri Vibrio harveyi
AHL
OHHL/IO-SM
Erwinia carotovora
AHLs
ODDHL/IO-6M; BHL/IO 6M; HHL/?; OHHL/?
AH Ls
OOH L / I O -6 M; 0 HHL / I O -6 M
Pseudomonas aeruginosa Agrobacterium tumefaciens
AH Ls
HHL/?; OHH L/?
Yersinia enterocolitica
AHL?
?/?
AHL?
?/?
'A-signal'
Mixture of peptides and amino acids/lO-6M
Pseudomonas aureofaciens Various Gram-negative species Myxococcus xanthus
'E-signal'
Branched-chain fatty acids?/(<10 3Mb)
M. xanthus
Lipid
? (200 Da)/lO 9M
Stigmatella aurantiaca
Competence factor
8 or 9 amino acids long, modified peptide/lO-9M
Bacillus subtilis
Many, e.g. A-factor
Butyrolactone/lO -9 M
Streptomyces species
SapB
18 amino acids long, modified peptide/?
Streptomyces coelicolor
Rap/Rip
Protein (38 kDa)/?; pentapeptide/?
Staphylococcus aureus
?/?
Anabaena species
? (5-Membered lactam ring fused to 5-membered thioketon ring)/<3 x IO-FM
Cylindrospermum licheniforme
Chemoattractant
Aspartate/lO 7M per cell; see Ref. 40
Escherichia coil
'Used medium'
undefined/?
Bacillus species
Sex pheromones
cOB1:V-A-V-L-V-L-G-A/2 x 10-1°M cAD1:L-P-S-L-V-L-A-G/5 x 10 11M lAD1: L-P-V-V-T-L-V-G/IO-l° M cPDI: P-L-V-M-P-L-S-G/5 x lO-11M iPDI: A-L-I-L-T-L-V-S/? cCFIO: L-V-T-L-V-P-V/2.5 x 10-11M iCFIO: A-I-T-L-I-P-I/IO I°M cAM373:A-I-P-I-L-A-S/5 x 10-11M iAM373:S-I-P-T-L-V-A/2 x 10 l°M
Enterococcus faecalis
Induce bacterial light emission Induce bacterial light emission Induces lytic enzymes and carbapenem antibiotic Induce elastase and other virulence factors Enhance conjugative transfer of Ti plasmids between bacterial cells Regulated proteins not yet physiologically characterized Induces phenazine antibiotics LuxR-activating activity described (see text) Induces fruiting-body formation Needed for completion of development Induces fruiting-body formation Induces competence for uptake of naked DNA Control secondary metabolism and sporulation Leads to formation of aerial hyphae Regulate the inducer for production of toxic exoproteins Determines spacing between heterocysts in filaments Induces formation of akinetes (other inducers of unknown structure needed) Induces stable regular growth patterns of chemotactic cells Induces colony patterns via 'chemotactic feedback' Induce sex-pheromoneplasmid-encoded adhesin, leading to enhanced conjugation. At least six, but probably more sex pheromones are produced by E. faecalis
"AHL, N-acyI-L-hOmoserinelactone; the abbreviations used for AHLs follow the nomenclature in Ref. 4; there the structure of some AHLs plus that of A-factor from Streptomyces griseus is given. The structure of sex pheromones and the inhibitory peptides of E. faecafis are given in the standard one-letter amino acid code. ? Means no data are available. bSee Ref. 25.
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In P. aeruginosa, expression of elastase and other extracellular virulence factors is regulated by AHLs. Again, it can be argued that it is advantageous for an opportunistic pathogen to synthesize virulence factors only if many bacteria are colonizing a tissue. Pseudomonas aeruginosa strain PAO1 synthesizes at least three AHLs 7and a total of four AHLs have been isolated from different strains. The LasR-LasI system regulates elastase, alkaline phosphatase, exotoxin A, exoenzyme S, heat-stable hemolysin and neuraminidase; the RhlRRhlI system regulates elastase, alkaline phosphatase, hemolysin, pyocyanin and HCN. The presence of these multiple regulatory circuits is thought to allow fine tuning of the regulation of P. aeruginosa exoproducts resulting in their optimal concentrations for varying growth conditions 7. Agrobacterium tumefaciens transfers its Ti plasmid conjugatively to susceptible plants and to members of its own species. Transfer (and expression of opinedegradation genes) is stimulated up to 10a-fold by at least two pheromones 14. Recently, the secretion of two AHLs by Y. enterocolitica has been reported 6. They are produced from a single protein, YenI, which belongs to the LuxI superfamily; the gene for a putative transcriptional regulator, YenR, is transcribed convergently to yenI. Regulation of virulence genes by these pheromones has not yet been shown, although AHL mutants have been made. This system awaits further analysis as, interestingly, yenI is not subject to autoinduction, but is expressed constitutively. Phenanzine antibiotics are produced by Pseudomonas aureofaciens and believed to play an important role in microbial competition and rhizosphere survival. Again, they are produced when cell density is reaches a threshold, and are regulated by PhzR, a member of the LuxR superfamily of response regulators. Although there is no evidence that P. aureofaciens synthesizes an AHL it is highly probable that it does, because P. aeruginosa autoinducer extract triggers phenanzine antibiotic production by P. aureofaciens. In one study using a lux-plasmid-based bioluminescence sensor system, pheromone production was demonstrated by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens 16. Only in E. carotovora has a role for AHL in pathogenesis been shown. Other bacteria that produce AHLs include Citrobacter species, Chromobacterium violaceum and four different Yersinia species, but it is not known if these organisms use the AHLs as pheromones. As the detection system relies on activation of LuxR by a cognate pheromone ~6, one might speculate that other AHL-derived pheromone systems (such as ones inducing LasR-homologous transcriptional activators) exist in bacteria. The pheromone in E. carotovora that acts as cell-density indicator is identical with 'factor 1' of P. fischeri. The totally different biotopes in which the two species are found excludes potential 'pheromone crosstalk' between these bacteria. It had been argued that the inducer(s) of P. fischeri could act in the P. aeruginosa system 13. The significance of this finding has been
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questioned by a recent report iv because again these two species do not occur in the same biotope. Although pheromone crosstalk has been shown for P. aeruginosa and P. aureofaciens under laboratory conditions is, it would appear to have no relevance in natural systems. Pheromones of fruiting-body-forming bacteria Detailed studies on the signals needed for the development of fruiting bodies are available only for the two species, Stigmatella aurantiaca and M. xanthus. The multicellular fruiting bodies of these species not only produce stress-resistant myxospores, but also maintain them in numbers high enough to allow successful regrowth. An early trigger for the development of fruiting bodies is the exhaustion of food supplies; other signals, including pheromones, are also needed. In the case of S. aurantiaca, an as-yet-incompletelycharacterized pheromone triggers the aggregation and swarming of bacterial cells on solid surfaces. The aggregates develop into stalks on which the myxosporecontaining sporangia are formed. The pheromone was characterized as a lipid of low molecular mass TM, extractable with chloroform and methanol from filter paper located below developing fruiting bodies of S. aurantiaca. Data from H. Schaierer's group (pers. commun.) indicate that a volatile substance can be purified by water-vapor distillation from developing S. aurantiaca cells incubated on solid surfaces. The pheromone is produced by cells early in development and elucidation of its structure will be a major breakthrough for studies on this system. Myxococcus xanthus excretes a 'diffusible fruiting factor '19 and studies on mutants have helped to define the series of 'signals' needed for the development of the fruiting bodies2°,21.Dworkin's 22detailed discussion of the various 'signals' required for the formation of fruiting bodies described most of the components as surface proteins or DNA-binding proteins. The socalled A-signal consists of two components; the heatlabile component contains two proteases and the heatstable component is a mixture of single amino acids and small peptides (produced by the action of the heatlabile component on extracellular substrates). If present in high concentrations (>10 3M), the heat-stable component supports growth; however, concentrations below 10-6M induce the formation of fruiting bodies. It was speculated recently23that the heat-labile A-signal might be connected to the inhibition of ADP-ribosylation of endogenous M. xanthus protein. Interestingly, the substrates for ADP-ribosylation seem to be located in the extracellular fibrils 24 that connect the cells during early development. It is tempting to speculate that, via this mechanism, a connection to other cellular regulatory circuits might exist. Branched-chain fatty acids are known to be released and are constituents of the E-signal 2s, which is needed to complete the development ofM. xanthus. This pheromone system has been discussed in detail elsewhere 26. Competence factor of B. subtilis In certain growth phases B. subtilis can take up 'naked DNA' triggered by a competence factor. Early data
