seudomonas putida

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Jan 7, 2015 - Rita A. Luu,1 Joshua D. Kootstra,1 Vasyl Nesteryuk,1. Ceanne N. Brunton,1 Juanito V. Parales,1. Jayna L. Ditty2 and Rebecca E. Parales1*.
Molecular Microbiology (2015) 96(1), 134–147 ■

doi:10.1111/mmi.12929 First published online 12 February 2015

Integration of chemotaxis, transport and catabolism in Pseudomonas putida and identification of the aromatic acid chemoreceptor PcaY Rita A. Luu,1 Joshua D. Kootstra,1 Vasyl Nesteryuk,1 Ceanne N. Brunton,1 Juanito V. Parales,1 Jayna L. Ditty2 and Rebecca E. Parales1* 1 Department of Microbiology and Molecular Genetics, College of Biological Sciences, University of California, Davis, CA, USA. 2 Department of Biology, University of St. Thomas, St. Paul, MN, USA.

Summary Aromatic and hydroaromatic compounds that are metabolized through the β-ketoadipate catabolic pathway serve as chemoattractants for Pseudomonas putida F1. A screen of P. putida F1 mutants, each lacking one of the genes encoding the 18 putative methyl-accepting chemotaxis proteins (MCPs), revealed that pcaY encodes the MCP required for metabolism–independent chemotaxis to vanillate, vanillin, 4-hydroxybenzoate, benzoate, protocatechuate, quinate, shikimate, as well as 10 substituted benzoates that do not serve as growth substrates for P. putida F1. Chemotaxis was induced during growth on aromatic compounds, and an analysis of a pcaYlacZ fusion revealed that pcaY is expressed in the presence of β-ketoadipate, a common intermediate in the pathway. pcaY expression also required the transcriptional activator PcaR, indicating that pcaY is a member of the pca regulon, which includes three unlinked gene clusters that encode five enzymes required for the conversion of 4-hydroxybenzoate to tricarboxylic acid cycle intermediates as well as the major facilitator superfamily transport protein PcaK. The 4-hydroxybenzoate permease PcaK was shown to modulate the chemotactic response by facilitating the uptake of 4-hydroxybenzoate, which leads to the accumulation of β-ketoadipate, thereby increasing pcaY expression. The results show that chemotaxis, transport and metabolism of aromatic compounds are intimately linked in P. putida. Accepted 7 January, 2015. *For correspondence. E-mail reparales@ ucdavis.edu; Tel. (+1) 530754 5233; Fax (+1) 530752 9014.

© 2015 John Wiley & Sons Ltd

Introduction Aromatic compounds are abundant in the environment as they comprise a major component of lignin and other plant-derived metabolites, and are commonly present as industrial pollutants (Kirk and Farrell, 1987; Samanta et al., 2002). The turnover of natural aromatic compounds in biogeochemical cycles and the need for remediation of polluted sites contaminated with aromatic compounds have led to the extensive study of bacterial biodegradation pathways for these compounds. Pathways for the aerobic dissimilation of aromatic compounds have been systematically studied, especially in Pseudomonas species and other closely related organisms (Harwood and Parales, 1996; Cao et al., 2009). Pseudomonas putida and other degraders of aromatic compounds such as Comamomas, Agrobacterium and some rhizobial species have been shown to exhibit chemotaxis toward aromatic compounds (Harwood et al., 1984; Parke et al., 1985; 1987; Lopez de Victoria and Lovell, 1993; Lacal et al., 2011; Ni et al., 2013). These and other motile bacteria are equipped with a conserved chemosensory system comprised of cell surface chemoreceptors, which are typically membrane-bound methyl-accepting chemotaxis proteins (MCPs), an adaptor protein (CheW), and the sensor kinase and response regulator CheA and CheY (Wadhams and Armitage, 2004). The binding of attractants or repellents to MCPs triggers a signaling cascade via the Che proteins to modulate flagellar rotation, effectively directing cell movement. The number of chemoreceptors varies widely among microbial species and may reflect the particular lifestyle and metabolic diversity of the organism (Lacal et al., 2010). While Escherichia coli is equipped with four MCPs and one MCP-like energy taxis receptor (Parkinson et al., 2005), metabolically versatile soil bacteria such as Pseudomonas species contain ≥ 25 MCP-like proteins (Parales et al., 2004; Sampedro et al., 2014). To date, the specific functions of relatively few Pseudomonas MCPs have been characterized (Sampedro et al., 2014). Although chemotaxis to aromatic acids by P. putida PRS2000 was first reported 30 years ago (Harwood et al., 1984), the mechanism by which these compounds are

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sensed has remained unclear. Chemotaxis to the aromatic acids benzoate and 4-hydroxybenzoate (4-HBA), as well as the nonmetabolizable compounds salicylate and chloro-, nitro-, amino- and methylbenzoates (toluates), was induced when PRS2000 cells were grown on aromatic acids. The inducer was identified as β-ketoadipate (β-KA) (Harwood et al., 1984; 1990; Parales, 2004), an intermediate of the pathway bearing its name. The widely distributed, and generally chromosomally encoded, β-KA pathway serves as a major funneling pathway for the aerobic degradation of a wide variety of aromatic compounds (Harwood and Parales, 1996). It consists of two parallel branches in which aromatic compounds are funneled to either protocatechuate (PCA) or catechol, which are subsequently converted to the common intermediate β-KA, and eventually to compounds that feed into the tricarboxylic acid (TCA) cycle (Harwood and Parales, 1996). In P. putida PRS2000, the transcriptional regulator PcaR, in response to β-KA, activates genes in the pca regulon, which are located in three unlinked clusters (one of which harbors the pcaR gene) in the P. putida genome (Parales and Harwood, 1993b; Romero-Steiner et al., 1994; Nichols and Harwood, 1995). A previous study revealed that an ∼ 60 kDa protein in P. putida PRS2000 was methylated in response to aromatic attractants, suggesting that an MCP was involved in the chemotactic response (Harwood, 1989). However, a genetic screen for mutant strains defective in chemotaxis to aromatic acids identified an inactivated pcaK gene, which encodes a member of the major facilitator superfamily (MFS) of transport proteins (Harwood et al., 1994). Several studies demonstrated a dual function for PcaK in P. putida PRS2000, mediating uptake of 4-HBA and PCA, as well as playing a role in chemotaxis to benzoate and 4-HBA (Harwood et al., 1994; Nichols and Harwood, 1997; Ditty and Harwood, 1999; 2002). It was proposed that PcaK functions directly as a new type of chemoreceptor that somehow interacts with a canonical membrane-bound MCP to transmit the signal (Harwood et al., 1994). Another possibility was that once transported inside the cell, aromatic acids were detected by a cytoplasmic MCP (Ditty and Harwood, 1999). However, the molecular mechanism of PcaK’s involvement in aromatic acid chemotaxis was never elucidated, and the participating MCP was not found. With the availability of a complete genome sequence and a previously constructed set of chemoreceptor mutants, we were able to investigate more thoroughly the chemotactic response to aromatic acids in a related bacterial strain, P. putida F1. The P. putida F1 genome encodes 18 putative MCPs (Liu, 2009; Parales et al., 2013) with predicted structures similar to those of the canonical MCPs in E. coli: a periplasmic ligand binding domain flanked by two transmembrane domains and a cytoplasmic signaling domain (Wadhams and Armitage, 2004). It also © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

