ABSTRACT
Escherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars and peptides. We envisioned modifying chemotaxis specificity of E. coli by expressing heterologous chemoreceptors from Pseudomonas putida enabling attraction either to toluene or benzoate. The mcpT gene encoding the type 40H methyl-accepting chemoreceptor for toluene from Pseudomonas putida MT53 and the pcaY gene for the type 40H receptor for benzoate and related molecules from P. putida F1 were expressed from the trg promoter on a plasmid in motile wild-type E. coli MG1655. E. coli cells expressing McpT accumulated in chemoattraction assays to sources with 60–200 μM toluene; less strongly than the response to 100 μM serine, but statistically significantly stronger than to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed in E. coli and yielding weak but distinguishable membrane and polar foci in 1% of cells. E. coli expressing PcaY showed weak attraction to 0.1–1 mM benzoate but 50–70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in the E. coli chemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.
IMPORTANCE Bacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis of Escherichia coli has been well-studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds from Pseudomonas putida at least partly functionally complement the E. coli chemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds in E. coli.
INTRODUCTION
Chemotaxis is a rapid (second-scale) behavior of motile organisms to swim towards an attractant or away from a repellent. Chemotactic bacteria can produce a variety of chemoreceptors, some of which with high chemical specificity and selectivity, and others reacting more broadly to related compound classes (1). Chemotaxis could thus be an interesting property for the development of bacterial-based biosensors, which might eventually be deployed to detect and quantify chemical targets in samples (2, 3).
Chemotaxis of Escherichia coli is strong and highly reproducible with known and potent chemoattractants, such as serine or aspartate, and has been widely studied (4, 5). Unfortunately, E. coli does not naturally display chemotaxis towards molecules of potential interest for environmental monitoring, such as aromatic or chlorinated solvents. Given its relatively narrow native chemo-attractant range, it is interesting to investigate whether the E. coli chemotaxis system can be complemented by heterologous chemoreceptors. One important characteristic of methyl-accepting chemotaxis proteins (MCPs) and chemotaxis effector proteins (e.g., CheY) is their structural conservation among bacteria (6–8). E. coli possesses five chemotaxis receptors, but other environmental bacteria frequently encode many more chemoreceptors albeit with often unknown effectors. For example, Pseudomonas species can encode more than 20 MCPs in their genomes (9, 10). A few studies have demonstrated successful expression of heterologous chemoreceptors in E. coli. For example, several MCPs from Shewanella oneidensis could be expressed in E. coli, enabling energy taxis with nitrate (11). Also, Aer-2, a soluble receptor from Pseudomonas aeruginosa involved in aerotaxis, and PctApp, a putative MCP for amino acids from Pseudomonas putida were shown to partially trigger chemotaxis response when expressed in E. coli (12, 13). However, no MCPs involved in sensing of environmental pollutants have to date been functionally expressed in E. coli.
As part of the characterization of bacterial biodegradation pathways, several bacteria were shown to be chemotactic to aromatic compounds, such as to naphthalene, toluene, benzoate or 2,4-dichlorophenoxyacetic acid (14–17). Some bacteria have been characterized in some detail as to their MCPs and chemical effector(s). For example, an MCP named McpT was identified on the self-transmissible plasmid pCRT1 in P. putida DOT-T1E, which enables chemotaxis to toluene and naphthalene (18, 19). This mcpT gene may be more widespread among pseudomonads, as it possesses 99.8% sequence similarity to coding sequences on the TOL plasmid pWW53 of P. putida MT53 (19). Strain MT53 was mentioned as a moderate chemotactic responder to toluene. Further chemoreceptors have been characterized in P. putida F1. As an example, the PcaY receptor was shown to be involved in chemotaxis towards vanillate, vanillin, 4-hydroxybenzoate, benzoate, protocatechuate, quinate and shikimate (20).
The primary goal of this work was to investigate whether chemotaxis specificity of E. coli could be expanded towards aromatic compounds. This could be used as proof of concept for the future development of biosensing strains of E. coli, selectively chemotactic towards environmental pollutants, for deployment in quantitative biosensor microfluidic platforms (3). Our strategy was to express the mcpT gene from P. putida MT53 (pWW53) or the pcaY gene from P. putida F1 on a selectable plasmid in motile E. coli wild-type MG1655 and in a mutant background in which the gene for the major chemoreceptor Tsr was deleted, and to compare chemotaxis to toluene or benzoate with chemotaxis to serine or to no attractant in strains expressing or not the mcpT or pcaY gene. Compound-specific chemotaxis was quantified in two manners: firstly, by microscopy and image analysis from cell accumulation nearby solid agarose sources containing the respective chemo-attractant; and secondly, by a recently developed in-situ chemotaxis microfluidic assay (ISCA) (21). Subcellular localization of the heterologous MCP receptors was assessed and quantified from expressed equivalent mCherry-fusion proteins in E. coli observed by epifluorescence microscopy, in comparison to that of a Tsr-mCherry fusion.
