Environmental Microbiology

Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp.

Kamini, Dasvit Shetty, Vikas D. Trivedi, Madhushri Varunjikar, Prashant S. Phale
Maia Kivisaar, Editor
DOI: 10.1128/AEM.02115-17

ABSTRACT

Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of Escherichia coli. The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of E. coli expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent Km and Vmax in the range of 98 to 111 μM and 69 to 73 μmol · min−1 · mg−1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of Pseudomonas spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.

IMPORTANCE Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of Pseudomonas spp. Predicted transmembrane domain and signal peptide sequences of Pseudomonas were found to be functional in Escherichia coli and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

INTRODUCTION

The use of pesticides has increased at an exponential rate due to the modernization of agriculture. Carbaryl (1-naphthyl-N-methylcarbamate) is a widely used carbamate family pesticide which acts by inhibiting acetylcholinesterase competitively, thus impairing the insect's nervous system (1). A number of soil organisms have been reported to degrade carbaryl partially or completely (2–9). Hydrolases are one of the key enzymes involved in the biodegradation of xenobiotics and naturally occurring compounds containing ester, amide, ether, or alkyl linkages (10–13). The carbaryl metabolic pathway is initiated by carbaryl hydrolase (CH) (EC 3.5.1.-), which catalyzes the conversion of carbaryl to 1-naphthol and methylamine. 1-Naphthol is more toxic and recalcitrant than carbaryl (7). Genes encoding CH have been found to be present on either the plasmid or chromosome (5, 6, 14, 15). CH has been purified and characterized from Blastobacter (4), Rhizobium (6), Arthrobacter sp. (16), and Pseudomonas (3, 17) strains, etc., and has been found to be localized in the cytoplasm (3) or speculated to be either membrane bound (17) or in the periplasm (6, 18). CH encoded by cehA in Rhizobium sp. strain AC100 and carbofuran hydrolase encoded by cfdJ in Novosphingobium sp. strain KN65.2 were predicted to be translocated to the periplasmic space via the Sec or TAT system (6, 18). However, their localization has not been demonstrated experimentally.

Soil isolate Pseudomonas sp. strain C5pp (7) and halotolerant Pseudomonas sp. strain C7 (9) utilize carbaryl as a sole source of carbon and energy via 1-naphthol, salicylate, and gentisate. Enzyme induction and metabolic studies in strain C5pp suggested that the carbaryl degradation pathway is divided into three segments and that the enzymes are encoded by genes located on the chromosome (14).

In this study, we demonstrate that in Pseudomonas sp. strains C5pp and C7, CH is localized in the periplasmic space. Bioinformatic analysis, studies on CH deletion mutants, and green fluorescent protein (GFP) fusion constructs suggest that a transmembrane domain (Tmd) and signal peptide (Sp) are involved in translocation of the enzyme across the inner membrane into the periplasmic space. Tmd and Sp together function efficiently in Escherichia coli to translocate proteins (CH and GFP) into the periplasm. Additionally, periplasmic CH from wild-type strains (C5pp and C7) and rCHΔ(Tmd)a expressed in the cytoplasm have been purified and characterized for kinetic constants and substrate specificity to understand the physiological significance/relevance of compartmentalization of the metabolic pathway enzymes.