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indicated that the development of competence and sporulation are somehow connected. The role of the competence factor in B. subtilis, its mode of synthesis, the sensory pathway and the role of the regulatory cascade triggered by the peptide have been described recently2v (see Fig. 5 in Ref. 27 for an excellent overview). Here, only a few aspects are discussed. Competence factor is a linear peptide of nine or ten amino acids, possessing an undefined modification (perhaps a fatty acid addition) that is essential for activity. The pheromone peptide sequence matches nine of the last ten amino acids predicted from the nucleotide sequence of comX. This gene, and the upstream gene comQ, are required for pheromone production, with ComQ possessing processing and/or modification activity. The excreted pheromone is sensed by the membrane-located SpoOK oligopeptide permease, which has ATPase activity. Pheromone sensing results, via an unknown mechanism, in phosphorylation of ComP. ComP might be associated with the SpoOK permease, because its amino-terminal domain contains eight putative membrane-spanning helices (the carboxy-terminal domain is similar to that of the histidine protein kinases). ComP-phosphate phosphorylates ComA, the pheromone-binding subunit of SpoOK. ComA-phosphate, in turn, directly activates the large operon (>25 kb) srfA, which encodes peptide synthetases needed for synthesis of the lipopeptide antibiotic surfactin. At present it is unclear whether ComA-phosphate induces only the srfA operon. Its effect on the competence system might be indirect because srfA expression leads to accumulation of ComK, a transcription factor that activates expression of the late competence operons (comC, comE, comF and comG). One connection between the development of competence and sporulation lies in comQ; a comQ mutant shows less efficient sporulation in minimal medium, up to a hundredfold reduction compared with rich media2v. Pheromones of Actinomycetes A-factor, produced by Streptomyces griseus, was the first pheromone identified in an Actinomycete; it is just one example of the very large group of butyrolactone autoregulators 28,29. The question of whether B-factor (butyl ester of 3'-AMP; active at 10-1°M) and C4actor (a 34.5kDa protein) are extracellularly acting signals remains open. A-factor triggers the expression of streptomycin production, streptomycin resistance, yellow pigment production and the formation of aerial hyphae (as a prerequisite for sporulation) via interaction with its receptor ArpA. On binding, A-factor relieves the repressing activity of ArpA, a protein possessing an (~-helix-turn-R-helix DNA-binding motif3°. The role of prokaryotic homologs of the eukaryotic serine/threonine kinases for the regulation of differentiation in this group of bacteria was discussed recently> . The formation of aerial hyphae by Streptomyces coelicolor is dependent on the peptide SapB, present in large amounts on cell surfaces and released into a zone surrounding the colonies31. For the production of SapB, diffusible signals seem to be needed3L
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Other systems The production of toxic exoproteins by Staphylococcus aureus depends on the global regulatory Agr system. The agr gene is autoinduced by secreted RNA III activating protein (Rap) (Ref. 33). Rap seems to interact with the putative receptor AgrC (a signal transducer of a two-component signal transduction system); AgrCphosphate then interacts with the response regulator AgrA. AgrA-phosphate activates transcription of RNA III, which in turn leads to expression of enterotoxin B, hemolysin and toxic-shock syndrome toxin 1. A naturally occurring mutant of S. aureus, excreting the inhibitor RNA-III-inhibiting protein (Rip) (which counteracts Rap activation), was also identified33; it is unclear whether Rap and Rip act via different receptors. In the filamentous cyanobacterium Anabaena, the distance between heterocysts appears to influence the excretion of an inhibitor of heterocyst formation 34 by existing heterocysts. For another cyanobacterium, Cylindrospermum licheniforme, the formation of akinetes seems to be triggered by a partially characterized pheromone3L Very clearly, these last two systems have to be further characterized. Inducers of growth patterns The formation of fractal-like colony patterns of Bacillus species occurs in used medium and is the product of chemotactic feedback36,3v.This means that extracellular signals are produced by the growing cells and sensed by them. The 'communicating walkers' approach 37has also been used to explain38the formation of growth patterns 39of motile E. coli (and Salmonella typhimurium) cells in low-viscosity agar media. The formation of these remarkable patterns depends on the excretion of aspartate, which acts as an attractant 4°, by the growing cells. Sex pheromones of E. faecalis The sex pheromone system of E. faecalis was discovered by Don Clewell's group 41 during experiments on conjugative plasmids. These bacteria have two different kinds of conjugative plasmids 42. Class I is about 30 kb, has a broad host range among Gram-positive bacteria and normally encodes antibiotic resistance. Class 2 is more or less restricted to E. faecalis, tends to be >60 kb and in only a few cases codes for drug resistance. In liquid medium the transfer of plasmids belonging to class 2 ('sex pheromone plasmids') is more efficient than for class 1 plasmids [up to 10 -1 for class 2 (per donor) compared with 10 -6 for class 11, because of the clumping of bacteria induced by the so-called sex pheromones excreted by plasmid-free strains. The sex pheromones are excreted by plasmid-free recipient strains of E. faecalis and sensed by donor strains carrying a sex pheromone plasmid. They induce the production of an adhesin (aggregation substance) encoded by the sex pheromone plasmid, thereby leading to a bacterial clumping reaction (see front cover of this issue); this, in turn, allows the high-efficiency conjugation of sex pheromone plasmids. Sex pheromones are named after the plasmid they induce (for example, sex pheromone plasmid pAD 1 is induced by sex pheromone
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pPD1
seal
I
III
asal
pAD1
1 kb
~-~
~
Fig. 1. Comparison of sequenced portions of sex pheromone plasmids pPD$, pAD1 and pCFIO, containing structural and regulatory genes for pheromone-induced aggregation. The solid lines indicate plasmid DNA; the broken line for pPD1 indicates that the sequence for sepl, the structural gene for surface-exclusion protein, has not yet been completed. Boxes indicate genes that have been translated into proteins. Transcription is usually from left to right; exceptions are indicated by the arrows. Sequence similarity is shown by different colors (pink, 30-50%; yellow, 50-75%; green, >90% identity): for all boxed genes this refers to the percentage identity of amino acid sequences but for the regulatory regions including stem-loop structures (closed circles on a stalk; see also Box 1), the sequence similarity refers to nucleotide sequence. A description of most genes can be found in Ref. 44 although some unpublished sequences have been included.