RESULTS
Chemotactic response of E. coli to attractants in agarose plug assays
In order to quantify E. coli chemotaxis to different molecules, we used two independent assays: microscopy observation of cell accumulation to chemoattractants diffusing from a solid agarose source, and a microfabricated in situ chemotaxis assay (ISCA). The agarose plug assays in microscope settings (22) embeds the test compound in a solidified cylinder (ø 4 mm, height 0.15 mm) of agarose (the source), while introducing a homogenous E. coli cell suspension in motility buffer around the source (Fig. S1 in the Supplemental Material). The bacteria accumulation nearby the source edge was recorded by phase-contrast microscopy after 15 min incubation at 21°C and quantified using image analysis (Fig. S1). Robust chemotaxis of E. coli MG1655 was detected to the known chemoattractants serine, aspartate and methylaspartate at 10 and 100 μM source concentration (Fig. 1A, B). In contrast, cell accumulation of E. coli MG1655 to the weaker chemoattractants ribose or galactose at 10 or 100 μM was not statistically significantly different from cells accumulating on the edge of agarose sources without any attractant added (Fig. 1C). These results indicate that the agarose plug assay protocol can be used to measure E. coli attraction to chemical targets with a ‘strength of attraction’ in between ribose/galactose and serine/aspartate/methylaspartate.
Chemotaxis of E. coli expressing the McpT protein of P. putida
Chemotaxis to toluene was tested in motile E. coli MG1655 expressing the mcpT gene from plasmid pWW53 of P. putida MT53 (23) on plasmid pSTV28. In first instance, the mcpT gene was expressed from the low constitutive synthetic PAA promoter (24), but this yielded only few viable transformants that always contained mutations in mcpT, causing frameshifts leading to a premature stop codon or a deletion. In contrast, expression of the mcpT gene on plasmid pSTV28 from the trg promoter (also controlling transcription of the native Trg chemoreceptor in E. coli (25)), resulted in many viable transformants with correct sequences of mcpT. This indicated that we could achieve expression of McpT in E. coli using the trg promoter.
Observing attraction to toluene is complicated by the technical difficulties to produce a solid source containing toluene, which is poorly soluble in water and volatile. First attempts using toluene dissolved in eicosane or dimethylsulfoxide before mixing with agarose were unsuccessful. We could improve consistency by mixing small volumes of liquid toluene directly with dissolved agarose at 55°C inside completely filled and closed glass vials. Indeed, E. coli cells expressing McpT from the trg promoter on plasmid pCRO20 incubated in motility buffer accumulated close to a solid agarose source with a 10−3 toluene dilution (equivalent to 60 μM, Fig. 2A, B). Accumulation of cells in response to toluene was less pronounced than in response to a 100 μM serine source but statistically significantly higher than with sources without any attractant added (Fig. 2A, B, one-way ANOVA and multiple comparison, p=0.0119). Accumulation was robust across fourfold replicates and experiments carried out independently on different days (Fig. 2B). The variation in the magnitude of accumulation was more important with toluene (±31% of the average) than with serine (±3%, Fig. 2B inset), which is likely due to the variation in preparing consistent sources with a volatile attractant. At a tenfold lower toluene source concentration (6 μM), cell accumulation of E. coli pCRO20 at the source edge was no different than to a source without anything added (Fig. 2B, inset). A tenfold higher source concentration of toluene (600 μM) did not result in cells accumulating near the source surface, even though the cells were visibly still motile and able to swim in the proximity of the source (data not shown).
In contrast to E. coli expressing McpT, cells of both E. coli containing the empty plasmid pSTV28 and E. coli carrying a plasmid with a frameshift mutation in the mcpT coding sequence causing premature ending (pCRO35) did not accumulate towards a 60 μM toluene source to a higher degree than to a source without attractant added (Fig. 2C, D and Fig. S2). Both strains, however, responded as expected to a 100 μM serine source and thus were chemotactic (Fig. 2C, D).