encodes nine MCP-like proteins, one of which has been identified as the energy taxis receptor Aer2 (Luu et al., 2013). In this study, we identified a single MCP in P. putida F1 that is responsible for the detection of multiple aromatic and hydroaromatic chemicals that feed into the β-KA pathway. This MCP also senses several nonmetabolizable aromatic compounds. Our investigation also identified the role of PcaK in chemotaxis and revealed an integrated response that involves direct links between aromatic acid transport, chemotaxis and catabolism.

Results Aromatic compounds that funnel into the β-KA pathway induce a chemotactic response in P. putida F1 Qualitative capillary assays were used to test whether aromatic compounds that funnel into the β-KA pathway (Fig. 1A) are detected as chemoattractants by P. putida F1. To assess whether the response required induction, cells were grown on pyruvate (uninduced) and on the test attractants (induced), with the exception of PCA; for these assays, cells were grown on the PCA precursor, 4-HBA. Qualitative capillary assays revealed that induced P. putida F1 cells responded to the aromatic compounds vanillin, vanillate, 4-HBA, benzoate and PCA, as shown by the accumulation of cells at the tip of the capillary (Fig. 1B). In contrast, uninduced cells did not respond to any of the aromatic compounds, although assays with Casamino acids as a positive control showed that all strains were motile and chemotactic to amino acids (Fig. 1B and data not shown). In addition, individual cultures grown on vanillin, vanillate, 4-HBA or benzoate exhibited chemotaxis to all five aromatic attractants (data not shown), suggesting a common induction mechanism and possibly a common chemoreceptor.

Identification of the chemoreceptor for vanillate As described previously for P. putida and Pseudomonas aeruginosa, aerotaxis can mask chemotaxis defects in soft agar swim plate assays that are based on catabolism of the chemoeffector of interest (Alvarez-Ortega and Harwood, 2007; Parales et al., 2013). Therefore, to identify the chemoreceptor for aromatic compounds, we screened a series of double mutants each lacking the energy taxis receptor Aer2 and one of the 18 putative MCP genes in P. putida F1 (Liu, 2009; Parales et al., 2013) using softagar swim plates containing 1 mM benzoate, 1 mM 4-HBA or 1 mM vanillate. A noticeably reduced response to vanillate was observed for a double mutant (strain GC006), in which both aer2 and the gene at locus tag Pput_2149 were deleted, relative to the strain lacking only aer2 (XLF010; Fig. 2A). Quantitative analysis of the responses on vanil-

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Fig. 1. Identification of aromatic attractants for P. putida F1. A. Pathway for the metabolism of selected aromatic and hydroaromatic compounds through the β-KA pathway in P. putida F1 based on growth studies (data not shown) and pathways predicted from genome comparison with the annotated P. putida KT2440 genome (Jiménez et al., 2002). Genes in the pca regulon are indicated at the relevant steps in the pathway. Genes shown in boxes were deleted in this study. B. Qualitative capillary assays of wild-type P. putida F1 grown on pyruvate (uninduced) or test attractants (induced), with the exception of the induced response to protocatechuate (PCA)*, which was tested following growth on 4-HBA. Chemotactic responses to Casamino acids (positive control) and buffer (negative control) are shown for pyruvate-grown cells. All cultures displayed similar responses to positive and negative controls. Images shown are representatives of at least triplicate experiments. All photographs were taken at 7 min.

late swim plates showed that the diameter of the chemotaxis ring of the strain lacking both aer2 and Pput_2149 was significantly smaller than that of the Δaer2 mutant (77 ± 7%, n = 9; p < 0.05, Student’s t-test). For reasons that will become clear below, the MCP encoded at locus tag Pput_2149 was designated PcaY.

Vanillate is sensed directly To rule out a role for energy taxis in sensing vanillate, we generated a catabolic mutant that was unable to grow on vanillate. Vanillate demethylase, encoded by vanAB (Fig. 1A), has been shown to catalyze the first step in vanillate catabolism in Pseudomonas sp. strain ATCC 19151 (Brunel and Davison, 1988). The genes in P. putida F1 at locus tags Pput_2027 and Pput_2026 were found to be 78% and 68% identical in nucleotide sequence to the vanA and vanB genes, respectively, which encode the demethylase in Pseudomonas sp. strain ATCC 19151. The ΔvanAB mutant (RJF1) was unable to grow on vanillate, and complementation with vanAB in trans restored growth on these substrates (data not shown). Qualitative capillary

Fig. 2. Identification of the vanillate chemoreceptor. A. Response of the P. putida F1 Δaer2 mutant (XLF019) and the Δaer2 ΔPput_2149 double mutant (GC006) in soft agar swim plates containing 1 mM vanillate. Plates were photographed after approximately 24 h of incubation at 30°C, and representative images of at least triplicate experiments are shown. Growth studies of both strains yielded similar growth rates in MSB medium with 5 mM vanillate (data not shown) indicating that the reduced colony size was solely a chemotaxis defect. The gene at locus tag Pput_2149 was subsequently designated pcaY. B. Qualitative capillary assays showing the response to 10 mM vanillate by wild-type P. putida F1 and a ΔvanAB mutant (RJF1) after growth on 5 mM 4-HBA. Images shown are representatives of at least triplicate experiments. All photographs were taken at 7 min. © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

