RESULTS

Finding the genes that determine the WS phenotype: To identify genes responsible for colonization of the air-broth interface we exploited the known correlation between undulate colony morphology on agar plate culture and colonization of the air-broth interface in spatially structured microcosms. Transposon mini-Tn5-km was introduced into the genome of WS and transconjugants were screened for loss of the wrinkled colony morphology. From a screen of 8000 kanamycin-resistant mutants, 40 (WS^-) mutants with colony phenotypes similar to the ancestral SM genotype were isolated. Subsequent phenotypic checks and elimination of siblings by Southern hybridization reduced the number of unique mutants to 27. With the exception of 2 of these (WS-5 and WS-39), all were defective in their ability to colonize the air-broth interface and behaved like the ancestral genotype. WS-5 and WS-39 formed a very weak mat, but grew primarily in the broth phase.

To verify that colonization of the air-broth interface was the primary cause of enhanced WS fitness (in spatially structured microcosms) all WS^- mutants were competed against the “wild-type” WS genotype. A fully balanced competition was not possible because of lack of a neutral marker in WS and carriage of the transposon by the WS^- mutants. Nevertheless, over the course of 24 hr all WS^- mutants suffered an average reduction in fitness of between 34 and 52%, which is considerably greater than the 3% reduction in fitness attributed to carriage of mini-Tn5-km.

Our ability to produce WS^- mutants by transposon mutagenesis suggested that the original mutation causing WS was a gain-of-function mutation. No WS phenotypes were detected following mini-Tn5-km mutagenesis of the ancestral SM genotype.

Phenotypic and genetic characterization of WS^- mutants: Colony morphology: The colony morphology of each mutant was examined on KB and LB agar after 24, 48, and 72 hr growth. The majority of WS^- mutants resembled the ancestral SM strain, which remained smooth irrespective of incubation time or environment. However, the colony phenotypes of mutants WS-6, WS-9, WS-12, WS-18, WS-36, and WS-39 showed plasticity. With the exception of WS-12 and WS-36, colonies formed by these mutants were initially smooth and indistinguishable from the ancestral type, but became gradually more WS-like after 48 hr. WS-12 and WS-36 produced WS-like colonies on LB agar, but produced SM-like colonies on KB. These plastic phenotypes bred true and showed the same patterns of differentiation when cultures were transferred to fresh media. Further examination of the nutrient-dependent expression of WS morphology in WS-12 and WS-36 showed that it was attributable to one or more components of proteose peptone and not due to the repression of this phenotype by a component of KB. WS-5 produced smooth, but slow-growing colonies on both KB and LB.

Cellular morphology: With the exception of WS-39, all WS^- mutants (and SM and WS controls) were motile, rod shaped, and indistinguishable from each other by light microscopy, scanning electron microscopy, or transmission electron microscopy (Kahn 1998). WS-39 cells were spherical.

Phenotypic diversification of WS^- mutants in static microcosms: To determine the importance of the loci identified by mini-Tn5-km mutagenesis in evolution of WS morphs during selection in a spatially structured microcosm, each of the WS^- mutants was inoculated into triplicate microcosms (supplemented with kanamycin to select for the transposon) and incubated without shaking. Patterns of diversity that evolved from each mutant were examined after 7 days of selection by plating onto KB agar supplemented with kanamycin.

Control SM populations diversified to produce a wide range of WS morphs (the dominant class), variant SM types, fuzzy spreader types, and other minor classes as previously described (Rainey and Travisano 1998). Mutants WS-4, WS-7, WS-11, WS-16, WS-29, and WS-40 generated patterns of diversity that were indistinguishable from the control, suggesting that mutations in these loci were readily compensated for by mutations elsewhere in the genome. A second group of 14 mutants (WS-1, WS-2, WS-13, WS-15, WS-20, WS-21, WS-22, WS-25, WS-27, WS-28, WS-30, WS-33, WS-34, and WS-35) underwent morphological diversification, but failed to generate WS morphs, while a third group of 7 mutants (WS-5, WS-6, WS-9, WS-12, WS-18, WS-36, and WS-39) did not diversify. Taken together, these phenotypic tests enabled the mutants to be subdivided into six different groups (Table 1), with most being assigned to Group II (14 mutants).

