Identification of acP-responsive genes
To identify genes that respond to acP, we used DNA macroarrays to compare E. coli wild-type cells (strain AJW678) with cells lacking AckA (ackA mutant; strain AJW1939) or both Pta and AckA (pta ackA double mutant; strain AJW2013). Cells that lack AckA can convert acCoA to acP but cannot degrade acP to acetate efficiently (Fig. 1). Therefore, these cells accumulate acP to a higher level than do wild-type cells, especially when grown on a carbon source that feeds into the bottom half of the glycolytic pathway (e.g. serine present in tryptone). In contrast, cells that lack Pta and AckA do not synthesize acP from either acCoA or acetate (Fig. 1) (McCleary and Stock, 1994; Prüß and Wolfe, 1994). The extracellular acetate concentrations and intracellular acCoA and acP concentrations associated with the single pta mutant resemble those of the double pta ackA mutant (Prüß and Wolfe, 1994). Thus, we chose the double mutant over the single mutant to avoid AckA-dependent activation to acP of any trace quantities of acetate that may be generated by non-Pta-dependent mechanisms.
We aerated cells in tryptone broth (TB) at 37°C. Wild-type cells grew about 40% more rapidly (doubling time = 32 min) than either of the mutants. However, the mutants doubled at almost identical rates (44 and 46 min respectively). Wild-type cells evolved ammonia and consumed amino acids in the following preferential order: l-serine, l-aspartate, l-tryptophan, l-glutamate, l-threonine, l-alanine, as reported previously (Prüßet al., 1994). Both mutants evolved ammonia and consumed amino acids preferentially in a manner that resembled that of wild-type cells, but with some significant differences: most notably, slower rates of consumption for tryptophan, glutamate and threonine. However, both mutants consumed amino acids in the same order at comparable rates and evolved ammonia similarly (data not shown). These similar behaviours gave us confidence to compare the ackA and pta ackA mutants directly.
We harvested cells during early exponential growth (A590 = 0.1), when acP levels peak in wild-type cells (Prüß and Wolfe, 1994). We purified the RNA, generated cDNA, hybridized a paired set of macroarrays (Sigma-GenoSys) and, for each sample, analysed and processed the data as described previously (Conway et al., 2002). We normalized the data by expressing the intensity of each gene-specific spot as a percentage of the sum of spot intensities, calculated the ratio of gene expression for each mutant relative to the control expression level (wild- type) and used Student's t-test to determine the probability that the ratio is significant (P-value). Our statistical analysis established significantly regulated genes meeting two criteria: ratio values ≥ 2.5 standard deviations from the mean of the log ratios (99% confidence) with P-values ≤ 0.05 (95% probability). A total of 146 genes [≈ 3.5% of 4290 total open reading frames (ORFs) identified in E. coli] met these criteria. We performed subsequent analyses only on this subset of genes.
To distinguish genes that respond to the status of acP from those that respond to a slower rate of growth or to a general defect in acetate excretion, we plotted the log10 of the expression ratio pta ackA/ WT against the log10 of the expression ratio ackA/ WT (Fig. 2A). Using this strategy, genes that respond identically in both mutants when compared with their wild-type parent map to a line with a slope of 1 and a y-intercept of 0. Presumably, such genes respond either to a slower rate of growth or to a general defect in the Pta-AckA pathway, e.g. the accumulation of the pathway substrate (acCoA) or the absence of its product (acetate). In contrast, genes that respond negatively to acP levels map above and to the left of the line, whereas those that respond positively to acP map below and to the right of that line.