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Fig. 3. Qualitative and quantitative capillary assays showing chemotactic responses to aromatic acids. Qualitative capillary assays showing the responses of wild-type [F1(pRK415Km)], ΔpcaY mutant [XLF010(pRK415Km)] and the complemented mutant [XLF010(pXLF210)] to (A) vanillate, 4-HBA, benzoate, PCA and vanillin; and (B) quinate and shikimate. Strains were grown in MSB containing 5 mM of the respective test attractant with the exception of PCA and shikimate; cells used in these assays were grown with 5 mM 4-HBA and quinate respectively. Attractants were provided at a concentration of 10 mM in capillaries. Images shown are representatives of at least triplicate experiments. All photographs were taken at 7 min. C. Concentration response curves for wild-type F1 in response to 4-HBA and quinate. The average number of cells in capillaries containing buffer only (120 ± 15) was subtracted from each data set. D. Responses of wild-type F1 and ΔpcaY mutant (XLF010) to 1 mM vanillate, 4-HBA, benzoate, PCA, vanillin, quinate and shikimate. The average numbers of cells in capillaries containing buffer only (120 ± 15 for wild type; 330 ± 120 for XLF010) were subtracted from each data set. Wild-type and the ΔpcaY mutant responded equally well to 0.2% Casamino acids (17,800 ± 3,300 and 19,700 ± 280 cells per capillary respectively). For data in C and D, results represent averages of at least three independent experiments (≥ 3 capillaries), and error bars represent the standard error of the mean.

assays with 4-HBA-grown cells revealed that the ΔvanAB mutant had a wild-type response to vanillate (Fig. 2B). These results demonstrate that the response to vanillate is metabolism independent, suggesting that vanillate itself is directly detected by a chemoreceptor. Since energy taxis does not appear to mediate the chemotactic response, strains with the Δaer2 mutation were not used in the following experiments. PcaY is also responsible for the detection of 4-HBA, benzoate, PCA and vanillin The role of PcaY in the detection of vanillate was confirmed using qualitative capillary assays. The pcaY mutant did not respond to vanillate, whereas the complemented strain exhibited a response similar to that of wild type (Fig. 3A). To © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

examine whether the product of pcaY is a chemoreceptor for other aromatic compounds, we tested the chemotactic responses of the wild type, ΔpcaY mutant, and the complemented ΔpcaY mutant to 4-HBA, benzoate, PCA and vanillin. The pcaY mutant [XLF010(pRK415Km)] showed markedly reduced responses to all compounds, and complementation [XLF010(pXLF210)] restored the wild-type responses (Fig. 3A). To test the sensitivity of the response to aromatic attractants, quantitative capillary assays were used to measure the response to a range of concentrations (0.01–10 mM) of 4-HBA and quinate. The peak response of P. putida F1 to both 4-HBA and quinate was observed at 1 mM, and the minimum concentrations detected were 100 and 10 μM respectively (Fig. 3C). The responses of wild-type F1 and the ΔpcaY mutant (XLF010) to 1 mM vanillate, 4-HBA, benzoate, PCA and vanillin were also

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measured using the quantitative assay. Compared with wild-type F1, the response of the ΔpcaY mutant to all five compounds was dramatically decreased (Fig. 3D). Although the mutant strain lacking pcaY still had slight residual responses to 4-HBA and PCA (Fig. 3A and D), the results nevertheless indicate that PcaY is the primary MCP responsible for detecting all five aromatic compounds. At this time, we do not know if these weak responses are due to the presence of one or more MCPs with overlapping specificity and/or a minor contribution of energy taxis. PcaY is required for chemotaxis to hydroaromatic compounds that funnel into the β-KA pathway Interestingly, hydroaromatic compounds that are degraded via the β-KA pathway were also shown to serve as attractants for P. putida F1. The cyclohexanes quinate and shikimate elicited chemotactic responses when the cells were induced by growth on these compounds (data not shown). The responses were eliminated when pcaY was deleted (Fig. 3B and D), and complementation with pcaY restored the wild-type response (Fig. 3B). Based on these findings, it appears that PcaY detects a broad range of compounds that are funneled through the β-KA pathway, and the receptor is not limited specifically to aromatic compounds (Fig. 1A). The catabolic pathways for quinate and shikimate degradation have not been elucidated in P. putida; therefore, catabolic mutants are not available, and we cannot rule out the possibility that downstream intermediates (i.e. 4-HBA or PCA) are sensed rather than the hydroaromatic compounds themselves. PcaY detects structurally related aromatic compounds To identify the range of aromatic compounds detected by PcaY, we broadened our screen to include aromatic acids that are not metabolized by P. putida F1, including salicylate, chloro-, nitro- and aminobenzoates, and 3- and 4-toluate. When cells were grown with either 4-HBA or benzoate, P. putida F1(pRK415Km) visibly responded to all compounds except 2-nitrobenzoate and 2chlorobenzoate (Fig. 4). Although responses to these two compounds were not detected with this vector-bearing strain, wild-type cells lacking the vector (which are generally more motile) displayed a very weak response to both compounds (data not shown). The responses to all compounds were diminished or eliminated in the ΔpcaY mutant, and when pcaY was provided in trans, responses were restored and in some cases appeared stronger (Fig. 4). The responses of wild-type and the ΔpcaY mutant to 4-aminobenzoate were verified using quantitative capillary assays. After correcting for the average numbers of cells in capillaries containing buffer only (120 ± 15 for wild-type and 330 ± 120 for the ΔpcaY mutant),

Fig. 4. Chemotactic responses to nonmetabolizable aromatic acids. The responses of wild-type [F1(pRK415Km)], ΔpcaY mutant [XLF010(pRK415Km)] and the complemented mutant [XLF010(pXLF210)] to 50 mM salicylate, chloro-, nitro-, amino- and methylbenzoates, in qualitative capillary assays. Strains were grown in MSB containing either 5 mM 4-HBA or benzoate. Images are representative of at least triplicate experiments, and all photographs were taken at 7 min. ABA, aminobenzoate; CBA, chlorobenzoate; NBA, nitrobenzoate.

© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

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Fig. 5. Expression of the pcaY-lacZ transcriptional fusion as represented by β-galactosidase activity. A. Activity in wild-type F1 in the absence of added inducer (none) or the presence of 5 mM 4-HBA, benzoate, vanillate or vanillin, and the ΔvanAB, ΔpcaD catabolic mutants and the complemented ΔpcaD mutant in the absence and presence of vanillate and/or 4-HBA. B. Induction of pcaY-lacZ by the β-KA analog adipate (20 mM) in wild type [F1(pRK415Km)], ΔpcaR mutant [RJF2(pRK415Km)] and complemented mutant [RJF2(pRJF2)]. Activities of least three independent experiments were measured in triplicate and error bars represent standard deviations.

6770 ± 1000 wild-type and 1090 ± 360 ΔpcaY mutant cells accumulated in capillaries containing 1 mM 4aminobenzoate. These results demonstrate that PcaY has broad specificity for a wide range of structurally related aromatic acids and provide further evidence that aromatic compounds are detected directly, rather than as degradation intermediates, since these compounds are not metabolized by P. putida F1. pcaY expression is induced by an intermediate in the β-KA pathway We showed above that the chemotactic response to aromatic compounds in P. putida F1 is inducible. To examine whether pcaY is the target of induction, a pcaY-lacZ transcriptional fusion was constructed, and β-galactosidase activity was examined after growth in the presence of 4-HBA, benzoate, vanillate and vanillin compared with pyruvate-grown cells. Cultures grown with each of the aromatic compounds had significantly higher (5- to 12-fold) β-galactosidase activity than pyruvate-grown cultures (Fig. 5A), which is consistent with our finding that the chemotactic response is induced during growth on each of the aromatic compounds. These results suggest that a common intermediate in the β-KA pathway is responsible for the induction of pcaY. We therefore tested the expression of the pcaY-lacZ fusion in the vanAB deletion mutant strain as this strain is unable to convert vanillate to downstream metabolites. β-Galactosidase activity in the ΔvanAB mutant grown in the presence of vanillate was similar to that of pyruvategrown cells; however, induction was seen when cells were © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

grown in the presence of 4-HBA (Fig. 5A). Complementation restored the level of β-galactosidase activity to that of wild-type induced cells (data not shown), indicating that a common metabolite downstream of vanillate serves as the actual inducer of pcaY. In P. putida PRS2000, the pca genes encode enzymes for the conversion of PCA to TCA cycle intermediates, and three of the Pca enzymes are shared between the PCA and catechol branches of the β-KA pathway (Fig. 1A). As just described, the pca genes are regulated by the transcriptional activator PcaR in response to β-KA (Harwood and Parales, 1996). Since PcaY is involved in the detection of compounds that funnel into this pathway, we examined whether β-KA is the inducer of pcaY. Adipate has been used as a stable nonmetabolizable analog of β-KA (Parke and Ornston, 1976) and has been shown to induce the pca genes as well as the chemotactic response to benzoate in P. putida PRS2000 (Harwood et al., 1990; Parales and Harwood, 1993b). When adipate was provided to pyruvate-grown P. putida F1 cells, a threefold increase in β-galactosidase activity from the pcaY-lacZ fusion was observed (Fig. 5B). The permeability of P. putida PRS2000 to adipate is limited (Parke and Ornston, 1976); low permeability of P. putida F1 to this compound may explain the lower fold induction in response to adipate compared with induction with aromatic compounds. To confirm that β-KA induces expression of pcaY, we generated a mutant that was blocked in the formation of β-KA. Strain RCF1 lacks a functional pcaD gene (locus tag Pput_4343), which in P. putida PRS2000 encodes β-KA enol-lactonase (Hughes et al., 1988), the enzyme that catalyzes the conversion of β-KA enol-lactone to β-KA

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(Fig. 1A). We tested the effect of a pcaD deletion on pcaY-lacZ expression. When the pcaD mutant was induced with 4-HBA, the β-galactosidase activity remained similar to that of pyruvate-grown (uninduced) wild-type cells (Fig. 5A). When provided in trans, the pcaD gene was able to complement the ΔpcaD mutant and resulted in β-galactosidase levels similar to those of wild-type cells (Fig. 5A). Together, these results indicate that β-KA is the actual inducer of pcaY.

pcaY expression is regulated by the transcriptional activator PcaR Because our results indicated that expression of pcaY is induced by β-KA, we tested whether the transcriptional activator of the pca regulon in PRS2000, PcaR (Parales and Harwood, 1993b; Romero-Steiner et al., 1994; Nichols and Harwood, 1995), was required for induction of pcaY. The gene encoding a predicted PcaR ortholog (locus tag Pput_4348; the deduced product shares 88% amino acid sequence identity with PcaR from PRS2000) was deleted. As expected, the ΔpcaR mutant was unable to grow on benzoate or 4-HBA. The strain was then tested for the ability to induce pcaY-lacZ expression. When the ΔpcaR mutant was grown on pyruvate in the presence of adipate, β-galactosidase activity was comparable with that of pyruvate-grown cells (Fig. 5B). However, when pcaR was used to complement the mutant, the addition of adipate resulted in fourfold induction, indicating that PcaR is required for transcriptional activation of pcaY. PcaR was also shown to be required for the induced chemotactic response to aromatic acids as the response of the pcaR mutant grown in the presence of pyruvate and adipate was no stronger than that of uninduced wild-type or the induced pcaY mutant cells (data not shown). These results demonstrate that the chemoreceptor gene is a member of the pca regulon, hence its designation, pcaY.