RESULTS

Prediction and analysis of Tmd and Sp in CH.Analysis of the amino acid sequence of the mcbA gene, encoding carbaryl hydrolase (CH), from strain C5pp using the TMHMM (19) and Phobius (20) servers predicted the presence of a transmembrane domain (Tmd) (amino acids [aa] 1 to 73) with a transmembrane helix (aa 7 to 29) followed by a signal peptide (Sp) (aa 74 to 96) with a positively charged “n region” at the N terminus (aa 74 to 78), followed by a hydrophobic “h region” (aa 79 to 88) and a short polar “c region” (aa 89 to 96) (see Fig. S1A and B in the supplemental material). The PrediSi server (21) predicted the signal peptidase cleavage site at aa 96 (Fig. 1A). The amino acid sequence of CH without Tmd (aa 74 to 769) was analyzed using SignalP4.1 (22), YRC Philius (23), and PRED-TAT (24). SignalP4.1 predicted a characteristic type I signal peptidase cleavage site, Ala-X-Ala, at the C terminus of Sp (Fig. 1A and S1C). The YRC Philius tool predicted CH to be noncytoplasmic, and PRED-TAT suggested it to be translocated across the membrane via the Sec pathway (Fig. S1D). The protein sequences of CH (from Rhizobium sp. strain AC100 and Arthrobacter sp. strain RC100) and carbofuran hydrolase (from Novosphingobium sp. strain KN65.2 and Achromobacter sp. strain WM111) were also analyzed. Interestingly, compared to the CH from strain C5pp, these sequences were predicted to have Sps with well-defined n, h, and c regions, but they lacked a Tmd region (Fig. 1B). These analyses indicate that the CH from strain C5pp is a novel protein consisting of the Tmd (73 aa long) region along with an Sp (23 aa long), followed by the mature protein (aa 97 to 769). To establish the functional significance of these domains in protein translocation, various CH deletion mutants lacking the Tmd and/or Sp region and (Tmd+Sp)-GFP fusion proteins were constructed and analyzed (Fig. 1C).

FIG 1
FIG 1

Prediction of transmembrane domain (Tmd) and signal peptide (Sp) of carbaryl hydrolase from Pseudomonas sp. strain C5pp. (A) Deduced amino acid sequence for the CH gene, mcbA. Amino acids in blue indicate the predicted transmembrane domain (Tmd), green represents the signal peptide (Sp) sequence, and the boxed amino acid sequence indicates the mature CH enzyme. The signal peptidase cleavage site (after ALA) is marked by an underline and an arrowhead. (B) Comparison of predicted domains (Tmd, Sp, and structural) of CH and carbofuran hydrolases from various carbaryl- and carbofuran-degrading microbes (numbers in parentheses indicate NCBI protein accession numbers). (C) Schematics of various deletion mutant constructs of the CH from C5pp and GFP fusion constructs encompassing the Tmd+Sp, Tmd, and/or Sp domain of CH from C5pp. The constructs are engineered with a His tag at either the N or C terminus and are denoted by “a” or “b,” respectively. GFP constructs carry a 6-aa-long linker, GAAGGG, to allow the proper folding of the expressed protein.

Periplasmic localization of carbaryl hydrolase.Several methods, viz., osmotic shock (25), lysozyme-sucrose (26, 27), lysozyme-MgCl2 (28), and MgCl2-cold osmotic shock (29), were attempted to extract periplasmic proteins from strains C5pp and C7. Among these, the MgCl2-cold osmotic shock method yielded good preparation of the periplasmic protein fraction without significant cell lysis of Pseudomonas and E. coli cells. The activity of gentisate 1,2-dioxygenase (GDO), a well-characterized cytoplasmic enzyme (predicted to be a cytoplasmic protein by PSORTb3.0) (30), was monitored to check the efficiency of periplasmic protein extraction without the cell lysis. Carbaryl-grown C5pp cells showed ∼4-fold-higher CH activity in the periplasm (1.33 ± 0.11 μmol · min−1 · mg−1) than in the cytoplasmic fraction (0.36 ± 0.06 μmol · min−1 · mg−1) (Table 1). The activity of GDO was found to be higher in the cytoplasm (0.24 ± 0.2 μmol · min−1 · mg−1) than in the periplasm (0.005 ± 0.006 μmol · min−1 · mg−1) (Table 1). In strain C7, the CH activity was 6-fold higher in the periplasm, while GDO activity was confined mainly to the cytoplasm. These results indicate that in C5pp and C7, CH is localized predominantly in the periplasmic space. Glucose-grown cells did not show a significant activity of CH in the periplasm or cytoplasm (Table 1), which rules out the possibility of the presence of nonspecific hydrolases acting on carbaryl. The detection of significantly higher activity of CH and GDO from the carbaryl- than from the glucose-grown cells suggests that both enzymes of the pathway are induced in the presence of carbaryl.