Figure 2. Scatter plots displaying genes significantly overexpressed or underexpressed relative to wild-type cells (AJW678) by mutant cells defective for pta ackA (AJW2013) or ackA (AJW1939). Cells were aerated in TB at 37°C, harvested at A590 = 0.1 and subjected to DNA macroarray analysis. The dotted line (m = 1, b = 0) represents the predicted position of genes identically expressed in cells of both mutants. A. All genes that are expressed differentially. B. Of the genes shown in (A), only those negatively influenced by acP are shown. Numbers refer to those in Table 1. C. Of the genes shown in (A), only those positively influenced by acP are shown. Open squares, genes involved in stress responses. Chequered squares, genes involved in type I pilus biosynthesis and assembly. Cross-hatched squares, genes involved in capsule biosynthesis. Grey squares, hypothetical genes and those of putative function. Numbers refer to those in Table 2.
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Under the experimental conditions tested, 50 genes appeared to respond negatively to high acP levels; those that responded most significantly (≥5 SD) function exclusively in flagellar biosynthesis and assembly (Fig. 2B, Table 1). These include genes involved in assembly of the filament and hook (fliC, flgE, fliD, flgK, flgD, flgL), the basal body (flgC, flgB, flgG, flgA, flgF, fliL, flgJ), the switching mechanism (fliM), the chemotactic signal transduction pathway (cheY, tar) and transcription of those genes (fliA). We confirmed these results, using quantitative reverse transcription polymerase chain reaction (RT-PCR) to analyse fliA and fliM (Fig. 3). Both genes were expressed at highest levels in the pta ackA mutant, followed by the wild type and then the ackA mutant, consistent with the array results. Other flagellar genes responded significantly, but to a lesser degree (≥2.5 SD): they include those involved in hook length control (fliK), flagellar-specific export (flgN, flhB, fliI, fliJ), chemotactic signalling and energy transduction (cheB, cheZ, cheR, trg, motB), regulation (flhD, flhC) and unknown functions (fliY, fliZ). Non-flagellar genes that responded negatively to high acP levels encode outer membrane porins (ompF, ompC), other proteins associated with or predicted to associate with the envelope (rbsB, b1966, yqiH, glpD, rfbX, rbsD), chaperones (dnaK, mopB, mopA), the regulators of the maltose regulon (malT) and the carnitine operon (caiF) and an enzyme involved in carnitine metabolism (caiD) (data not shown, see http:www.ou.edumicroarray).
Table 1. . Genes significantly upregulated in a pta ackA mutant compared with an ackA mutant .ab
| ||Gene||b no.|| pta ackA/ackA|| pta ackA/WT|| ackA/WT ||Gene product|
| 1|| fliC ||1923||30.9||1.5||11.8||1.1||−2.6||−0.4||Flagellin, major filament component|
| 2|| flgE ||1076|| 8.9||1.0|| 4.0||0.6||−2.3||−0.4||Hook|
| 3|| fliD ||1924|| 5.8||0.8|| 4.1||0.6||−1.4||−0.2||Filament cap|
| 4|| flgC ||1074|| 5.5||0.7|| 3.1||0.5||−1.8||−0.3||Basal body-rod|
| 5|| flgB ||1073|| 5.4||0.7|| 2.6||0.4||−2.1||−0.3||Basal body-rod|
| 6|| flgK ||1082|| 4.1||0.6|| 3.2||0.5||−1.3||−0.1||Hook–filament junction|
| 7|| flgG ||1078|| 4.1||0.6|| 2.5||0.4||−1.7||−0.2||Basal body-rod|
| 8|| flgD ||1075|| 3.6||0.6|| 2.2||0.3||−1.6||−0.2||Hook assembly initiation|
| 9|| flgL ||1083|| 3.4||0.5|| 2.4||0.4||−1.4||−0.2||Hook–filament junction|
|10|| fliA ||1922|| 3.4||0.5|| 1.9||0.3||−1.8||−0.3||Flagellar-specific sigma factor|
|11|| fliL ||1944|| 3.1||0.5|| 1.4||0.1||−2.3||−0.4||Basal body-rod|
|12|| flgA ||1072|| 2.7||0.4|| 1.6||0.2||−1.7||−0.2||Basal body assembly|
|13|| flgF ||1077|| 2.5||0.4|| 1.8||0.3||−1.4||−0.1||Basal body-rod|
|14|| fliM ||1945|| 2.5||0.4|| 1.5||0.2||−1.7||−0.2||Motor switch; energy transduction|
|15|| cheY ||1882|| 2.3||0.4|| 2.3||0.4||1.0||0||Chemotactic signalling|
|16|| tar ||1886|| 2.2||0.3|| 1.9||0.3||−1.1||−0.1||Chemoreceptor|
|17|| flgJ ||1801|| 2.1||0.3|| 1.8||0.3||−1.2||−0.1||Basal body-rod|
Figure 3. Mean log expression ratios ± SEM of a subset of genes. Cells were aerated in TB at 37°C, harvested at A590 = 0.1 and subjected to quantitative RT-PCR.