Physical mapping of WS^- mutants: Southern hybridization was used to determine the genomic distribution of candidate loci identified by mini-Tn5-km insertions. Genomic DNA from each WS^- mutant was digested with ApaI, ClaI, and KpnI (which do not cut within mini-Tn5-km) and with SalI and SphI (which have restriction sites within the transposon) and hybridized to a DNA probe derived from mini-Tn5-km. The majority of transposon insertions were located within three large fragments: an 18.5-kb KpnI fragment (5 mutants), a 15.8-kb ClaI fragment (14 mutants), or an 8.6-kb ClaI fragment (3 mutants). These groupings corresponded closely to the previously identified phenotypic groups (Tables 1 and 2).

To determine whether the three groups of loci were clustered, SpeI-digested genomic DNA from representative mutants was hybridized with a DNA probe derived from mini-Tn5-km. The genomic location of each SpeI fragment was then checked against the existing physical map (Rainey and Bailey 1996). Mutants from Groups I and V were located on SpeI fragment SpA, Groups II and III were located on SpH 1, and Group IV was located on SpT. None of these fragments lie adjacent to any other; no mapping information was obtained for Group VI mutants.

Identity of disrupted loci: Genomic DNA from each transposon mutant was digested with ApaI, CalI, or KpnI and cloned into pSK⁺. Clones containing the transposon and flanking DNA were selected by plating on media supplemented with kanamycin. A total of eight transposon-carrying clones were obtained and the flanking DNA was sequenced from each using primers to either end of the transposon.

Sequence from a single Group I mutant, WS-4, revealed an open reading frame (ORF; the entire 1002-nucleotide ORF was sequenced; GenBank accession no. AY074937) that was 37% identical at the amino acid level to PleD, a GGDEF-type response regulator from Caulobacter crescentus, which in conjunction with PleC and DivK couples polar differentiation and cell cycle processes (Hecht and Newton 1995; Jenal 2000). The WS-4 ORF also showed 33% identity to CelR2, which is a putative regulator of cellulose biosynthesis in Rhizobium leguminosarum (Ausmeeset al. 1999) and 34% identity to AdrA, which is a regulator of cellulose and thin aggregative fimbriae in E. coli and Salmonella typhimurium (Romlinget al. 2000; Zogajet al. 2001). Further in silico analysis showed the predicted protein to be comprised of two clearly defined domains separated by a short linker. The N-terminal domain is typical of response regulators and has a conserved aspartate at residue 67 that is the predicted site of phosphorylation. The C-terminal domain is widespread in bacteria, but has no known function. However, recent in silico analyses predict that the conserved GGDEF motif is an active site for nucleotide cyclization or hydrolysis (Pei and Grishin 2001). Given a probable regulatory function we named this gene wspR (WS phenotype regulator). Subsequent sequence analysis has shown that wspR is the terminal gene in an operon of seven genes that shows substantial similarity to the “frz-like” chemosensory signal transduction pathway of P. aeruginosa (Stoveret al. 2000; E. Bantinaki and P. B. Rainey, unpublished data).

The transposon and flanking DNA were cloned from five Group II mutants. In each instance the DNA sequence showed significant similarity to the yhj locus from E. coli (Sofiaet al. 1994), recently shown to be required for cellulose biosynthesis (Zogajet al. 2001), and to genes from Acetobacter xylinum, also required for the biosynthesis of cellulose (Wonget al. 1990; Saxenaet al. 1994). The possibility that this locus encoded the primary structural determinant of the WS morphology led us to name this locus the WS structural locus, wss (see below).

