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C-factor: A cell-cell signaling protein required for fruiting body morphogenesis of M. Xanthus
Cell, Vol. 61, 19-26,
April 6, 1990, Copyright
0 1990 by Cell Press
C-Factor: A Cell-Cell Signaling Protein Required for Fruiting Body Morphogenesis of M. xanthus Seung K. Kim and Dale Kaiser Department of Biochemistry and Department of Developmental Biology Stanford University School of Medicine Stanford, California 94305
Summary During fruitfng body development, the product of the csgA gene lo n eceeeary for cellular aggregation, for spore dffferentlatlon, and for gene expreeeion that is initiated after 6 hr of atawation. From nascent wildtype fruiting bodies we have purified a polypeptide of 17 kd caffed C-factor, whkh, at approximately 1 to 2 nM, restores normal development to csgA mutant cells. C-factor activity is not recovered from extracts of unstarved, growing cells or csgA mutant cells. The amino acid sequence from purified C-factor demonatrates that it is the product of the csgA gene. C-factor is activeoveranamwrangeofconcentret ion and has properties of a morphogenetic paracrine signal. Introduction Cell interactions are necessary to establish differentiated cell fates in the development of multicellular organisms. Well-studied examples include CAMP relay in Dictyostelium (Devreotes, 1983) vulva1 development in Caenorhabditis elegans (Perguson et al., 1987), insect neurogenesis (Doe and Goodman, 1985), and vertebrate mesoderm induction (Smith, 1987). Cell interactions also appear to govern multicellular development and cellular differentiation of the Gram-negative bacterium Myxococcus xanthus, an organism whose prokaryotic genetic system and cellular organization offer benefits of experimental accessibility (Rosenberg, 1984). Following starvation at high cell density on a solid surface, rod-shaped M. xanthus cells glide to aggregation centers where they build a structure of family-specific shape, called a fruiting body. Early in aggregation, ridgelike accumulations of cells move coordinately and rhythmically over the substrate like ripples on a water surface (Reichenbach, 1965; Shimkets and Kaiser, 1982). Some cells lyse, while other cells within a nascent fruiting body differentiate to dormant, ovoid spores (Wireman and Dworkin, 1975). Roughly lo5 cells participate in constructing a fruiting body, and the process requires about 20 hr from the removal of nutrient to the beginning of sporulation. Environmentally resistant spores mature over the next few days. Control of M. xanthus fruiting body development by several sequential cell-cell interactions is implied by the existence of four classes of developmental mutants that are not cell autonomous (Hagen et al., 1978; Shimkets and Dworkin, 1981; LaRossa et al., 1983; Janssen and Dwor-
kin, 1985; Kroos and Kaiser, 1987). Mutants of the asg, bsg, csg, and dsg (a signal, b signal, c signal, and d signal, respectively) classes cannot sporulate alone, but can be rescued for sporulation by codevelopment with wildtype cells or with cells of another mutant class. The rescue does not involve genetic exchange but rather occurs extracellularly. Each cell interaction mutant class modifies the pattern of developmentally regulated gene expression in a different way (Kuspa et al., 1988; Kroos and Kaiser, 1987). The patterns have been revealed using a transposable promoter probe, Tn5lac (Kroos and Kaiser, 1984) which can generate transcriptional fusions of lat.? to developmentally regulated genes. Assay of &galactosidase activity in such fusion strains indicates that gene expression during development, which is disrupted in asg, bsg, and csg mutants, is rescued by codevelopment with wildtype cells (Kuspa et al., 1986; Gill and Cull, 1986; Kroos and Kaiser, 1987). The csg class of developmental mutations is the subject of this paper. All existing members of this class have resulted from mutation at a single genetic locus named csgA (Shimkets et al., 1983; Shimkets and Asher, 1988). Unlike wild-type cells, which aggregate into compact translucent mounds by 12 hr on solid agar substrate, csgA mutants aggregate only after 18 hr and then into larger, less compact mounds and ridges than csgA+ cells. Under conditions of submerged culture (Kuner and Kaiser, 1982; see Experimental Procedures), the aggregation defect of csgA mutants is even more severe, and csgA cells fail to construct any stable multicellular structures. csgA mutants also fail to ripple, to lyse, or to sporulate (Shimkets and Kaiser, 1982). A mutation in csgA does not alter the expression of /acZ transcriptional fusion strains that produce P-galactosidase during the first 8 hr of development (C-independent fusions), but does reduce or abolish 6-galactosidase synthesis from /acZ transcriptional fusion strains that normally express after 6 hr (C-dependent fusions; Kroos and Kaiser, 1987). Thus, the csgA product may be involved in production or transmission of a developmental signal that is crucial for normal fruiting body morphogenesis, cellular differentiation, and developmentally regulated gene expression. The csgA gene has been cloned (Shimkets et al., 1983; Shimkets and Asher, 1988) and sequenced (Hagen and Shimkets, 1990). Based on codon usage, the best possible reading frame indicates that csgA could specify a 17.7 kd protein (Hagen and Shimkets, 1990). Does csgA specify the signal molecule itself or some other essential component of the signaling system? To identify the csg signal molecule(s), we have constructed a bioassay based on the completion of development by a csgA mutant. We report the characterization of C-factor, a polypeptide of approximately 17 kd that has been extracted from immature fruiting bodies and that rescues development of csgA mutants. Its amino acid sequence analysis demonstrates that C-factor is encoded by the csgA gene.
Cell 20
Table
1. C-Factor
Rescues
Developmental
Sporulation
of csgA
Unsupplemented Spores
Plus C-Factor
Strain
Genotype
DK1622
Wild-type
DK5246
csgA,
D4499
<5
x 102
<0.02
DK5253
csgA,
R4435
<5
x 102
DK5267
csgA,
94414
<5
x 102
DK4499
G4499
2.5 x lo6
100
DK5204
a4435
2.5
x 10s
100
DK5279
a4414
5.0 x 104
2
2.5
per Milliliter x 106
Mutants
% Wild-Type
Spores
per Milliliter
Plus DK1622
Cells
% Wild-Type
Spores
per Milliliter
% Wild-Type
2.5 x IO6
100
3 x 106
120
<0.02
3.0 x 10s
120
3 x 106
120
<0.02
1 x 105
5 x 103
2
(100)
4
Two units of purified C-factor were added to the responder strains indicated. Sporulation tests were performed as described in Experimental Procedures. In extracellular complementation tests, approximately equal numbers of wild-type strain DKl622 and mutant cells were mixed and plated for development.
Results Production and Assay of C-Factor Wild-type ceils mixed with csgA mutant cells in equal proportion rescue fruiting body morphogenesis and sporulation of these mutants to wild-type levels (Table 1, lines 1, 2, and 3; Hagen et al., 1978; LaRossa et al., 1983). Developmental gene expression in the csgA mutant cells, as monitored by a panel of /acZ transcriptional fusions, is also restored to an approximately wild-type pattern in such cell mixtures (Kroos and Kaiser, 1987; present study). A simple interpretation of these results is that wild-type cells provide an intercellular signal molecule that is missing from the csgA mutants. If so, csgA mutant cells could be used to monitor purification of signal molecules from csgA+ cells. Several experiments suggested that csgA-rescuing activity is tightly cell associated. Wild-type source cells, if separated from responder csgA cells by a 0.45 urn nucleopore membrane filter, failed to rescue mutant cells (data not shown). Extracts made by extensive washings of wildtype cells at various developmental stages also had no effect on csgA responder cells. However, cell-free extracts made from lysed M. xanthus cells were found to restore the ability of csgA mutants to form fruiting bodies and spores and, as shown below, to express C-dependent genes. Ripples, however, were not detected. A bioassay that measures development-rescuing activity, or C-factor, is diagrammed in Figure 1A. Cell-free extracts of wild-type M. xanthus in a series of P-fold dilutions were incubated with csgA cells prepared for development (see Experimental Procedures). The choice of a limiting P-fold dilution assay proved critical to initially detecting C-factor activity because undiluted extracts were inactive (Figure 16). As purification progressed and specific activity rose, activity was consistently detected only within an approximately 4-fold concentration range (Figure 16). Another characteristic of the assay is that fruiting bodies, spores, and C-dependent gene expression (see below) are first detected about 40 hr after nutrient deprivation initiates development-about 24 hr later than seen in wildtype development.
