How and why bacteria talk to each other

Cell, Vol. 73, 873-885,

June 4, 1993, Copyright

0 1993 by Cell Press

Review

How and Why Bacteria Talk to Each O ther Dale Kaiser’ and Richard Losickt Department of Developmental Biology Stanford University School of Medicine Stanford, California 94305 tThe Biological Laboratories Harvard University Cambridge, Massachusetts 02138 l

Importance

of Cell-Cell

Interactions

As a vertebrate animal develops from a fertilized egg, progressive differentiation of cell structure and function occurs, resulting in fibroblasts, neurons, liver cells, muscle cells, and many other types of cells. This diversity of differentiated cells is believed to represent different regulatory states of the same set of DNA sequences. How can different states arise from a single cell? Viewing an early embryo from any one of its cells allows us to distinguish two sources of developmental information. One is internal, the cells own inherited program. The second is external, including the surrounding cells that provide the cell with physical or chemical cues and create for it a unique microenvironment. The general importance of the external sources in vertebrate development is illustrated by two kinds of experiments. Usually a fertilized egg becomes one individual, but sometimes at an early multicellular stage, a human embryo dissociates into two pieces. In that case, identical twins are born, each complete despite its origin in half an embryo. Nowadays, this process is carried out commercially to produce many offspring from the matings of prize cattle (Brotman, 1983). What amounts to acomplementary experiment has been performed with mice. Two early embryos were pressed together; there issued not a twoheaded monster, but one mouse of normal size and physiology that had inherited qualities from four parents (Mintz, 1974; Petters and Market?, 1980). More than 3000 viable tetraparental mice, as well as a few rabbits, rats, and sheep, have been born. Clearly, the fate of cells in the early vertebrate embryo is not fixed but depend8 on interactions between cells, some of which are known as tissue inductions. Interactions can be direct, or indirect through the extracellular matrix laid down by other cells. Specification of both the anteroposterior and dorsoventral axes in vertebrates depends on tissue inductions (Sive, 1993; Slack and Tannahill, 1992). Initial steps in the organization of the nervous system-the formation of the lens of the eye, the limbs, the exocrine pancreas and salivary glands, and the kidney and liver-all involve interactions between different cell types (Gurdon, 1987; Saxen et al., 1978). Even in the simplest of organisms, the bacterium, development is frequently governed by intricate systems of intercellular communication. Information transfer between cells determines differentiation and morphogenesis in a wide variety of bacterial systems, ranging in complexity from the induction of luminescence in Vibrio by bacterially

produced cell density signals to elaborate systems of cellcell interaction that govern sporulation in Bacillus, fruiting body formation in Myxococcus, and the erection of aerial hyphae by Streptomyces. What is the molecular basis of information flow between cells, and how is it used to govern development? Here we address this question with representative examples drawn from diverse forms of bacterial cell-cell interaction. Despite the variety, our examples reveal common themes, of relevance to developing organisms of many kinds, in the strategies by which cell-cell interactions are used to regulate differentiation, behavior, and morphogenesis. Signals in Myxococcus

Multicellular

Development

In Myxococcus xanthus, four sequential intercellular signals control the differentiation of vegetative cells into spores, the culmination of a 24 hr program of multicellular development. The developmental program of this gramnegative soil bacterium is initiated by starvation. Early morphological changes are evident about 4 hr later, as the cells begin to congregate. About lo5 cells eventually accumulate at each focus in a dense, orderly, moundshaped structure that isvisible to the unaided eye. Starting about 20 hr after starvation, cells inside this mound differentiate into myxospores, which are heat- and desiccationresistant dormant cells, thereby creating the fruiting body. Many genes increase their expression coordinately with the morphological changes (Shimkets, 1990). These changes in morphology and gene expression are controlled by at least four sequential cell-cell interactions; four classes of non-cell-autonomous mutants (named A, B, C, and D) have been isolated (Kroos and Kaiser, 1987). None of these mutants is able to sporulate on its own, but when wild-type cells are added, spores of mutant genotype are formed. In some cases, half the spores in such chimeras are mutant. Evidently, wild-type cells provide the extracellular signal missing in the mutant. Each mutant class arrests with a different state of gene expression (see Figure 1). The four states are resolved morphologically (whether or not aggregation or sporulation takes place) and biochemically (which of the developmentally dependent, sequentially active promoters become active in the mutant, as recognized by transcriptional fusions to /acZ; Kroos and Kaiser, 1987). Two signals have been chemically identified: the A signal, which is needed prior to aggregation, and the C signal, which is needed to complete aggregation and to initiate sporulation. The focus here will be on the nature and activity of the A and C signals. A Signal A signal molecules were identified by bioassay for substances in wild-type cells that allow A- mutants (asg mutants) to express a /acZ transcriptional fusion dependent on A signal. A factor activity was found in the medium conditioned by development of wild-type cells. Half the A factor activity in cell-free, conditioned medium was found