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TABLE 1

Carbaryl hydrolase activity in Pseudomonas sp. strains C5pp and C7

To ascertain the functional significance of the predicted Tmd and Sp, the CH activity was monitored from E. coli BL21 cells carrying the constructs [rCH, rCHΔ(Tmd)a, rCHΔ(Tmd+Sp)a, or rCHΔ(Tmd+Sp)b] (Fig. 1C) or pET28a(+) alone (vector without insert) as a control. The periplasmic fraction of E. coli carrying pET28a(+) alone showed background hydrolytic activity with carbaryl as a substrate (0.26 ± 0.6 μmol · min−1 · mg−1), which could be due to the broad substrate specificity of hydrolases (31). E. coli cells carrying the mcbA gene cloned in pET28a(+) (rCH having both Tmd and Sp with an N-terminal His tag) showed ∼3.6-fold-higher activity (0.64 ± 0.09 μmol · min−1 · mg−1) in the periplasmic fraction than in the cytoplasmic fraction (0.11 μmol · min−1 · mg−1) (see Table S1 in the supplemental material). The periplasmic protein preparation from these cells failed to bind to an Ni-nitrilotriacetic acid (NTA) matrix, which suggests the lack of a His tag in the mature CH. This may be due to the processing of the polypeptide during translocation resulting in cleavage of the His tag plus Tmd plus Sp from the N terminus to yield the mature protein. The CH construct lacking the Tmd domain, rCHΔ(Tmd)a, showed expression of the protein in the soluble fraction as well as in the inclusion bodies (see Fig. S2B in the supplemental material). The soluble fraction showed an activity of 0.09 μmol · min−1 · mg−1. Construct rCHΔ(Tmd+Sp)a or -b, where the protein lacks both Tmd and Sp (Tmd+Sp) and carries a His tag at either the N or C terminus, respectively, showed expression of the inactive protein in the soluble (minor quantity) as well as in the inclusion bodies (major quantity) (Fig. S2C and D). These results suggest that Tmd and Sp together are responsible for the translocation of CH across the inner membrane into the periplasmic space and that the Sp region potentially has a role in the proper folding of CH.

Tmd+Sp translocates GFP into the periplasmic space of E. coli.To confirm the roles of Tmd and Sp in the translocation of proteins into the periplasmic space, the Tmd+Sp, Sp, or Tmd region of CH from C5pp was fused to the N terminus of GFP and expressed in E. coli. The confocal microscopic analysis of E. coli cells expressing GFP (without Tmd+Sp) showed the distribution of green fluorescence in the cytoplasm (Fig. 2A), whereas the cells expressing (Tmd+Sp)-GFP showed distinct green fluorescence in the periplasmic space, thus visualizing the cells as green fluorescent rings (Fig. 2B). Cells expressing Sp-GFP displayed fluorescence throughout the cytoplasm, with few cells showing a polar fluorescent signal (Fig. 2C). Cells expressing Tmd-GFP displayed a green fluorescent signal in the cytoplasm (Fig. 2D). These results suggest that Tmd and Sp are required together to translocate GFP into the periplasmic space of E. coli.

FIG 2
FIG 2

Periplasmic localization of rGFP(Tmd+Sp) fusion protein. E. coli cells carrying construct GFP (A), (Tmd+Sp)-GFP (B), (Sp)-GFP (C), or (Tmd)-GFP (D) were induced with IPTG (500 μM, for 16 h at 25°C). Cells were processed and analyzed by confocal microscopy for fluorescence as described in Materials and Methods. The scale bars equal 2 μm. The experiments were done independently at least three times, and the best images are shown here.

