Cell density-dependent gene expression controls luminescence in marine bacteria and virulence in several pathogens

Transcription

1 Cell density-dependent gene expression controls luminescence in marine bacteria and virulence in several pathogens E. Peter Greenberg t has been said that every novel idea in science passes through three stages. First people say it isn t true, then they say it s true but not important, and finally they say it s true and important, but not new. Over the past several years, there has been an increasing appreciation among microbiologists that bacteria can perceive and respond to other bacteria. This capability is often important in the colonization of animal and plant hosts by symbiotic or pathogenic bacterial species. Although different bacterial groups have different mechanisms for monitoring their own abundance in a local environment, one mechanism that has emerged as common in gram-negative bacteria is that initially discovered by J. W. (Woody) Hastings and his collaborators in the luminescent bacterium Vibrio fischeri (see box p. 376). The phenomenon originally termed autoinduction has become known as quorum sensing and response, and has been extensively reviewed recently (see Suggested Reading). The term quorum sensing first appeared in a Journal of Bdcteriology minireview written by Clay Fuqua, Steve Winans, and myself in It originated with Steve Winans brother-in-law, a lawyer who was trying to understand what we were studying as Steve explained it to him during a family gathering at Christmas. Stage 1, the 1970s: the Discovery and the Ecological Explanation Hastings was driven by his curiosity to explain why luminous bacteria like VI fischeri contain high levels of luciferase only when cultures reach the late-logarithmic phase of growth. The basic framework for quorum sensing was established in the early 1970s by Hastings, Terry Platt, Ken Nealson, and Anatol Eberhard. They showed that V. fischeri and another luminous species, Vibrio harveyi, produce diffusible compounds, termed autoinducers, that accumulate in the medium during growth. These autoinducer signals can accumulate to sufficient concentrations only when there is a critical mass of cells in a confined environment. The signals from V. fischeri and VI harveyi do not crossreact, showing species specificity. When I was a student in the MBL Summer Microbiology Program at Woods Hole, Mass., in 1973, Ken Nealson, then an instructor in the course, explained to me that V. fischeri occurs at very high densities ( 1 Ol to 1 O1 l cells per ml) as a specific symbiont in light organs of certain fish and is also found free in seawater at much lower densities (perhaps 5 cells per ml). Autoinduction allows VI fischeri to sense its elevated density in the light organ and express the luminescence system there, where it is required for the symbiosis, but not in seawater, where luminescence, which is energetically expensive, would be frivolous. The concept that bacteria produce pheromones and communicate with one another was met with considerable skepticism by many and disinterest by others at the time. My own curiosity was stimulated, however, and upon completion of my Ph.D. in 1977 I moved directly to Woody Hastings laboratory to begin my postdoctoral research on autoinduction. In 1978 Hastings, Shimon Ulitzur, and I showed that although V fischeri did not make a signal that activates the luminescence genes in V. harveyi, many other gram-negative marine bacteria do. This was the first suggestion that cell-to-cell signaling within and perhaps between bacterial E. Peter Greenberg is professor of microbiology at the University of Iowa, Iowa City. Volume 63, Number 7 / ASM News 371

2 FIGURE 1 1 Quorum sensing in Vibrio fischeri. (A) The luminescence gene cluster. The /ux/? gene encodes an autoinducer-dependent. transcriptional activator of the luxl-g operon. The luxl product is the autoinducer synthase, /uxc, 0, and E form a complex responsible for generation of one of the substrates for the luciferase reaction, the long chain fatty aldehyde, /uxa and B encode the two subunits of luciferase, and the function of /uxg remains unknown. (B) Cartoon of L: fischeri cells, each producing the diffusible autoinducer signal. At low cell densities the luminescence operon is transcribed at a basal level. At high cell densities the autoinducer signal can reach a sufficient concentration and bind to the cellular LuxR protein, which will then activate transcription of the luminescence operon. 372 ASM News /Volume 63, Number 7