No transposons were cloned from Group III mutants, but the three insertions were subsequently mapped by PCR and DNA sequence analysis and shown to be contiguous with the locus identified by the Group II mutants (see below).

The transposon from WS-12, a Group IV mutant, was located in a gene that showed 52% identity to phosphoglucose isomerase (pgi) from Haemophilus influenzae (Fleischmannet al. 1995). PGI is a highly conserved enzyme responsible for the interconversion of glucose-6-phosphate and fructose-6-phosphate.

DNA flanking the transposon in WS-39 (Group V) encodes a putative protein that is 62% identical to MreB from E. coli. The mre locus in E. coli is involved in cell shape determination and progression to cell division (Doiet al. 1988; Wachi and Matsuhashi 1989). No sequence information was obtained for any Group VI mutants.

The wss locus: Complementation of Group II and III mutants: The similarity of the wss locus to genes involved in cellulose biosynthesis and the phenotypic data that showed Group II mutants incapable of generating compensatory mutations led us to postulate that this was the primary determinant of the WS phenotype and the innovation responsible for initiating phenotypic divergence. We therefore carried out a detailed analysis of this locus. In the absence of a genome sequence, cosmid clones flanking the region were obtained by screening an existing cosmid library made from wild-type (ancestral SM) genomic DNA (Rainey 1999) with DNA probes derived from the wss locus. Two cosmids (pCSA and pCSB) that were shown by restriction analysis to overlap by ∼25 kb were obtained. Both cosmids contained restriction fragments that matched the predicted fragment sizes for the disrupted loci of all Group II mutants; both cosmids were able to restore the WS morphology to all Group II mutants. Interestingly, both cosmids were also able to complement the three Group III mutants, suggesting that the locus disrupted in these mutants was closely linked to the wss locus. Group I, IV, V, and VI mutants were not complemented by either cosmid.

The wss operon: The DNA sequence of the central region of cosmid pCSA was obtained from a series of overlapping subclones. Analysis of the nucleotide sequence using FramePlot (Ishikawa and Hotta 1999), which identifies ORFs on the basis of the G + C bias at the third codon position, revealed the presence of 10 ORFs. These ORFs were designated wssA-J and form a putative operon (Figure 1). A combination of PCR and sequencing, using primers to both the I- and O-ends of the transposon and to specific sequences within the wss operon, allowed all transposons from Group II and III mutants to be mapped to the wss operon (Figure 1). A 20-kb nucleotide sequence is available from the GenBank database under accession no. AY074776.

Overall, the wss operon shows strong similarity to cellulose biosynthetic clusters from A. xylinum and E. coli although there are notable differences, particularly at the downstream end (Figure 2). Upstream of the wss operon (Figure 1) is a single precursor tRNA encoding tRNA^Thr (Cove score = 94.51; Lowe and Eddy 1997), a truncated ORF encoding the C-terminal two-thirds of an oxidoreductase, and a gene showing 77% identity at the amino acid level to outer membrane protein porin Q (oprQ) from P. aeruginosa (Okamotoet al. 1999). The truncated ORF shows greatest similarity at the amino acid level (between 36 and 38% identity) to FabG from a wide range of eubacteria. Downstream from wssJ the operon is flanked by genes showing strong similarity to an arginine and ornithine transport operon (aot; Nishijyoet al. 1998) and a second, closely homologous putative transport operon found in P. aeruginosa (Stoveret al. 2000). The gene immediately adjacent to wssJ encodes a protein that is 68% identical to the hypothetical protein PA5156 of unknown function, and the gene upstream of this is 65% identical to PA5155 and 47% identical to the membrane protein component AotM. Between wssJ and aotM is an ∼1-kb stretch of AT-rich DNA sequence that contains no obvious repeat motifs or terminator structures.