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In contrast, 46 genes appeared to respond positively to high acP concentrations; the most significant (≥4.8 SD) function primarily in stress protection (osmB, ivy, osmY, osmC, dps, hslJ), colanic acid biosynthesis (rcsA, wcaE, gmd), type 1 pilus assembly (fimG, fimI, fimA, fimC, fimH, fimF) and the assembly of putative non-type 1 pili (b0943, sfmC) (Fig. 2C, Table 2). We confirmed these results, using quantitative RT-PCR to analyse osmY, osmB, osmC, fimA, fimG, rcsA, gmd and wcaE (Fig. 3). These genes were expressed at highest levels in the ackA mutant, followed by the pta ackA mutant and then by the wild-type parent, consistent with the array results. Apparently, these genes respond not only to acP but also, to a lesser degree, to slowed growth or a general pathway defect. Other genes responded significantly, but to a lesser degree (≥2.5 SD): among these are two other genes involved in colanic acid biosynthesis (wcaD, ugd) and one of unknown function located within the colanic acid biosynthetic locus (orf1.3); also included are genes that encode an outer membrane protein (ompX), a putative regulator of maltose metabolism (sfsA), a component of formate dehydrogenase-H (fdhF) and glutathione oxidoreductase (gor) (data not shown, see http:www.ou.edumicroarray).
Table 2. . Genes significantly upregulated in an ackA mutant compared with a pta ackA mutant. ab
| ||Gene||b no.|| pta ackA/ackA|| pta ackA/WT|| ackA/WT ||Gene product|
| 1|| osmB ||1283||−10.9||−1.0||−1.1||<−0.05||10.1||1.0||Osmotically inducible lipoprotein|
| 2|| ivy ||0220||−5.5||−0.7|| 2.0||0.3||10.8||1.0||Inhibitor of vertebrate lysozyme, previously yfkE|
| 3|| osmY ||4376||−4.6||−0.7|| 1.8||0.3|| 8.2||0.9||Hyperosmotically inducible periplasmic protein|
| 4|| yjbE ||4026||−3.6||−0.6|| 1.0||<0.05|| 3.6||0.6||Hypothetical protein|
| 5|| rcsA ||1951||−3.5||−0.6|| 1.0||<0.05|| 3.6||0.6||Colanic acid biosynthesis (CAB), positive regulator|
| 6|| wcaE ||2055||−3.5||−0.5||−1.1||<−0.05|| 3.3||0.5||CAB, putative glycosyl transferase|
| 7|| yafP ||0234||−3.4||−0.5|| 1.2||0.1|| 4.1||0.6||Hypothetical protein|
| 8|| gmd ||2053||−3.4||−0.5||−1.1||−0.1|| 3.0||0.5||CAB, GDP-d-mannose dehydratase|
| 9|| b0943 ||0943||−3.0||−0.5||−1.3||0.1|| 2.4||0.4||Putative fimbrial protein|
|10|| sfmC ||0531||−2.7||−0.4|| 1.3||0.1|| 3.4||0.5||Putative fimbrial chaperone|
|11|| fimG ||4319||−2.7||−0.4|| 1.8||0.3|| 4.9||0.7||Type 1 pilus, morphology|
|12|| b1172 ||1172||−2.6||−0.4|| 1.3||0.1|| 3.5||0.5||Hypothetical protein|
|13|| osmC ||1482||−2.6||−0.4|| 1.4||0.1|| 3.7||0.6||Osmotically inducible protein|
|14|| ycfJ ||1110||−2.4||−0.4|| 1.0||<0.05|| 2.6||0.4||Hypothetical protein|
|15|| dps ||0812||−2.3||−0.4|| 3.5||0.6|| 8.3||0.9||Starvation, global regulator|