Purification and kinetic characterization of wild-type CH and rCHΔ(Tmd)a.Wild-type CH was partially purified from the periplasmic fraction of carbaryl-grown cells of strains C5pp and C7 using conventional column chromatography (see Fig. S3 and S4 in the supplemental material). The CH from strain C5pp was partially purified to 46.4-fold with a specific activity of 30 μmol · min−1 · mg−1 and a yield of 28% (see Fig. S4A and Table S2 in the supplemental material), while that from strain C7 was partially purified to 62-fold with a specific activity of 26 μmol · min−1 · mg−1 and a yield of 12.7% (see Fig. S4B and Table S3 in the supplemental material). Further attempts to purify these proteins to homogeneity were unsuccessful, as the protein yields were very low. Based on gel filtration chromatography, the native molecular mass was found to be ∼68 kDa (Fig. S3D) with a subunit molecular mass of ∼70 kDa, suggesting that the active protein is a monomer. The N-terminal sequence of the partially purified CH from strain C5pp was found to be (K/H/Q)(S/G)TGPLGKIE, which suggests that the mature periplasmic protein lacks the Tmd+Sp region (aa 1 to 96) and that the cleavage takes place after Ala-Leu-Ala as predicted by bioinformatic tools (Fig. 1A).

Due to the poor yield of CH from strains C5pp and C7, the gene mcbA was cloned and overexpressed in E. coli. The cytosolic rCHΔ(Tmd)a was purified to near homogeneity using Ni-NTA affinity chromatography, with a specific activity of 30 μmol · min−1 · mg−1 and a subunit molecular mass of ∼76 kDa, as determined by SDS-PAGE (Fig. 3A). The observed high molecular mass for rCHΔ(Tmd)a could be due to the extra 23 amino acid residues present at the N terminus of the expressed protein compared to the mature CH expressed in the periplasm of strain C5pp or C7.

FIG 3
FIG 3

Purification of recombinant carbaryl hydrolase. (A) SDS-PAGE analysis of protein eluted during the purification of rCHΔ(Tmd)a from E. coli cells. Lanes 1, cell extract; 2, pooled and concentrated Ni-NTA-purified enzyme; M, protein molecular mass markers phosphorylase B (97.4 kDa), BSA (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa). The arrowhead indicates the purified rCH with a molecular mass of ∼76 kDa. (B) Substrate saturation plot. The initial velocity v versus carbaryl concentration for the purified enzyme is shown. The experiment was done independently at least three times with measurements in triplicate at each concentration. The best profile is depicted here with standard error bars.

The purified enzymes displayed a typical Michaelis-Menten substrate saturation profile with carbaryl as the substrate (Fig. 3B and S4C and D). All three enzymes showed similar Km, Vmax, kcat, and catalytic efficiency (kcat/Km) (Table 2). Divalent metal cations, viz., Ca2+, Mg2+, Zn2+, and Mn2+ (at a 1 mM concentration) as well as metal chelators such as EDTA and EGTA (1 or 10 mM concentration) did not show any significant effect on the CH activity (see Table S4 in the supplemental material), suggesting that metal ions probably do not play any significant role in catalysis.

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TABLE 2

Kinetic properties of carbaryl hydrolase

CH from strain C7 showed activity on carbaryl (55 μmol · min−1 · mg−1, considered to be 100%) (Fig. 4A), 1-naphthylacetate (∼23%; 13.2 μmol · min−1 · mg−1) (Fig. 4B), and p-nitrophenylacetate (1.1%; 0.24 μmol · min−1 · mg−1) (Fig. 4C). All these substrates have an ester bond. The time-dependent spectral scan data indicate the formation of 1-naphthol (increase in absorbance at 322 nm) from carbaryl and 1-naphthylacetate and the formation of p-nitrophenol (increase in absorbance at 405 nm) from p-nitrophenylacetate. The enzyme failed to show any time-dependent spectral changes with 1-naphthalene acetamide (absence of ester bond in the structure) (Fig. 4D). Similar spectral changes were observed with rCHΔ(Tmd)a (see Fig. S5 in the supplemental material). These results suggest that the enzyme acts only on the ester bond (as an esterase type) and not on the amide bond of the substrates.