3 I I species might be a common phenomenon. However, because we lacked an understanding of the genes, proteins, and signal molecules involved in autoinduction, this idea was not further developed until the 1990s. FIGURE Regulator domain - I I e Activator domain e! I Stage 2, the 1980s: Proof of the Model The 1980s brought many important scientific discoveries about autoinduction in V. fischeri, first physiological, then chemical, and finally genetic. A careful chemostat study confirmed that luminescence required high cell density. The structure of the autoinducer signal, N-3-( oxohexanoyl) homoserine lactone, was solved. This molecule was shown to move out of and into cells by passive diffusion. The genes for luminescence were cloned from VI fischeri into E. coli. Fortunately, the genes for autoinduction are linked to the luminescence structural genes (Fig. l), and E. coli cells containing this lux gene cluster produce light in a cell density-dependent fashion. Thus, quorum sensing could be analyzed with the tools of E. coli genetics. It was found that the regulatory region that enables autoinduction of luminescence consists of two genes: luxr, which encodes an autoinducer-responsive transcriptional activator, and luxl, which encodes a protein required for autoinducer synthesis. The region between luxr and 1~x1 contains the regulated lux promoter elements. Because 1~x1 is positively autoregulated, basal levels of 1uminescence operon transcription lead to low rates of autoinducer production, and quite high densities of cells are necessary for activation of the luminescence genes. Once activation has occurred, the rate of autoinducer synthesis increases, and cell density must drop considerably before the rate of transcription of the luminescence operon returns to the basal level. Not surprisingly, autoinduction is just one of the regulatory systems that come to bear on luminescence gene expression. 1uxR requires activation by cyclic AMP (CAMP) and the CAMP receptor protein. Iron can influence expression of luminescence. FNR seems to exert an effect on luxr, and there may be other cellular regulatory elements that affect expression of the luminescence genes of K fischeri. Since the late 198Os, more details of the mechanism of autoinduction have become known. Multimerization Helix-turn-helix IL fischeri MJI /UX box ACC CGTA /UX box-like consensus sequence RNS NXTR Elements of autoinducible lux gene expression. (Top) Key regions of LuxR, the activator of luminescence gene transcription. The polypeptide consists of two domains. There is a C-terminal helix-turn-helix (H-T-H) containing activator domain extending from about residue 160 to the C-terminal residue, 250. This domain interacts with the transcription initiation complex. The region from residue 230 to 250 is thought to be required for transcriptional activation but not for DNA binding. There is an N-terminal regulatory domain extending to about residue 160. A region of this domain is involved with autoinducer binding, residues 79-l 27, and a region is involved in multimer formation, around 120 to 160. In the absence of autoinducer the regulatory domain interferes with the activity of the activator domain. (Bottom) The lux box from L: fischeri strain MJ I and a consensus sequence for lux-box like elements found in promoter regions of acylhomoserine lactone-regulated genes from other bacterial species. The 20-bp lux box is centered at about -40 from the start of /ux/ transcription. Consensus sequence abbreviations: N = A, T, C, or G: R = A or G; S = C or G; Y = T or C; X = N or a gap in the sequence. Since the VI fischeri autoinducer is free to diffuse out of and into cells, the cellular and environmental concentrations of this signal are equivalent. For this reason, the transcriptional activator LuxR, which is located on the cytoplasmic side of the cell membrane, can respond to the environmental concentration of the autoinducer, which increases with V. fischeri cell density. LuxR is a 250-amino acid polypeptide that consists of two domains and functions as a homomultimer, likely a dimer, with o70-rna polymerase to activate lux gene expression. It is a member of the LuxR superfamily*of transcription factors, all of which contain somewhat similar H-T-H motifs in their DNA binding regions. This superfamily includes LuxR homologs (see below) and other regulatory proteins such as MalT, GerE, NarL, and region 4 of the u subunit of bacterial RNA polymerase. The N-terminal 160 amino acids or so of LuxR constitute Volume 63, Number 7 / ASM News 373