Chemotactic response of E. coli expressing the PcaY receptor for benzoate
In separate experiments, we expressed in motile E. coli MG1655 the gene for the PcaY receptor from P. putida F1, which has been reported to induce chemotaxis to molecules such as vanillate, vanillin, 4-hydroxybenzoate, benzoate, protocatechuate, quinate and shikimate (20). Cells of E. coli MG1655 (pCRO33) expressing PcaY from the trg promoter accumulated nearby a source plug with 1 mM benzoate. The response was weaker than the response to 100 μM serine but stronger than to a source without added attractant (Fig. 3A). A lower concentration of benzoate (0.1 mM) decreased cell accumulation to a level no different from that observed without attractant added (Fig. 3A). Cells of E. coli MG1655 pSTV28 (without pcaY) also accumulated nearby a 1 mM benzoate source, with similar intensity to MG1655 (pCRO33) (Fig. 3B). Cell accumulation of E. coli MG1655 (pCRO33) to sources of 4-hydroxybenzoate and vanillate (at 0.1 and 1 mM) was not significantly different than to a source without attractant added.
Chemotactic cell accumulation in microfabricated wells
As a second independent method for chemotaxis quantification we deployed the recently developed ISCA assay (21). The ISCA consists of five replicates of ~110–μl wells, fabricated out of the biocompatible polymer polydimethylsiloxane (PDMS) bonded to a glass slide. The wells are filled with a chemoattractant solution and then immersed in a dilute cell suspension (2–4×106 cells ml−1). A single acentrically placed port (ø 0.8 mm) functions as inlet channel, through which the chemoattractants diffuse out to form gradients and through which motile, chemotactically attracted cells can enter into the wells. Washed E. coli MG1655 wild-type motile cells suspended in motility buffer (strain 4498) accumulated up to five-fold inside the ISCA cavities within a 35–min incubation period with 100 or 300 μM serine as chemoattractant, in comparison to motility buffer (MB) alone (Fig. 4A). In contrast, cells did not statistically significantly accumulate to benzoate at 100 or 300 μM concentrations in comparison to MB, but were statistically significantly repelled at higher benzoate concentrations (300 and 1000 μM) and by toluene at 60 and 200 μM dosages (Fig. 4A). E. coli MG1655 cells expressing the McpT receptor (plasmid pCRO20, strain 5197) consistently accumulated inside ISCA wells filled with serine (100 and 300 μM), as well as with toluene at 60 and 200 μM (~3-fold), but not with benzoate (300 μM), in comparison to MB (Fig. 4B). Cells were not attracted to a higher concentration of toluene (600 μM, Fig. 4B). E. coli MG1655 cells expressing PcaY from plasmid pCRO33 (strain 5447) were attracted to serine, as expected, and slightly (1.2-fold) to 300 μM benzoate, although this was not statistically significant from MB alone (Fig. 4C) as a result of larger variation across replicates. Strain 5447 cells were repelled by high benzoate concentrations, but not by toluene (Fig. 4C). However, in an E. coli MG1655 motile background in which the major chemoreceptor Tsr (for serine) was deleted and PcaY was expressed, accumulation to serine was largely absent, and a statistically significant response to benzoate was observed (1.5–fold; Fig. 4D). Expression of McpT from plasmid pCRO20 in the E. coli Δtsr background yielded cells no longer accumulating to serine, but attraction to toluene did not further improve (1.7-times accumulation at a 200 μM toluene dosage, Fig. 4E).