Cell 074

to be heat labile and nondialyzable, and when purified it was shown to be a mixture of proteases (Plamann et al., 1992). The remaining half of the A factor activity in cellfree, conditioned medium was shown to be a mixture of 8 amino acids (Tyr, Pro, Phe, Trp, Leu, Ile, Ala, and Ser) and peptides containing these (mainly hydrophobic) amino acids (Kuspa et al., 1992b). Peptides have activity because they can be cleaved by cellular peptidases to release the A factor amino acids. Proteases have A factor activity because they cleave extracellular proteins, releasing active peptides and amino acids. M. xanthus A factor is a cell density signal: the amount of A factor released into conditioned medium is proportional to the number of cells per unit volume (Kuspa et al., 1992a). Knowing the minimum quantity of A factor required to produce wild-type levels of developmental gene expression from an asg mutant, one can calculate the minimum cell density to provide sufficient A factor for the population to signal to itself. Below the critical density of 3 x 10’ to 5 x lOa cells per milliliter, wild-type cells should fail to develop, because they are unable to signal to themselves. Indeed, expression of an early A-dependent gene is defective at this low cell density. Also, below the critical density for self-signaling, the wild-type cells behave as phenocopies of asg mutants, and their development can be rescued either by the 8 amino acids or by proteases. Thus, 1 to 2 hr into development, cells release the 8 amino acids and peptides containing them. If at this stage the cell density is more than about 5 x lOa cells per milliliter, the concentration of A factor amino acids is high enough to trigger the population to begin aggregation, assured of sufficient cells to build a fruiting body (Kuspa et al., 1992a). The function of M. xanthus A factor reflects the cell density; it is not an autoinducer. In somewhat similar behavior, chemotactic strains of Escherichia coli swarm outward from a spot inoculum in a pattern of concentric rings. However, when subjected to oxidative stress, the ring pattern becomes a symmetrical array of point aggregates (Budrene and Berg, 1991). These cells aggregate because they release autoattractants. This response is mediated by the aspartate chemotaxis receptor, tsr, and addition of a high level of aspartate eliminates the pattern. Complex cell arrangements and patterns of differential gene expression have also been observed within single E. coli colonies (Shapiro and Higgins, 1989; Shapiro and Hsu, 1989). Bacillus subtilis releases two small peptide-like substances, called extracellular differentiation factors. Both factors are dialyzable, and they stimulate spore formation of cells at low density under normal starvation conditions (Grossman and Losick, 1988). B. subtilis also secretes one or more peptide factors that stimulate entry into the developmental state of genetic competence. (Conceivably, extracellular differentiation factors and the competence factors are the same [A. D. Grossman, personal communication].) The multiprotein oligopeptide permease, encoded by spoOK, is necessary to initiate sporulation and entry into competence and is implicated in the response to the extracellular differentation factor and competence factor (Rudner et al., 1991; Perego et al., 1991).

C Signal and Morphogenesis Once Myxococcus has begun to aggregate, it requires the C signal to complete fruiting body morphogenesis and to initiate sporulation within it. C factor has been identified by means of a bioassay employing a csg mutant that carries a C signal-dependent IacZ fusion as a reporter. A 17 kd protein was purified that has the capacity to rescue fruiting body formation, sporulation, and the expression of Cdependent genes. Purified C factor is hydrophobic and is active at a concentration of 1 nM. Amino acid sequence analysis showed that C factor is the product of csgA, the gene that is mutated in the C class of signaling-deficient mutants (Hagen and Shimkets, 1990; Kim and Kaiser, 1990a). Unlike the A signal, the C signal cannot pass through the medium but requires a special kind of contact between cells. The aqueous nature of A factor and the association of C factor with the cell surface are appropriate to the conditions in which each signal is passed: A signal at a relatively low cell density, C signal when the cells are within a nascent fruiting body and touching each other. Appropriate cell contact is normally produced by the movement of cells, explaining why nonmotile mutants do not sporulate. Packing cells by centrifugation to simulate the high cell density inside a fruiting body increases sporulation efficiency, but only to 1% of that of motile cells. The nonmotile mutants begin to develop, then arrest at the same stage as the C factor-less mutants (Figure 1). The apparent signaling defect of nonmotile mutants is specific to C signaling: they express genes that are dependent on the earlier A , B, and D signals and therefore must be able to transmit these signals (Figure 1). How might motility be involved specifically in C signaling? Direct tests were made of the possibilities that motility was necessary to produce C factor or to receive it. However, nonmotile mutants were shown to produce C factor with normal potency in normal amounts and to have normal capacity to receive and respond to C factor (Kim and Kaiser, 1990b). These results emphasized a third possibility: a defect in the process of C signal exchange. This possibility was tested by mixing motile with nonmotile cells. C factor-less mutants (which are motile) can respond to C factor donated in vivo by motile wild-type cells, but nonmotile (csg+) cells do not respond. The important difference is that C factor-less mutants are motile, which argues that C factor transmission requires that the responder cell be motile. Likewise, nonmotile mutants are impotent donors of C signal to motile C factor-less cells. Motility is thus required of both partners-donor and responder-to transfer the C signal. Microscopic examination shows that cells in the outer parts of a nascent fruiting body are aligned (Sager and Kaiser, 1993). Cell alignment, rather than motility itself, was shown to be the key factor by a mechanical forcing of nonmotile cells into a closely packed arrangement. The elongated rod shape of Myxococcus cells (0.5 urn diameter, 7 urn length) was used to orient them lengthwise as they fell into narrow troughs (produced by scoring agar with a fine-grained emery paper; Kim and Kaiser, 199Oc). Only the mechanically aligned cells in the troughs, which are nonmotile but csg*, activated C-dependent gene ex-

Review: How and Why Bacteria Talk to Each Other 075

I

0

+ Statve

I 10

I 5

I 15

I

I 20 I

I 25 hours

I

Aggregate

Sporulate

-

Mutant Class B

Figure 1. Four Classes of Signal-Defective Developmental Mutants of M. xanthus The horizontal bars indicate the period prior to which the indicated mutants exhibit a developmentaldefect.Thecolumnsto the right indicate the phenotype with respect to aggregation and sporulation of the indicated mutants.