The role of the MFS transporter PcaK in chemotaxis to aromatic compounds revealed PcaK, a member of the MFS class of transporters (Pao et al., 1998), was previously shown to be required for chemotaxis to 4-HBA and other structurally related aromatic acids in P. putida PRS2000, but its specific function in chemotaxis remained unresolved (Harwood et al., 1994; Ditty and Harwood, 1999). A pcaK ortholog (locus tag Pput_4347) whose deduced amino acid sequence shares 98% identity with the PRS2000 PcaK was identified in P. putida F1. Similar to P. putida PRS2000 (Nichols and Harwood, 1997), the growth rate of a P. putida F1 ΔpcaK mutant on 4-HBA at pH 8.1 was significantly slower than at neutral pH (Fig. 6A), indicating that the transport

Fig. 6. Characterization of the 4-HBA transport mutant ΔpcaK. A. Growth of wild type (F1) and ΔpcaK (RCF2) in MSB with 5 mM 4-HBA at pH 7.3 and pH 8.1. Error bars represent standard deviations from three independent experiments. Means with different letters are significantly different. p < 0.05, one-way analysis of variance (ANOVA) interaction, Tukey multiple comparison test. B. Chemotactic responses of wild type [F1(pRK415Km)], ΔpcaK mutant [RCF2(pRK415Km)], complemented ΔpcaK mutant [RCF2(pRCF2)] and the ΔpcaK mutant expressing pcaY [RCF2(pXLF210)] to 4-HBA after growth on MSB with 40 mM pyruvate and 5 mM 4-HBA at pH 8.1. C. Chemotactic responses of uninduced (pyruvate grown; pH 7.3) wild type [F1(pRK415Km)], ΔpcaK mutant [RCF2(pRCF2)] and the ΔpcaK mutant expressing pcaY [RCF2(pXLF210)] to 4-HBA. D. Chemotactic responses of wild-type [F1(pRK415Km)] and ΔpcaK [RCF2(pRK415Km)] to 4-HBA after growth on MSB containing 40 mM pyruvate and 5 mM benzoate at pH 8.1. Images shown are representatives of at least triplicate experiments, and all photographs were taken at 7 min.

function of PcaK is critical at high pH when less 4-HBA is in the protonated form. To determine whether a defect in the uptake of 4-HBA influences chemotaxis, capillary assays with wild-type F1(pRK415Km), the ΔpcaK mutant RCF2(pRK415Km) and the complemented ΔpcaK mutant RCF2(pRCF2) were tested for their responses to 4-HBA at pH 8.1. After growth in the presence of 4-HBA, the pcaK deletion mutant was no longer responsive to 4-HBA; however, the complemented strain had a response similar to that of wild type (Fig. 6B). In addition, a ΔpcaK mutant that was provided pcaY in trans responded to 4-HBA © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

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(Fig. 6B), suggesting that the inability of the ΔpcaK mutant to respond to 4-HBA was due to the absence of PcaY. We also compared the uninduced responses of the ΔpcaK mutant in which pcaK or pcaY was constitutively expressed from a plasmid promoter. As expected, wildtype F1(pRK415Km) did not respond to 4-HBA after growth on pyruvate, and the complemented pcaK mutant RCF2(pRCF2) also failed to respond. However, the uninduced pcaK mutant that constitutively expressed pcaY [RCF2(pXLF210)] exhibited a strong chemotactic response to 4-HBA (Fig. 6C). These results demonstrate that the expression of pcaY in the 4-HBA transport mutant was sufficient to generate a chemotactic response similar to the induced wild-type response, and indicate that PcaK is not in and of itself a chemoreceptor. PcaK facilitates the induction of pcaY expression Taken together, our results suggest that inefficient uptake of 4-HBA results in an inadequate intracellular accumulation of β-KA, the inducer of pcaY, thereby affecting chemotaxis. To examine this further, we tested the βgalactosidase activity of a ΔpcaK mutant carrying the pcaY-lacZ fusion at pH 8.1. At this pH, β-galactosidase activity was almost twofold lower in the ΔpcaK mutant compared with wild type (7480 ± 170 vs. 13,340 ± 1200 Miller units respectively). To further demonstrate that the chemotaxis defect is due to the inability to induce pcaY when transport of 4-HBA is limited, the ΔpcaK mutant was grown on benzoate, as benzoate uptake should not be affected in this mutant (Harwood et al., 1994), and sufficient amounts of β-KA should be generated to induce pcaY expression. As expected, the chemotactic response of benzoate-grown cells to 4-HBA was unaffected at pH 8.1 (Fig. 6D).

Discussion In this study, we identified the MCP-encoding gene pcaY as a member of the pca regulon, which includes genes encoding five enzymes of the PCA branch of the β-KA pathway [pcaBCD, pcaIJ and pcaF (Harwood and Parales, 1996)], and the MFS transporter of 4-HBA and PCA [pcaK (Nichols and Harwood, 1995)]. The pcaY gene is predicted to encode a canonical 541-amino acid MCP with two transmembrane domains flanking a periplasmic ligand binding domain (LBR), a HAMP domain and a cytoplasmic signaling domain (Falke and Hazelbauer, 2001). The LBR is 156 amino acids in length, placing it in Cluster 1 as defined by Lacal et al. (2010); it is one of six Cluster 1 MCPs in P. putida F1. The only other functionally characterized Cluster 1 MCP is McfR, which senses succinate, fumarate and malate (Parales et al., 2013). A BLAST search for PcaY homologs revealed that PcaY is widespread in Pseu© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