4 FIGURE 3 1 Amino acid number I I I I I I I I I I I I I I I I I I ll N Active site Acyl-ACP substrate specificity? A scheme for autoinducer synthesis and key regions of the Luxl protein. (Top) Luxl binds an acyl-acp and S-adenosylmethionine (SAM). The acyl group is transferred from the bound ACP forming amide bond with the SAM. The acyl-sam is converted to acylhomoserine lactone with release of 5 -methylthioadenosine (MTA) and release of the acylhomoserine lactone. (Bottom) Luxl is 193 amino acids in length. There is a region extending from about residue 25 to residue 104 that appears to represent the active site for amide bond formation. There is limited evidence to suggest that a region from about residue 133 to residue 164 is involved in selection of the appropriate acyl-acp from the cellular pools. C We are beginning to understand the mechanism by which the 1~x1 gene directs the synthesis of the autoinducer signal. We now know that LuxI is an autoinducer synthase that catalyzes the formation of an amide bond between its two substrates, a six-carbon fatty acyl-acyl carrier protein (acyl-acp) and S-adenosylmethionine (Fig. 3). Although it has been suggested that the acyl group forms a covalent bond with an active site cysteine in LuxI, recent studies of cysteine substitution mutants indicate that this is not the case. Studies of LuxI mutants have revealed that the active site in which amide bond formation is catalyzed is roughly in the region of residues 2.5-l 10 of this 19%amino-acid protein. A region in the C terminus may be involved in selection of the appropriate acyl-acp from those existing in the cellular pools (Fig. 3). Stage 3, the 1990s: the Discovery of LuxR-Luxl-Type Systems in Other Bacteria In the early l99os, several groups made key discoveries that led to our current view that quorum sensing is common to many gram-negative bacterial species. First, LuxR homologs were discovered in Pseudomonas aeruginosa and Agrobacterium tumefaciens, and several bacte- an autoinducer-binding, regulatory domain which, in the absence of sufficient autoinducer, interferes with the C-terminal domain (the last 90 or so amino acids), which binds to RNA polymerase and the lux regulatory DNA to activate transcription of the luminescence operon (Fig. 2). Tom Baldwin and his coworkers at Texas A&M University in College Station, Tex., identified a 20-bp inverted repeat at about -40 from the start of transcription of the luminescence operon (Fig. 2) which is required for autoinduction of luminescence. In vitro studies of LuxR have been difficult and slow, but from such studies we believe LuxR and ~70 RNA polymerase are the only transcription factors required for activation of the 1~x1 promoter and that these two factors bind synergistically to the promoter region. Many autoinducer analogs with alterations in the acyl side chain can bind to LuxR. Some serve weakly as autoinducers, but others inhibit the activity of the natural autoinducer, presumably by competition for the autoinducer binding site. 374 ASM News /Volume 63, Number 7

5 rial species were shown to produce N-3-(0x0- hexanoyl) homoserine lactone. Shortly thereafter, it was found that the A. tumefaciens and P. aeruginosa autoinducers are analogs of the VI fischeri autoinducer. For A. tumefaciens the autoinducer is N-3-( oxooctanoyl) homoserine lactone. I? aeruginosa has at least two quorum sensing systems, one that uses N-3-(oxododecanoyl)homoserine lactone and one that uses N- butyrylhomoserine lactone. The genes responsible for autoinducer production were sequenced, and their products were found to be homologous to LUXI. There are now over a dozen LuxI homologs and over a dozen LuxR homologs in the protein sequence data bases. Furthermore, lux box-like sequences can be found in the promoter regions of many of the genes regulated by LuxR homologs in bacteria other than VI fischeri. The LuxI homologs direct the synthesis of acylhomoserine lactones with saturated or unsaturated acyl chains of 4 to 14 carbons with either a hydroxyl group, a carbonyl group, or hydrogens on the third carbon from the amide bond. The constant among these