Localization of the expressed P. putida MCPs in E. coli
In order to further demonstrate whether McpT and PcaY are functionally produced in E. coli, their coding regions were translationally fused with that for mCherry (without start codon itself). The fusion genes were cloned and again expressed under the trg promoter on plasmid pSTV28 (Fig. S2 and S3) in either E. coli MG1655 motile wild-type or the Δtsr deletion background. As a positive control, we used E. coli MG1655 cells expressing a Tsr-mCherry fusion protein from the trg promoter on plasmid pSTV28. These cells showed bright fluorescence, which was enriched in the membrane and in broad zones near the cell poles (Fig. 5B). An E. coli control expressing Tsr alone (without mCherry) was not fluorescent (Fig. 5A). Projection of detectable foci (see Materials and Methods for ‘foci’ detection) as well as overall pixel intensities across all imaged cells normalized to a standardized E. coli cell length as in Figure 6A and 6B showed the strong overall polar localization of Tsr-mCherry fusion protein. This is in agreement with previous studies and what is expected for the localization of the major E. coli chemoreceptors (26, 27). E. coli cells expressing McpT-mCherry were on average more fluorescent than E. coli MG1655, MG1655 expressing McpT alone (without mCherry), or MG1655 expressing a frameshifted mcpT-mCherry (Fig. 5C-E, note fluorescence scales). A small proportion of cells (~1%) contained confined (but rather weak) fluorescent foci in the membrane (Fig. 5C, arrows). Superposed projection of all detected fluorescent foci across imaged cells showed that McpT-mCherry expression was localized in the membrane area of the cells and the poles (Fig. 6A). Cells expressing truncated McpT-mCherry still displayed some fluorescence, which might be the result of a start codon downstream the frameshift position in mcpT, but never produced any visible foci (Fig. 5D). Projection of detected foci produced very few and spurious spots across many cells of both E. coli expressing McpT without mCherry fusion or the frame-shifted McpT-mCherry (Fig. 6A). Enrichment of McpT-mCherry foci near the cell poles was clearer in an E. coli Δtsr background (Fig. 6A). We further quantified mCherry-fusion protein expression by recording the mean intensity of the top–10% brightest pixels per cell, normalized to the mean fluorescent brightness over all individual cells (Fig. 6C). Mean top–10% fluorescence was statistically significantly higher in E. coli wild-type and Δtsr background expressing McpT-mCherry than in E. coli expressing McpT or the frame-shifted McpT-mCherry (Fig. 6C).
E. coli expressing pcaY-mCherry showed on average brighter fluorescence localization in the cellular membrane and frequently at the cell poles (Fig. 5F, 6A), in a higher proportion of cells (50–70%), and the top–10% fluorescence was clearly higher than E. coli expressing McpT-mCherry (Fig. 6C). Fluorescence of the expressed PcaY-mCherry was less bright than in case of Tsr-mCherry (Fig. 5B, 6C), but its localisation was similar (Fig. 6A). Expression of PcaY-mCherry in a Δtsr background increased the top–10% fluorescence level of cells, suggesting higher expression and or more appropriate oligomerization.
These results thus confirmed that the McpT- and PcaY-mCherry receptors are expressed in E. coli and are preferentially localised to the cell membrane and poles. In contrast to expression of Tsr- and PcaY-mCherry, the proportion of E. coli cells with visibly localised McpT-mCherry fluorescence was low (~1%).
DISCUSSION
Heterologous expression of chemoreceptors and functional complementation of chemotaxis in E. coli is not straightforward, and relatively few studies have examined it (11, 13). The major aim of this work was to investigate the possibility to functionally express chemoreceptors for detection of aromatic compounds from P. putida in motile E. coli. We focused on two chemoreceptors, McpT (18, 19) and PcaY (20), which from studies in their native host or by analogy, were reported to detect and signal the presence of toluene and benzoate (plus a further range of substituted aromatic compounds), respectively. By using two different chemotaxis assays and by studying expression and subcellular localisation of chemoreceptor-mCherry fusion proteins, we conclude that both chemoreceptors are functionally expressed in E. coli and can lead to chemotaxis of motile E. coli towards toluene or benzoate at source concentrations in the range of 60–300 μM. Accumulation was concentration dependent, which is a hallmark of chemotaxis. But the range of source concentrations yielding measurable cell accumulation was relatively narrow, which may be due to toxicity or repellent-response at higher chemoattractant concentrations.
Chemotaxis of E. coli expressing the heterologous chemoreceptors McpT or PcaY is relatively weak compared to its major chemoattractant serine. This may be due to the small proportion of cells correctly expressing and localising the McpT or PcaY chemoreceptors (Fig. 6A–H), and to a general poor compatibility of this class of chemoreceptors with E. coli downstream signaling proteins CheA and CheW. According to the chemoreceptor classification of Alexander et al. (6), McpT and PcaY belong to the 40–helical bundle (40H) type, whereas the E. coli MCPs (like Tsr and Tar) are part of the 36H type. Although the two chemoreceptor types have strong sequence conservation in the signaling domain (Fig. S4), with conserved amino acids at positions known to be contacted by CheA and CheW (28), notably the positions of methylation sites are partly different or absent in the 40H–type, and also the nature of the residue for inter-dimer trimerization of Tsr (Phe-373) is charged instead of hydrophobic in the 40H–type (6). The consequence of this may be different oligomerization arrangements of expressed 40H-type chemoreceptors in E. coli and poorer downstream signaling.