cAD1). A strain carrying one sex pheromone plasmid can act as recipient for (all?) others. This is probably why plasmid-free strains of E. faecalis are rare. Recent reviews 43~s provide a more detailed picture. Sex pheromones are linear hydrophobic peptides of seven or eight amino acids, which are probably synthesized on ribosomes. They are highly active: 1-10 molecules per cell (about 10 -15M) are sufficient for induction. Inhibitory peptides encoded by sex pheromone plasmids have 25-50% identity to the corresponding chromosomally encoded inducing peptides, suggesting that there are specific receptors for pheromones in E. faecalis. Genetic and biochemical data indicate that, in addition to the receptors TraC (Ref. 46) and PrgZ (Ref. 47) encoded by sex pheromone plasmids, chromosomally encoded proteins also interact with sex pheromones. The inhibitory peptides iPD1, iAD1 and iCF10 are encoded by ipd, iad and prgQ on the sex pheromone plasmids pPD1, pAD1 and pCF10, respectively48-s°. They are produced from precursors of 21, 22 and 23 amino acid residues, with the carboxy-terminal amino acids comprising the active peptide. Interestingly, recombinant E. coli strains can secrete the inhibitory peptides; possibly the amino-terminal amphiphilic helices are involved in transport across bacterial cell membranes. The genetic organization of the genes for the activating peptides is unknown, although the pheno-
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type of mutant strains indicates processing from a common precursor. Alternatively, processing from the 'core' of a signal peptide might occur; indeed, processing and excretion of a cADl-like peptide from the signal peptide of an S. aureus gene was observed sl. A peptide-inducing sex pheromone plasmid, pAM373, is produced by E. faecalis strains and by some other Gram-positive bacteria s2 but, except for E. faecalis, it is unclear whether these peptides serve any functional role for the producers. Sex pheromones are active in eukaryotic systems 44-~3 but the question of whether or not they are virulence factors, as are the adhesins induced by them, is open to speculation. Sex pheromone plasmids have a highly conserved DNA region coding for surface exclusion protein and aggregation substance s4y. Sequence data are available for sex pheromone plasmids pAD1, pPD1 and pCF10 (Refs 44,48,56-58) (Fig. 1). DNA sequence identities for the structural genes are in the range of 50 to >95%. This is not the case for DNA regions coding for regulatory functions (for example, traA and prgX show <15% similarity; data on the function of orf5 from pPD1 are lacking); on the other hand, a region of 350 bp containing strong stem-loop structures is nearly identical in pAD1 and pCF10. It can be concluded from this that a common ancestor should exist for sex pheromone plasmids and that evolution of the various plasmids has involved combinations of different regulatory
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genes and open reading frames of unknown functions. Some ideas on the evolution of the sex pheromone system have recently been formulated 44. At present it is not possible to define clearly distinct sex pheromone plasmids (although at least 20 different restriction profiles exist) but they can generally be distinguished by their inducibility by different sex pheromones where the cognate pheromone structure is known 44. The DNA differences of pAM373 set it apart from all other sex pheromone plasmids 44. Aggregation substance was long ago reported to be proteinaceous and located on the surface of induced cells. Using immunological and electronmicroscopic studies we showed that this adhesin appears as hair-like structures incorporated mainly in 'old' cell wall 59'6°. The DNA sequence of asal, the structural gene of pADl-encoded aggregation substance, revealed the presence of two RGDS motifs 56. This indicated possible interactions with eukaryotic cells via surface receptors belonging to the integrin class. Indeed, an aggregation-substance-dependent interaction occurs with in vitro cultured pig kidney tubular cells61;interestingly, addition of the peptide RGD reduced aggregation-substance-mediated binding to eukaryotic cells. Binding of E. faecalis to other eukaryotic cells via this adhesin has also been observed. Unpublished work from our group indicates that the amino-terminal region of aggregation substance is probably the domain interacting with E. faecalis cells; interaction with eukaryotic cells is mainly via the carboxy-terminal domain, which also contains the RGD m o t i f s 44.
Box 1. Model for regulation of sex pheromone plasmid pAD1
The upper part shows an uninduced cell of Enterococcus faecalis, the lower part a cell induced by sex pheromone cAD1. The solid black line represents pAD1 DNA, above which the names of relevant genes are given; their length and direction of transcription are indicated below. Under non-induced conditions a negative-regulatory gene traA prevents transcription proceeding from a promoter located directly upstream of lad1 through the stem-loop structures between iadl and traE1 (indicated by the red line). Upon induction by sex pheromone cAD1 this negative effect is relieved, allowing transcription to proceed beyond traE1 (indicated by the yellow arrow). The positive regulator TraE1 acts in trans and allows transcription of at least three mRNAs (indicated by the green arrows). The first one starts at the promoter located directly upstream of iadl and ends after traE1, thereby creating an autoregulatory circuit. (This idea is supported by our data that cells of E. faecalis show an 'all or nothing' induction effect; no intermediate levels of adhesin can be found63.) The second transcript starts upstream of a small open reading frame (with unknown function) located just before seal and ends directly downstream of seal. (Under non-inducing conditions seal is expressed constitutively from a -10/-35-type promoter; induction leads to activation of a second, sex pheromone-dependent promoter.) The third, also strictly cADl-dependent transcript, starts upstream of a small open reading frame (with unknown function), located just before asal, the structural gene for the adhesin. This third transcript seems to possess a prominent termination signal after the small open reading frame downstream of asal. A small portion of it, however, extends some 4 - 5 kb beyond the terminator, to allow transcription of genes needed for 'stabilization of aggregates'. The main product of the asal-specific transcript clearly is the adhesin, appearing as hair-like structure (blue color) on the surface of induced cells.
A model for the regulation of pAD 1 is shown in Box 1. It is based on analysis of mutations in regulatory genes62 and of transcripts before and after induction 63,6a,and on the finding that the positive regulator TraE1 can function in trans 57,63,64. The traE1 gene is expressed only after induction, and synthesis of TraE1 precedes that of Asal. The regulatory mechanisms leading to the induction of pAD1 and pCF10 are probably completely different44. For pCF10 the existence of a cisacting positive control element has been reported 65. A model for the regulation of pCF10 postulates direct interaction between transcripts from the regulatory region and RNA polymerase to allow transcription of structural genes66. This implies that regulation of pCF 10
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could be mainly at the level of RNA stability and translation. It is expected that in both cases only an in vitro system driving specific expression of sex-pheromoneregulated promoters will allow understanding of the underlying regulatory circuits.
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Questions for future research • How widespread are bacterial pheromone systems? For example, does an earlier report ~8 on excretion of insulin-like signal(s) by Escherichia coli indicate the existence of an as-yet-unidentified pheromone system in this extensively studied species? • How do the signal transduction chains function? Do connections exist to other regulatory circuits? Which other regulatory systems influence genes activated by N-acyl-L-hOmoserine lactones (AHLs)? For Erwinia carotovora, does inactivation of the global represser locus rsmA lead to overproduction of extracellular enzymes in an AHL-independent manner69? • Are parameters other than cell density indicated by pheromones? • AHLs supposedly diffuse freely over bacterial membranes. What mechanism ensures that the signals have to act from the outside and that newly synthesized AHLs do not constitutively induce the producing cell? Is AHL production merely constitutive or might their synthesis be regulated by factors like the growth stage/ nutritional status of the producer or by growth temperature? • How has the Enter•coccus faecalis sex pheromone system evolved? What is the genetic organization of structural genes for the signals and what is the role of sex-pheromone-like peptides excreted by other species?
Concluding remarks
It is evident from the data discussed here that so far pheromones have only been described in systems in which their effect can easily be detected (for example, light emission by bacteria, production of virulence factors, formation of fruiting bodies and aggregation). It is tempting to speculate that other systems exist that enable bacteria to obtain information on others of their kind. To date AHLs, for example, have been detected only in Gram-negative bacteria; might they also be present in Gram-positive bacteria ? Likewise, no pheromones have been described for Archaeans; probably because no one has looked yet. Swarming behavior of, for example, P r o t e u s mirabilis very well might be regulated by pheromones and, indeed, extracellular glutamate seems to play a role here 67. Advances are occurring rapidly and, during the preparation of this review, the S. aureus system was described and data on quorum sensing, the regulation of bacteriocin production in L a c t o b a c i l l u s and the production of N - ( 3 R hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone acting as both a bacteriocin and as an autoinducer of rhi genes in R h i z o b i u m l e g u m i n o s a r u m were described at the Beijerinck Centennial meeting*. For some systems, at least, ideas exist on how the signal transduction chain might function. It is tempting to speculate that pheromonal signals produced by bacteria might be received and transduced in a way similar to other environmental signals. Therefore, crosstalk between pheromone transduction chains and other signaling systems is a possibility. The B. subtilis system discussed above may offer an example; it seems unlikely that competence and sporulation are regulated solely via one common system, c o m Q . All the pheromones described to date can be viewed as signals for the cell density of a bacterial species. *The Beijerinck Centennial meetingwas held at The Hague, The Netherlands, 10-14 December 1995.