FIG 4
FIG 4

Time-dependent spectral changes observed for the purified CH from Pseudomonas sp. strain C7. The enzyme reaction was performed as described in Materials and Methods, and spectral scans were recorded using the substrates carbaryl (400 μM, 10 cycles every 1-min interval) (A), 1-naphthyl acetate (400 μM, 10 cycles every 3-min interval) (B), p-nitrophenyl acetate (400 μM, 10 cycles every 2-min interval) (C), or 1-naphthalene acetamide (400 μM, 10 cycles every 3-min interval) (D). The amount of enzyme used per reaction was 1 μg. The spectral changes observed for rCHΔ(Tmd)a with various substrates are similar and are shown in Fig. S5 in the supplemental material.

DISCUSSION

Proteins present in the periplasmic space of bacteria play important roles in various cellular functions such as solute transport, antibiotic resistance, xenobiotic detoxification, etc. (32, 33, 34). Carbaryl hydrolase (EC 3.5.1.-), the first enzyme involved in carbaryl degradation, has been reported to be present in the cytoplasm (3) and speculated to be either membrane bound (17) or present in the periplasm (6, 18). This report demonstrates the localization of CH in the periplasmic space of carbaryl-degrading Pseudomonas sp. strains C5pp and C7. Both strains showed 4- to 6-fold-higher activity of CH in the periplasmic fraction of the cells. Polypeptides destined to be transported across the inner membrane generally contain a signal peptide with a peptidase cleavage site, such as Ala-x-Ala-Ala, Leu-x-x-Cys, or Ala-x-Ala (35). Bioinformatic analysis of the full-length CH polypeptide predicted the presence of Tmd and Sp consisting of n, h, and c regions with signal peptidase cleavage sequence Ala-Leu-Ala (aa 94 to 96). The Tmd+Sp (aa 1 to 96) is cleaved after Ala96, as evident from the N-terminal sequencing of the purified mature periplasmic CH from strain C5pp. Secretory proteins are generally known to be translocated to the periplasm via either TAT (twin arginine translocation) or Sec (secretory) pathway. Proteins translocated via the TAT pathway have a conserved twin-arginine motif, are translocated in their folded forms, and predominantly require a cofactor for their activity (35, 36). In the Sec pathway, proteins are transported in their unfolded state (35). The absence of the twin Arg motif in the Sp sequence of CH suggests that the enzyme in strain C5pp is probably transported via the Sec pathway. The interesting and unique feature observed is the presence of the Tmd domain (aa 1 to 73) at the N terminus of the polypeptide, prior to Sp (aa 74 to 96). This arrangement is a highly unusual, and Sec signal peptides are usually only 25 to 27 amino acids in length. Both these sequences function efficiently in E. coli, as evident from the 4-fold-higher activity of rCH observed in the periplasm. The CH construct having Sp but lacking the Tmd region [rCHΔ(Tmd)a] showed functionally active CH in the cytoplasm, suggesting that the presence of Sp alone failed to translocate the enzyme into the periplasm. Based on these observations, we speculate that the Tmd with transmembrane helix (aa 7 to 23) is probably essential for anchoring/targeting CH (769 aa long) to the inner membrane, followed by translocation. However, the presence of the Sp region alone does not interfere with folding of the polypeptide, thus yielding an active CH with kinetic properties similar to those of the wild-type CH from strain C5pp. The absence of the Sp region [rCHΔ(Tmd+Sp)] led to the expression of inactive CH, suggesting that Sp probably plays an important role in the folding of CH. Further, the expression of (Tmd+Sp)-GFP fusion protein in the periplasm of E. coli supports the role of Tmd+Sp in the translocation. Overall, the activity results with various CH constructs, N-terminal sequencing of mature periplasmic CH, and localization of GFP fusion proteins are in agreement with the bioinformatic predictions indicating the periplasmic localization of CH in strain C5pp. However, it is currently unclear whether Tmd is cleaved off prior to Sec transport or perhaps prior to the cleavage of the Sec signal sequence during periplasmic localization of CH.