Despite this, detectable fluorescent foci and fluorescent membrane zones in E. coli cells (Fig. 6) indicated that McpT-mCherry and PcaY-mCherry are mostly localized in the membrane and cell poles. Expression of McpT- and PcaY-mCherry was much weaker than that of Tsr-mCherry expressed from the same trg promoter on plasmid pSTV, even in an E. coli host devoid of Tsr, although the average top–10% pixel fluorescence further increased in E. coli Δtsr compared to wild-type (pCRO33) with pcaY-mCherry (Fig. 6C). When assuming that the localisation of McpT and PcaY is analogous to their -mCherry counterparts, these results are a further sign that their folding or membrane oligomerization is not optimal for E. coli. The relatively small proportion of cells with visible McpT-mCherry foci (~1%, Fig. 5C) might be an indication that only a small subpopulation of E. coli actually is responsive to toluene, which would explain the relatively poor overall accumulation of motile cells in suspensions. Our results are thus in agreement with previous studies demonstrating successful heterologous expression in E. coli of other non-native type 40H–receptors, such as the PctA serine-receptor of P. putida (13) or the nitrate energy-taxis MCPs from S. oneidensis (11). Expression of the PctA receptor from a salicylate-inducible system yielded at least partially properly protein-protein interactions to E. coli CheA and CheY, although cell accumulation was only observed at 10 mM serine (13). Type 40H chemoreceptors thus seem to connect to the E. coli chemotaxis signaling pathways, but with lesser efficiency in attractant-biased motility.
One of the issues when studying heterologous chemoreceptor expression in E. coli and its correspondingly weaker or different chemotactic behaviour, is the poor sensitivity and reproducibility of most traditional chemotaxis assays, such as capillary assays, swimming plates or source accumulation assays. We showed here how cell accumulation to chemoattractants at concentrations of the order of 100 μM concentrations can be more accurately quantified from microscopic agarose plug assays with standard errors in the order of 5% of the mean (Fig. 1). Cell accumulation in the wells of the ISCA device across five replicates was slightly more variable (standard error ~15% of the mean), which is most likely due to the lower concentration of cells used (2–4×106 versus 109 cells ml−1), or, possibly, to small differences in the geometry of the wells or fluid motion while incubating the cell suspension. We noted additional effects on the outcome of the ISCA assays related different growth temperatures of the E. coli culture (30°C or 37°C), the cell treatment procedure (washing in motility buffer or not), and assay incubation temperature (preheating culture media). ISCA assays confirmed previous studies that benzoate is a repellent for E. coli and Salmonella (29), but only at concentrations above 300 μM (Fig. 4). Toluene acts as repellant for wild-type E. coli MG1655 already in the range of 60–200 μM (Fig. 4A). At lower benzoate concentrations E. coli MG1655 chemotaxis is not significantly perturbed, and cells expressing the PcaY receptor showed a net positive attraction to 300 μM benzoate (1.5–2.0 fold when compared to accumulation of E. coli MG1655 on 300 μM benzoate).
We conclude, albeit carefully, that the E. coli chemoattractant repertoire can be expanded to aromatic compounds by heterologous expression of P. putida type 40H chemoreceptors. It could potentially be interesting to use E. coli chemotaxis for quantitative sensing of chemicals, because of the relatively rapid response time (5–30 min in microfluidic assays, Ref. (3)) and its potentially narrower detection selectivity than the original host bacteria. For instance, P. putida encodes 20 potential chemoreceptors with partially overlapping chemoattractants (9, 10) compared to E. coli with only five. Quantification of cell accumulation as a function of chemoattractant concentration may further be improved by using microfluidic platforms in which stable chemical gradients can be produced, as we have recently shown (3). However, if heterologously expressed chemoreceptors in E. coli are to be used for quantitative sensing of chemoattractants, their compatibility with the existing E. coli chemotaxis machinery has to be improved. For that matter, an alternative and successful approach recently showed that functional hybrid receptors can be expressed in E. coli by fusing non-cognate ligand-binding domains to the signaling domain of its chemoreceptors. Reactions to ligands can be measured by Föster–resonance energy transfer between CheY and CheZ (30, 31). The existing E. coli chemotaxis machinery can thus be expanded both by hybrid as well as heterologous chemoreceptors and could pave the way for future faster and selective biosensors.