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However, one can imagine how other situations such as nutritional status might be communicated to other cells of the same species. Very clearly work on such interactions needs to be done under more natural circumstances than growth in rich media under laboratory conditions. Acknowledgements
Space constraints have limited detailed discussions in this rapidly developing field; it is hoped that access to relevant primary data is possible from the literature cited. We want to thank all present and past members of our laboratories for their dedicatedwork on the E. faecalis sex pheromonesystem.The financialsupportof the authors by DFG (SFB145, B8 and Y1) is gratefullyacknowledged. References
1 Karlson,P. and Liischer,M. (1959) Nature 183, 55-56 2 Williams,P. etal. (1992) FEMS Microbiol. Lett. 100, 161-168 3 Fuqua,W.C., Winans,S.C. and Greenberg,E.P. (1994) J. Bacteriol. 176,269-275 4 Swift,S., Bainton,N.J. and Wins•n, M.K. (1994) Trends Microbiol. 2, 193-198 5 Salmond,G.P.C.et al. (1995)Mol. Microbiol. 16, 615-624 6 Throup,J.P. et al. (1995)Mol. Mitt•biol. 17, 345-356 7 Latifi,A. et al. (1995) Mol. Microbiol. 17, 333-343 8 Sun,W. et aL (1994)J. Biol. Chem. 269, 20785-20790 9 Kuo,A., Blough,N.Y. and Dunlap,P.V. (1994)J. Bacteriol. 176, 7558-7565 10 Bassler,B.L.et al. (1993) Mol. Microbiol. 9, 773-786 11 Stevens,A.M., Dolan, K.A.and Greenberg,E.P. (1994) Proc. Na.tl Acad. Sci. USA 91, 12619-12623 12 Miyamoto,C.M. et al. (1994)Mol. Microbiol. 14, 255-262 13 Jones, S. et al. (1993) EMBO J. 12, 2477-2482 14 Zhang,L. et al. (1993) Nature 362, 446-448 15 Pierson,L.S.,III, Keppenne,V.D. and Wood,D.W. (1994) J. Bacleriol. 176, 3966-3974 16 Swift,S. el al. (1993) Mol. Microbiol. 10, 511-520 17 Gray,K.M.et al. (1994)J. Bacteriol. 176, 3076-3080 18 Stephens,K., Hegeman,G.D. and White,D. (1982)J. Bacteriol. 149, 739-747 19 Lev,M. (1954) Nature 173, 501 20 Hagen,D.C. et al. (1978)Dev. Biol. 64, 284-296 21 Downard,J., Ramaswamy,S.V. and Kil,K-S.(1993)J. Bacteriol. 175, 7762-7770 22 Dworkin,M. (1991) in Microbial Cell-Cell Interactions (Dworkin, M., ed.), pp. 179-216, AmericanSocietyfor Microbiology 23 Eastman,D. and Dworkin,M. (1994) Microbiology, 140, 3167-3176 24 Dworkin,M. (1993) in Myxobacteria II (Dworkin,M. and Kaiser, D., eds), pp. 63-83, AmericanSocietyfor Microbiology 25 Toal, D.R. et al. (1995) Mol. Microbiol. 16, 177-189 26 Downard,J. and Toai, D. (1995) Mol. Microbiol. 16, 171-175 27 Magnus•n,R., Solomon,J. and Grossmann,A.D. (1994) Cell 77, 207-216 28 Horinouchi,S. and Beppu,T. (1992) Annu. Rev. Microbiol. 46, 377-398 29 Beppu,T. (1995) Trends Biotechnol. 13, 264-269 30 Onaka, H. et al. (1995)J. Bacteriol. 177, 6083-6092 31 Willey,J. et al. (1991) Cell 65, 641-650 32 Willey,J., Schwedock,J. and Losick,R. (1993) Genes Dev. 7, 895-903 33 Balaban,N. and Novick,R.P. (1995) Proc. Nail Acad. Sci. USA 92, 1619-1623 34 Kaiser,D. and Losick,R. (1993) Cell 73, 873-885 35 Hirosawa,T. and Wolk, C.P. (1979)]. Gen. Microbiol. 114, 433-441 36 Schindler,J. (1993) Trends Mitt•biol. 1~333-338 37 Ben-Jacob,E. et al. (1994) Nature 368, 46-49
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38 Ben-Jacob,E. et al. (1995) Nature 373, 566-567 39 Budrene,E.O. and Berg,H.C. (1991) Nature 349, 630-633 40 Budrene,E.O. and Berg,H.C. (1995) Nature 376, 49-53 41 Dunny,G.M.et al. (1978) Proc. Natl Acad. Sci. USA 75, 3479-3483 42 Clewell,D.B. (1981) Microbiol. Rev. 45,409-436 43 Clewell,D.B. (1993) Cell 73, 9-12 44 Wirth,R. (1994) Eur. J. Biochem. 222, 235-246 45 Dunny,G.M. (1995)J. Bacteriol. 177, 871-876 46 Tanimoto,K., An, F.Y. and Clewell,D.B. (1993)J. Bacteriol. 175, 5260-5264 47 Ruhfel,R.E., Manias,D.A. and Dunny,G.M. (1993)J. Bacteriol. 175, 5253-5259 48 Fujimoto,S. et al. (1995)J. Bacteriol. 177, 5574-5581 49 Clewell,D.B.et al. (1990) Plasmid 24, 156-161 50 Nakayama,J. etal. (1994)J. Bacteriol. 176, 7405-7408 51 Firth,N. et al. (1994)J. Bacteriol. 176, 5871-5873 52 Clewell,D.B.et al. (1985)J. Bacteriol. 162, 1212-1220 53 Ember,J.A. and Hugli,T.A. (1989)Am.J. Pathol. 134, 797-805 54 Galli,D. and Wirth, R. (1991)J. Bacteriol. 173, 3029-3033 55 Hirt, H. et al. (1993) Eur. J. B iochem. 211,711-716 56 Galli,D., Lottspeicb,F. and Wirth, R. (1990) Mol. Microbiol. 4, 895-904 57 Galli,D., Friesenegger,A. and Wirth, R. (1992) Mol. Microbiol. 6, 1297-1308
58 Kao,S-M.et al. (1991)J. Bacteriol. 173, 7650-7664 59 Galli,D., Wirth, R. and Wanner,G. (1989) Arch. Microbiol. 151,486-490 60 Wanner,G. et al. (1989) Arch. Microbiol. 151,491-497 61 Kreft,B. et al. (1992) Infect. Immun. 60, 25-30 62 Weaver,K.E.and Clewell,D.B. (1998)J. Bacteriol. 170, 4343-4352 63 Muscholl,A. et al. (1993) Eur. J. Biochem. 214, 333-338 64 Tanimoto,K. and Clewell,D.B. (1993)J. Bacteriol. 175, 1008-1018 65 Chung,J.W. and Dunny,G.M. (1992) Proc. Natl Acad. Sci. USA 89, 9020-9024 66 Chung,J.W.and Dunny,G.M.(1995)J. Bacteriol. 177, 2118-2124 67 Allison,C. et al. (1993) MoI. MicrobioI. 8, 53-60 68 LeRoith,D. et al. (1980)J. Biol. Chem. 256, 6533-6536 69 Chatterjee,A. et al. (1995) Appl. Environ. Microbiol. 61, 1959-1967 70 Bassler,B.L.and Silverman,M.R. (1995) in Two-component Signal Transduction (Hoch,J.A. and Silhavy,T.J., eds), pp. 431-445, AmericanSocietyfor Microbiology Note added in proof An outstanding comparative discussion of regulatory functions in the P. fischeri and V. harveyi quorumsensingsystemscan be foundin Ref. 70.