I

A D

abnormal

C

abnormal

Nonmotile

pression (Figure 2). When the plates were incubated longer, sporulation was seen, but again only in the troughs. Sporulation, which requires expression of many C dependent genes, shows that C factor transfer had been efficient. A requirement for extensive alignment of cells explains why both the donors and responders of C signaling must normally be motile. Transmission of C signal certifies that the aggregative cell movements have brought them into alignment at high density. Aligned cells can be closely packed and thus have the highest possible cell density. A high spore density may be the biological objective of constructing a fruiting body. In a volume of regular closely packed cells, effective exchange of C factor would activate the late sporulation functions, and the final irreversible steps of spore differentiation would then proceed. There are many examples of gene expression controlling morphology, but a requirement for cell alignment illustrates how a morphological test can regulate gene expression. Within a nascent fruiting body, this alignment condition is met in an outer shell of cells but not in the central region inside this shell (Sager and Kaiser, 1993). Cells in the outer shell are moving in circular orbits about the center of the fruiting body and apparently signal as they move. Suc-

cessful C signaling initiates the differentiation of rodshaped cells into spherical but nonmotile spores. As cells become spherical and lose their motility, it is thought that they are passively transported by the circulating cells into the center, where they accumulate and complete their maturation into environmentally resistant myxospores (B. Sager, unpublished data). Fruiting body morphogenesis represents a dialogue between morphology and gene expression. Expression of aggregation and motility control genes brings cells into alignment within nascent fruiting bodies, which triggers C signal-dependent gene expression. Expression of sporulation genes, most of which are C dependent, leads to loss of motility and transport of the sporulating cells into the center of the fruiting body. As C signaling continues in the outer shell, more spores are formed, eventually filling the entire fruiting body center outward. As the fruiting body dries, it shrinks, becoming a dense, closely packed array of spherical spores (Kuner and Kaiser, 1982).

Autoinducer

in Vibrio

A molecular mechanism for cell density dependence been elucidated in the symbiotic luminous bacteria

has that

Figure 2. C Factor-Dependent Gene Expression and Cell Differentiation in Nonmotile Cells Depends on Cell Position in M. xanthus Mutant nonmotile cells containing a C factordependent /acZ fusion were allowed to settle from suspension onto an agar surface containing microscopic troughs made by scratching an agar suface with emery paper. The stripes of blue color indicate C factor-dependent B-galactosidase activity from aligned cells in the troughs. Cells on the flat surfaces between troughs remain yellow (M. xanthus produces yellow carotenoids). Photograph was taken 3 days after starvation had initiated development (Kim and Kaiser, 199oc).

Cell 076

inhabit the light organs of bony fishes and the squid. Light is emitted when the oxidation of FMNH, and tetradecanal is catalyzed by luciferase (Meighen, 1991). In dilute culture, the bacterial cells are very dim. As cell density increases, however, the luminescence per cell rises as much as lOO-fold (Nealson and Hastings, 1979). The levels of both luciferase and enzymes that synthesize the aldehyde substrate increase, as well as the cellular specific activity of luciferase. In Vibrio fischeri, the structural genes responsible for the synthesis and activity of luciferase (/uxA and lux6) and of tetradecanal (/uxC, /uxD, and /uxE) (Boylan et al., 1969; Engebrecht and Silverman, 1966) are under the transcriptional control of two regulatory genes: 1~x1,which encodes the synthesis of a membrane-permeable small molecule called autoinducer (Byers and Meighen, 1964; Devine et al., 1966); and /uxR, which encodes a DNA-binding protein that mediates the effect of autoinducer (Kaplan and Greenberg, 1967; Shadel et al., 1990a). The luminescence of dilute cultures of V. fischeri can be increased by adding medium conditioned by higher density cultures or purified autoinducer (Eberhard, 1972; Eberhard et al., 1961). When the autoinducer is prevented from accumulating, luminescence remains low. In the confines of the light organ of a fish or squid, the concentration of autoinducer is believed to increase, luminescence is thereby induced, and the animals are provided with light (Ruby and McFall-Ngai, 1992). The V. fischeri autoinducer has been identified as 6-ketocaproyl homoserine lactone (Eberhard et al., 1961). It is capable of diffusing freely across the membrane and is excreted into the medium. The V. harveyi inducer is a closely related compound, 6-hydroxybutryl homoserine lactone (Cao and Meighen, 1969). Positive feedback explains the induction of luminescence in V. fischeri (Engebrecht et al., 1963). Autoinducer is produced at a low constitutive rate during the early stages of growth. When a sufficient level of autoinducer has accumulated, interaction with LuxR stimulates transcription of the luxlCDAi3EG operon and /uxR (Engebrecht and Silverman, 1967). Because &xl encodes the autoinducer synthase, this transcription increases autoinducer concentration. It also creates a positive transcriptional loop, increasing the specific activities of the enzymes required for light production (Shadel and Baldwin, 1991, 1992). Mutational analysis of /uxR defines the N-terminal region as the site of autoinducer binding (Shade1 et al., 1990b; Slack et al., 1990). The C-terminal portion of LuxR belongs to the response-regulator superfamily of two-component systems with a proposed helix-turn-helix DNA-binding motif (Bainton et al., 1992; Parkinson, 1993 [this issue of Cc//j). In addition to being produced by V. fischeri, j&ketocaproyl homoserine lactone is produced by a diverse groupof terrestrial bacteria, including Pseudomonas aeruginosa, Erwinia carotovora, Erwinia herbicola, and Serratia marcescens (Bainton et al., 1992). In E. carotovora, it induces the expression of genes controlling the biosynthesis of carbapenem, an antibiotic. Another structurally related compound, isocapryld butryl lactone (called A factor; see below), induces sporulation, streptomycin synthe-

sis, and streptomycin resistance in Streptomyces griseus (Horinouchi and Beppu, 1990, 1992) Intercompartmental Communication Sporulation in 8. subtilis