MATERIALS AND METHODS
Cloning procedures
The gene for the methyl-accepting chemotaxis receptor (mcpT) was amplified from P. putida MT53 pWW53 (23) (Table 1) genomic DNA by using Q5 proofreading polymerase (New England Biolabs) and primers 141001 and 141002 (Table S1). The forward primer 141001 contained a BamHI restriction site and the reverse primer 141002 was elongated with a ClaI restriction site. The PCR fragment was cloned into pGEM-t-Easy® (Promega) and the insert was verified by sequencing (Fig. S3). The trg promoter of the E. coli chemoreceptor gene for Trg was amplified from E. coli MG1655 genomic DNA using a Q5 proofreading polymerase and primers 150613, elongated with a BamHI site, and 150612, with a SacI restriction site and including the 17-bp 5’-part of mcpT until its internal NheI site (Fig. S3). The trg promoter fragment was inserted upstream of the mcpT sequence in pGEM-t-Easy® by digestion with BamHI and NheI, taking advantage of mcpT-internal NheI site (Fig. S2). The correct fragment was finally inserted into pSTV28 by digestion with SacI and ClaI, and the plasmid was renamed pCRO20 (Table 1, Fig. S2, S3). This plasmid was inserted into a verified motile strain of E. coli MG1655 (E. coli Genetic Center, Yale, CGSC#8237, Table 1).
A frameshift mutation was introduced in mcpT to disrupt its coding sequence, by digestion of pCRO20 with NsiI, removal of the 3’-overhangs by treatment for 20 min at 12 °C with T4 DNA polymerase (New England Biolabs), and recircularization of the plasmid with T4 DNA ligase. After transformation, this plasmid was renamed pCRO35 (Table 1, Fig. S2). A mcpT-mcherry fusion was produced by amplification of a ‘linker-mcherry’ fragment from plasmid pBAM-link-mcherry (32) using primers 170239 and 170240 elongated with a BglII restriction site and the C-terminal part of mcpT until the internal MfeI site, respectively (Table S1, Fig. S2). The ‘link-mcherry’ fragment was inserted in pCRO20 by digestion with BglII and MfeI. This plasmid was renamed pCRO36 (Table 1, Fig. S2).
The receptor gene for PcaY from P. putida F1 was also cloned under the control of the trg promoter on pSTV28. Its coding sequence (Pput2149) was amplified from P. putida F1 genomic DNA using primers 160306 and 160307, whereas the trg promoter was amplified using primers 150613 and 160305 (Table S1). Both PCR fragments were fused by sewing PCR and cloned back into pSTV28 by digestion with SacI and ClaI. This plasmid was renamed pCRO33 (Table 1, Fig. S2). To fuse the pcaY with mcherry reading frame, pCRO33 was digested with ClaI and EcoRI and the backbone was recovered. The trg-promoter-pcaY fragment was reamplified and combined with the ‘link-mcherry’ fragment by sewing PCR, using primers 170931 and 170932. This fragment was then reinserted into the pCRO33-ClaI-EcoRI backbone using In-Fusion HD cloning (Takara).
The tsr coding sequence was amplified from E. coli MG1655 genomic DNA using primer 160309 and 160310, whereas the trg promoter fragment was amplified using primers 150613 and 160308 (Table S1). Both fragments were fused by sewing PCR and subcloned into pGEM-t-Easy®. The complete part was then recovered and introduced into pSTV28 by digestion with SacI and PstI (localized in pGEM-t-Easy®). This plasmid was renamed pCRO34 (Table 1).
A tsr-mcherry fusion was produced by amplification of the ‘link-mcherry’ fragment from pBAM-link-mcherry using primers 101003 and 101004, and a ‘Ptrg-tsr’ fragment from pCRO34 using primers 070418 and 160308 (sTable S1). Both fragments were fused by sewing PCR, subcloned into pGEM-t-Easy® and cloned back into pCRO34 by digestion with SacI and SpeI. This plasmid was named pCRO38 (Table 1). Relevant plasmids were then further transformed into E. coli MG1655-Δtsr (strain 5396) with a complete deletion of tsr by double recombination.
Preparation of E. coli cultures for chemoattraction assays
E. coli strains were grown overnight at 37°C with 180 rpm shaking in M9 minimal medium supplemented with 4 g l-1 of glucose, 1 g l-1 of Bacto™ casamino acids (BD difco), Hutner’s trace metals (33), 1 mM of MgSO4 and 30 μg ml-1 of chloramphenicol (hereafter called M9-Glc-Cm30). The cultures were diluted 100-fold in the morning in fresh M9-Glc-Cm30 and incubated for 3 h at 37°C with 180 rpm shaking until they reached exponential phase (culture turbidity at 600 nm of between 0.5 and 0.6). For chemoattraction assays, 1–5 milliliter of culture was centrifuged at 2,400×g for 5 min, the upper 0.9 ml of liquid were carefully removed (note that motile cells do not really sediment), and replenished with 1 mL of motility buffer (motility buffer is 10 mM potassium phosphate, 0.1 mM EDTA, 10 mM lactate, pH 7.0) (34). This procedure was repeated once more and finally the cells were resuspended in 500 μl of motility buffer, yielding a density of ~109 cells ml-1.