The contribution of pneumolysin to the pathogenicity of
Streptococcuspneumoniae James C. Paton The antiphagocytic polysaccharide capsule charides themselves (of which treptococcus pneumoniae there are over 80 serotypes) are of Streptococcus pneurnoniae has long is a human pathogen.of completely nontoxic and cannot been been considered the principal malor importance, causing alone account for death from virulence determinant of this organism. invasive diseases such as pneupneumococcal infection. Host However, there is growing evidence that monia, meningitis and bacterthe toxin pneumolysin plays an important inflammatory responses to cellaemia, as well as otitis media wall components undoubtedly role in the pathogenesis of pneumococcal and sinusitis. Morbidity and contribute to pathogenesis, but disease and may thus be a significant mortality from pneumococcal so too does direct attack on host disease remain high, even in additional target for vaccine development. cells and tissues by the toxin countries where antimicrobial J.C. Paton is in the Molecular Microbiology Unit, pneumolysin. therapy is readily available. Women's and Children's Hospital, North Adelaide, The growing problem of drugSA 5006, Australia. tek +61 8 204 6302, Structure and mode of action resistant pneumococci, coupled fax: +61 8 204 6051, e-mail: [email protected] Pneumolysin is a potent toxin with the suboptimal clinical efproduced by virtually all clinificacy of purified pneumococcal cal isolates of S. p n e u m o n i a e 1. This 53 kDa protein is polysaccharide vaccines in high-risk groups (particularly located in the cytoplasm, but is released when pneuyoung children), has highlighted the need to understand mococci undergo spontaneous autolysis. Pneumolysin the mechanisms underlying pneumococcal disease better. is a member of a family of structurally related toxins Such information is an essential prerequisite for the called the thiol-activated cytolysins, which are prodevelopment of improved preventative strategies. duced by representatives of several Gram-positive Although the antiphagocytic capsule of S. p n e u genera 2. Their mode of action involves interaction with m o n i a e is essential for virulence, the capsular polysac-
S
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The role of pheromones in bacterial interactions Reinhard Wirth, Albrecht Muscholl and Gerhard Wanner
p
heromones were defined (Agrobacterium tumefaciens), An increasing number of bacterial species in 1959 by Karlson and LasR (Pseudomonas aerugiare found to communicate via excreted Liischer ~ as substances pheromones. These signals trigger various nosa) and YenR (Yersinia enterocolitica), to define a LuxR which are secreted to the outresponses, including luminescence, side by an individual and resuperfamily of response reguproduction of virulence factors, lators. A recent comparison 6 ceived by a second individual development of fruiting bodies, lists ten regulators known to be of the same species, in which competence and sporulation, secondary they release a specific action, for induced by AHLs. In addition, metabolism and plasmid transfer. the transcriptional activators example, a definite behaviour R. Wirth * and A. Muscholl are in or a developmental process. The FixJ (Rhizobium meliloti), the Universitat Regensburg, Lehrstuhl fiir principle of minute amounts NarL, MalT, RpoD (EscherMikrobiologie-Archaebakterienzentrum, ichia coli) and GerE (Bacillus being effective holds...', to difUniversit#tstrafle 31, D- 93053 Regensburg, subtilis) belong to this superferentiate these extracellular Germany,; G. Wanner is in the Universit~it M~inchen, Institut f~ir Botanik, Menzinger Strafle 67, signals from intracellularly actfamilyL Likewise, a LuxI superD- 80638 Miinchen, Germany. family of signal-generating (i.e. ing hormones. Pheromones are '=tel: +49 941 943 3182, fax: +49 941 943 2403, AHL-synthesizing) proteins can one of the few means by which [email protected] be defined and recently ten of a single bacterial cell can obtain these have been documented 7. information from others of the Why bacteria like P. fischeri should emit light when same species (the extracellular fibrils of Myxococcus xanthus constitute another mechanism; see below). their critical cell density is above 107ml 1 (indicated by AHL concentrations > ] 0 - 9 M ) remains unknown. Communication via such signals is important for the survival of bacteria in nature, not only as defense Some bioluminescent bacteria live free in the ocean at against predators and competitors but also to supply concentrations of about 10 z cells m1-1, but in the symbiotic organs of luminous fish and squids they exceed information about members of the same species. Pherothe critical cell density, reaching 1011cells ml-'. It could mones belonging to several chemical classes have been be speculated that pheromone-induced enzymes conidentified for a great variety of bacteria. This review tribute to bacterial nutrition in the symbiotic light ordiscusses the biology of some of these systems with the gans. In these organs, doubling times of about 20 h are emphasis on N-acyl-L-homoserine lactones (AHLs), observed, compared with <1 h for growth in nutrientfound in a great variety of Gram-negative bacteria, and rich medium. Interestingly, it was shown recently s that the sex pheromone system of Enterococcus faecalis, a pheromone of Vibrio harveyi not only regulates biowhich is very probably restricted to that Gram-positive luminescence, but also induces the formation of polyspecies (Table t). 3-hydroxybutyrate, which might act as an energy source for maintaining cell viability in the stationary stage of AHL-regulatedsystems AHLs were first identified in luminous bacteria. If growth in this species. For P. fischeri, regulation is more complex than prepresent above a certain concentration, these excreted viously thought 9, as two pheromones are produced substances induce an operon of genes including those from luxI and a third from ain. Similarly, more than for their own production (AHLs are also called autoone pheromone seems to be produced by V. harvey? °. inducers). For Vibrio and Photobacterium species the induced proteins enable the bacteria to emit light. AHLs LuxR of P. fischeri is composed of two domains, of also act in systems totally unrelated to bioluminescence which the amino-terminal domain of 160 amino acids inhibits the DNA-binding activity of the carboxy-ter(see Table 1). AHLs are produced by LuxI-type prominal domain (amino acids 160-250) ~1. Binding of the teins, diffuse freely over the cell membrane and accumulate in the medium. If sufficiently large amounts of autoinducer relieves the inhibition. In contrast, LuxR AHLs are accumulated extracellularly these signals acti- of V. harveyi does not bind an autoinducer 12 and has its own distinct DNA-binding sites. vate an intracellular response regulator of the LuxR The nutritional needs of Erwinia carotovora (a plant type to induce transcription of at least one operon, including a luxI-type gene. Excellent recent reviews 2 pathogen) seem to be a plausible explanation for the pheromone-stimulated expression of enzymes that lyse cover this field. Sufficient sequence similarities exist between differ- plant cell walls 13. The effectiveness of these enzymes is increased if many bacterial cells of the same species ent transcriptional activators, including LuxR (Photobacterium fischeri; also known as Vibrio fischeri), TraR attack susceptible plants. Copyright © 1996 Elsevier Science Ltd. All rights reserved. 0966 842X/96t$15.00 -|'I
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Table 1. Characterization of bacterial pheromone systems" Signal(s)
Structure/minimal active concentration
Bacterial species
Comments
AHLs AHLs
OHHL/IO 9M; HHL/? (prob. 10-8M); OOHL/? (prob. 10 7M) HBHL/IO-6M; ?/?
Photobacterium fischeri Vibrio harveyi
AHL
OHHL/IO-SM
Erwinia carotovora
AHLs
ODDHL/IO-6M; BHL/IO 6M; HHL/?; OHHL/?
AH Ls
OOH L / I O -6 M; 0 HHL / I O -6 M
Pseudomonas aeruginosa Agrobacterium tumefaciens
AH Ls
HHL/?; OHH L/?
Yersinia enterocolitica
AHL?
?/?
AHL?
?/?
'A-signal'
Mixture of peptides and amino acids/lO-6M
Pseudomonas aureofaciens Various Gram-negative species Myxococcus xanthus
'E-signal'
Branched-chain fatty acids?/(<10 3Mb)
M. xanthus
Lipid
? (200 Da)/lO 9M
Stigmatella aurantiaca
Competence factor
8 or 9 amino acids long, modified peptide/lO-9M
Bacillus subtilis
Many, e.g. A-factor
Butyrolactone/lO -9 M
Streptomyces species
SapB
18 amino acids long, modified peptide/?
Streptomyces coelicolor
Rap/Rip
Protein (38 kDa)/?; pentapeptide/?
Staphylococcus aureus
?/?
Anabaena species
? (5-Membered lactam ring fused to 5-membered thioketon ring)/<3 x IO-FM
Cylindrospermum licheniforme
Chemoattractant
Aspartate/lO 7M per cell; see Ref. 40
Escherichia coil
'Used medium'
undefined/?
Bacillus species
Sex pheromones
cOB1:V-A-V-L-V-L-G-A/2 x 10-1°M cAD1:L-P-S-L-V-L-A-G/5 x 10 11M lAD1: L-P-V-V-T-L-V-G/IO-l° M cPDI: P-L-V-M-P-L-S-G/5 x lO-11M iPDI: A-L-I-L-T-L-V-S/? cCFIO: L-V-T-L-V-P-V/2.5 x 10-11M iCFIO: A-I-T-L-I-P-I/IO I°M cAM373:A-I-P-I-L-A-S/5 x 10-11M iAM373:S-I-P-T-L-V-A/2 x 10 l°M
Enterococcus faecalis
Induce bacterial light emission Induce bacterial light emission Induces lytic enzymes and carbapenem antibiotic Induce elastase and other virulence factors Enhance conjugative transfer of Ti plasmids between bacterial cells Regulated proteins not yet physiologically characterized Induces phenazine antibiotics LuxR-activating activity described (see text) Induces fruiting-body formation Needed for completion of development Induces fruiting-body formation Induces competence for uptake of naked DNA Control secondary metabolism and sporulation Leads to formation of aerial hyphae Regulate the inducer for production of toxic exoproteins Determines spacing between heterocysts in filaments Induces formation of akinetes (other inducers of unknown structure needed) Induces stable regular growth patterns of chemotactic cells Induces colony patterns via 'chemotactic feedback' Induce sex-pheromoneplasmid-encoded adhesin, leading to enhanced conjugation. At least six, but probably more sex pheromones are produced by E. faecalis
"AHL, N-acyI-L-hOmoserinelactone; the abbreviations used for AHLs follow the nomenclature in Ref. 4; there the structure of some AHLs plus that of A-factor from Streptomyces griseus is given. The structure of sex pheromones and the inhibitory peptides of E. faecafis are given in the standard one-letter amino acid code. ? Means no data are available. bSee Ref. 25.