during

At least three intercompartmental signals govern the metamorphosis of cells of 8. subtilis into spores in a 7-10 hr program of development. Entry into sporulation is triggered by nutrient limitation and by cell density peptides called extracellular differentiation factors (see above). Here, we are concerned with three signals that govern development after the process of sporulation has begun. In contrast with signaling in the multicellular systems considered so far, these signals are transmitted between a single pair of cells, the dissimilar cellular compartments of the sporangium in which the spore is produced. The signals govern the activation of three cell type-specific transcription factors called dK, dG, and dE and are exchanged in crisscross fashion between the two compartments of the sporangium. B. subtilis is a gram-positive soil bacterium. A hallmark of its developmental cycle is the formation of a septum near one pole of a cell that has begun to sporulate (Figure 3) (Losick et al., 1966; Piggot and Coote, 1976). This asymmetrically positioned septum partitions the developing cell or sporangium into unequally sized cellular compartments. These are the forespore (the smaller compartment) and the mother cell, which are initially lined up end by end within the sporangium. Shortly after the formation of the polar septum, the septal layer of cell wall material (peptidoglycan) that separates the two compartments is removed, at which time the mother cell membrane begins to migrate around and eventually wholly engulfs the forespore. In this way, the forespore is pinched off as a free protoplast within the mother cell (Figure 3). Initially, the forespore protoplast and the engulfment membrane of the mother cell are in tight juxtaposition, with little or no cell wall material in between. At late stages of development, however, the space between the mother cell and forespore membranes enlarges as it becomes filled with a thick layer of peptidoglycan known as the cortex. Thus, from the stage of engulfment until the process of spore formation is complete, the sporangium can be considered a cell within a cell. The inner cell (that is, the forespore) is a germline cell that will become the spore and hence give rise to subsequent progeny. The outer or mother cell is a terminally differentiating cell, which is discarded by lysis when development is complete. This strategy of forming a spore (or more properly, an endospore) entirely within a larger cell is a distinctive feature of sporulation in Bacillus and related genera. The conversion of the forespore into a mature spore is mediated by events occurring both within the forespore and in the mother cell. Thus, proteins produced within the forespore-such as a family of small acid-soluble proteins called SASPs-contribute to the inner structure (the core) of the spore. Conversely, a complex set of proteins that are produced within the mother cell is deposited around

Review: How and Why Bacteria Talk to Each Other 077

Figure 3. Electron Micrograph Sporangium

of a 8. subtilis Sporangium

is shown at the stage of polar septation

(top) and engulfment

the outside of the mother cell membrane that envelops the forespore to create the tough protein shell known as the coat, which will encase the spore. The mother cell also produces the enzymes that are responsible for the synthesis of the cortex. Gene expression in the forespore and the mother cell is regulated in two principal phases: an early phase, when the forespore and mother cell compartments are lying end by end, and a late phase, when the forespore is a free protoplast within the mother cell (reviewed by Errington, 1993; Losick and Stragier, 1992; Shapiro, 1993 [this issue of CeNj). In the early phase, gene expression is largely controlled by the action of transcription factors oF in the

(bottom). The picture was kindly provided by A. Driks

forespore and crE in the mother cell. In the late phase, aF and crE are replaced by transcription factors cG and cK, respectively. Developmental Checkpoint Governing the Activation of gK The most striking and extensively studied example of intercellular communication occurs just after engulfment, when compartment-specific gene expression is differentially regulated by the action of oG and crK.The oG factor directs the transcription of forespore-expressed genes, such as those that encode the SASP family. Conversely, oK dictates the transcription of mother cell-expressed genes,

Cdl 078

Mother

Cell

RO-UK

such as those encoding the coat proteins, and one or more enzymes involved in synthesis of the cortex. The forespore and the mother cell are not, however, wholly independent of one another. Mutations that block gene expression in the forespore, such as a mutation in the gene spa///G, encoding oG, prevent the transcription in the mother cell of genes under the control of oK (Cutting et al., 1990). The basis for this effect is an intricate, intercompartmental signal-transduction pathway that links the activity of oK in the mother cell to the expression of a gene under the control of crGin the forespore. The activity of oK is controlled at the level of the proteolytic processing of a precursor to the transcription factor (Cutting et al., 1990; Lu et al., 1990). The primary product of the gene (called sigK) that encodes oK is an inactive proprotein (pro-o”) that contains 20 more amino acids at its N-terminus than uK. Conversion of the proprotein to oK depends on a protein in the mother cell called SpolVFB, which is the proprotein-processing enzyme or its regulator (Cutting et al., 1991 b). The activity of SpolVFB is negatively regulated by two other mother cell proteins called BofA and SpolVFA (Cutting et al., 1990, 1991 b; Ricca et al., 1992). The putative processing enzyme depends on a signal from the forespore in order to escape the effect of these inhibitory proteins. This signal is provided by the product (SpolVB, not to be confused with SpolVFB) of a gene under the control of oG (Cutting et al., 1991a). Somehow, SpolVB acts across the two membranes that separate the forespore from the mother cell to stimulate the activity of the processing enzyme. It is not known how SpolVB stimulates SpolVFB or whether this interaction is direct. However, as illustrated in Figure 4, one simple possibility (but not the only one) is that activation occurs by protein-protein interaction between SpolVB, which is inserted into the forespore membrane, and SpolVFB, which is inserted into the mother cell membrane that surrounds the forespore protoplast. The model is based on the observation that all four components (BofA, SpolVFA, SpolVFB, and SpolVB) of the signal

Forespore

Figure 4. Speculative Model for Intercompartmental Signaling between the Forespore and the Mother Cell of the 6. subtilis Sporangium See text for details. The figure was kindly provided by S. Alper.