As judged either by restoration of csgA cell aggregate formation or sporulation, C-factor activity is only produced by wild-type cells during the interval between 12 and 18 hr poststarvation (Figure 2). At this time, source cells have formed compact mounds but have not yet begun to differentiate into spores. Developing source cells harvested before 12 hr or after 18 hr showed little or no rescuing activity. Extracts made from csgA mutant cells harvested at intervals between 0 and 48 hr poststarvation and assayed over a 500~fold concentration range failed to rescue fruiting body or spore formation of csgA mutants. Wild-type cells developing on starvation agar medium for 18 hr were chosen as the standard source for further purification and characterization of C-factor. Purification of C-Factor When unfractionated cell lysates containing C-factor activity were sedimented at 100,000 x g for 1 hr, activity was found only in the pelleting material. Crucial to the purification of C-factor was the finding that the zwitterionic detergent 3-([3-cholamidopropyI]dimethylammonio)-l-propanesulfonate (CHAPS) solubilizes C-factor while maintaining it in a biologically active form. Cholate, deoxycholate, and octylglucoside, detergents with a critical micelle concentration similar to CHAPS, failed to solubilize active material over a wide range of concentrations (S. K. K., unpublished data). The limiting 2-fold dilution assay, shown in Figure 1, was used to follow C-factor activity during fractionation of CHAPS-solubilized cell extracts, and progress in purification was monitored by SDS-polyacrylamide gel etectrophoresis (SDS-PAGE). Following a P-fold enrichment of activity by ammonium sulfate fractionation, homogeneous C-factor was obtained after anion-exchange chromatography, gel filtration, and a final anion-exchange step. The ability of purified material to restore sporulation is demonstrated in the column marked “Plus C-Factor” in Table 1. Activity that rescues sporulation, fruiting body formation, and, as shown below, C-dependent gene expression copurified at each step, indicating that the same molecule possesses all these activities. The final degree of purification is over 1000~fold with an
r$Factor
Is Required
for M. xanthus
C-Factor
A
Differentiation
Assay
C-Factor Souro (Cells, cxtrrcts)
1 48 tws.
32’
Fruiting bodies Spr,S
0
2
.
8
8
1012141(1182022242(12830
G-Galactosidasc
B
The assay for C factor activity is a limiting two-fold dilution series
10’
1:6
0
Figure
8
8
1012141518202224262830
2. Time Course
of C-Factor
Activity
in Developing
Cells
Cultures of wild-type M. xanthus cells were plated on developmentinducing media (see Experimental Procedures) for defined periods. Cells were then harvested and lysed as described in Experimental Procedures, and cell-free fractions were tested for their ability to restore fruiting or sporulation to a csgA mutant as described in Figure 1.
1:24
1. C-Factor
.
nmoofaulooafth
132
Figure
2
Assay
for Development
Rescuing
Activity
(A) The scheme used to detect rescued development of csgA mutants is outlined. csgA responder cells (2.5 x 108) were resuspended in a developmental buffer (see Experimental Procedures) in a series of culture dish wells. After cells settle and firmly adhere to the bottom of the culture dish, cell extracts are added in a 2.fold dilution series. Responder cells contain a /acZ transcriptional fusion, which is expressed during development if C-factor is present. Activity is monitored during incubation for 3 days by scoring fruiting body formation, sporulation, and developmental p-galactosidase production. (B) Fruiting body rescue from a typical C-factor assay dilution series. Fruiting bodies are evident as dark punctate forms in 1:6 and 1:12 dilutions within a 44old concentration range of added cell fractions. Concentrations of extracts greater or less than this optimum showed little or no detectable rescuing activity. Fruiting bodies were scored after 3 days at 32%. Scale bar = 1 mm.
overall yield of 10%. The purification procedure is detailed elsewhere (Kim and Kaiser, 1990). C-factor is recovered as a single, sharp symmetric peak on the final step of ionexchange chromatography. Analysis of this material by SDS-PAGE and silver staining revealed a single species with a nominal molecular mass of 17 kd (Figure 3). This 17 kd species, when excised and eluted from a nondenaturing gel, retains the ability to rescue csgA fruiting body formation and sporulation. ldcntlflcation of C-Factor As a Product of the csgA Gene A number of observations were consistent
with the idea
that C-factor is encoded by the csgA gene. All known csg class mutations are found in the csgA gene (Shimkets et al., 1963). Wild-type cells produce C-factor activity, but csgA mutant cells do not, as described above. The deduced molecular mass of the primary translation product of the csgA gene, as determined by DNA sequence analysis (Hagen and Shimkets, 1990) is 17.7 kd, similar to the observed molecular weight of biologically active C-factor. Moreover, polyclonal antibodies raised against a /acZcsgA fusion protein produced in Escherichia coli and purified by binding to the fusion protein react with purified C-factor (Kim and Kaiser, 1990; L. Shimkets, personal communication). A partial amino acid sequence of C-factor was obtained to confirm that it is indeed the product of the csgA gene. The base sequence of the csgA gene predicts a unique lysine as residue 92 of a 166 residue csgA gene product (Figure 4; Hagen and Shimkets, 1990). In agreement with this, cleavage of purified C-factor with lysine-specific endoproteinase-Lys-C (Jekel et al., 1983) resulted in only two proteolytic products whose combined size was 17 kd. The two peptides were separated by gel electrophoresis and subjected to sequential Edman degradation (see Experimental Procedures). The smaller proteolytic fragment yielded the sequence, reading from its N-terminus, of AlaAla-LeuAsn-Met-Ala-Val-Arg-Ser-Met, which corresponds
Cell
22
a b c
. ..
96 67
VRAFATNVCTGPVDVLINNAGV
22
SGLWCALGDVDYADMARTFTIN
44
ALGPLRVTSAMLPGLRQGALRR
66
VAHVTSRMGSLAANTDGGAYAY
88
RMSFAAt
NMAVRSMSTDLRPEG
FVTVLLHPGWVQTDMGGPDATL
132
PAPDSVRGMLRVIDGLNPEHSG
154
RFFDYQGTEVPW
166
Figure 4. Amino Acid Sequence of C-Factor, a Product Gene, as Deduced from Nucleotide Sequence Analysis Gene Figure
3. C-Factor
Is a 17 kd Polypeptide
Fractions eluting near the activity peak in the final chromatographic step of C-factor purification were analyzed by SDS-PAGE and with silver staining. Lane a, purified C-factor that restores csgA fruiting body formation, sporulation, and geneexpression (see text). Lane b, inactive material etuling during the final chfomatographic step in the fraction immediately following elution of peak C-factor activity. Lane c, protein molecular weight standards. Minor bands of material of high molecular weight in lanes a and b arise from the sample buffer.
exactly with the amino acid sequence following the unique lysine residue predicted from the csgA gene sequence (Figure 4). An amino acid sequence from the other fragment generated by endoproteinase-Lys-C cleavage could not be obtained, suggesting that the N-terminus of C-factor is blocked. Thus, C-factor is encoded by the csgA gene, known to be necessary for proper fruiting body morphogenesis. C-Factor Reecue of Gene Expression Transcriptional fusions of lacZ to developmentally regulated genes in Myxococcus have been generated with the transposable promoter probe, TnS/ac (Kroos and Kaiser, 1984). P-galactosidase activity in these fusion strains measures expression of those particular transcription units and their constituent genes during development. A mutation in csgA does not affect expression of any IacZ fusion that begins to be expressed during the first 6 hr of development, but does reduce or abolish expression of all fusions normally expressed after 8 hr of development (Kroos and Kaiser, 1987). Both the normal time of increased expression and the proper levels of expression by these C-dependent /acZfusions are restored in mixtures of csgA mutant and wildtype cells (Figure 5). All five C-dependent /acZ fusions tested, -499, SWI14, Q4403, 04435, and SWlOl, were rescued by addition of wild-type cells, confirming the observations of Kroos and Kaiser (1987). These fusions include two whose fi-galactosidase expression is reduced by a csgA mutation (a4499 and Q4414) and three whose expression is abolished (Q4403, Q4435, and Q4401). Purified C-factor was added to each of these five csgA strains containing IacZ fusions, and the data in Figure 5 show that the level of l3-galactosidase expression eventually rose to wild-type levels in all five cases. This observa-
110
of the csgA of the csgA
Derived from Hagen and Shimkets (1990). Numbers at the right indicate positions in the amino acid sequence of residues at the end of each line. The unique lysine residue predicted in the sequence is marked by an asterisk. Amino acid residues in C-factor identified by C-factor purification and sequential Edman degradation are underlined. The single letter amino acid code is used.