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In P. aeruginosa, expression of elastase and other extracellular virulence factors is regulated by AHLs. Again, it can be argued that it is advantageous for an opportunistic pathogen to synthesize virulence factors only if many bacteria are colonizing a tissue. Pseudomonas aeruginosa strain PAO1 synthesizes at least three AHLs 7and a total of four AHLs have been isolated from different strains. The LasR-LasI system regulates elastase, alkaline phosphatase, exotoxin A, exoenzyme S, heat-stable hemolysin and neuraminidase; the RhlRRhlI system regulates elastase, alkaline phosphatase, hemolysin, pyocyanin and HCN. The presence of these multiple regulatory circuits is thought to allow fine tuning of the regulation of P. aeruginosa exoproducts resulting in their optimal concentrations for varying growth conditions 7. Agrobacterium tumefaciens transfers its Ti plasmid conjugatively to susceptible plants and to members of its own species. Transfer (and expression of opinedegradation genes) is stimulated up to 10a-fold by at least two pheromones 14. Recently, the secretion of two AHLs by Y. enterocolitica has been reported 6. They are produced from a single protein, YenI, which belongs to the LuxI superfamily; the gene for a putative transcriptional regulator, YenR, is transcribed convergently to yenI. Regulation of virulence genes by these pheromones has not yet been shown, although AHL mutants have been made. This system awaits further analysis as, interestingly, yenI is not subject to autoinduction, but is expressed constitutively. Phenanzine antibiotics are produced by Pseudomonas aureofaciens and believed to play an important role in microbial competition and rhizosphere survival. Again, they are produced when cell density is reaches a threshold, and are regulated by PhzR, a member of the LuxR superfamily of response regulators. Although there is no evidence that P. aureofaciens synthesizes an AHL it is highly probable that it does, because P. aeruginosa autoinducer extract triggers phenanzine antibiotic production by P. aureofaciens. In one study using a lux-plasmid-based bioluminescence sensor system, pheromone production was demonstrated by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens 16. Only in E. carotovora has a role for AHL in pathogenesis been shown. Other bacteria that produce AHLs include Citrobacter species, Chromobacterium violaceum and four different Yersinia species, but it is not known if these organisms use the AHLs as pheromones. As the detection system relies on activation of LuxR by a cognate pheromone ~6, one might speculate that other AHL-derived pheromone systems (such as ones inducing LasR-homologous transcriptional activators) exist in bacteria. The pheromone in E. carotovora that acts as cell-density indicator is identical with 'factor 1' of P. fischeri. The totally different biotopes in which the two species are found excludes potential 'pheromone crosstalk' between these bacteria. It had been argued that the inducer(s) of P. fischeri could act in the P. aeruginosa system 13. The significance of this finding has been
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questioned by a recent report iv because again these two species do not occur in the same biotope. Although pheromone crosstalk has been shown for P. aeruginosa and P. aureofaciens under laboratory conditions is, it would appear to have no relevance in natural systems. Pheromones of fruiting-body-forming bacteria Detailed studies on the signals needed for the development of fruiting bodies are available only for the two species, Stigmatella aurantiaca and M. xanthus. The multicellular fruiting bodies of these species not only produce stress-resistant myxospores, but also maintain them in numbers high enough to allow successful regrowth. An early trigger for the development of fruiting bodies is the exhaustion of food supplies; other signals, including pheromones, are also needed. In the case of S. aurantiaca, an as-yet-incompletelycharacterized pheromone triggers the aggregation and swarming of bacterial cells on solid surfaces. The aggregates develop into stalks on which the myxosporecontaining sporangia are formed. The pheromone was characterized as a lipid of low molecular mass TM, extractable with chloroform and methanol from filter paper located below developing fruiting bodies of S. aurantiaca. Data from H. Schaierer's group (pers. commun.) indicate that a volatile substance can be purified by water-vapor distillation from developing S. aurantiaca cells incubated on solid surfaces. The pheromone is produced by cells early in development and elucidation of its structure will be a major breakthrough for studies on this system. Myxococcus xanthus excretes a 'diffusible fruiting factor '19 and studies on mutants have helped to define the series of 'signals' needed for the development of the fruiting bodies2°,21.Dworkin's 22detailed discussion of the various 'signals' required for the formation of fruiting bodies described most of the components as surface proteins or DNA-binding proteins. The socalled A-signal consists of two components; the heatlabile component contains two proteases and the heatstable component is a mixture of single amino acids and small peptides (produced by the action of the heatlabile component on extracellular substrates). If present in high concentrations (>10 3M), the heat-stable component supports growth; however, concentrations below 10-6M induce the formation of fruiting bodies. It was speculated recently23that the heat-labile A-signal might be connected to the inhibition of ADP-ribosylation of endogenous M. xanthus protein. Interestingly, the substrates for ADP-ribosylation seem to be located in the extracellular fibrils 24 that connect the cells during early development. It is tempting to speculate that, via this mechanism, a connection to other cellular regulatory circuits might exist. Branched-chain fatty acids are known to be released and are constituents of the E-signal 2s, which is needed to complete the development ofM. xanthus. This pheromone system has been discussed in detail elsewhere 26. Competence factor of B. subtilis In certain growth phases B. subtilis can take up 'naked DNA' triggered by a competence factor. Early data
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indicated that the development of competence and sporulation are somehow connected. The role of the competence factor in B. subtilis, its mode of synthesis, the sensory pathway and the role of the regulatory cascade triggered by the peptide have been described recently2v (see Fig. 5 in Ref. 27 for an excellent overview). Here, only a few aspects are discussed. Competence factor is a linear peptide of nine or ten amino acids, possessing an undefined modification (perhaps a fatty acid addition) that is essential for activity. The pheromone peptide sequence matches nine of the last ten amino acids predicted from the nucleotide sequence of comX. This gene, and the upstream gene comQ, are required for pheromone production, with ComQ possessing processing and/or modification activity. The excreted pheromone is sensed by the membrane-located SpoOK oligopeptide permease, which has ATPase activity. Pheromone sensing results, via an unknown mechanism, in phosphorylation of ComP. ComP might be associated with the SpoOK permease, because its amino-terminal domain contains eight putative membrane-spanning helices (the carboxy-terminal domain is similar to that of the histidine protein kinases). ComP-phosphate phosphorylates ComA, the pheromone-binding subunit of SpoOK. ComA-phosphate, in turn, directly activates the large operon (>25 kb) srfA, which encodes peptide synthetases needed for synthesis of the lipopeptide antibiotic surfactin. At present it is unclear whether ComA-phosphate induces only the srfA operon. Its effect on the competence system might be indirect because srfA expression leads to accumulation of ComK, a transcription factor that activates expression of the late competence operons (comC, comE, comF and comG). One connection between the development of competence and sporulation lies in comQ; a comQ mutant shows less efficient sporulation in minimal medium, up to a hundredfold reduction compared with rich media2v. Pheromones of Actinomycetes A-factor, produced by Streptomyces griseus, was the first pheromone identified in an Actinomycete; it is just one example of the very large group of butyrolactone autoregulators 28,29. The question of whether B-factor (butyl ester of 3'-AMP; active at 10-1°M) and C4actor (a 34.5kDa protein) are extracellularly acting signals remains open. A-factor triggers the expression of streptomycin production, streptomycin resistance, yellow pigment production and the formation of aerial hyphae (as a prerequisite for sporulation) via interaction with its receptor ArpA. On binding, A-factor relieves the repressing activity of ArpA, a protein possessing an (~-helix-turn-R-helix DNA-binding motif3°. The role of prokaryotic homologs of the eukaryotic serine/threonine kinases for the regulation of differentiation in this group of bacteria was discussed recently> . The formation of aerial hyphae by Streptomyces coelicolor is dependent on the peptide SapB, present in large amounts on cell surfaces and released into a zone surrounding the colonies31. For the production of SapB, diffusible signals seem to be needed3L