transduction pathway have features characteristic of integral membrane proteins. So, in summary, intercompartmental communication is mediated by the following chain of events. The oG factor causes the synthesis in the forespore of SpolVB, which acts across the forespore and mother cell membranes to enable SpolVFB to escape the inhibitory effect of BofA and SpolVFA. This, in turn, triggers processing of pro-oK and hence the transcription of genes under oK control. (The recent discovery of a new locus at which mutations influence pro-oK processing suggests that BofA, SpolVFA, SpolVFB, and SpolVB may not constitute a complete list of the components in the signal transduction pathway [S. Cutting, personal communication].) What is the function of this signal transduction pathway? A possible answer is revealed by the use of mutations that bypass the dependence of UK-directed gene expression on the action of oG and SpolVB (Cutting et al., 1990; Ricca et al., 1992). Examples of such bypass mutations are mutations in the genes for the inhibitory proteins BofA and SpolVFA and a deletion of the pro-amino acid-coding sequence from the structural gene for oK (such that the primary product of the deletion-mutated gene is the active transcription factor). These bypass mutations cause oK-directed gene expression (in otherwise wild-type cells) to commence an hour earlier than normal, and this in turn has deleterious effects on spore formation. Thus, the prooK processing system delays the action of oK until (and unless) the forespore signals the mother cell, by means of aG-directed synthesis of SpolVB, that it has attained an appropriate stage of development (characterized by an engulfed forespore in which oG is active) in order for developmental events in the mother cell to proceed. Intercompartmental communication is therefore a kind of checkpoint that coordinates the timing of events in the mother cell with differentiation (and maybe morphogenesis) of the forespore. This is reminiscent of the case of the Myxococcus C signal, whose action certifies that the developing cells have reached the morphological stage of being

ggview:

How and Why Bacteria Talk to Each Other

Donor (PA) Figure 5. Pheromone

Donor (~6)

Action in E. faecalis

In the center, a plasmid-free recipient synthesizes several different sex pheromones encoded by different chromosomal genes; only two are shown, called CA and CC. The pheromones are released to the medium. The cell on the left, carrying plasmid pA, responds specifically to pheromone CA; the cell on the right, carrying plasmid pB, responds specifically to pheromone c6. Upon receipt of the pheromone, a signal (denoted RCA) is transduced to the resident plasmid. RCA induces expression of a plasmid gene-encoding surface adhesin on the donor cells. The adhesin (AS) is shown to bind to ES, a normal constituent of the wall of all E. faecalis cells. Clumps containing donor and recipient are thus formed. Provided by P. Christie.

closely packed into an intimate array of side by side and end to end associations. Crisscross Regulation Pro-aK processing is not the only example of intercellular communication during spore formation. There is evidence that the activity of oG in the forespore is governed by the action of the early phase, mother cell transcription factor cE (Stragier, 1992). The gene encoding oG is induced in the forespore prior to engulfment, but aG-directed gene expression does not commence until the forespore is pinched off as a free protoplast within the mother cell. The aE factor in the mother cell directs the transcription of genes that govern the process of engulfment and the eight-cistron .spolllA operon, whose products are thought to be involved in activating oG (A. M. Guerout-Fleury, G. Gonzy-Treboul, and P. Stragier, unpublished data). So the activation of oG in the forespore is determined by events under the control of cE in the mother cell. Finally, the activation of oE in the mother cell is dependent upon the action of the early phase forespore transcription factor, oF. Like oK, oE is generated by the proteolytic processing of an inactive proprotein called pro-oE (LaBelI et al., 1987). Just as the processing of pro-OK is controlled by the action of crG, so too the processing of pro-oE in the mother cell is determined by of-directed gene expression in the forespore (Jonas and Haldenwang, 1989; Stragier et al., 1988). The forespore and the mother cell communicate back and forth with each other in crisscross fashion in at least three steps during the course of morphogenesis (Losick and Stragier, 1992). First, the action of qF in the forespore triggers the processing of pro-oE in the mother cell. Next, aE orchestrates events

in the mother cell that lead to the activation of oG in the forespore. Finally, oG-directed gene expression in the forespore leads to the conversion of pro-oK to mature crK in the mother cell. Intercompartmental communication is therefore a device for coordinating gene expression between the mother cell and the forespore at each stage of development. Pheromone-Inducible Gene Expression Enterococcus faecalis

in

Conjugation between sexually differentiated bacterial cells requires their interaction. Enterococcus faecalis, a nonmotile gram-positive species, needs cell interactions for sex plasmid transfer during conjugation. Mating mixtures of E. faecalis form heterogenous multicellular aggregates of donor cells, which carry a plasmid, and recipient cells, which are initially plasmid free (Dunny et al., 1978). Prior to conjugation, the recipient cells have excreted small, heat- and nuclease-stable but trypsin- and chymotrypsin-sensitive pheromones. The peptide pheromones signal the donor cells to synthesize adhesin (Dunny et al., 1978) which causes them to clump with recipient cells and other donor cells. Conjugation takes place within the resulting multicellular aggregate, and the plasmid is transferred to the recipient cells. In a sense, the clumping reaction substitutes for the adhesive F pili of gram-negative bacteria in gathering cells together. At least 18 plasmids that encode a pheromone response have been described (Galli et al., 1992). The pheromones are plasmid specific, and their specificity is illustrated in Figure 5. Several pheromones have been purified and shown to