tion argues that purified C-factor has the same activity as whole cells in respect to the control of expression of this set of /acZ fusions. The absence of C-factor activity in csgA mutants as tested by gene expression is illustrated in Figure 5f. In principle, the developmental stage at which the addition of rescuing cells restores the normal developmental process to a csgA mutant should be indicated by the pattern of j3-galactosidase expression in this set of /acZ transcriptional fusions, which in effect monitors the progress of a cell through its development. The fact that added whole wild-type cells restored expression by the earliest identified C-dependent fusion, m499, argues that development never arrests at the stage that would otherwise be blocked by the csgA mutation. Two experiments argue that purified C-factor also restores fruiting body development at or close to the point at which it is arrested due to mutation in csgA. First, purified C-factor eventually restores P-galactosidase expression by the earliest known C-dependent fusion, &X499, to the same level achieved during cell-cell rescue of 04499. The kinetics of rescued increase in P-galactosidase specific activity was not identical to wild type, however (Figure 5a; see Discussion below). Unlike the other four C-dependent /acZ fusions tested, insertion of Tnfjlac at a4414 disrupts the function of a developmentally essential gene (Kroos et al., 1990). This function appears to be required later than normal C-factor signaling because strains carrying TnSlac at 524414 arrest development as compact translucent mounds. Sporulation, however, only reaches 2% of wild-type sporulation (Table 1, last line). The developmental defect caused by at 04414 is cell autonomous; sporulation of an insertion strains containing this insertion cannot be restored by extracellular complementation (Table 1, line 4; compare lines 1,5, and 6). If, rather than allowing bypass to a terminal stage, external C-factor restores normal development to a csgA mutant at the point blocked by the csgA muta-
C-Factor 23
Is Required
0
for M. xanthus
20
t
40
Differentiation
60
20 da 60 Hours of Development
6
d
20 .a 69 Hours of Development Figure
5. C-Factor
Rescues
Developmental
Gene
Expression
in csgA
Mutants
csgA strains containing TnBlac insertions were conditioned on developmental media and harvested at various times for determination of b-galactosidase specific activity (nmol of o-nitrophenol per minute per milligram of protein) as described in Experimental Procedures. csgA strains mixed with an equal number of wild-type strain DK1622 (solid diamonds) and csgA strains (open squares) containing Tntilac insertions M499 (a), 04414 (b), MO3 (c), &I401 (d), and 04435 (e) illustrate the effects of the csgA mutation on developmentally regulated expression of &galactosidase. DK1622, having no /acZ fusion, does not express P-galactosidase. Addition of 2 U of C-factor to csgA strains (solid squares) restores developmental @galactosidase expression to the proper wild-type levels. (f) Illustrates the comparative inability of csgA cell fractions to rescue developmental gene expression; in this case, extracts made at different times from developing csgA source cells fail to rescue expression from TnSlac Q4435. Results summarize data from at least two independent sets of experiments.
tion, then C-factor addition to a csgA mutant carrying a4414 should rescue P-galactosidase production from the fusion and aggregation, but should not bypass the sporulation defect caused by the a4414 insertion. As expected, C-factor addition to a csgA 04414 double mutant restores both aggregation (data not shown) and P-galactosidase production (Figure 5b). C-factor addition to this double mutant brings sporulation to about the level produced in strain DK5279, which contains the a4414 insertion but is csgA+ (Table 1, lines 4 and 7). This observation, together with restoration of all other C-dependent gene expression, suggests that external C-factor allows development to pass through what would otherwise be the developmental block in a csgA mutant.
Discussion Rescue of certain admixed wild-type
classes of developmental mutants by cells has led to the proposal that M.
xanthus passes essential signal molecules from cell to cell to coordinate formation of its multicellular fruiting body. If the proposal is correct, then it should be possible to isolate the molecules involved in each specific signaling event. A 17 kd polypeptide has been isolated from csgA+ cells by means of its ability to restore proper development to csgA mutants. csgA mutants fail to form proper multicellular aggregates, to differentiate from rod-shaped cells to environmentally resistant spores, and to express development-specific genes that are detected by transcriptional fusions to /acZ. Purified C-factor can restore each of these aspects of development that are defective in csgA mutants. Partial amino acid sequencing of C-factor shows that it is a product of the csgA gene. These data support the proposal that exchange of external C-factor between cells is a necessary part of normal fruiting body development.
Based on a 2-fold limiting dilution assay, 1 U of C-factor
is defined as the minimal amount that restores csgA mutant sporulation and aggregation to wild-type levels. One unit of purified C-factor corresponds to about 24 ng of purified protein. From this we calculate that aggregating wildtype cells should contain at least 9000 molecules of C-factor per cell when harvested at 18 hr of development. Similarly, it appears that about 8000 molecules of C-factor are required per cell to rescue c.sgA fruiting body development. Given a molecular mass of 17 kd, this implies that C-factor functions at a concentration of approximately 1 to 2 x 10dg M. This concentration is typical of the low effective concentrations measured for other small morphogenetic signals, including retinoic acid (Thaller and Eichele, 1987), Dictyostelium differentiation-inducing factor (Kay et al., 1983), and Hydra head activator peptide (Schaller and Bodenmiiller, 1981). Earlier studies have revealed that, under certain conditions, millimolar amounts of purified components of M. xanthus peptidoglycan (Shimkets and Kaiser, 1982) and development-stimulating factor, a low molecular weight, partially purified glycoconjugate (Janssen and Dworkin, 1985), can restore aggregation and sporulation to mutant strains carrying a csgA mutation. However, peptidoglycan components and development-stimulating factor are present in CsgA cells and therefore may provide a metabolic bypass of the normal signaling event, instead of supplying the signal itself. In contrast, C-factor was found only in developing wild-type cells; cells that are either csgA or are growing fail to produce C-factor activity (Figure 2). The biological activity of peptidoglycan and development-stimulating factor, both derived from the cell envelope, suggest that one outcome of C-factor signaling may be altered envelope metabolism. Our inability to reconstitute rippling of csgA mutants with purified C-factor may have several explanations. For example, rippling may require a separate signal. A more intriguing possibility is that the synchronous, pulsatile, wave-like cell movements in rippling require local oscillation of C-signal, a requirement probably not fulfilled by our method of adding C-factor in a single step (see Experimental Procedures). A temporal or spatial requirement for C-factor addition may also explain why rescue of b-galactosidase expression by C-factor is delayed relative to rescue by whole cells. CAMP chemotaxis in Dictyostelium provides a well-characterized precedent for an oscillatory signal that triggers coordinated cell movement (Devreotes, 1983). Evidence for a signal relay system involving C-factor arises from the observation that cell motility is required for the csgA-mediated cell interaction (Kroos et al., 1988). Nonmotile mutants arrest their development at a stage similar, if not identical, to csgA mutants. Kroos et al. (1988) hypothesized that if, in turn, C-signaling caused cells to move, then a positive feedback loop would be created (movement, C-signaling, movement, etc.), which in theory could produce a signal oscillation manifest as ripples. The feedback loop would be broken by loss of motility or by csgA mutation, and, indeed, both functions have been shown to be required for rippling (Shimkets and Kaiser, 1982; Shimkets et al., 1983). To the extent that restored
aggregation implies cell movement, our finding that purified C-factor allows csgA cells to build aggregation centers that become normal fruiting bodies provides experimental support for the proposal that C-factor can induce cell movement. The isolation of C-factor and the demonstration of its activity in development raises the question of how it functions. Although it remains to be demonstrated directly, our observations support the proposal that C-factor is transferred from cell to cell. Exogenous, solubilized C-factor restores csgA mutant cell development, suggesting that C-factor functions at the cell surface. The apparent tight association of C-factor with membrane suggests that intercellular signaling between wild-type cells might occur only over a very short range, perhaps restricted to immediately contiguous cells. It seems likely that C-factor acts as a paracrine signal that must bind to a receptor on the external surface of a cell. Given a procedure to isolate biologically active C-factor, it should be possible to investigate its production, transmission, and reception. Experimental Materials