domonas species and is conserved in P. putida strains. A BLAST search identified putative orthologs sharing 99% amino acid sequence identity with PcaY in strains that have been previously shown to be chemotactic to aromatic compounds, including P. putida DOT-T1E and P. putida KT2440 (Lacal et al., 2011). It is therefore likely that many soil pseudomonads utilize PcaY orthologs to sense aromatic compounds. PcaY is the newest addition to the small group of MCPs known to detect aromatic compounds. NahY was shown to sense naphthalene in P. putida G7 (Grimm and Harwood, 1999), NbaY senses 2-nitrobenzoate in Pseudomonas fluorescens KU-7 (Iwaki et al., 2007), McpT detects toluene and related compounds in P. putida DOT-T1E (Lacal et al., 2011), and CtpL senses 4-chloroaniline and catechol in P. aeruginosa PAO1 (Vangnai et al., 2013). The LBRs of all of these aromatic compound receptors are in Cluster 1 (Lacal et al., 2010), except for CtpL, which is in Cluster 2. Amino acid sequence comparisons of the LBRs revealed minimal sequence conservation, with the highest identity between PcaY and McpT (16%). Low sequence identities between aromatic compound receptors with similar ligand binding profiles (McpT and NahY) have been previously reported (Lacal et al., 2012). Unfortunately, the large sequence divergence among LBRs poses a challenge in using sequence comparisons to predict attractant profiles. The response of P. putida F1 to nonmetabolizable aromatic acids and the ability of a catabolic mutant to detect vanillate indicate that aromatic acids are directly detected in P. putida F1. We expect that PcaY binds aromatic acids directly, but we cannot rule out the participation of a periplasmic binding protein, such as those involved in chemotaxis to sugars in E. coli (Wadhams and Armitage, 2004). In contrast to the metabolism-independent sensing strategy of P. putida F1, some bacteria use alternative strategies for the detection of aromatic compounds. A recent study in Comamonas testosteroni CNB-1 revealed that the aromatic compounds 4-HBA, vanillate, vanillin and PCA are strong chemoattractants for this strain (Ni et al., 2013). Unlike in P. putida F1, however, these compounds are not sensed directly in C. testosteroni CNB-1 but rather by the chemoreceptor MCP2201, which senses the TCA cycle intermediates generated during aromatic compound metabolism. Another type of sensory response, termed energy taxis, in which the energy generated from metabolism of the attractant drives the response (Alexandre and Zhulin, 2001; Alexandre et al., 2004), has also been shown to be a mechanism used by some bacteria to sense aromatic compounds. For example, energy taxis mediates responses to phenylacetic acid in P. putida F1 (Luu et al., 2013), (methyl)phenols in P. putida CF600 (Sarand et al., 2008) and 2-nitrotoluene in Acidovorax sp. strain JS42 (Rabinovitch-Deere and Parales, 2012).

142 R. A. Luu et al. ■

MFS transporters are secondary active membrane transporters for a diverse group of substrates. PcaK belongs to the family of aromatic acid/H+ symporters within the MFS (Pao et al., 1998), and additional putative members of this family of symporters are present in P. putida F1. VanK (locus tag Pput_2023), which is encoded in a cluster with the vanillate degradation genes, presumably transports vanillate and/or PCA based on functional characterization of the orthologs in Corynebacterium glutamicum (Chaudhry et al., 2007), and Acinetobacter (D’Argenio et al., 1999), respectively. P. putida F1 also has a putative BenK ortholog (locus tag Pput_2550) associated with the benzoate degradation gene cluster. BenK has been shown to transport benzoate in Acinetobacter sp. strain ADP1 (Collier et al., 1997) and C. glutamicum (Chaudhry et al., 2007). As shown in Fig. 6, PcaK is only required for chemotaxis to 4-HBA when cells are grown on 4-HBA. Since vanillate, PCA and benzoate feed into the same pathway, which results in formation of the inducer of pcaY, it is possible that VanK and BenK may be required for chemotaxis to aromatic acids when cells are grown on these compounds. However, experiments to investigate this possibility have not yet been carried out. Previous studies identified a dual-function MFS transporter (TfdK) in Cupriavidus necator (formerly Ralstonia eutropha) JMP134(pJP4). TfdK was shown to aid in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) at low substrate concentrations (Leveau et al., 1998) and was also necessary for chemotaxis of strain JMP134 to 2,4-D (Hawkins and Harwood, 2002). Similarly, the MFS transporter of gallic acid, GalT, was required for chemotaxis to gallic acid in P. putida KT2440 (Nogales et al., 2011). However, like PcaK, the specific roles of TfdK and GalT in chemotaxis were not determined. Expression of tfdK is induced by an intermediate in the degradation of 2,4-D (Leveau et al., 1998), and it is therefore possible that TfdK plays an indirect role in 2,4-D chemotaxis similar to that of PcaK; it may facilitate the uptake of a substrate necessary for inducing the gene encoding the MCP required for 2,4-D chemotaxis either directly or following conversion to the relevant inducing intermediate. The studies reported here demonstrate intimate physiological links between transport, chemotaxis and metabolism of 4-HBA, which are highlighted in the proposed model (Fig. 7). In the absence of permeases or under conditions in which the MFS transporter is not induced, aromatic compounds have been found to diffuse into the cell at rates sufficient to support wild-type rates of growth (Harwood et al., 1994; Hawkins and Harwood, 2002). Once in the cytoplasm, 4-HBA is slowly catabolized by low constitutive levels of Pca enzymes (Harwood and Parales, 1996) until sufficient β-KA accumulates to up-regulate genes of the pca regulon (Fig. 1A) by at least 10-fold (Nichols and Harwood, 1995; Harwood and

Parales, 1996). The increased production of PcaK facilitates entry of 4-HBA, which is particularly important at low concentrations and at high pH. Higher rates of 4-HBA catabolism, resulting from higher Pca enzyme levels, increase the intracellular concentration of β-KA, which further induces the system (Fig. 7). Increased production of PcaY allows cells to detect and respond to higher concentrations of this carbon and energy source via canonical chemotactic signal transduction pathways, providing an added layer of efficiency and potential growth and survival advantage over nonmotile and nonchemotactic microbes. It may be an evolutionary advantage for soil bacteria such as P. putida to have fine-tuned their ability to sense, acquire and catabolize aromatic compounds by simultaneously activating genes for their detection, uptake and degradation. This is likely to be only one example of a common competition mechanism in motile soil bacteria.

Experimental procedures Bacterial strains, plasmids and culture conditions Strains and plasmids used in this study are listed in Table 1. E. coli strain DH5α λpir was used for cloning and plasmid propagation (White and Metcalf, 2004), while HB101(pRK2013) (Figurski and Helinski, 1979) was used for triparental matings. All E. coli strains were grown on lysogeny broth agar at 37°C (Sambrook et al., 1989). P. putida F1 and its derivatives were grown in phosphate-buffered minimal medium (MSB) (Stanier et al., 1966) containing 40 mM pyruvate with (induced) or without (uninduced) 5 mM aromatic compounds at 30°C. Adipate was added as a fortuitous inducer at a final concentration of 20 mM. For plasmid selection and maintenance, gentamicin was provided at 15 μg ml−1 and kanamycin at 50 μg ml−1.