The wss operon consists of 10 ORFs (wssA-J). The predicted product of wssA is a protein of 344 amino acids. It shows greatest similarity to YhjQ from E. coli (Table 3), but is also between 23 and 27% identical to MinD from a range of bacteria including Helicobacter pylori (27%) and Bacillus subtilis (29%). MinD is an ATPase, which in concert with MinC is responsible for determining cell polarity and the positioning of the septum by FtsZ during cell division (Sullivan and Maddock 2000). In common with other ATPases of this class, WssA also contains a clearly defined Walker Box. Mutant WS-1 contains a transposon insertion in wssA that abolishes the WS phenotype and cellulose production (see below), indicating that this gene, although with no previously recognized role in cellulose biosynthesis (Zogajet al. 2001), is part of the wss operon.

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Figure 1.

—Genetic structure of the wss operon. The wss operon is a cluster of 10 genes predicted to encode a CLP. The cellulose synthase is predicted to be composed of WssB, WssC, and WssE; WssD is an associated cellulase; WssG, WssH, and WssI are predicted to play a role in acetylation of the basic cellulose polymer. WssA and WssJ have homology with MinD. Upstream of the wss operon is tRNA^Thr (open arrow), a truncated gene (’fabG) encoding an oxidoreductase and a gene predicted to encode a membrane protein porin Q (oprQ). Downstream of the operon is a putative arginine and ornithine transport gene (aotM) and a hypothetical protein of no known function. The locations of Group II and III mini-Tn5-km transposon insertions are indicated; the position of the wssE-’lacZ fusion is shown by a rightward facing arrow; H, HindIII; K, KpnI.

WssB, WssC, and WssE are predicted to encode proteins of 739, 689, and 1279 amino acids, respectively. Each shows similarity to proteins from A. xylinum (Table 3) that together form a cellulose synthase complex (Saxenaet al. 1994). Significant similarity also exists at the amino acid level to genes of the yhj locus of E. coli (Blattneret al. 1997; Figure 2 and Table 3), recently shown to be involved in cellulose biosynthesis (Zogajet al. 2001). On the basis of biochemical analyses of the cellulose synthase complex in A. xylinum (Saxenaet al. 1994) and Agrobacterium tumefaciens (Matthysseet al. 1995) WssB is predicted to be the catalytically active subunit of a membrane-bound β-glucan synthase that polymerizes UDP-glucose into cellulose. Consistent with this prediction is the presence of eight putative membrane-spanning regions in WssB and conservation of DNA sequence surrounding the conserved asparate residues (U1-4), including the signature motif QXXRW that is characteristic of repetitive transferases involved in the formation of β-glycosidic linkages (Saxenaet al. 1994). Also present are the conserved KAG and QTP motifs that are found in both plant and bacterial cellulose synthases and other glycosyltransferases (Stasinopouloset al. 1999; Blanton et al. 2000). WssC and WssE lack transmembrane helices, but are likely to be membrane associated because they are predicted to be closely associated with the catalytic subunit. The amino acid sequence of each protein was analyzed for the existence of signal peptides using both neural networks and hidden Markov chains (Nielsenet al. 1997). Neither WssB nor WssC showed any evidence of a signal sequence; however, WssE is highly likely to have a signal peptide (P = 1.00) and to be secreted.

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Figure 2.