Procedures and Bacterial
Strains
MOPS (3[N-morpholino]propanesulfonate), kanamycinsulfate,CHAPS, o-nitrophenolgalactoside, and gel electrophoresis standards were from Sigma. Dialysis tubing (8000 dalton nominal cutoff) was from Spectrum. Lysobacter enzymogenes endoproteinase-Lys-C was from Boehringer Mannheim. All M. xanthus strains used in this study were derived from DK1822 (wild type). Sites of TnBlac transposon insertions are designated by the Greek letter 61 followed by a four digit numeral (Kroos et al., 1988). C-factor was prepared from DK5204, a kanamycin-resistant (Km’), developmentally competent strain that contains TmYac at position 04435. The csgA mutation used to construct csgA Tn5lac derivatives is created by insertion of Tn5-132 into the csgA gene at site QLS205 (Shimkets and Asher, 1988). This transposon insertion contains the tetracycline resistance (Tc’) element from TnlO (Foster et al., 1981), which allowS introduction of the csgA mutation into Km’ TnBlac strains by generalized transduction and selection for Tc’ as described in Kroos and Kaiser (1987). In this way, strains that were csgA and contained the Tn5lac insertions R4499 (DK5246). 514414 (DK5287), 04403 (DK5270), Q4435 (DK5253), and Q4401 (DK5229) were constructed (Kroos and Kaiser, 1987). Source of C-Factor Cells growing exponentially in casitone-Tris liquid medium (Hodgkin and Kaiser, 1977) were sedimented at 10,000 x g for 10 min at 5% and suspended in TPM buffer (10 mM Tris-HCI, 1 mM K2HP04.KH2PO4, 8 mM MgSOd [pH 7.51) at a density of 5 x log cells per ml. Aliquots (-50 ~1) were spotted on TPM agar (TPM plus 1.5% agar) in 25 x 25 cm tissue culture plates (Nunc) and incubated at 32% for 18 hr when C-factor activity is most abundantly recovered. Agar. rather than submerged culture, was chosen for conditioning cells because the yield of cells per cm2 was greater on agar than on plastic. At 18 hr of development on TPM agar, cells have aggregated but have not formed spores. Cells were scraped with a razor blade into MC7 buffer (10 mM MOPS, 1 mM CaC12 (pH 7.01) at a calculated concentration of 1.5 x lOlo cells per ml. This suspension was homogenized by vortex mixing, flash-frozen in liquid nitrogen, and stored at -80°C. C-Factor Assay C-factor was assayed by its ability to restore fruiting body formation and sporulation to csgA mutant cells. The procedure is based on the assay for A-factor described by Kuspa et al. (1988), but modified to provide a solid surface for aggregation and to permit counting of fruiting bodies and spores. The csgA strain DK5253 was routinely used. Each sample of C-factor to be assayed was dialyzed against 4 I of 10 mM MOPS, 1 mM CaCl*, 4 mM MgClp, 50 mM NaCl (pH 7.2) (buffer A) for
C-Factor 25
Is Required
for M. xanthus
Differentiation
12-18 hr at 4%. Aliquots of the dialyzed samples were serially diluted in P-fold steps, typically through 6 to 8 such steps; 400 PI of each dilution was warmed to 32%. then added to responder csgA cells that had been developing in submerged culture as illustrated in Figure 1. When exponentially growing M. xanthus is resuspended in microtiter wells containing nutrient-free buffer and Ca*+, the cells settle to the bottom of the well and form an adherent mat as described by Kuner and Kaiser (1962). During normal development, fruiting body morphogenesis, sporulation, and gene expression occur over the next 72 hr. In the C-factor assay, exponentially growing csgA assay cells were centrifuged at 10,000 x g for 10 min and resuspended in buffer A at a concentration of 5 x lb cells per ml; 400 ~1 of this suspension was transferred into the wells of a 24 well microtiter dish (Falcon) and incubated at 32% for 6 hr in a humid chamber. At this time, when the morphological defects of csgA mutants are first manifest, the buffer A overlying the confluent mat of adherent cells is gently removed, and prewarmed fractions to be assayed are added. One unit of activity (Figure 1) is defined as the amount of C-factor that restores wild-type level fruiting body formation (200-300 per 2.5 x 10s input cells) and sporulation (2.5 x 106 per 2.5 x lo* input cells) to csgA mutants developing in submerged culture. Other Methods C-factor purification was analyzed by SDS-PAGE using 15% acrylamide and 0.12% bisacrylamide (Laemmli, 1970; Pfeffer, 1987). Silver staining was performed by the method of Merril et al. (1981). except that 0.004% KMnO, was substituted for K&&O,. Measurement of developmental 6galactosidase expression was performed as described by Kmos and Kaiser (1987). One unit of &galactosidase specific activity is equal to 1 nmol of o-nitrophenol produced per minute per milligram of protein. Heat-resistant. sonication-resistant, Km’ spores were quantified as described by Kroos et al. (1966). Fruiting bodies were scored visually at 6x magnification using a dissecting microscope (Wild-Heerbrug). The presence of ovoid, refractile spores within fruiting bodies at the bottom of a microtiter well was confirmed with a Leitz inverted light microscope using a 40x objective. In preparation for microsequencing. purified C-factor was treated with endoproteinase Lys-C, which specifically cleaves polypeptides on the carboxy side of lysine residues (Jekel et al., 1963). Endoproteinase Lys-C was mixed with substrate at a ratio of 1:100 in 150 mM Tiis-HCI (pH 8.7) for 4 hr a1 37%. A second aliquot of protease equal to the initial amount was then added and the mixture allowed to stand at 37% for 16 hr. Purification of protein for amino acid sequence analysis was performed by the method of Matsudaira (1987). Samples were analyzed by the Protein Structure Laboratory, University of California at Davis School of Medicine. Acknowledgments We thank Drs. L. Kroos. A. Kuspa, B. Glick, H. Kaplan, and K. Irvine and J. Rodriguez for helpful discussions. We especially thank Drs. L. Shimkets and T Hagen for their critical discussion, for providing affinity-purified antibodies tocsgA fusion protein, and for communicating their csgA sequence data prior to its publication. We thank C. Tate, R. de la Cruz, N. Malimban, J. Vizyak, and B. Scott for excellent technical assistance and J. Gardner, A. Smith, and the Protein Structure Laboratory at the University of California, Davis for protein sequence determinations. This investigation was supported by National Institutes of Health research grant GM23441 to D. K. and stipend to S. K. K. by training grant GMO7365, both from the National Institute of General Medical Sciences. S. K. K. is a trainee of the Medical Scientist Training Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
April 28, 1989; revised
December
29, 1969.
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nucleotides and cell-cell communicaIn Advances in Cyclic Nucleotide Re.
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Doe, C. Q., and Goodman, C. S. (1965). Early events in insect neurogenesis. II. The role of cell interactions and cell lineage in the determination of neuronal precursor cells. Dev. Biol. 771, 206-219. Ferguson, E. L., Sternberg. P W., and Horvitz, H. R. (1987). A genetic pathway for the specification of the vulva1 cell lineages of Ceenorhebditis e&ens. Nature 326, 259-267. Foster. T. J., Davis, M. A., Roberts, D. E., Takeshita, K., and Kleckner, N. (1961). Genetic organization of transposon TnlO. Cell 23, 201-213. Gill, R. E., and Cull, M. G. (1966). Control of developmental gene expression by cell-to-cell interactions in Myxococcus xanthus. J. Bacteriol. 168, 341-347. Hagen, D. C., Bretscher, A. I?, and Kaiser, D. (1976). Synergism between morphogenetic mutants of Myxococcus xanthus. Dev. Biol. 64, 284-296. Hagen, T., and Shimkets, L. J. (1990). transcriptional products of the csg locus Bacterial. 172, 15-23.
Nucleotide sequence and of Myxncoccus xanthus. J.
Hodgkin, J., and Kaiser, D. (1977). Cell to cell stimulation in nonmotile mutants of Myxococcus. Proc. Natl. Acad. 2936-2942.
of movement Sci. USA 74,
Janssen, G. R., and Dworkin, M. (1965). Cell-cell interactions in developmental lysis of Myxococcus xanthus. Dev. Biol. 712, 194-202. Jekel, F! A., Wither, J. W., and Beintema, J. J. (1963). Use of endoproteinase Lys-C from Lyso6acfef enzymogenes in protein sequence analysis. Anal. B&hem. 134, 347-354. Kay, R. R., Dhokia, B., and Jermyn, K. A. (1963). Purification of stalkcell-inducing morphogens from Dictyostelium disco&urn. Eur. J. Biothem. 736, 51-56. Kim, S. K.. and Kaiser, D. (1990). Purification ococcus xanthus C-factor, an intercellular Natl. Acad. Sci. USA, in press.
and properties of Myxsignaling protein. Proc.
Kroos, L., and Kaiser, D. (1984). Construction of TnSlac, a transposon that fuses lecZ expression to exogenous promoters, and its introduction into Myxococcus xanthus. Proc. Natl. Acad. Sci. USA 81, 58165820. Kroos, L., and Kaiser, D. (1987). Expression of many developmentally regulated genes in Myxococcus depends on a sequence of cell interactions. Genes Dev. 7, 840-854. Kroos, L., Kuspa, A., and Kaiser, D. (1966). velopmentally regulated genes in Myxococcus 252-266.