Cloning and DNA manipulations Primers used in this study are shown in Table S1. Genomic DNA was isolated using the 5′ ArchivePure DNA kit (5 Prime, Gaithersburg, MD). PCR products and plasmids were purified using a commercially available gel extraction kit (Bio Basic Inc., Markham, Ontario) and a plasmid miniprep kit (Fermentas, Glen Burnie, MD) respectively. Restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs (Beverly, MA). Standard methods for the manipulation of plasmids and DNA fragments were followed (Sambrook et al., 1989). Sequences of all cloned PCR products were verified at the University of California Davis Sequencing Facility using fluorescent automated DNA sequencing with an Applied Biosystems 3730 automated sequencer. Escherichia coli strains were transformed with plasmid DNA (Sambrook et al., 1989), and plasmids were mated into P. putida strains by conjugation in the presence of E. coli HB101(pRK2013) (Simon et al., 1983). Exconjugants were selected on MSB plates containing 10 mM succinate and the © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

Chemotaxis to aromatic acids in Pseudomonas putida 143

Fig. 7. Model of 4-HBA chemotaxis linking transport, metabolism and chemotaxis in P. putida F1. 4-HBA presumably enters the periplasm via nonspecific outer membrane porins. When pcaK is absent (under uninduced conditions or in a pcaK mutant), protonated 4-HBA diffuses across the cytoplasmic membrane. Low levels of Pca enzymes convert 4-HBA to the inducer β-KA; once sufficient β-KA builds up, it binds to the transcriptional activator PcaR to turn up transcription of the pca regulon (pcaK, pcaY, pcaBDC, pcaIJ and pcaF), resulting in more Pca enzymes as well as increased levels of the MFS transporter PcaK and the MCP PcaY. Increased levels of PcaK in the membrane result in rapid entry of 4-HBA into the cell, and higher levels of PcaY allow the cells to sense and move toward environments with higher concentrations of 4-HBA. PcaR negatively regulates its own synthesis independent of β-KA (Guo and Houghton, 1999). This integrated control of sensing, uptake and catabolism provides cells an optimized mechanism for acquiring carbon and energy, and likely enhances the competitiveness of P. putida in the soil environment. appropriate antibiotic. Deletion mutants that arose from double-crossover events were isolated by counterselection in MSB containing 10 mM succinate and 20% sucrose. Mutants were screened for antibiotic sensitivity to confirm the loss of plasmid, and deletions were verified by PCR using the appropriate primers (Table S1).

Construction of the pcaY deletion mutant and complementation plasmid The pcaY (locus tag Pput_2149) deletion mutant was constructed using primers containing restriction sites for SpeI and SacI as listed in Table S1 (Liu et al., 2009). The 1 kb regions upstream and downstream of pcaY were amplified by PCR, and the resulting fragments were fused by overlap extension PCR (Horton et al., 1993). The product was further amplified by PCR, and the resulting 2 kb DNA fragment with an in-frame deletion of pcaY was cloned into the suicide vector pAW19 (White and Metcalf, 2004). The construct was introduced into DH5α λpir and then moved into P. putida F1 © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

by triparental mating with HB101(pRK2013), and isolation and verification of exconjugants were carried out as previously described. To complement the mutant, the pcaY gene was amplified by PCR using primers 2149_HindIII_For and 2149_XbaI_Rev (Table S1) and then directionally cloned into pRK415Km (Luu et al., 2013) to generate the plasmid pXLF210.

Construction of vanAB, pcaD, pcaR and pcaK deletion mutants and complementation plasmids To construct the vanAB, pcaD, pcaR and pcaK deletion mutants, the 1 kb regions upstream and downstream of vanAB (locus tags Pput_2027 and Pput_2026), pcaD (locus tag Pput_4343), pcaR (locus tag Pput_4348), and pcaK (locus tag Pput_4347) were amplified by PCR using primers listed in Table S1. The resulting fragments were gel purified and directionally cloned into the pEX18Gm vector using an In-Fusion HD cloning kit (Clonetech, Mountain View, CA). The resulting constructs were introduced into P. putida F1 via

144 R. A. Luu et al. ■

Table 1. Strains and plasmids used in this study. Strain or plasmid

Relevant characteristics

Reference or source

E. coli DH5αλpir HB101

Cloning host Host for plasmid mobilization

White and Metcalf (2004) Sambrook et al. (1989)

P. putida F1

Wild type

Gibson et al. (1970); Finette et al. (1984) This study This study This study This study This study This study Luu et al. (2013)

GC006 RCF1 RCF2 RJF1 RJF2 XLF010 XLF019 Plasmids pAW19 pEX18Gm pHRP309 pHRP310 pIFAB pIFPD pIFPK pIFPR pRCF1 pRCF2 pRJF1 pRJF2 pRJF3 pRJF4 pRK415Km pRK2013 pXLF010 pXLF019 pXLF210 R

F1 F1 F1 F1 F1 F1 F1

ΔpcaY Δaer2 (parental strain XLF010) ΔpcaD (locus tag Pput_4343) ΔpcaK (locus tag Pput_4347) ΔvanAB (locus tag Pput_2027 and Pput_2026) ΔpcaR (locus tag Pput_4348) ΔpcaY (locus tag Pput_2149) Δaer2 (locus tag Pput_3628)

sacB containing cloning vector, ApR, KmR sacB containing cloning vector, GmR Broad-host-range lacZ transcriptional fusion vector, GmR pK19 with Ω SmR/SpR cassette vanAB deletion construct: 1-kb PCR fragments from upstream and downstream of vanAB fused and cloned into pEX18Gm pcaD deletion construct: 1-kb PCR fragments from upstream and downstream of pcaD fused and cloned into pEX18Gm pcaK deletion construct: 1-kb PCR fragments from upstream and downstream of pcaK fused and cloned into pEX18Gm pcaR deletion construct: 1-kb PCR fragments from upstream and downstream of pcaR fused and cloned into pEX18Gm pRK415Km carrying pcaD pRK415Km carrying pcaK pRK415Km carrying vanAB pRK415Km carrying pcaR pHRP310 containing cloned pcaY promoter region pHRP309 containing cloned pcaY promoter region upstream of promoterless lacZ Broad-host-range cloning vector, KmR ColE1 ori, RP4 mobilization function, KmR 1-kb PCR fragments from upstream and downstream of Gene Pput_2149 (pcaY) fused and cloned into SpeI-SacI sites of pAW19 1-kb PCR fragments from upstream and downstream of Gene Pput_3628 (aer2) fused and cloned into SpeI-SacI sites of pAW19 pRK415Km containing pcaY R