|16|| yiaD ||3552||−2.3||−0.4||−1.3||−0.1|| 1.9||0.3||Putative outer membrane protein|
|17|| hslJ ||1379||−2.3||−0.4|| 1.4||0.1|| 3.1||0.5||Heat shock protein|
|18|| fimI ||4315||−2.3||−0.4|| 1.6||0.2|| 3.7||0.6||Type 1 pilus, minor protein|
|19|| fimA ||4314||−2.2||−0.3|| 1.7||0.2|| 3.7||0.6||Type 1 pilus, major subunit, pilin|
|20|| ybdG ||0577||−2.2||−0.3||−1.1||<−0.05|| 1.9||0.3||Putative transport protein|
|21|| fimC ||4316||−2.1||−0.3||−1.1||<−0.05|| 2.0||0.3||Type 1 pilus, periplasmic chaperone|
|22|| fimH ||4320||−2.1||−0.3|| 1.5||0.2|| 3.2||0.5||Type 1 pilus, d-mannose-specific adhesin|
|23|| b2833 ||2833||−2.1||−0.3|| 1.1||<0.05|| 2.2||0.3||Hypothetical protein|
|24|| fimF ||4318||−2.0||−0.3|| 1.4||0.2|| 2.9||0.5||Type 1 pilus, morphology|
|25|| eco ||2209||−2.0||−0.3||−1.1||<−0.05|| 1.9||0.3||Ecotin, serine protease inhibitor|
Twenty-seven genes appeared to respond positively to a slower growth rate or a general pathway defect; these genes function primarily in stress protection and the TCA cycle (Fig. 2A, see http:www.ou.edumicroarray). In contrast, 23 genes responded negatively; most remain hypothetical or putative, whereas others do not fall into clearly delineated functional categories (Fig. 2A, see http:www.ou.edumicroarray).
These results agree with our previous report that pta ackA mutants synthesize far more flagella than ackA mutants (Prüß and Wolfe, 1994). As predicted from our array data, ackA mutants produced mucoid colonies. In contrast, wild-type cells and pta ackA mutants produced non-mucoid colonies (Fig. 4A). Also as predicted, ackA mutants exhibited numerous pili, as do wild-type cells. In contrast, pta ackA mutants displayed few pili (Fig. 4B). At first glance, it might be puzzling that pta ackA mutants display substantially fewer pili than wild-type cells. Like ackA mutants, pta ackA mutants express all the fim structural genes (fimAIFGH) at higher levels than wild-type cells (Table 2, Fig. 2C). We suspect the difference lies with the behaviour of fimC, which encodes the type 1 pilus-specific chaperone that delivers pilus subunits to the pilus-specific usher, an assembly platform encoded by fimD (Sauer et al., 2000). Unlike ackA mutants, pta ackA cells apparently do not upregulate fimC (Table 2, Figs 2C and 5). This result leads us to suspect that an excess of pilus subunits might overload the FimC chaperone and that this overload results in reduced pilus assembly. If so, we do not suspect the usher, FimD, because both ackA and pta ackA mutants appear to express similar amounts of fimD (Fig. 5). Regardless of the underlying mechanism responsible for the pta ackA pilus assembly defect, taken together, our results support the argument that high acP levels enhance colanic acid production and type 1 pilus assembly, whereas low acP levels favour flagellar expression.