A global analysis of dexanfttus. Dev. Biol. 717,
Kroos, L., Hartzell, t?, Stephens, K., and Kaiser, D. (1988). A link between cell movement and gene expression argues that motility is required for cell-cell signaling during fruiting body formation. Genes Dev. 2, 1677-1685. Kroos, L., Kuspa, A., and Kaiser, D. (1990). Defects in fruiting body development caused by Tn5lac insertions in Myxococcus xanthus. J. Bacteriol. 172, 484-487. Kuner, J. M., and Kaiser, D. (1982). submerged cultures of Myxococcus 461.
Fruiting body morphogenesis in xanthus. J. Bacterial. 757. 4513-
Kuspa, A., Kroos, L., and Kaiser, D. (1986). Intercellular signalling is required for developmental gene expression in Myxococcus xanthus. Dev. Biol. 177, 267-276. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 660-685. LaRossa, R.. Kuner. J., Hagen, D., Manoil, C., and Kaiser, D. (1983). Developmental cell interactions of Myxococcus xanthus. analysis of mutants. J. Bacterial. 153, 1394-1404. Matsudaira, I? (1967). Sequence elect&lotted onto polyvinylidene 262, 10035-10038.
from picomole quantities of proteins difluoride membranes. J. Biil. Chem.
Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1981). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437-1436. Pfeffer,
S. R. (1987).
The
endosomal
concentration
of a mannose
Bphosphate receptor is unchanged J. Cell Biol. 705, 229-234.
in the absence
of ligand synthesis.
Reichenbach, H. (1965). Rhythmische vorgange bei der schwarmentfaltung von myxobakterien. Berichte der deutschen Botanischen Gesellschaft 78, 102-105. Rosenberg, E., ed. (1984). Myxobacteria: actions (New York: Springer-Verlag).
Development
and Cell Inter-
Schaller, H. C., and Bodenmiiller, H. (1981). Isolation and amino acid sequence of a morphogenetic peptide from hydra. Proc. Natl. Acad. Sci. USA 78, 7000-7004. Shimkets, L. J., and Asher, S. J. (1988). Use of recombination niques to examine the structure of the csg locus of Myxococcus thus. Mol. Gen. Genet. 211, 83-71.
tech-
xan-
Shimkets, L. J., and Dworkin, M (1981). Excreted adenosine is a cell density signal for the initiation of fruiting body formation in Myxococcus xanthus. Dev. Biol. 84, 51-80. Shimkets. L. J., and Kaiser, D. (1982). developmental sporulation of Myxococcus 462-470.
Murein components rescue xanthus. J. Bacterial. 752.
Shimkets, L. J., Gill, Pt. E., and Kaiser, D. (1983). Developmental interactions in Myxococcus xanthus and the spoC locus. Proc. Acad. Sci. USA 80, 1406-1410.
cell Natl.
Smith, J. C. (1987). A mesoderm-inducing Xenopus cell line. Development 99, 3-14.
by a
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Thaller, C., and Eichele, G. (1987). Identification and spatial distribution of retinoids in the developing chick limb bud. Nature 327, 625-628. Wireman, J. W., and Dworkin, M (1975). Morphogenesis and developmental interactions in myxobacteria. Science 189, 516-523.
April 6, 1990, Copyright
0 1990 by Cell Press
C-Factor: A Cell-Cell Signaling Protein Required for Fruiting Body Morphogenesis of M. xanthus Seung K. Kim and Dale Kaiser Department of Biochemistry and Department of Developmental Biology Stanford University School of Medicine Stanford, California 94305
Summary During fruitfng body development, the product of the csgA gene lo n eceeeary for cellular aggregation, for spore dffferentlatlon, and for gene expreeeion that is initiated after 6 hr of atawation. From nascent wildtype fruiting bodies we have purified a polypeptide of 17 kd caffed C-factor, whkh, at approximately 1 to 2 nM, restores normal development to csgA mutant cells. C-factor activity is not recovered from extracts of unstarved, growing cells or csgA mutant cells. The amino acid sequence from purified C-factor demonatrates that it is the product of the csgA gene. C-factor is activeoveranamwrangeofconcentret ion and has properties of a morphogenetic paracrine signal. Introduction Cell interactions are necessary to establish differentiated cell fates in the development of multicellular organisms. Well-studied examples include CAMP relay in Dictyostelium (Devreotes, 1983) vulva1 development in Caenorhabditis elegans (Perguson et al., 1987), insect neurogenesis (Doe and Goodman, 1985), and vertebrate mesoderm induction (Smith, 1987). Cell interactions also appear to govern multicellular development and cellular differentiation of the Gram-negative bacterium Myxococcus xanthus, an organism whose prokaryotic genetic system and cellular organization offer benefits of experimental accessibility (Rosenberg, 1984). Following starvation at high cell density on a solid surface, rod-shaped M. xanthus cells glide to aggregation centers where they build a structure of family-specific shape, called a fruiting body. Early in aggregation, ridgelike accumulations of cells move coordinately and rhythmically over the substrate like ripples on a water surface (Reichenbach, 1965; Shimkets and Kaiser, 1982). Some cells lyse, while other cells within a nascent fruiting body differentiate to dormant, ovoid spores (Wireman and Dworkin, 1975). Roughly lo5 cells participate in constructing a fruiting body, and the process requires about 20 hr from the removal of nutrient to the beginning of sporulation. Environmentally resistant spores mature over the next few days. Control of M. xanthus fruiting body development by several sequential cell-cell interactions is implied by the existence of four classes of developmental mutants that are not cell autonomous (Hagen et al., 1978; Shimkets and Dworkin, 1981; LaRossa et al., 1983; Janssen and Dwor-
kin, 1985; Kroos and Kaiser, 1987). Mutants of the asg, bsg, csg, and dsg (a signal, b signal, c signal, and d signal, respectively) classes cannot sporulate alone, but can be rescued for sporulation by codevelopment with wildtype cells or with cells of another mutant class. The rescue does not involve genetic exchange but rather occurs extracellularly. Each cell interaction mutant class modifies the pattern of developmentally regulated gene expression in a different way (Kuspa et al., 1988; Kroos and Kaiser, 1987). The patterns have been revealed using a transposable promoter probe, Tn5lac (Kroos and Kaiser, 1984) which can generate transcriptional fusions of lat.? to developmentally regulated genes. Assay of &galactosidase activity in such fusion strains indicates that gene expression during development, which is disrupted in asg, bsg, and csg mutants, is rescued by codevelopment with wildtype cells (Kuspa et al., 1986; Gill and Cull, 1986; Kroos and Kaiser, 1987). The csg class of developmental mutations is the subject of this paper. All existing members of this class have resulted from mutation at a single genetic locus named csgA (Shimkets et al., 1983; Shimkets and Asher, 1988). Unlike wild-type cells, which aggregate into compact translucent mounds by 12 hr on solid agar substrate, csgA mutants aggregate only after 18 hr and then into larger, less compact mounds and ridges than csgA+ cells. Under conditions of submerged culture (Kuner and Kaiser, 1982; see Experimental Procedures), the aggregation defect of csgA mutants is even more severe, and csgA cells fail to construct any stable multicellular structures. csgA mutants also fail to ripple, to lyse, or to sporulate (Shimkets and Kaiser, 1982). A mutation in csgA does not alter the expression of /acZ transcriptional fusion strains that produce P-galactosidase during the first 8 hr of development (C-independent fusions), but does reduce or abolish 6-galactosidase synthesis from /acZ transcriptional fusion strains that normally express after 6 hr (C-dependent fusions; Kroos and Kaiser, 1987). Thus, the csgA product may be involved in production or transmission of a developmental signal that is crucial for normal fruiting body morphogenesis, cellular differentiation, and developmentally regulated gene expression. The csgA gene has been cloned (Shimkets et al., 1983; Shimkets and Asher, 1988) and sequenced (Hagen and Shimkets, 1990). Based on codon usage, the best possible reading frame indicates that csgA could specify a 17.7 kd protein (Hagen and Shimkets, 1990). Does csgA specify the signal molecule itself or some other essential component of the signaling system? To identify the csg signal molecule(s), we have constructed a bioassay based on the completion of development by a csgA mutant. We report the characterization of C-factor, a polypeptide of approximately 17 kd that has been extracted from immature fruiting bodies and that rescues development of csgA mutants. Its amino acid sequence analysis demonstrates that C-factor is encoded by the csgA gene.