homologs is the homoserine lactone component, while the acyl group provides species specificity. Different LuxI homologs produce different autoinducers, and the cognate LuxR homologs respond best to the appropriate autoinducer. Table 1 lists some bacteria that we now know to produce acylhomoserine lactones. What does autoinduction control in these other bacteria? Quorum sensing in A. tumefaciens controls conjugal transfer genes, probably to ensure that the catabolic Ti plasmid is present when cells are at a high density in a crown gall tumor. l? aeruginosa, an opportunistic human pathogen, uses quorum sensing to regulate expression of a battery of extracellular virulence genes, including enzymes and exotoxins. Autoinduction of extracellular enzymes is a common theme. Production of extracellular enzymes at low cell densities would be of no value; the enzymes would diffuse away from the cell and convert relatively little substrate to product, and because the environmental concentration of the product would not change appreciably, the bacterial cells would not benefit. When the bacteria have achieved a high enough density, production of an extracellular enzyme could have an impact on the environment. Why might quorum sensing control exotoxin production? Here we can use the analogy of an invading army. The bacterial pathogen first masses its troops, but it does not reveal its weapons until they can be deployed in sufficient quantity to overwhelm the opposition. By not producing exotoxins at low cell densities and waiting until the host defenses can be overwhelmed, I? aeruginosa deprives the host of the chance to respond immunologically. In fact, R aeruginosa quorum sensing mutants can colonize neonatal mouse lungs, but the progression of the disease is impaired; in contrast to the wild type, infection with the quorum sensing mutants does not lead to death. Another example of LuxR-LuxI-type quorum sensing occurs in Erwinia carotovora, in which not only extracellular enzymes, but also carbepenem antibiotic synthesis and in fact virulence of this plant pathogen are controlled by this mechanism. The significance of some quorum sensing systems is more difficult to picture. For example, the autoinduction of a set of Rhizobium leguminosarum genes that is expressed just prior to root hair penetration and of genes that lead to stationary phase. One general theme that has emerged is that the bacteria that exhibit this type of cell density-dependent gene regulation experience a plant or animal host association as part of their lifestyle. Thus, we were quite surprised to discover in collaboration with Agnes Puskus and Sam Kaplan of the University of Texas Medical School, Houston, Tex., that the free-living photosynthetic bacterium Rhodobacter sphaeroides has a quorum sensing system. Although the divergently transcribed 1~x1 and 1uxR genes in VI fischeri are linked to each other and to the genes they regulate (Fig. l), this is not always the case. In fact, every sort of arrangement imaginable has been reported. The 1~x1 and luxr genes can regulate unlinked genes, and in some cases are not even linked to each other. There can even be multiple luxr and 1~x1 homologs in a single bacterium. There is now some evidence that Burkholderia cepacia, an opportunistic pathogen that can colonize lungs of cystic fibrosis patients, may sense and respond to the density of another bacterial species infecting the cystic fibrosis lung, I? aeruginosa. It appears that the elements of the LuxR-LuxI system evolved in gram-negative bacteria early or have moved from species to species by gene transfer Volume 63, Number 7 / ASM News

6 and that each species has adapted these elements to its own needs. A Case of Convergent Evolution. Understanding autoinduction of luminescence in VI harveyi has come more slowly than our understanding of autoinduction in VI fischeri, in large part because the K harveyi system is more complicated. For instance, there are two signaling systems that can function independently of each other. One of the systems involves an acyl- homoserine lactone, N-( 3-hydroxybutyryl)homoserine lactone; the structure of the signal for the other system remains unknown and it may not be an acylhomoserine lactone. LuxR homologs have 376 l ASM News /Volume 63, Number 7 not been identified in VI harveyi. Rather, the signal sensors are complex proteins with sequence similarities to both components of two-component regulatory proteins. Two genes, 1uxL and luxm, are required for synthesis of N-(3-hydroxybutyryl) homoserine lactone. Neither of these genes encode a proteins with similarity to the VI fischeri LuxI protein or any of its homologs. Paul Dunlap and coworkers, then at the Woods Hole Oceanographic Institute in Woods Hole, Mass., discovered that VI fischeri 1~x1 mutants produce octanoylhomoserine lactone, which serves as a very poor substitute for the LuxI-produced 3-oxohexanoylhomoserine lac-