Figure 4. A. Mucoidy phenotype of colonies formed by cells wild type (AJW678) or deficient for either ack (AJW1939) or pta and ackA (AJW2013). Cells were grown at 37°C on defined medium (M63) supplemented with 0.2% glucose. B. Transmission electron micrographs of wild-type cells (AJW678), ackA mutant cells (AJW1939) or pta ackA mutant cells (AJW2013) grown at 37°C in TB and harvested at A590 = 0.4. Each bar represents 1 µm.
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Figure 5. Schematic showing expression profiles of the fim operon. P/A, log10(pta ackA/ackA); P/+, log10(pta ackA/WT); A/+, log10(ackA/WT). NS, not significantly different.
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Pellicle and abiotic surface-adherent biofilm formation
Escherichia coli cells can simultaneously form two different structured communities during growth in static culture: a pellicle that forms on the surface of the culture medium and an abiotic surface-adherent biofilm that forms at the air–liquid interface. Formation of the pellicle depends, in part, upon the presence of intact type 1 pili that permit cell–cell interactions (Harris et al., 1990). As others have previously implicated type 1 pili and other adhesins, flagella, colanic acid and several stress responses in the formation of surface-adherent biofilms (reviewed by Stoodley et al., 2002), we compared the pellicle- and surface-adherent biofilm-forming ability of wild-type cells with that of cells that either accumulate acP (ackA) or do not synthesize it (pta ackA). For these experiments, we grew cells at 37°C in static TB cultures. At 2 h intervals, we monitored the A590 as a measure of biomass. Wild-type cells grew more rapidly (doubling time = 56 min) than either of the mutants. The mutants, however, doubled at identical rates (doubling time = 121 min). Wild-type cells consumed amino acids and evolved ammonia in a manner that resembles that observed under aerated conditions (data not shown). Although they did so more slowly than their wild-type parent, both mutants consumed amino acids and evolved ammonia similarly (data not shown). Biofilm development requires growth through exponential phase (Danese et al., 2001). Thus, the difference between wild-type and mutant growth rates may make direct comparison between wild-type and mutant biofilm development problematic. However, the virtually identical rates of growth and ammonia evolution and the similar profiles of amino acid consumption exhibited by the mutants permits their direct comparison.
To observe the ability of each strain to form a surface-adherent biofilm at the liquid–air interface, we stained 18 h cultures with crystal violet, and rinsed away non-adherent cells (Fig. 6A). The morphology of each biofilm appeared to be distinctly different. Both wild-type and pta ackA mutant cells produced substantial surface-adherent biofilms in contrast to ackA mutant cells, which constructed a sparse biofilm. To examine these biofilms more closely, we performed differential interference contrast (DIC) microscopy on biofilms grown on thin strips of microscope slides (Fig. 6B). After 6 h incubation, wild-type cells produced large microcolonies. In contrast, pta ackA mutants formed smaller microcolonies, whereas ackA mutants formed even smaller ones.
Figure 6. A. Surface-adherent biofilms formed by cells wild-type (WT) or deficient for either ackA or pta ackA. Cells were grown without aeration at 37°C in TB. After 18 h incubation, the triplicate cultures were stained with crystal violet, the non-adherent cells were rinsed away, and the adherent cells that formed a biofilm at the air–liquid interface were visualized using an Alphaimager 2000 documentation and analysis system. Representative images are shown. B. DIC micrographs of microcolonies formed by wild-type cells (AJW678), ackA mutant cells (AJW1939) or pta ackA mutant cells (AJW2013). Duplicate cultures were grown without aeration for 6 h at 37°C on thin strips of microscope slides partially immersed in TB. Representative images are shown. C. Pellicles formed by cells wild type (AJW678) or defective for either ackA (AJW1939) or pta ackA (AJW2013). Cells were grown without aeration at 37°C in TB. After 18 h incubation, the triplicate cultures were photographed using a Wild dissecting microscope at a magnification of 25×. Representative images are shown. Note cracks at the bottom left of the wild-type pellicle and the upper right of the pta ackA pellicle.