Cell 880

be different hydrophobic octapeptides, or in one case a heptapeptide (Clewell and Weaver, 1989). Chemical synthesis has confirmed the structures of pheromones cPD1, cAD1, cAM373, and cCFl0. Pheromones are typically active at concentrations below 5 x lo-” M, and as few as 2 molecules per donor cell may be sufficient to induce the transcription of genes on the target plasmids (Galli et al., 1992; Mori et al., 1988; Weaver and Clewell, 1990). Not only is the response to pheromone very sensitive, but its specificity is also high. Pheromone cAD1 is unable to induce expression (indicated by clumping by cells) from the heterologous plasmid pPD1, even at a concentration of 1 PM, which is 105-fold higher than that needed to induce expression from the homologous plasmid. The pheromone-induced surface-bound adhesins specified by plasmids pAD1 and pCFl0 have been purified, and their structural genes have been cloned and sequenced (Galli et al., 1990, 1992). These adhesins are large, closely related proteins that may form dense, hairlike structures on the cell wall of the induced bacteria (Galli et al., 1989). The ligand for the adhesin on E. faecalis cells is a surface constituent present on all cells, whether or not they carry a plasmid. Available evidence strongly favors involvement of lipoteichoic acid, the major wall antigen of gram-positive cells (Ehrenfeld et al., 1988; Trotter and Dunny, 1990). These and other experiments imply that the adhesion system is a heterophilic, adhesin-lipoteichoic acid binding system (Dunny, 1990). Once transferred from donor to recipient, the plasmid directs the synthesis of a plasmid-encoded inhibitor that specifically blocks the inducing action of the cognate sex pheromone (Clewell et al., 1982). The inhibitors for cAD1 and cPD1 have been purified and shown to be hydrophobic octapeptides that have sequences weakly related to their corresponding pheromones (Clewell and Weaver, 1989). In one case, the inhibitor has 3 identical residues among 7 total. The inhibitor peptide neutralizes its cognate pheromone, probably by competition, thus preventing a donor cell from responding to its own pheromone. A puzzling aspect of the pheromone system is that at least five different pheromones can be produced by a single recipient cell. Do potential recipients just happen to secrete specific pheromones for several potential donors? Pheromones are encoded by as yet unidentified chromosomal genes. Production of the adhesins and of the pheromone inhibitors are encoded by the plasmids. A signal transduction system that recognizes pheromone and causes genes to be expressed on the plasmid is currently being defined (D. B. Clewell, personal communication; G. M. Dunny, personal communication). Extracellular Signaling during Multicellular Development in Streptomyces coelicolor Morphological differentiation in Streptomyces coelicolor under certain nutritional conditions involves theextracellular accumulation of a small morphogenetic protein. Production of this protein is governed by a hierarchical cascade of extracellular signaling molecules in a 3- to 5-day program of multicellular development.

S. coelicolor is a filamentous, fungus-like soil bacterium that grows as a branching network of multinucleoid hyphae, which are occasionally interrupted by septa (Chater, 1993). The hyphae penetrate the stratum upon which the bacteria feed to form a mat of cells known as the substrate mycelium. Differentiation occurs by the formation of an aerial mycelium, which consists of a dense lawn of aerial hyphae that grow perpendicular to the substrate mycelium to impart a fuzzy white appearance to the colony surface (see cover of this issue). The aerial hyphae must escape from the aqueous environment of the colony surface in order to grow into the air. In the next phase of development, the aerial mycelium undergoes massive septation into uninucleoid units, which metamorphose into chains of gray-pigmented, hydrophobic spores. The aerial mycelium is a device for the dispersal of the spores. Mutants blocked in erecting aerial hyphae are known as bald (b/d) mutants because of the smooth, hairless appearance of their colonies. Mutants blocked in spore formation are known as white (w/r/) mutants because they produce an aerial mycelium but fail to display the gray color characteristic of colonies of wild-type cells undergoing spore formation. The capacity of certain b/d mutants to produce aerial hyphae can be restored by growth of the mutants near colonies of whi mutant or wild-type cells (Willey et al., 1991). This phenomenon is similar to extracellular complementation in M. xanthus, in which wild-type cells rescue the ability of certain developmental mutants to sporulate in mixtures of wild-type and mutant bacteria (see above). As in M. xanthus, extracellular complementation in S. coelicolor has been interpreted to indicate that the wild-type bacteria produce an extracellular molecule(s) that restores the capacity of the mutant bacteria to undergo morphological differentiation. Such a molecule, known as SapB (for sporulation-associated protein), has been purified from S. coelicolor cells undergoing development on rich sporulation medium (a SapB-independent pathway of aerial mycelium formation is believed to occur during sporulation on minimal medium) (Guijarro et al., 1988). SapB is an approximately 18 residue peptide of unusual structure in that it evidently contains viscinal hydroxyl groups (Willey et al., 1991). Because it is synthesized in the presence of chloramphenicol (Willey et al., 1993). it is probably produced by a nonribosomal mechanism, as is known to be the case for certain peptide antibiotics. SapB is produced in copious amounts during aerial mycelium formation. It is found on the surface of aerial hyphae and mature spores and is also exported into the agar. Production of SapB is impaired in many different b/d mutants but not in colonies of whi mutant cells. Extracellular supplementation experiments indicate that SapB plays a direct role in the erection of aerial hyphae (Willey et al., 1991, 1993). Addition of the purified protein to several different b/d mutants restores their capacity to produce aerial hyphae. Because of its high abundance and its presence on the surface of cells, SapB is likely to be a morphogenetic protein that coats certain hyphae on the top of colony, enabling them to break surface tension and grow into the air. A high local concentration of SapB

Review: How and Why Bacteria Talk to Each Other 881

Figure 6. Extracellular coelicolor

Complementation

of S.

The photomicrograph shows extracellular complementation of a bldA mutant by a MG mutant. Bar, 500 mm. Reproduced from Willey et al. (1993), with permission.

is achieved by the large number of cells involved in the process of morphological differentiation. This can be likened to a Quaker-style barn raising in which many members of the community cooperate in a task of mutual benefit. Strikingly, extracellular complementation can also be observed between certain pairs of b/d mutants that are incapable individually of producing SapB (Willey et al., 1993). When pairs of certain b/d mutants are grown near each other, one member of the pair produces a fringe of aerial mycelium on the side of the colony closest to the other colony (Figure 6). Such extracellular complementation, when it is observed, is always unidirectional; that is, one member of a complementing pair is the donor and the other is the responder. Not all pairs complement each other; thus certain mutants, such as b/dA and b/d/+, fall into the same extracellular complementation group. A mutant that is a donor to one b/d mutant can be a responder to another b/d mutant. These results have been interpreted to indicate that S. coelicolor produces at least four different extracellular signals. These signals seem to be arranged in a hierarchical cascade, in which signal A induces the production of signal B, and so forth. At least one such signal (produced by a bldD mutant) can be detected in conditioned agar; it is heat stable and protease resistant. If SapB is involved in erecting aerial hyphae and if b/d mutants are defective in SapB production, then by what mechanism does extracellular complementation between b/d mutants restore morphological differentiation? When pairs of complementing b/d mutants are tested for the presence of SapB, production of SapB is restored, but its presence is confined to the fringe of aerial mycelium on the responder colony (Willey et al., 1993). Thus, extracellular complementation between b/d mutants restores the capacity of the responder mutant to produce SapB. SapB in the responder mutant then causes aerial hyphae to form. Thus, the cascade of b/d-dependent signals evidently serves to control the production of a morphogenetic protein that is directly involved in the formation of aerial hyphae. S. coelicolor seems to have evolved an elaborate