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Other systems The production of toxic exoproteins by Staphylococcus aureus depends on the global regulatory Agr system. The agr gene is autoinduced by secreted RNA III activating protein (Rap) (Ref. 33). Rap seems to interact with the putative receptor AgrC (a signal transducer of a two-component signal transduction system); AgrCphosphate then interacts with the response regulator AgrA. AgrA-phosphate activates transcription of RNA III, which in turn leads to expression of enterotoxin B, hemolysin and toxic-shock syndrome toxin 1. A naturally occurring mutant of S. aureus, excreting the inhibitor RNA-III-inhibiting protein (Rip) (which counteracts Rap activation), was also identified33; it is unclear whether Rap and Rip act via different receptors. In the filamentous cyanobacterium Anabaena, the distance between heterocysts appears to influence the excretion of an inhibitor of heterocyst formation 34 by existing heterocysts. For another cyanobacterium, Cylindrospermum licheniforme, the formation of akinetes seems to be triggered by a partially characterized pheromone3L Very clearly, these last two systems have to be further characterized. Inducers of growth patterns The formation of fractal-like colony patterns of Bacillus species occurs in used medium and is the product of chemotactic feedback36,3v.This means that extracellular signals are produced by the growing cells and sensed by them. The 'communicating walkers' approach 37has also been used to explain38the formation of growth patterns 39of motile E. coli (and Salmonella typhimurium) cells in low-viscosity agar media. The formation of these remarkable patterns depends on the excretion of aspartate, which acts as an attractant 4°, by the growing cells. Sex pheromones of E. faecalis The sex pheromone system of E. faecalis was discovered by Don Clewell's group 41 during experiments on conjugative plasmids. These bacteria have two different kinds of conjugative plasmids 42. Class I is about 30 kb, has a broad host range among Gram-positive bacteria and normally encodes antibiotic resistance. Class 2 is more or less restricted to E. faecalis, tends to be >60 kb and in only a few cases codes for drug resistance. In liquid medium the transfer of plasmids belonging to class 2 ('sex pheromone plasmids') is more efficient than for class 1 plasmids [up to 10 -1 for class 2 (per donor) compared with 10 -6 for class 11, because of the clumping of bacteria induced by the so-called sex pheromones excreted by plasmid-free strains. The sex pheromones are excreted by plasmid-free recipient strains of E. faecalis and sensed by donor strains carrying a sex pheromone plasmid. They induce the production of an adhesin (aggregation substance) encoded by the sex pheromone plasmid, thereby leading to a bacterial clumping reaction (see front cover of this issue); this, in turn, allows the high-efficiency conjugation of sex pheromone plasmids. Sex pheromones are named after the plasmid they induce (for example, sex pheromone plasmid pAD 1 is induced by sex pheromone
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pPD1
seal
I
III
asal
pAD1
1 kb
~-~
~
Fig. 1. Comparison of sequenced portions of sex pheromone plasmids pPD$, pAD1 and pCFIO, containing structural and regulatory genes for pheromone-induced aggregation. The solid lines indicate plasmid DNA; the broken line for pPD1 indicates that the sequence for sepl, the structural gene for surface-exclusion protein, has not yet been completed. Boxes indicate genes that have been translated into proteins. Transcription is usually from left to right; exceptions are indicated by the arrows. Sequence similarity is shown by different colors (pink, 30-50%; yellow, 50-75%; green, >90% identity): for all boxed genes this refers to the percentage identity of amino acid sequences but for the regulatory regions including stem-loop structures (closed circles on a stalk; see also Box 1), the sequence similarity refers to nucleotide sequence. A description of most genes can be found in Ref. 44 although some unpublished sequences have been included.
cAD1). A strain carrying one sex pheromone plasmid can act as recipient for (all?) others. This is probably why plasmid-free strains of E. faecalis are rare. Recent reviews 43~s provide a more detailed picture. Sex pheromones are linear hydrophobic peptides of seven or eight amino acids, which are probably synthesized on ribosomes. They are highly active: 1-10 molecules per cell (about 10 -15M) are sufficient for induction. Inhibitory peptides encoded by sex pheromone plasmids have 25-50% identity to the corresponding chromosomally encoded inducing peptides, suggesting that there are specific receptors for pheromones in E. faecalis. Genetic and biochemical data indicate that, in addition to the receptors TraC (Ref. 46) and PrgZ (Ref. 47) encoded by sex pheromone plasmids, chromosomally encoded proteins also interact with sex pheromones. The inhibitory peptides iPD1, iAD1 and iCF10 are encoded by ipd, iad and prgQ on the sex pheromone plasmids pPD1, pAD1 and pCF10, respectively48-s°. They are produced from precursors of 21, 22 and 23 amino acid residues, with the carboxy-terminal amino acids comprising the active peptide. Interestingly, recombinant E. coli strains can secrete the inhibitory peptides; possibly the amino-terminal amphiphilic helices are involved in transport across bacterial cell membranes. The genetic organization of the genes for the activating peptides is unknown, although the pheno-
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type of mutant strains indicates processing from a common precursor. Alternatively, processing from the 'core' of a signal peptide might occur; indeed, processing and excretion of a cADl-like peptide from the signal peptide of an S. aureus gene was observed sl. A peptide-inducing sex pheromone plasmid, pAM373, is produced by E. faecalis strains and by some other Gram-positive bacteria s2 but, except for E. faecalis, it is unclear whether these peptides serve any functional role for the producers. Sex pheromones are active in eukaryotic systems 44-~3 but the question of whether or not they are virulence factors, as are the adhesins induced by them, is open to speculation. Sex pheromone plasmids have a highly conserved DNA region coding for surface exclusion protein and aggregation substance s4y. Sequence data are available for sex pheromone plasmids pAD1, pPD1 and pCF10 (Refs 44,48,56-58) (Fig. 1). DNA sequence identities for the structural genes are in the range of 50 to >95%. This is not the case for DNA regions coding for regulatory functions (for example, traA and prgX show <15% similarity; data on the function of orf5 from pPD1 are lacking); on the other hand, a region of 350 bp containing strong stem-loop structures is nearly identical in pAD1 and pCF10. It can be concluded from this that a common ancestor should exist for sex pheromone plasmids and that evolution of the various plasmids has involved combinations of different regulatory
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genes and open reading frames of unknown functions. Some ideas on the evolution of the sex pheromone system have recently been formulated 44. At present it is not possible to define clearly distinct sex pheromone plasmids (although at least 20 different restriction profiles exist) but they can generally be distinguished by their inducibility by different sex pheromones where the cognate pheromone structure is known 44. The DNA differences of pAM373 set it apart from all other sex pheromone plasmids 44. Aggregation substance was long ago reported to be proteinaceous and located on the surface of induced cells. Using immunological and electronmicroscopic studies we showed that this adhesin appears as hair-like structures incorporated mainly in 'old' cell wall 59'6°. The DNA sequence of asal, the structural gene of pADl-encoded aggregation substance, revealed the presence of two RGDS motifs 56. This indicated possible interactions with eukaryotic cells via surface receptors belonging to the integrin class. Indeed, an aggregation-substance-dependent interaction occurs with in vitro cultured pig kidney tubular cells61;interestingly, addition of the peptide RGD reduced aggregation-substance-mediated binding to eukaryotic cells. Binding of E. faecalis to other eukaryotic cells via this adhesin has also been observed. Unpublished work from our group indicates that the amino-terminal region of aggregation substance is probably the domain interacting with E. faecalis cells; interaction with eukaryotic cells is mainly via the carboxy-terminal domain, which also contains the RGD m o t i f s 44.