Cell 20
Table
1. C-Factor
Rescues
Developmental
Sporulation
of csgA
Unsupplemented Spores
Plus C-Factor
Strain
Genotype
DK1622
Wild-type
DK5246
csgA,
D4499
<5
x 102
<0.02
DK5253
csgA,
R4435
<5
x 102
DK5267
csgA,
94414
<5
x 102
DK4499
G4499
2.5 x lo6
100
DK5204
a4435
2.5
x 10s
100
DK5279
a4414
5.0 x 104
2
2.5
per Milliliter x 106
Mutants
% Wild-Type
Spores
per Milliliter
Plus DK1622
Cells
% Wild-Type
Spores
per Milliliter
% Wild-Type
2.5 x IO6
100
3 x 106
120
<0.02
3.0 x 10s
120
3 x 106
120
<0.02
1 x 105
5 x 103
2
(100)
4
Two units of purified C-factor were added to the responder strains indicated. Sporulation tests were performed as described in Experimental Procedures. In extracellular complementation tests, approximately equal numbers of wild-type strain DKl622 and mutant cells were mixed and plated for development.
Results Production and Assay of C-Factor Wild-type ceils mixed with csgA mutant cells in equal proportion rescue fruiting body morphogenesis and sporulation of these mutants to wild-type levels (Table 1, lines 1, 2, and 3; Hagen et al., 1978; LaRossa et al., 1983). Developmental gene expression in the csgA mutant cells, as monitored by a panel of /acZ transcriptional fusions, is also restored to an approximately wild-type pattern in such cell mixtures (Kroos and Kaiser, 1987; present study). A simple interpretation of these results is that wild-type cells provide an intercellular signal molecule that is missing from the csgA mutants. If so, csgA mutant cells could be used to monitor purification of signal molecules from csgA+ cells. Several experiments suggested that csgA-rescuing activity is tightly cell associated. Wild-type source cells, if separated from responder csgA cells by a 0.45 urn nucleopore membrane filter, failed to rescue mutant cells (data not shown). Extracts made by extensive washings of wildtype cells at various developmental stages also had no effect on csgA responder cells. However, cell-free extracts made from lysed M. xanthus cells were found to restore the ability of csgA mutants to form fruiting bodies and spores and, as shown below, to express C-dependent genes. Ripples, however, were not detected. A bioassay that measures development-rescuing activity, or C-factor, is diagrammed in Figure 1A. Cell-free extracts of wild-type M. xanthus in a series of P-fold dilutions were incubated with csgA cells prepared for development (see Experimental Procedures). The choice of a limiting P-fold dilution assay proved critical to initially detecting C-factor activity because undiluted extracts were inactive (Figure 16). As purification progressed and specific activity rose, activity was consistently detected only within an approximately 4-fold concentration range (Figure 16). Another characteristic of the assay is that fruiting bodies, spores, and C-dependent gene expression (see below) are first detected about 40 hr after nutrient deprivation initiates development-about 24 hr later than seen in wildtype development.
As judged either by restoration of csgA cell aggregate formation or sporulation, C-factor activity is only produced by wild-type cells during the interval between 12 and 18 hr poststarvation (Figure 2). At this time, source cells have formed compact mounds but have not yet begun to differentiate into spores. Developing source cells harvested before 12 hr or after 18 hr showed little or no rescuing activity. Extracts made from csgA mutant cells harvested at intervals between 0 and 48 hr poststarvation and assayed over a 500~fold concentration range failed to rescue fruiting body or spore formation of csgA mutants. Wild-type cells developing on starvation agar medium for 18 hr were chosen as the standard source for further purification and characterization of C-factor. Purification of C-Factor When unfractionated cell lysates containing C-factor activity were sedimented at 100,000 x g for 1 hr, activity was found only in the pelleting material. Crucial to the purification of C-factor was the finding that the zwitterionic detergent 3-([3-cholamidopropyI]dimethylammonio)-l-propanesulfonate (CHAPS) solubilizes C-factor while maintaining it in a biologically active form. Cholate, deoxycholate, and octylglucoside, detergents with a critical micelle concentration similar to CHAPS, failed to solubilize active material over a wide range of concentrations (S. K. K., unpublished data). The limiting 2-fold dilution assay, shown in Figure 1, was used to follow C-factor activity during fractionation of CHAPS-solubilized cell extracts, and progress in purification was monitored by SDS-polyacrylamide gel etectrophoresis (SDS-PAGE). Following a P-fold enrichment of activity by ammonium sulfate fractionation, homogeneous C-factor was obtained after anion-exchange chromatography, gel filtration, and a final anion-exchange step. The ability of purified material to restore sporulation is demonstrated in the column marked “Plus C-Factor” in Table 1. Activity that rescues sporulation, fruiting body formation, and, as shown below, C-dependent gene expression copurified at each step, indicating that the same molecule possesses all these activities. The final degree of purification is over 1000~fold with an
r$Factor
Is Required
for M. xanthus
C-Factor
A
Differentiation
Assay
C-Factor Souro (Cells, cxtrrcts)
1 48 tws.
32’
Fruiting bodies Spr,S
0
2
.
8
8
1012141(1182022242(12830
G-Galactosidasc
B
The assay for C factor activity is a limiting two-fold dilution series
10’
1:6
0
Figure
8
8
1012141518202224262830
2. Time Course
of C-Factor
Activity
in Developing
Cells
Cultures of wild-type M. xanthus cells were plated on developmentinducing media (see Experimental Procedures) for defined periods. Cells were then harvested and lysed as described in Experimental Procedures, and cell-free fractions were tested for their ability to restore fruiting or sporulation to a csgA mutant as described in Figure 1.
1:24
1. C-Factor
.
nmoofaulooafth
132
Figure
2
Assay
for Development
Rescuing
Activity
(A) The scheme used to detect rescued development of csgA mutants is outlined. csgA responder cells (2.5 x 108) were resuspended in a developmental buffer (see Experimental Procedures) in a series of culture dish wells. After cells settle and firmly adhere to the bottom of the culture dish, cell extracts are added in a 2.fold dilution series. Responder cells contain a /acZ transcriptional fusion, which is expressed during development if C-factor is present. Activity is monitored during incubation for 3 days by scoring fruiting body formation, sporulation, and developmental p-galactosidase production. (B) Fruiting body rescue from a typical C-factor assay dilution series. Fruiting bodies are evident as dark punctate forms in 1:6 and 1:12 dilutions within a 44old concentration range of added cell fractions. Concentrations of extracts greater or less than this optimum showed little or no detectable rescuing activity. Fruiting bodies were scored after 3 days at 32%. Scale bar = 1 mm.
overall yield of 10%. The purification procedure is detailed elsewhere (Kim and Kaiser, 1990). C-factor is recovered as a single, sharp symmetric peak on the final step of ionexchange chromatography. Analysis of this material by SDS-PAGE and silver staining revealed a single species with a nominal molecular mass of 17 kd (Figure 3). This 17 kd species, when excised and eluted from a nondenaturing gel, retains the ability to rescue csgA fruiting body formation and sporulation. ldcntlflcation of C-Factor As a Product of the csgA Gene A number of observations were consistent
with the idea
that C-factor is encoded by the csgA gene. All known csg class mutations are found in the csgA gene (Shimkets et al., 1963). Wild-type cells produce C-factor activity, but csgA mutant cells do not, as described above. The deduced molecular mass of the primary translation product of the csgA gene, as determined by DNA sequence analysis (Hagen and Shimkets, 1990) is 17.7 kd, similar to the observed molecular weight of biologically active C-factor. Moreover, polyclonal antibodies raised against a /acZcsgA fusion protein produced in Escherichia coli and purified by binding to the fusion protein react with purified C-factor (Kim and Kaiser, 1990; L. Shimkets, personal communication). A partial amino acid sequence of C-factor was obtained to confirm that it is indeed the product of the csgA gene. The base sequence of the csgA gene predicts a unique lysine as residue 92 of a 166 residue csgA gene product (Figure 4; Hagen and Shimkets, 1990). In agreement with this, cleavage of purified C-factor with lysine-specific endoproteinase-Lys-C (Jekel et al., 1983) resulted in only two proteolytic products whose combined size was 17 kd. The two peptides were separated by gel electrophoresis and subjected to sequential Edman degradation (see Experimental Procedures). The smaller proteolytic fragment yielded the sequence, reading from its N-terminus, of AlaAla-LeuAsn-Met-Ala-Val-Arg-Ser-Met, which corresponds
Cell
22
a b c
. ..