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The morphology of each pellicle also appeared to be distinctly different. Wild-type cells formed a film-like pellicle that covered the entire surface of the static culture (Fig. 6C). Upon this film, complex swirls of colony-like structures formed. When vortexed, the wild-type pellicle did not disperse easily; instead, it formed tight aggregates (data not shown). In contrast, ackA mutant cells formed isolated rafts of cells. When vortexed, these cells also formed aggregates (data not shown). Like wild-type cells, pta ackA mutant cells formed a film that covered the entire surface of the culture. In contrast to wild-type and ackA mutant pellicles, pta ackA mutant cells formed few colony-like structures and dispersed easily into a homogeneous suspension when vortexed (data not shown).
Finally, we began to explore the molecular basis that underlies the distinct characteristics displayed by biofilms and pellicles formed by ackA and pta ackA mutants. We introduced a mutant allele (fliA) that precludes the expression of flagella into cells either wild type or deficient for ackA alone or both pta and ackA. As reported previously (Pratt and Kolter, 1998), the lack of flagella eliminated the ability of otherwise wild-type cells (fliA) to form a biofilm (Table 3). Similarly, pta ackA mutants that cannot synthesize flagella (pta ackA fliA) formed, at best, very poor biofilms. In contrast, ackA fliA mutants formed biofilms indistinguishable from those produced by their ackA fliA+ parent. Thus, biofilm formation by the ackA mutant does not require flagella.
Table 3. . Biofilm phenotypes.
|AJW678||Wild type||Wild-type standard|
|AJW1939|| ackA::Km|| ackA standard|
|AJW2067|| ackA::TnphoA′-2|| ackA standard|
|AJW2013||Δ(ackA pta hisJ hisP dhu)|| pta ackA standard|
|AJW2149|| ackA::Km fliA::Tn5|| ackA standard|
|AJW2153||Δ(ackA pta hisJ hisP dhu) fliA::Tn5||Trace|
|AJW2070|| ackA::TnphoA′-2 ΔfimA::Km||0|
|AJW2064||Δ(ackA pta hisJ hisP dhu) ΔfimA::Km||0|
|AJW2069|| ackA::TnphoA′-2 fimH::Km||0|
|AJW2062||Δ(ackA pta hisJ hisP dhu) fimH::Km||0|
The tendency of wild-type and ackA mutant biofilms to resist dispersal by agitation correlates well with their expression of numerous pili, surface structures intimately involved in cell–cell contact. In contrast, the easy dispersal of pta ackA mutant biofilms reflects their relative lack of pili. Both mannose and its analogue methyl-α-d-mannopyranoside inhibit wild-type pellicle formation by binding a d-mannose-specific adhesin, encoded by fimH and used for cell–cell contact (Harris et al., 1990). Both inhibitors interfered with the formation of wild-type and ackA mutant pellicles and surface-adherent biofilms (Table 4), but not with those formed by pta ackA mutants. However, when we introduced mutant alleles that eliminate the expression of the entire type 1 pilus (Δfim) or simply the adhesin (fimH), cells formed neither pellicles nor surface-adherent biofilms, regardless of the status of ackA and/or pta (Table 3). Taken together, our observations suggest that type 1 pili must play some important role in biofilm development by pta ackA mutants, but that the small number they exhibit cannot resist dispersal.
Table 4. . Effect of mannose and a mannose analogue on pellicle formation. a
|Wild type||17.5 (A)||17.5 (A)||20 (B)||50 (A)|
| ackA ||17.5 (A)||ND||20 (A)||30 (A)|
| pta ackA ||>100||>100||>100||>100|