—Genetic organization of bacterial cellulose biosynthetic gene clusters. The cellulose biosynthetic operons of A. xylinum (bcsI and bcsII), Aquifex aeolicus (putative operon), E. coli (yhj), and P. fluorescens (wss) are shown schematically. Each cluster contains the three subunits of the cellulose synthase, although in bcsII the first two subunits are fused. Solid arrows, cellulose synthase subunits (A. xylinum bcsAI, BI, and CI; bcsABII-A and CII; A. aeolicus bcsA, 1400, and 1402/3; E. coli yhjO, N, and L; P. fluorescens wssB, C, and E). Shaded arrows, cellulase [A. xylinus orf1 (CMCase), A. aeolicus celY, E. coli yhjM, and P. fluorescens wssD]. Open arrows, genes associated with the cellulose synthase (A. xylinum ccpA and bcsX are of unknown function; bcsD is involved in cellulose crystallization; bcsY is a putative acetylase; A. aeolicus 1404, E. coli yhjQ, and P. fluorescens wssA and wssJ are minD homologs; P. fluorescens wssF is a homolog of bcsX; wssG, wssH, and wssI show similarity to genes encoding proteins involved in acetylation of alginate and are unrelated to bcsY). Dotted arrows, genes with no apparent association with cellulose production (A. aeolicus 1405 and E. coli yhjK; Wonget al. 1990; Rawlings and Cronan 1992; Saxenaet al. 1994; Sofiaet al. 1994; Standalet al. 1994; Saxena and Brown 1995; Deckertet al. 1998; Okamotoet al. 1999).

The deduced product of the wssD gene (436 amino acids) is a member of the D-family cellulases (β1-4 glucosidase; Table 2). In A. xylinum (Saxenaet al. 1994) and A. tumefaciens (Matthysseet al. 1995) the cellulase forms an essential part of the cellulose biosynthetic complex. WssF is predicted to encode a protein of 221 amino acids. The amino acid sequence is similar to that encoded by a small ORF (bcsX) of unknown function in A. xylinum (Umedaet al. 1999; Figure 2).

The genes wssG, wssH, and wssI are predicted to encode proteins of 221, 468, and 374 amino acids, respectively. Homologs of these genes are present in P. aeruginosa PAO1 (Table 3) where they are responsible for acetylation of the polysaccharide polymer alginate (Franklin and Ohman 1996). WssH, like AlgI, has 10 predicted membrane-spanning domains and is likely to be an integral transmembrane protein. WssG possesses a putative signal peptide (P = 0.976) and is probably secreted.

The predicted product of the wssJ gene is a protein of 324 amino acids. It is similar to WssA at the amino acid level (51% identical), but shows low nucleotide sequence similarity, with only three short stretches of 60-130 nucleotides showing significant (>70%) similarity. Like WssA, WssJ also contains a Walker Box motif.

The wss operon encodes a cellulose-like polymer that provides structural integrity to WS mats: Cellulose is a structurally simple polymer of (1-4)-β-linked linear glucose chains. Given the similarity of several genes of the wss operon to those involved in the biosynthesis of cellulose in bacteria and plants, we reasoned that the product of the wss operon was cellulose, or at least had a core of (1-4)-β-linked glucose. To test this, the affinity of two dyes, Congo Red and Calcofluor, for WS, SM, and Group II WS^- mutants was determined. Congo Red binds (1-4)-β-d-glucopyranosyl units, basic or neutral extracellular polysaccharides, and some proteins, whereas Calcofluor is a more specific dye for cellulose and binds to (1-3)-β- and (1-4)-β-d-glucopyranosyl units. WS colonies bound Congo Red after 24 hr growth on agar plates containing the dye (the colonies turned bright orange) and, when stained with Calcofluor, copious amounts of filamentous material were observed by fluorescent microscopy. Colonies of the ancestral SM strain showed weak evidence of Congo Red binding and small amounts of Calcofluor-stained material were sometimes observed within cell clumps; however, the Group II WS^- mutants bound neither Congo Red nor Calcofluor. In light of this result we hereafter refer to the product of the wss operon as “cellulose-like polymer” (CLP).

The Congo Red and Calcofluor binding ability of the remaining WS^- mutants were also examined and while all mutants failed to produce wrinkled colonies on agar and colonize the air-broth interface, not all mutants were defective in the production of CLP (Table 2). The most notable exceptions were the Group III strains that contain transposons in the wss cluster (wssG, wssH, and wssI), and the mreB mutant, WS-39.