system of intercellular communication that could govern when and where aerial mycelium formation takes place. Other Streptomyces species are known to produce moleculesthatstimulatetheformationof aerial hyphae. Examples are A factor and C factor in the species S. griseus and pamamycin in S. alboniger (Beppu, 1992; Biro et al., 1960; Khokhlov, 1965; Khokhlov et al., 1973; McCann and Pogell, 1979). It is not known whether these species use a SapB-like molecule in erecting aerial hyphae or whether these factors act by stimulating the production of a morphogenetic protein. Pattern Formation in Anabaena Is Mediated by Intercellular Interactions Anabaena is a filamentous cyanobacterium that forms a linear array of cells. Certain cells at intervals along this filament undergo differentiation over the course of about a day into a nitrogen-fixing cell known as a heterocyst. The one-dimensional pattern of heterocyst formation is determined by intercellular communication between cells in the filament. In the presence of an abundant nitrogen source, vegetative Anabaena cells divide by binary fission to form long filaments. When depleted of its nitrogen source, however, Anabaena undergoes differentiation into heterocysts, thick-walled cells that are capable of fixing atmospheric NZ (Buikema and Haselkorn, 1993). This differentiation does not occur uniformly in all cells along the filament but rather in a semiregular spatial pattern within the filament, in which single cells separated from each other by 10 to 20 cells (depending on growth conditions) undergo conversion into heterocysts. The developing cell or proheterocyst undergoes a variety of distinctive morphological, physiological, and genetic changes that adapt it to the specialized task of fixing atmospheric NP. These include the removal from the chromosome of two DNA elements that interrupt genes encoding components of the nitrogenase system (in the species strain PCC 7120; Golden et al., 1966, 1965) the synthesis and assembly of the nitroge-

Cell 882

nase system, and the formation of a thick and oxygenimpermeable outer envelope that protects the highlysensitive nitrogenase system from oxygen poisoning. The proheterocyst can dedifferentiate into a vegetative cell (Wilcox et al., 1973a, 1973b), but the mature heterocyst is a terminally differentiated cell type that is incapable of growing or of reverting to the vegetative state. It exists in a symbiotic relationship with its neighboring vegetative cells, from which it receives carbon compounds and to which it transports fixed nitrogen in the form of glutamine. Classic experiments by Wolk (1967) and Wilcox et al. (1973a, 1973b), based on an analysis of the behavior of broken filaments, indicate that the development and maintenance of heterocysts is determined by interactions between neighboring cells. Wolk (1967) found that breakage of filaments into short chains of cells results in an overall increase in the number of heterocysts produced. An interpretation of this observation was that heterocysts release an inhibitor of differentiation that diffuses to neighboring cells in the filament and prevents them from undergoing heterocyst formation. Thus, when separated from the source of the inhibitor by breakage of the filament, vegetative cells exhibit a greater tendency to undergo differentiation. Wilcox et al. (1973a, 1973b) found that proheterocysts revert back to the vegetative state when the filament is broken on one or both sides of the developing cell. Thus, a certain number of neighboring vegetative cells is required in order to sustain differentiation. This observation was interpreted to indicate that breakage of the filament near the proheterocyst leads to a high local concentration of the inhibitor, thereby causing the proheterocyst todedifferentiate into a vegetative cell. Models based on the existence of an inhibitor of differentiation have been proposed to account for the patterned formation of heterocysts. In one such model, cells along the filament change at random into prospective heterocysts, which then prevent nearby cells from differentiating by release of the inhibitor (Wolk, 1989). Diffusion sets up a gradient of inhibitor around each heterocyst that determines the spacing of heterocyst formation. To date, direct evidence for the existence of an inhibitor remains elusive. If an inhibitor exists, it is unlikely to be a metabolic product of nitrogen fixation, because proheterocysts are incapable of fixing nitrogen, and mutants defective in nitrogen fixation exhibit pattern formation (R. Haselkorn, personal communication). Also, it is possible to imagine alternative models that do not posit the existence of an inhibitor. For example, in a scavenging model, the first cells to become starved for nitrogen scavenge nitrogen from neighboring cells, which, in turn, propagate the scavenging response (Wolk, 1989). Eventually, the most nitrogen-depleted cells, which have been scavenged from both sides, are induced to differentiate. The patterned formation of heterocysts reflects an underlying one-dimensional spatial pattern in the expression of genes involved in the differentiation. This is most strikingly demonstrated by the use of a luciferase-encoding /uxAB reporter to monitor the spatial localization of gene activity (Elhai and Wolk, 1990). For example, filaments of cells bearing a IuxAB fusion to the nitrogenase-encoding