For ISCA assays, 5 ml of washed exponentially growing culture in M9-Glc-Cm30 was diluted in 300 ml preheated (37°C) motility buffer to obtain a cell concentration of 2–4 ×106 cells ml-1, and this suspension was used within 30 min. Note that we kept the washing procedure the same between both chemotaxis assays, although we noticed that directly diluting exponentially growing cells in motility buffer (without any centrifugation) increases the proportion of cells responsive to 100 and 300 μM serine in the ISCA assay by almost a factor of ten. This did not measurably influence the cell accumulation to toluene and benzoate.
Preparation of the chemoattractant sources
As positive control for E. coli chemotaxis, 1.4 ml of 2% dissolved agarose (LE, Analytical grade, Promega) solution at 55°C was supplemented with 0.15 ml of 1 mM serine solution in water (final serine concentration = 100 μM). The negative control consisted of 1.8% agarose solution in tap water. Further test sources for E. coli consisted of aspartate, N-Methyl-D-aspartate, D-ribose and D-Galactose with final concentrations of 10 and 100 μM.
To prepare the source of toluene, 1.8 % of agarose was dissolved in tap water and kept at 55°C. 2 ml glass vials with Teflon-lined screw-cap (Supelco Analytical) were filled with 1.6 ml of melted 55°C-warm agarose solution, into which was dissolved 10 μl of pure toluene. The toluene density is 0.87 g mL-1 and its molecular mass is 92.14 g mol-1; therefore, adding 10 μl toluene to 1.6 ml volume is equivalent to 8.7 mg per 1.6 ml = 5.4 mg ml-1. This corresponds to 60 mM. This toluene stock was serially diluted in prewarmed agarose by adding and mixing 0.15 ml of the agarose with the pure toluene source into 1.4 ml of 55°C-warm agarose solutions, and from there to further agarose solutions. The 10−3 dilution is thus equivalent to 60 μM. Toluene stocks were prepared fresh for every experiment.
Sources of benzoate were prepared by 100-fold dilution of a 1 M sodium benzoate stock in 1.8 % 55°C-warm agarose, which corresponds to a concentration of 10 mM benzoate. From here, benzoate was serially diluted in 55°C-warm agarose to obtain stocks of 1 and 0.1 mM. All vials were kept tightly closed in a water bath at 55°C until preparing the chambers. Agarose solutions were prepared fresh for every experiment.
For ISCA assays, the chemoattractants were diluted in motility buffer without agarose.
Chemoattraction assays using agarose plugs on microscope slides
While washing the cell cultures, the microscope source chambers were prepared (Fig. S1). Chambers consisted of a standard microscopy glass slide (Menzel Gläser, Thermo Scientific), onto which two small coverslips (24×24 mm, 0.13–0.17 mm thick, MGF-Slides) were deposited on both sides and maintained in place with ~10 μl of tap water. A drop of 4 μl of 55°C agarose solution with the chemoattractant source (see above) was deposited in the middle and immediately covered by a cleaned large coverslip (24×50 mm, Menzel Gläser) that bridges over the side coverslips and thus creates a chamber with a height of 0.17 mm.
A freshly grown and washed bacterial suspension in motility buffer was inserted around the agarose plug by pipetting 150 μl of cell suspension between the glass slide and the large coverslip. E. coli standard assays with serine and other known chemoattractants were carried out in triplicate in independent chambers. Toluene and benzoate assays were repeated in fourfold replicates (one prepared E. coli culture, four independent chambers) in conjunction with positive (serine) and negative (no attractant added) controls. Toluene assays were further repeated on at least four independent occasions.
Bacterial accumulation was imaged after 15 min incubation at room temperature (20±2 °C) using a DFC 350 FX R2 Leica camera mounted on an inverted DMI 4000 Leica microscope using a N PLAN 10× objective. This timing was based on parallel video-imaging of agarose source assays with a Dino-Lite digital microscope at 50× magnification (AnMo Electronics Corporation, Taiwan) (Video S1). For each replicate, one image was taken at each side of the agarose plug. Images were analyzed with ImageJ software (v. 1.49r, http://imagej.nih.gov/ij). Cells were identified using the “find edges” routine in ImageJ and the accumulated intensity values were quantified per zones of 25 pixels width (corresponding to 2.5 μm) parallel to the plug border (3 zones in the plug and 27 zones outside the plug, Fig. S1). Chemotactic responses were then averaged from four replicates. Intensity values were summed and averaged across the three zones closest to the source edge, and intensity variations among chemoattractants were analyzed in one-way ANOVA statistics.