Given the overproduction of CLP in the WS genotype (and all other WS genotypes examined) we reasoned that this polymer might be responsible for the structural integrity and rigidity of WS mats. To test this, WS mats were allowed to form by incubation in static broth microcosms; mats were then removed after 3 days and incubated with the enzyme cellulase. Overnight incubation in cellulase caused the WS mats to disintegrate, whereas the integrity of control mats was unaffected (Figure 3).

Transcription of wss in WS and evidence of a regulatory role for WspR: To determine whether CLP overproduction was caused by enhanced transcription of the wss locus, the level of wss transcription was compared in SM and WS. A chromosomal fusion between wssE and the reporter ’lacZ was constructed in both SM and WS by integrating, via homologous recombination, a modified version of an existing wssE-’lacZ fusion plasmid (Gal 1999) into the chromosomal copy of wssE (Figure 1). Correct integration was confirmed by PCR. The level of wss expression was determined by monitoring β-galactosidase levels of each fusion strain after growth in LB. A small but significant (P < 0.01) increase in the level of wss expression was observed in the WS morph at 17 hr; at 29 hr, expression of wss in WS was approximately fourfold greater than in SM (Table 4).

An identical wssE-’lacZ fusion was constructed in the wspR::mini-Tn5-km mutant strain, WS-4, to test the putative regulatory role of WspR in transcription of wss. The level of wss transcription was also measured after 17 and 29 hr growth in LB (Table 4). In the wspR-defective background the level of wss transcription reverted to that observed in the ancestral SM genotype, suggesting that WspR plays a regulatory role in wss transcription.

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Figure 3.

—Mat integrity is a result of CLP. Single WS colonies were removed from LB agar plates and incubated in the presence or absence of cellulase. (A) Without cellulase. (B) With cellulase. Colonies were incubated overnight at 37° and then gently agitated before photography.

Elevated transcription of wss does not cause WS: The relatively small (two- to fourfold) difference between transcription of wss in SM vs. WS suggested that increased transcription of wss was not the primary cause of the WS phenotype. To test this hypothesis more rigorously, the level of wss transcription was experimentally elevated in the ancestral SM genotype by introducing (by double homologous recombination) the constitutive promoter from the neomycin phosphotransferase gene (nptII promoter) 107 nucleotides upstream from the start codon of wssA. Enhanced transcription was confirmed by monitoring β-galactosidase levels and CLP production was checked by examining the ability of bacterial strains to bind Congo Red. When grown on agar plates containing Congo Red, the SM nptII-wss fusion strain produced bright-orange colonies indicative of CLP production, but did not express the wrinkled colony morphology typical of WS genotypes; growth was primarily in the broth phase and only a weak mat formed at the air-broth interface.

The fact that WS morphology and CLP production were separable traits led to further experiments to determine whether CLP production per se was the cause of niche specialization and enhanced ecological performance of WS. To do this, the fitness of SM nptII-wss was measured relative to pantothenate-marked ancestral SM (PR1252) and WS in both spatially structured and unstructured microcosms. Overexpression of wss slightly increased fitness in the spatially structured microcosm, but had no effect in the spatially unstructured microcosm (Table 5). The fitness effects were significantly less beneficial than those measured for the WS genotype; moreover, SM nptII-wss did not suffer the marked fitness costs that WS experiences when grown in a spatially unstructured microcosm (Table 5). This result clearly showed that, while necessary, overproduction of CLP alone is not sufficient to generate WS.

Phenotypic divergence, fitness, and the significance of wss during adaptive radiation: The results of our molecular-level analysis led to the hypothesis that overproduction of CLP by the wss locus is a critical component of the WS adaptive phenotype and a primary cause of the phenotypic innovation upon which selection acts to cause WS to increase in frequency. If true, then an ancestral SM genotype carrying a mutation in the wss locus should be competitively inferior, in comparison with the ancestral genotype, during the course of adaptive radiation in the spatially structured microcosms, but not in the spatially unstructured microcosms.