R

White and Metcalf (2004) Hoang et al. (1998) Parales and Harwood (1993a) Parales and Harwood (1993a) This study This study This study This study This study This study This study This study This study This study Luu et al. (2013) Figurski and Helinski (1979) Liu (2009); Parales et al. (2013) Luu et al. (2013) This study

Ap , ampicillin resistance; Gm , gentamicin resistance; Km , kanamycin resistance; Sm streptomycin resistance; SpR, spectinomycin resistance.

conjugation, and the deletion mutants (Table 1) were isolated and verified as just described. To complement the mutants, the vanAB, pcaD, pcaR and pcaK genes were amplified by PCR using primers listed in Table S1, and the products were cloned into pRK415Km to generate the plasmids listed in Table 1.

R

SalI and EcoRI restriction sites of the lacZ transcriptional fusion vector pHRP309 (Parales and Harwood, 1993a) to generate plasmid pRJF4. The plasmid was introduced into E. coli DH5α and then mated into wild-type P. putida F1 as just described.

Soft agar swim plate assays Construction of a pcaY-lacZ transcriptional fusion A pcaY-lacZ transcriptional fusion was constructed using primers 2149_PromFor and 2149_PromRev (Table S1) to amplify the promoter region of pcaY. The resulting PCR fragment was digested and cloned into the KpnI and EcoRI restriction sites of the cohort vector pHRP310 (Parales and Harwood, 1993a), generating plasmid pRJF3. The plasmid containing the correct construct was verified by DNA sequence analysis, and then purified and digested with SalI and EcoRI. The resulting fragment was then cloned into the

Soft agar swim plates were used to screen for mutants defective in chemotaxis to vanillate. MSB soft agar medium (Harwood et al., 1994) contained 5 mM vanillate in 0.3% Noble agar. Strains were harvested after overnight growth on MSB containing 5 mM vanillate. Pellets were washed and resuspended in chemotaxis buffer (CB; 50 mM potassium phosphate buffer at pH 7.0, 0.05% glycerol, 10 μM EDTA) to a final OD660 of ∼ 0.4; 2 μl of each suspension was inoculated into the soft agar, and plates were incubated at 30°C for approximately 24 h. The diameters of colonies of the mutant © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

Chemotaxis to aromatic acids in Pseudomonas putida 145

strains (n = 9) were measured and compared with those of the wild type, which was normalized to 1. The plates were observed under backlighting (Parkinson, 2007), and photographs were taken using a Canon EOS Rebel T2i camera.

Qualitative capillary chemotaxis assays Qualitative capillary assays were carried out as previously described (Grimm and Harwood, 1997) with slight modifications. Cells were grown in MSB medium with the appropriate carbon source(s) as indicated and harvested during midexponential phase [optical density at 660 nm (OD660), 0.3– 0.45]. Pellets were washed and resuspended in CB (50 mM potassium phosphate buffer at pH 7.0, 0.05% glycerol, 10 μM EDTA) to an OD660 of approximately 0.1. The resuspended cells were placed into a chamber formed by a glass slide, glass U-tube and cover slip. Microcapillaries (1 μl) filled with attractants, 2% Casamino acids (positive control) or CB (negative control) solidified in 2% low-melting-temperature agarose (NuSieve GTG; Lonza, Rockwell, ME) were introduced into the cell suspension. Responses were monitored at room temperature for up to 30 min under 40× total magnification on a Nikon Eclipse TE2000-S microscope (Melville, NY) and photographed using an Evolution Micropublisher 3.3 RTV camera and EVOLUTION MP/QIMAGING software (Media Cybernetics Inc., Silver Springs, MD).

Quantitative capillary assays Quantitative capillary assays were carried out as previously described (Liu et al., 2009). For these assays, cells were grown to an OD660 of approximately 0.4 in MSB containing 5 mM 4-HBA, harvested by centrifugation and resuspended in CB to a final OD660 of approximately 0.15. In all experiments, the response to 0.2% Casamino acids and CB was tested as positive and negative controls respectively. Responses to concentrations of 0.01, 0.1, 1, and 10 mM 4-HBA and quinate were tested. Responses to all other attractants were tested at 1 mM.

β-Galactosidase enzyme assays Assays were carried out as previously described by Miller (Miller, 1975). Cells were grown to an OD660 of between 0.45 and 0.55 in MSB containing either 40 mM pyruvate (uninduced) or 40 mM pyruvate plus 5 mM test aromatic compound (induced).

Growth studies For growth at pH 8.1, minimal medium was buffered with 1.92 mM KH2PO4 and 38.08 mM Na2HPO4. Strains were pregrown in MSB at either pH 7.3 or 8.1 containing 40 mM pyruvate at 30°C. To start the growth assay, precultures were inoculated in 96-well plates containing MSB at pH 7.3 or 8.1 and 5 mM 4-HBA in a total volume of 200 μl. Plates were incubated at 30°C on a Synergy 1 Biotek plate reader with constant shaking. Growth was monitored as increasing turbidity at 660 nm. © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 134–147

Acknowledgements This work was supported by a grant from the National Science Foundation to REP and JLD (MCB0919930). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. We thank Xianxian Liu and Grischa Chen for constructing strains and clones, and Pamela Lin and Victoria Wu for carrying out mutant screens. We also thank Shota Atsumi for the use of his equipment.

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