In situ chemotaxis assay (ISCA)
As an alternative, independent approach to the agarose plug assays, we measured chemotaxis in the ISCA assay (21). An ISCA device consists of a polydimethylsiloxane (PDMS) structure bonded to a glass slide, forming five replicate circular we1ls, each having a volume of ~110 μl that connects to the outside through an acentrically placed, 0.8–mm diameter inlet port. Wells were filled through the inlet port with a chemoattractant solution to the top, with care to leave a small (5 μl) droplet on the surface of the inlet. The ISCA was then placed in a Petri dish, which was very slowly filled with 55 ml of a suspension of E. coli at a density of 2–4×106 cells ml-1 (in motility buffer, preheated at 37°C), until the ISCA was completely submerged. After 35 min of incubation at room temperature (22°C), the external cell suspension was removed by pipetting and the ISCA surface was wiped with a clean tissue. The contents of each ISCA well were collected with a 1 ml syringe and a clean needle, transferred to a 200–μl well of a flat-bottom 96-well culture plate, and mixed with 1 μl of a 1:100 dilution of SYBR Green I for cell staining. Stained cell suspensions were kept on ice until all samples were obtained and then aspired into a Becton Dickinson Flow Cytometer, operated at 30 μl min-1 and counted over 60 sec. From the cell counts (number of cells μl-1) determined by flow cytometry for each ISCA well, we computed the mean and the standard deviation across the five replicate wells. Results presented in Fig. 4 were then obtained by normalizing to the mean cell count obtained with the ISCA for the same strain on the same day over five replicate wells containing only motility buffer (no-chemoattractant control), to quantify the enhancement in cell concentration due to chemotaxis (‘Normalized accumulation’).
Epifluorescence microscopy of fusion proteins
In order to visualize the localisation of McpT-, Tsr- and PcaY-mCherry expressed in E. coli MG1655, strains were precultured with the same protocol as for the agarose plug assays. However, cells were resuspended in 50 μl of motility buffer after the final washing step. A drop of 7 μl of this cell suspension was spotted on a 1% (w/v) agarose (in motility buffer) coated microscopy slide (layer thickness 1 mm) and then covered with a regular 0.17-mm thick glass coverslip. Cells were imaged at an exposure time of 50 ms (phase-contrast) or 750 ms (mCherry) with a Nikon Eclipse Ti-E inverted microscope, equipped with an ORCA-flash4.0 camera (Hamamatsu) and a Plan Apo λ 100×1.45 oil objective (Nikon). Images were recorded in ImageJ, saved as 8-bit grayscale for reproduction, opened and cropped to their final size in Adobe Photoshop (v. CC2017), and finally saved as. TIF with 300 dpi resolution for display. Cells were automatically segmented using SuperSegger and standard E. coli parameter settings (35), and both cellular fluorescence as well as the fluorescence intensities, scores and positions of up to 9 foci in individual cells were extracted. Foci surpassing a focus score of 9 were listed using an in-house MatLab script (version 2016a) and their positions were normalized to a standardized E. coli cell for accumulated display. For expression quantification, cells with outlier mean fluorescence levels (<5% and >95% percentiles) were removed, after which the top–10% pixel intensities per cell were extracted (assuming this would correspond to the mCherry fusion protein positions in foci or fluorescent bands) and averaged per cell, and further normalized by the cell’s mean fluorescence. This list of normalized average top 10% pixels per cell was then multiplied by the average of all mean individual cellular fluorescence values for that strain and incubation, in order to allow for inter-strain expression comparisons. Lists were randomly subsampled in ten individual replicates, the means of which were used for ANOVA comparison among strains, followed by Tukey’s post hoc testing of statistical significance, using the program R.
ACKNOWLEDGMENTS
We thank Vitali Maffenbeier for his help in cloning the PcaY-mCherry fusion construct. This work was supported by the Swiss National Science Foundation NanoTera project 20NA21-143082, by financing from the Herbette Foundation (2018-1-D-26), and by a grant from the Gordon and Betty Moore Foundation (grant #3801 to RS). The authors declare no conflict of interest. We thank the Stocker Lab at the ETH Zürich for advice and training in the ISCA assay.