To test this hypothesis a mutation was created in the wss locus of the ancestral SM genotype. This was achieved by inserting the mini-Tn5-km transposon into the second gene of the wss operon, wssB, by allelic replacement, using the transposon and flanking sequence cloned from WS-13 as the source of the recombined DNA. The resulting strain (SM wssB::mini-Tn5-km) was named SM-13.

Carriage of mini-Tn5-km by P. fluorescens incurs a fitness cost that could potentially be greater than any cost caused by the mutation it generates upon insertion. A valid measure of fitness (SM-13 relative to SM) requires that this cost be taken into account. This was achieved by using strain PR1253 as the competing ancestral SM genotype. PR1253 is a pantothenate auxotroph of SM that carries an insertion of mini-Tn5-km-’xylE within the first gene of the pantothenate biosynthetic pathway (panB). The mutation itself is neutral in an environment supplemented with pantothenate and cost of carriage of mini-Tn5-Km-’xylE balances the cost of carriage of mini-Tn5-Km by SM-13. PR1253 is readily distinguished from SM-13 on account of auxotrophy and also yellow coloration of colonies after exposure to 1% catechol.

SM-13 (four replicates of three independent SM wssB:: mini-Tn5-km isolates) was competed directly against PR1253 in both spatially structured and unstructured microcosms. After 7 days of propagation, microcosms were harvested and the population density of the two strains determined. In the spatially unstructured microcosm, SM (PR1253) and SM-13 exhibited equal competitive ability [selection rate constant of SM-13 relative to SM (PR1253): 2.78 ± 3.45 (95% C.I.)] whereas, in the spatially structured microcosm, SM-13 was at a significant fitness disadvantage [selection rate constant of SM-13 relative to SM (PR1253): -5.54 ± 1.93 (95% C.I.)]. No WS morphs were detected in populations derived from SM-13 in either spatially structured or unstructured microcosms.

A notable outcome of competition between SM (PR1253) and SM-13 in spatially structured microcosms was a greater-than-expected fitness deficit in SM-13-derived populations and fewer-than-expected SM-13-derived SM morphs. After 7 days of competition there were approximately two orders of magnitude fewer SM morphs in the SM-13 populations compared with SM morphs derived from SM (PR1253). Our initial expectation was that the fitness of the two evolving populations would be indistinguishable until day 3, when, in the absence of WS morphs from SM-13-derived populations, WS morphs derived from SM (PR1253) would begin to increase in frequency and eventually dominate after 7 days. Given such an outcome, the reduction in fitness of SM-13-derived populations could be directly attributed to the inability of SM-13 to generate WS during the course of the adaptive radiation. However, the smallerthan-expected number of SM-13-derived SM morphs suggests that wss is necessary not only for evolution of WS in spatially structured microcosms but also for optimal performance of the ancestral genotype in this environment. To examine this in more detail, growth curves of SM-13 and SM (PR1253) were determined in both spatially structured and unstructured microcosms during the first 3 days of selection (Figure 4). During this initial period SM morphs are dominant, with evolved WS genotypes being detected only after 48 hr. These data showed that SM-13 had a growth disadvantage relative to SM (PR1253) in spatially structured microcosms, but had no such growth disadvantage over the same period in unstructured microcosms. This indicates that the wss operon contributes to fitness of the SM genotype in the spatially structured microcosm and, as a consequence, the fitness loss observed in derived populations of SM-13 can be only partly attributed to the inability of this genotype to generate the WS phenotype during the course of the adaptive radiation.

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Figure 4.

—Growth rates of SM, SM-13, and pantothenate-marked SM (PR1253) in spatially unstructured (A) and spatially structured (B) microcosms. Data are means and 95% confidence intervals. Growth rates of all three strains were determined for four replicates at each time point. Circles, SM; triangles, SM-13; squares, SM (PR1253).