96 67
VRAFATNVCTGPVDVLINNAGV
22
SGLWCALGDVDYADMARTFTIN
44
ALGPLRVTSAMLPGLRQGALRR
66
VAHVTSRMGSLAANTDGGAYAY
88
RMSFAAt
NMAVRSMSTDLRPEG
FVTVLLHPGWVQTDMGGPDATL
132
PAPDSVRGMLRVIDGLNPEHSG
154
RFFDYQGTEVPW
166
Figure 4. Amino Acid Sequence of C-Factor, a Product Gene, as Deduced from Nucleotide Sequence Analysis Gene Figure
3. C-Factor
Is a 17 kd Polypeptide
Fractions eluting near the activity peak in the final chromatographic step of C-factor purification were analyzed by SDS-PAGE and with silver staining. Lane a, purified C-factor that restores csgA fruiting body formation, sporulation, and geneexpression (see text). Lane b, inactive material etuling during the final chfomatographic step in the fraction immediately following elution of peak C-factor activity. Lane c, protein molecular weight standards. Minor bands of material of high molecular weight in lanes a and b arise from the sample buffer.
exactly with the amino acid sequence following the unique lysine residue predicted from the csgA gene sequence (Figure 4). An amino acid sequence from the other fragment generated by endoproteinase-Lys-C cleavage could not be obtained, suggesting that the N-terminus of C-factor is blocked. Thus, C-factor is encoded by the csgA gene, known to be necessary for proper fruiting body morphogenesis. C-Factor Reecue of Gene Expression Transcriptional fusions of lacZ to developmentally regulated genes in Myxococcus have been generated with the transposable promoter probe, TnS/ac (Kroos and Kaiser, 1984). P-galactosidase activity in these fusion strains measures expression of those particular transcription units and their constituent genes during development. A mutation in csgA does not affect expression of any IacZ fusion that begins to be expressed during the first 6 hr of development, but does reduce or abolish expression of all fusions normally expressed after 8 hr of development (Kroos and Kaiser, 1987). Both the normal time of increased expression and the proper levels of expression by these C-dependent /acZfusions are restored in mixtures of csgA mutant and wildtype cells (Figure 5). All five C-dependent /acZ fusions tested, -499, SWI14, Q4403, 04435, and SWlOl, were rescued by addition of wild-type cells, confirming the observations of Kroos and Kaiser (1987). These fusions include two whose fi-galactosidase expression is reduced by a csgA mutation (a4499 and Q4414) and three whose expression is abolished (Q4403, Q4435, and Q4401). Purified C-factor was added to each of these five csgA strains containing IacZ fusions, and the data in Figure 5 show that the level of l3-galactosidase expression eventually rose to wild-type levels in all five cases. This observa-
110
of the csgA of the csgA
Derived from Hagen and Shimkets (1990). Numbers at the right indicate positions in the amino acid sequence of residues at the end of each line. The unique lysine residue predicted in the sequence is marked by an asterisk. Amino acid residues in C-factor identified by C-factor purification and sequential Edman degradation are underlined. The single letter amino acid code is used.
tion argues that purified C-factor has the same activity as whole cells in respect to the control of expression of this set of /acZ fusions. The absence of C-factor activity in csgA mutants as tested by gene expression is illustrated in Figure 5f. In principle, the developmental stage at which the addition of rescuing cells restores the normal developmental process to a csgA mutant should be indicated by the pattern of j3-galactosidase expression in this set of /acZ transcriptional fusions, which in effect monitors the progress of a cell through its development. The fact that added whole wild-type cells restored expression by the earliest identified C-dependent fusion, m499, argues that development never arrests at the stage that would otherwise be blocked by the csgA mutation. Two experiments argue that purified C-factor also restores fruiting body development at or close to the point at which it is arrested due to mutation in csgA. First, purified C-factor eventually restores P-galactosidase expression by the earliest known C-dependent fusion, &X499, to the same level achieved during cell-cell rescue of 04499. The kinetics of rescued increase in P-galactosidase specific activity was not identical to wild type, however (Figure 5a; see Discussion below). Unlike the other four C-dependent /acZ fusions tested, insertion of Tnfjlac at a4414 disrupts the function of a developmentally essential gene (Kroos et al., 1990). This function appears to be required later than normal C-factor signaling because strains carrying TnSlac at 524414 arrest development as compact translucent mounds. Sporulation, however, only reaches 2% of wild-type sporulation (Table 1, last line). The developmental defect caused by at 04414 is cell autonomous; sporulation of an insertion strains containing this insertion cannot be restored by extracellular complementation (Table 1, line 4; compare lines 1,5, and 6). If, rather than allowing bypass to a terminal stage, external C-factor restores normal development to a csgA mutant at the point blocked by the csgA muta-
C-Factor 23
Is Required
0
for M. xanthus
20
t
40
Differentiation
60
20 da 60 Hours of Development
6
d
20 .a 69 Hours of Development Figure
5. C-Factor
Rescues
Developmental
Gene
Expression
in csgA
Mutants
csgA strains containing TnBlac insertions were conditioned on developmental media and harvested at various times for determination of b-galactosidase specific activity (nmol of o-nitrophenol per minute per milligram of protein) as described in Experimental Procedures. csgA strains mixed with an equal number of wild-type strain DK1622 (solid diamonds) and csgA strains (open squares) containing Tntilac insertions M499 (a), 04414 (b), MO3 (c), &I401 (d), and 04435 (e) illustrate the effects of the csgA mutation on developmentally regulated expression of &galactosidase. DK1622, having no /acZ fusion, does not express P-galactosidase. Addition of 2 U of C-factor to csgA strains (solid squares) restores developmental @galactosidase expression to the proper wild-type levels. (f) Illustrates the comparative inability of csgA cell fractions to rescue developmental gene expression; in this case, extracts made at different times from developing csgA source cells fail to rescue expression from TnSlac Q4435. Results summarize data from at least two independent sets of experiments.
tion, then C-factor addition to a csgA mutant carrying a4414 should rescue P-galactosidase production from the fusion and aggregation, but should not bypass the sporulation defect caused by the a4414 insertion. As expected, C-factor addition to a csgA 04414 double mutant restores both aggregation (data not shown) and P-galactosidase production (Figure 5b). C-factor addition to this double mutant brings sporulation to about the level produced in strain DK5279, which contains the a4414 insertion but is csgA+ (Table 1, lines 4 and 7). This observation, together with restoration of all other C-dependent gene expression, suggests that external C-factor allows development to pass through what would otherwise be the developmental block in a csgA mutant.
Discussion Rescue of certain admixed wild-type
classes of developmental mutants by cells has led to the proposal that M.
xanthus passes essential signal molecules from cell to cell to coordinate formation of its multicellular fruiting body. If the proposal is correct, then it should be possible to isolate the molecules involved in each specific signaling event. A 17 kd polypeptide has been isolated from csgA+ cells by means of its ability to restore proper development to csgA mutants. csgA mutants fail to form proper multicellular aggregates, to differentiate from rod-shaped cells to environmentally resistant spores, and to express development-specific genes that are detected by transcriptional fusions to /acZ. Purified C-factor can restore each of these aspects of development that are defective in csgA mutants. Partial amino acid sequencing of C-factor shows that it is a product of the csgA gene. These data support the proposal that exchange of external C-factor between cells is a necessary part of normal fruiting body development.