operon nif/-/DK emit light in a periodic pattern that corresponds to the location of heterocysts (Elhai and Wolk, 1990) (Figure 7). A periodic pattern is also seen with a IuxAB fusion to hepA (Walk et al., 1993) a gene whose product is involved in the formation of the envelope of the heterocyst (Holland and Wolk, 1990). When the luciferase signal is amplified in an alternative strategy (hepA is fused to the gene for the phage T7 RNA polymerase, which, in turn, directs transcription of /uxAB fused to aT7 promoter; Wolk et al., 1993) spatially patterned light emission can be detected even before the earliest morphological manifestations of heterocyst formation. An important insight into the control of heterocyst formation has come from the discovery of regulatory gene hefr?, in which mutations block differentiation at an early stage (and prevent the induction of the hepA gene consideredabove)(Buikemaand Haselkorn, 1991). Interestingly, the presence of the wild-type hetR gene in multiple copies stimulates heterocyst formation; cells bearing extra copies of hetR produce heterocysts under high ammonium conditions (when differentiation is normally suppressed), and, under conditions of low concentrations of nitrogen sources, such cells produce supernumerary heterocysts in clusters of 2-5 cells. These observations indicate that the hefR gene product is both necessary and sufficient to induce heterocyst formation and suggest that the conversion of a vegetative cell into a heterocyst is triggered by an increase in the amount or activity of the HetFl protein. The use of /uxAB as a reporter indicates that heft? is uniformly transcribed at a low level in all cells along the filament but that, shortly after a shift to nitrogen limitation conditions, its transcription is strongly enhanced in spatially separated cells (Walk, 1991). Since this enhanced expression depends on the hetR gene product itself, HetR is apparently positively autoregulatory and recalls the Vibrio autoinducer (above). Apparently, then, intercellular communication between cells in the filament determines the spatial pattern of enhanced hefR expression. How this occurs is a central unsolved mystery, but a possible clue comes from the recent discovery of p&A, in which mutations impair intrafilament heterocyst formation but not the formation of heterocysts at the ends of filaments (Liang et al., 1992). ThepafA gene product contains a region of similarity to the responseregulator member of prokaryotic two-component regulatory systems. Conceivably, therefore, the PatA protein is part of a signal transduction pathway that couples the activation of hetR to the local concentration of a diffusible inhibitor of differentiation or other morphogen. Conclusions As the examples we have considered illustrate, diverse kinds of bacteria communicate with each other in a wide variety of ways to coordinate their behavior during differentiation and morphogenesis. Bacteria are endowed with intricate internal programs that dictate complex developmental events, but external cues impinge on the genetic instructions to elicit particular patterns of gene expression at appropriate times and appropriate places. Some cues

Review: How and Why Bacteria Talk to Each Other 003

Figure 7. Periodic Light Emission from an Anabaena Filament of Cells Bearing a /uxAS Fusion to the nifHDK Operon (A) is a video image, and (B) is an image of luminescence. Bar, 10 mm. Reproduced from Elhai and Wolk (1990) with permission.

come from the environment, such as nutrients and their depletion, but often cues are provided by the bacteria themselves. Communication between bacteria can involve a single pair of cells, as in the Bacillus sporangium, a one-dimensional array of cells, as in an Anabaena filament, or a community of cells, as in a Streptomyces mycelium or a Myxobacterium fruiting body. Communication serves several kinds of developmental functions. V. fischeri, B. subtilis, and M. xanthus secrete and monitor the concentration of different kinds of small molecules to measure cell density. This information is used to govern light emission, entry into sporulation and the state of genetic competence, and the early stages of fruiting body formation, respectively. M. xanthus and B. subtilis also relay signals between cells to coordinate gene expression with the course of morphological development. Transmission of a protein signal informs developing cellsof M. xanthus that the fruiting body has reached a morphological stage of intimate cell packing, thereby triggering the expression of genes that transform previously motile bacteria into myxospores. Similarly, in B. subtilis, a signal transduction pathway operating at the level of the activation of a proprotein transcription factor coordinates the timing of gene expression in the mother cell with forespore development. Intimate cell packing and forespore formation are examples of developmental checkpoints in which cells communicate with each other in order to tie their internal developmental programs to the overall course of morphogenesis. Some kinds of signals (pheromones) elicit developmental responses that facilitate genetic exchange between cells, as in the examples of the mating peptides of E. faecalis and the competence factor of B. subtilis. Yet another purpose of intercellular interaction is illustrated by morphological differentiation in S. coelicolor, in which large numbers of cells coordinately produce a morphogenetic protein that facilitates the erection of aerial hyphae in response to a cascade of intercellular signals. Multicellular cooperation enables the colony to attain a high local concentration of a morphogenetic protein. Finally, an intercellular gradient of morphogen enables Anabaena to establish a one-dimensional pattern of differentiation in which only certain cells, separated from each

other at intervals along the filament, are allowed to metamorphose into heterocysts. In the Anabaena example, intercellular communication is a device for subjecting cellular differentiation to spatial regulation. Of course, bacteria communicate not only with each other, but with other kinds of organisms as well, as illustrated by the exchange of signals between the nodule-forming bacterium Rhizobium and the root hairs of its plant host (reviewed by Long and Staskawicz, 1993 [this issue of Cell]). Far from being solitary creatures, bacteria are colonial organisms that exploit elaborate systems of intercellular interaction and communication to facilitate their adaptation to changing environmental circumstances. In contrast with the traditional view of a bacterial cell as a selfcontained entity, it seems likely that cooperative behavior is prevalent in the prokaryotic world. Even the best known of all bacteria, E. coli, turns out to be capable of intercellular interactions that result in intricate multicellular patterns (Budrene and Berg, 1991; Shapiro and Higgins, 1989; Shapiro and Hsu, 1989). The application of traditional and molecular methods of genetic analysis to prokaryotic organisms both familiar and foreign is likely to yield yet more surprises in and illuminating examples of the ways in which the simplest of organisms talk to each other. Acknowledgments Wethank G. Dunny, A. Grossman, G. Shadel, L. Shapiro, and P. Wolk for advice on the manuscript. This work was supported by grants from the National institutes of Health and American Cancer Society to D. K. and from the National Institutes of Health to R. L.

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in Proof

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