Box 1. Model for regulation of sex pheromone plasmid pAD1
The upper part shows an uninduced cell of Enterococcus faecalis, the lower part a cell induced by sex pheromone cAD1. The solid black line represents pAD1 DNA, above which the names of relevant genes are given; their length and direction of transcription are indicated below. Under non-induced conditions a negative-regulatory gene traA prevents transcription proceeding from a promoter located directly upstream of lad1 through the stem-loop structures between iadl and traE1 (indicated by the red line). Upon induction by sex pheromone cAD1 this negative effect is relieved, allowing transcription to proceed beyond traE1 (indicated by the yellow arrow). The positive regulator TraE1 acts in trans and allows transcription of at least three mRNAs (indicated by the green arrows). The first one starts at the promoter located directly upstream of iadl and ends after traE1, thereby creating an autoregulatory circuit. (This idea is supported by our data that cells of E. faecalis show an 'all or nothing' induction effect; no intermediate levels of adhesin can be found63.) The second transcript starts upstream of a small open reading frame (with unknown function) located just before seal and ends directly downstream of seal. (Under non-inducing conditions seal is expressed constitutively from a -10/-35-type promoter; induction leads to activation of a second, sex pheromone-dependent promoter.) The third, also strictly cADl-dependent transcript, starts upstream of a small open reading frame (with unknown function), located just before asal, the structural gene for the adhesin. This third transcript seems to possess a prominent termination signal after the small open reading frame downstream of asal. A small portion of it, however, extends some 4 - 5 kb beyond the terminator, to allow transcription of genes needed for 'stabilization of aggregates'. The main product of the asal-specific transcript clearly is the adhesin, appearing as hair-like structure (blue color) on the surface of induced cells.
A model for the regulation of pAD 1 is shown in Box 1. It is based on analysis of mutations in regulatory genes62 and of transcripts before and after induction 63,6a,and on the finding that the positive regulator TraE1 can function in trans 57,63,64. The traE1 gene is expressed only after induction, and synthesis of TraE1 precedes that of Asal. The regulatory mechanisms leading to the induction of pAD1 and pCF10 are probably completely different44. For pCF10 the existence of a cisacting positive control element has been reported 65. A model for the regulation of pCF10 postulates direct interaction between transcripts from the regulatory region and RNA polymerase to allow transcription of structural genes66. This implies that regulation of pCF 10
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could be mainly at the level of RNA stability and translation. It is expected that in both cases only an in vitro system driving specific expression of sex-pheromoneregulated promoters will allow understanding of the underlying regulatory circuits.
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Questions for future research • How widespread are bacterial pheromone systems? For example, does an earlier report ~8 on excretion of insulin-like signal(s) by Escherichia coli indicate the existence of an as-yet-unidentified pheromone system in this extensively studied species? • How do the signal transduction chains function? Do connections exist to other regulatory circuits? Which other regulatory systems influence genes activated by N-acyl-L-hOmoserine lactones (AHLs)? For Erwinia carotovora, does inactivation of the global represser locus rsmA lead to overproduction of extracellular enzymes in an AHL-independent manner69? • Are parameters other than cell density indicated by pheromones? • AHLs supposedly diffuse freely over bacterial membranes. What mechanism ensures that the signals have to act from the outside and that newly synthesized AHLs do not constitutively induce the producing cell? Is AHL production merely constitutive or might their synthesis be regulated by factors like the growth stage/ nutritional status of the producer or by growth temperature? • How has the Enter•coccus faecalis sex pheromone system evolved? What is the genetic organization of structural genes for the signals and what is the role of sex-pheromone-like peptides excreted by other species?
Concluding remarks
It is evident from the data discussed here that so far pheromones have only been described in systems in which their effect can easily be detected (for example, light emission by bacteria, production of virulence factors, formation of fruiting bodies and aggregation). It is tempting to speculate that other systems exist that enable bacteria to obtain information on others of their kind. To date AHLs, for example, have been detected only in Gram-negative bacteria; might they also be present in Gram-positive bacteria ? Likewise, no pheromones have been described for Archaeans; probably because no one has looked yet. Swarming behavior of, for example, P r o t e u s mirabilis very well might be regulated by pheromones and, indeed, extracellular glutamate seems to play a role here 67. Advances are occurring rapidly and, during the preparation of this review, the S. aureus system was described and data on quorum sensing, the regulation of bacteriocin production in L a c t o b a c i l l u s and the production of N - ( 3 R hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone acting as both a bacteriocin and as an autoinducer of rhi genes in R h i z o b i u m l e g u m i n o s a r u m were described at the Beijerinck Centennial meeting*. For some systems, at least, ideas exist on how the signal transduction chain might function. It is tempting to speculate that pheromonal signals produced by bacteria might be received and transduced in a way similar to other environmental signals. Therefore, crosstalk between pheromone transduction chains and other signaling systems is a possibility. The B. subtilis system discussed above may offer an example; it seems unlikely that competence and sporulation are regulated solely via one common system, c o m Q . All the pheromones described to date can be viewed as signals for the cell density of a bacterial species. *The Beijerinck Centennial meetingwas held at The Hague, The Netherlands, 10-14 December 1995.
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However, one can imagine how other situations such as nutritional status might be communicated to other cells of the same species. Very clearly work on such interactions needs to be done under more natural circumstances than growth in rich media under laboratory conditions. Acknowledgements
Space constraints have limited detailed discussions in this rapidly developing field; it is hoped that access to relevant primary data is possible from the literature cited. We want to thank all present and past members of our laboratories for their dedicatedwork on the E. faecalis sex pheromonesystem.The financialsupportof the authors by DFG (SFB145, B8 and Y1) is gratefullyacknowledged. References
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58 Kao,S-M.et al. (1991)J. Bacteriol. 173, 7650-7664 59 Galli,D., Wirth, R. and Wanner,G. (1989) Arch. Microbiol. 151,486-490 60 Wanner,G. et al. (1989) Arch. Microbiol. 151,491-497 61 Kreft,B. et al. (1992) Infect. Immun. 60, 25-30 62 Weaver,K.E.and Clewell,D.B. (1998)J. Bacteriol. 170, 4343-4352 63 Muscholl,A. et al. (1993) Eur. J. Biochem. 214, 333-338 64 Tanimoto,K. and Clewell,D.B. (1993)J. Bacteriol. 175, 1008-1018 65 Chung,J.W. and Dunny,G.M. (1992) Proc. Natl Acad. Sci. USA 89, 9020-9024 66 Chung,J.W.and Dunny,G.M.(1995)J. Bacteriol. 177, 2118-2124 67 Allison,C. et al. (1993) MoI. MicrobioI. 8, 53-60 68 LeRoith,D. et al. (1980)J. Biol. Chem. 256, 6533-6536 69 Chatterjee,A. et al. (1995) Appl. Environ. Microbiol. 61, 1959-1967 70 Bassler,B.L.and Silverman,M.R. (1995) in Two-component Signal Transduction (Hoch,J.A. and Silhavy,T.J., eds), pp. 431-445, AmericanSocietyfor Microbiology Note added in proof An outstanding comparative discussion of regulatory functions in the P. fischeri and V. harveyi quorumsensingsystemscan be foundin Ref. 70.
The contribution of pneumolysin to the pathogenicity of
Streptococcuspneumoniae James C. Paton The antiphagocytic polysaccharide capsule charides themselves (of which treptococcus pneumoniae there are over 80 serotypes) are of Streptococcus pneurnoniae has long is a human pathogen.of completely nontoxic and cannot been been considered the principal malor importance, causing alone account for death from virulence determinant of this organism. invasive diseases such as pneupneumococcal infection. Host However, there is growing evidence that monia, meningitis and bacterthe toxin pneumolysin plays an important inflammatory responses to cellaemia, as well as otitis media wall components undoubtedly role in the pathogenesis of pneumococcal and sinusitis. Morbidity and contribute to pathogenesis, but disease and may thus be a significant mortality from pneumococcal so too does direct attack on host disease remain high, even in additional target for vaccine development. cells and tissues by the toxin countries where antimicrobial J.C. Paton is in the Molecular Microbiology Unit, pneumolysin. therapy is readily available. Women's and Children's Hospital, North Adelaide, The growing problem of drugSA 5006, Australia. tek +61 8 204 6302, Structure and mode of action resistant pneumococci, coupled fax: +61 8 204 6051, e-mail: [email protected] Pneumolysin is a potent toxin with the suboptimal clinical efproduced by virtually all clinificacy of purified pneumococcal cal isolates of S. p n e u m o n i a e 1. This 53 kDa protein is polysaccharide vaccines in high-risk groups (particularly located in the cytoplasm, but is released when pneuyoung children), has highlighted the need to understand mococci undergo spontaneous autolysis. Pneumolysin the mechanisms underlying pneumococcal disease better. is a member of a family of structurally related toxins Such information is an essential prerequisite for the called the thiol-activated cytolysins, which are prodevelopment of improved preventative strategies. duced by representatives of several Gram-positive Although the antiphagocytic capsule of S. p n e u genera 2. Their mode of action involves interaction with m o n i a e is essential for virulence, the capsular polysac-
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