7 tone in luminescence gene activation. The gene required for octanoylhomoserine lactone synthesis was cloned and sequenced, and although its product is not a LuxI homolog, there is a 38 % sequence identity between its amino-terminal region and the V. harveyi LuxM protein. This suggests that there is a second family of acylhomoserine lactone-synthesizing enzymes. The mechanism of acylhomoserine lactone synthesis by this family has not yet been investigated. It s True and Important but Not New. The phenomenon of autoinduction noticed by Woody Hastings 30 years ago is well established, and has been found to be common among a diverse group of gram-negative bacteria. It plays a role not only in the curious light organ relationship between V fischeri and its marine animal hosts but also in the virulence of certain human and plant pathogens. Although quorum sensing is no longer considered new, the field of research is young and there are many important areas to investigate. It is clear that LuxI and LuxR homologs in pathogens are targets for development of novel antimicrobial factors, but we need more knowledge about how to inhibit them. Staffan Kjelleberg and his collaborators at the University of New South Wales in Sydney, Australia, have reported recently that at least one marine alga produces furanone compounds that can inhibit autoinduction. This may provide an explanation as to why luminescent marine bacteria, which can be isolated from a variety of marine habitats, are not found on the surface of algae. We know little about quorum sensing in natural environments and in the biofilms in which bacteria often grow. Is communication between bacterial species in complex natural environments common or important? Why do bacteria have multiple quorum sensing systems, and how many can be found in an individual strain? What is the significance of the second family of autoinducer synthesis enzymes? Finally, one might expect that with the intimate associations known to exist between mutualistic and pathogenic quorum sensing bacteria and their plant and animal hosts, the hosts may have evolved systems that can sense and respond to acylhomoserine lactone signals. It has been reported by Alice Prince and collaborators at Columbia University, New York City, N.Y., that one of the I? aeruginosa autoinducers stimulates epithelial cell production of interleukin-8, but in general host detection and response to autoinducers remains an untapped avenue of investigation. ACKNOWLEDGMENTS I thank the Office of Naval Research for continued support of my research on quorum sensing from the early years of the field up to the present. The originator of the opening lines of this article (about the three stages through which scientific ideas pass) is unknown, but the statement appeared in print in F. M. Harold s work cited below. SUGGESTED READING Bassler, B. L., and M. R. Silverman Intercellular communication in marine Vibrio species: density-dependent regulation of the expression of bioluminescence, p J. A. Hoch and T. J. Silhavy (ed.), Two-component signal transduction. ASM Press, Washington, D.C. Fuqua, W. C., S. C. Winans, and E. I? Greenberg Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbial. 50: Fuqua, W. C., S. C. Winans, and E. I! Greenberg Quorum sensing in bacteria: the LuxR-LuxI family of cell densityresponsive transcriptional regulators. J. Bacterial. 176: Harold, F. M The vital force: a study of bioenergetics. W. H. Freeman and Co., New York. Kaiser, D., and R. Losick How and why bacteria talk to each other. Sci. Am. 276: Salmond, G. I? C., B. W. Bycroft, G. S. A. B. Stewart, and P. Williams The bacterial enigma : cracking the code of cell-cell communication. Mol. Microbial. 16: Sitnikov, D., J. B. Schineller, and T. 0. Baldwin Transcriptional regulation of bioluminescence genes from Vibrio fischeri. Mol. Microbial. 17: Volume 63, Number 7 / ASM News 377