Based on a 2-fold limiting dilution assay, 1 U of C-factor
is defined as the minimal amount that restores csgA mutant sporulation and aggregation to wild-type levels. One unit of purified C-factor corresponds to about 24 ng of purified protein. From this we calculate that aggregating wildtype cells should contain at least 9000 molecules of C-factor per cell when harvested at 18 hr of development. Similarly, it appears that about 8000 molecules of C-factor are required per cell to rescue c.sgA fruiting body development. Given a molecular mass of 17 kd, this implies that C-factor functions at a concentration of approximately 1 to 2 x 10dg M. This concentration is typical of the low effective concentrations measured for other small morphogenetic signals, including retinoic acid (Thaller and Eichele, 1987), Dictyostelium differentiation-inducing factor (Kay et al., 1983), and Hydra head activator peptide (Schaller and Bodenmiiller, 1981). Earlier studies have revealed that, under certain conditions, millimolar amounts of purified components of M. xanthus peptidoglycan (Shimkets and Kaiser, 1982) and development-stimulating factor, a low molecular weight, partially purified glycoconjugate (Janssen and Dworkin, 1985), can restore aggregation and sporulation to mutant strains carrying a csgA mutation. However, peptidoglycan components and development-stimulating factor are present in CsgA cells and therefore may provide a metabolic bypass of the normal signaling event, instead of supplying the signal itself. In contrast, C-factor was found only in developing wild-type cells; cells that are either csgA or are growing fail to produce C-factor activity (Figure 2). The biological activity of peptidoglycan and development-stimulating factor, both derived from the cell envelope, suggest that one outcome of C-factor signaling may be altered envelope metabolism. Our inability to reconstitute rippling of csgA mutants with purified C-factor may have several explanations. For example, rippling may require a separate signal. A more intriguing possibility is that the synchronous, pulsatile, wave-like cell movements in rippling require local oscillation of C-signal, a requirement probably not fulfilled by our method of adding C-factor in a single step (see Experimental Procedures). A temporal or spatial requirement for C-factor addition may also explain why rescue of b-galactosidase expression by C-factor is delayed relative to rescue by whole cells. CAMP chemotaxis in Dictyostelium provides a well-characterized precedent for an oscillatory signal that triggers coordinated cell movement (Devreotes, 1983). Evidence for a signal relay system involving C-factor arises from the observation that cell motility is required for the csgA-mediated cell interaction (Kroos et al., 1988). Nonmotile mutants arrest their development at a stage similar, if not identical, to csgA mutants. Kroos et al. (1988) hypothesized that if, in turn, C-signaling caused cells to move, then a positive feedback loop would be created (movement, C-signaling, movement, etc.), which in theory could produce a signal oscillation manifest as ripples. The feedback loop would be broken by loss of motility or by csgA mutation, and, indeed, both functions have been shown to be required for rippling (Shimkets and Kaiser, 1982; Shimkets et al., 1983). To the extent that restored
aggregation implies cell movement, our finding that purified C-factor allows csgA cells to build aggregation centers that become normal fruiting bodies provides experimental support for the proposal that C-factor can induce cell movement. The isolation of C-factor and the demonstration of its activity in development raises the question of how it functions. Although it remains to be demonstrated directly, our observations support the proposal that C-factor is transferred from cell to cell. Exogenous, solubilized C-factor restores csgA mutant cell development, suggesting that C-factor functions at the cell surface. The apparent tight association of C-factor with membrane suggests that intercellular signaling between wild-type cells might occur only over a very short range, perhaps restricted to immediately contiguous cells. It seems likely that C-factor acts as a paracrine signal that must bind to a receptor on the external surface of a cell. Given a procedure to isolate biologically active C-factor, it should be possible to investigate its production, transmission, and reception. Experimental Materials
Procedures and Bacterial
Strains
MOPS (3[N-morpholino]propanesulfonate), kanamycinsulfate,CHAPS, o-nitrophenolgalactoside, and gel electrophoresis standards were from Sigma. Dialysis tubing (8000 dalton nominal cutoff) was from Spectrum. Lysobacter enzymogenes endoproteinase-Lys-C was from Boehringer Mannheim. All M. xanthus strains used in this study were derived from DK1822 (wild type). Sites of TnBlac transposon insertions are designated by the Greek letter 61 followed by a four digit numeral (Kroos et al., 1988). C-factor was prepared from DK5204, a kanamycin-resistant (Km’), developmentally competent strain that contains TmYac at position 04435. The csgA mutation used to construct csgA Tn5lac derivatives is created by insertion of Tn5-132 into the csgA gene at site QLS205 (Shimkets and Asher, 1988). This transposon insertion contains the tetracycline resistance (Tc’) element from TnlO (Foster et al., 1981), which allowS introduction of the csgA mutation into Km’ TnBlac strains by generalized transduction and selection for Tc’ as described in Kroos and Kaiser (1987). In this way, strains that were csgA and contained the Tn5lac insertions R4499 (DK5246). 514414 (DK5287), 04403 (DK5270), Q4435 (DK5253), and Q4401 (DK5229) were constructed (Kroos and Kaiser, 1987). Source of C-Factor Cells growing exponentially in casitone-Tris liquid medium (Hodgkin and Kaiser, 1977) were sedimented at 10,000 x g for 10 min at 5% and suspended in TPM buffer (10 mM Tris-HCI, 1 mM K2HP04.KH2PO4, 8 mM MgSOd [pH 7.51) at a density of 5 x log cells per ml. Aliquots (-50 ~1) were spotted on TPM agar (TPM plus 1.5% agar) in 25 x 25 cm tissue culture plates (Nunc) and incubated at 32% for 18 hr when C-factor activity is most abundantly recovered. Agar. rather than submerged culture, was chosen for conditioning cells because the yield of cells per cm2 was greater on agar than on plastic. At 18 hr of development on TPM agar, cells have aggregated but have not formed spores. Cells were scraped with a razor blade into MC7 buffer (10 mM MOPS, 1 mM CaC12 (pH 7.01) at a calculated concentration of 1.5 x lOlo cells per ml. This suspension was homogenized by vortex mixing, flash-frozen in liquid nitrogen, and stored at -80°C. C-Factor Assay C-factor was assayed by its ability to restore fruiting body formation and sporulation to csgA mutant cells. The procedure is based on the assay for A-factor described by Kuspa et al. (1988), but modified to provide a solid surface for aggregation and to permit counting of fruiting bodies and spores. The csgA strain DK5253 was routinely used. Each sample of C-factor to be assayed was dialyzed against 4 I of 10 mM MOPS, 1 mM CaCl*, 4 mM MgClp, 50 mM NaCl (pH 7.2) (buffer A) for
C-Factor 25
Is Required
for M. xanthus
Differentiation
12-18 hr at 4%. Aliquots of the dialyzed samples were serially diluted in P-fold steps, typically through 6 to 8 such steps; 400 PI of each dilution was warmed to 32%. then added to responder csgA cells that had been developing in submerged culture as illustrated in Figure 1. When exponentially growing M. xanthus is resuspended in microtiter wells containing nutrient-free buffer and Ca*+, the cells settle to the bottom of the well and form an adherent mat as described by Kuner and Kaiser (1962). During normal development, fruiting body morphogenesis, sporulation, and gene expression occur over the next 72 hr. In the C-factor assay, exponentially growing csgA assay cells were centrifuged at 10,000 x g for 10 min and resuspended in buffer A at a concentration of 5 x lb cells per ml; 400 ~1 of this suspension was transferred into the wells of a 24 well microtiter dish (Falcon) and incubated at 32% for 6 hr in a humid chamber. At this time, when the morphological defects of csgA mutants are first manifest, the buffer A overlying the confluent mat of adherent cells is gently removed, and prewarmed fractions to be assayed are added. One unit of activity (Figure 1) is defined as the amount of C-factor that restores wild-type level fruiting body formation (200-300 per 2.5 x 10s input cells) and sporulation (2.5 x 106 per 2.5 x lo* input cells) to csgA mutants developing in submerged culture. Other Methods C-factor purification was analyzed by SDS-PAGE using 15% acrylamide and 0.12% bisacrylamide (Laemmli, 1970; Pfeffer, 1987). Silver staining was performed by the method of Merril et al. (1981). except that 0.004% KMnO, was substituted for K&&O,. Measurement of developmental 6galactosidase expression was performed as described by Kmos and Kaiser (1987). One unit of &galactosidase specific activity is equal to 1 nmol of o-nitrophenol produced per minute per milligram of protein. Heat-resistant. sonication-resistant, Km’ spores were quantified as described by Kroos et al. (1966). Fruiting bodies were scored visually at 6x magnification using a dissecting microscope (Wild-Heerbrug). The presence of ovoid, refractile spores within fruiting bodies at the bottom of a microtiter well was confirmed with a Leitz inverted light microscope using a 40x objective. In preparation for microsequencing. purified C-factor was treated with endoproteinase Lys-C, which specifically cleaves polypeptides on the carboxy side of lysine residues (Jekel et al., 1963). Endoproteinase Lys-C was mixed with substrate at a ratio of 1:100 in 150 mM Tiis-HCI (pH 8.7) for 4 hr a1 37%. A second aliquot of protease equal to the initial amount was then added and the mixture allowed to stand at 37% for 16 hr. Purification of protein for amino acid sequence analysis was performed by the method of Matsudaira (1987). Samples were analyzed by the Protein Structure Laboratory, University of California at Davis School of Medicine. Acknowledgments We thank Drs. L. Kroos. A. Kuspa, B. Glick, H. Kaplan, and K. Irvine and J. Rodriguez for helpful discussions. We especially thank Drs. L. Shimkets and T Hagen for their critical discussion, for providing affinity-purified antibodies tocsgA fusion protein, and for communicating their csgA sequence data prior to its publication. We thank C. Tate, R. de la Cruz, N. Malimban, J. Vizyak, and B. Scott for excellent technical assistance and J. Gardner, A. Smith, and the Protein Structure Laboratory at the University of California, Davis for protein sequence determinations. This investigation was supported by National Institutes of Health research grant GM23441 to D. K. and stipend to S. K. K. by training grant GMO7365, both from the National Institute of General Medical Sciences. S. K. K. is a trainee of the Medical Scientist Training Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
April 28, 1989; revised
December
29, 1969.
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