Carbaryl-degrading Rhizobium sp. strain AC100, which is speculated to have a periplasmic carbaryl hydrolase (CehA) (794 aa) (6), showed 100% identity with the speculated membrane-bound carbofuran hydrolase (CfdJ) (794 aa) (18) from Novosphingobium sp. strain KN 65.2. Both these enzymes shared 27% identity with CH from strain C5pp (15). CehA and CfdJ possess a 29-aa-long Sp with defined n, h, and c regions but lack Tmd. Comparatively, the Sp of CH from C5pp is shorter (23 aa long) and shows significantly lower identity (5/23 amino acids) (see Fig. S6 in the supplemental material). CehA and CfdJ have two Arg residues at positions 9 and 10 (Fig. S6) and have been predicted to be translocated via the Sec or TAT pathway (18). These findings suggest that the smaller Sp in CH from strain C5pp probably is assisted by the Tmd to allow successful anchoring and translocation of the polypeptide across the inner membrane. The Sp region has been reported to contribute toward activity and stability of esterases of the GDSL family (37). This was recently observed for carboxyl esterase (EstPS1), where the presence of Sp yielded a soluble and active enzyme, while EstPS1ΔSp protein was insoluble (37).

The purified active rCHΔ(Tmd)a protein from the cytoplasm of E. coli had a higher molecular mass (∼76 kDa) than the purified wild-type CH (70 kDa) from strain C5pp; this could be due to the presence of uncleaved Sp in the former protein. Purified CH from strains C5pp, C7, and rCHΔ(Tmd)a showed very similar kinetic constants (Km, 97 to 116 μM; Vmax, 69 to 72 μmol · min−1 · mg−1) which are moderate compared to the reported Km (9 to 213 μM) and Vmax (8 to 931 μmol · min−1 · mg−1) for CH from other organisms (3–6, 17). All the three enzymes catalyze the hydrolysis of an ester bond and not an amide bond, and externally provided divalent metal ions or chelators do not affect their activity.

Based on these results, we propose the compartmentalization of the carbaryl degradation pathway in Pseudomonas sp. strains C5pp and C7 as shown in Fig. 5. Compared to other bacterial isolates (2, 5, 8, 38, 39), strains C5pp and C7 showed faster (12 to 14 h) and efficient degradation of carbaryl at higher concentrations (up to 1% [wt/vol] tested so far). Carbaryl is speculated to be transported/taken up by partitioning in the outer membrane and/or through general diffusion porins. In the periplasm, carbaryl is hydrolyzed by CH to yield 1-naphthol, which is more recalcitrant and toxic than carbaryl. This is evident, as strains C5pp and C7 showed poor growth on 1-naphthol (250 mg · liter−1) in minimal salt medium (MSM) containing yeast extract (0.025%, wt/vol), while in the absence of yeast extract, they failed to grow. 1-Naphthol at ≥50 mg · liter−1 has been reported to stop the growth of Pseudomonas putida S12 (40). The localization of low-affinity CH in the periplasm leads to the controlled formation of 1-naphthol, thus preventing its accumulation and subsequent toxicity to the organism. The generated 1-naphthol, which is more hydrophobic than carbaryl, gets transported via partition and/or diffusion across the inner membrane. Carbaryl which is able to partition and diffuse through the inner cell membrane in to the cytoplasm can be metabolized by the CH present in the cytosol. The presence of high-affinity 1-naphthol 2-hydroxylase (1NH) (41) in the cytoplasm catalyzes the rapid conversion of 1-naphthol to 1,2-dihydroxynaphthalene, which is further metabolized to the central carbon metabolic pathway intermediates by various ring-cleaving and ring-hydroxylating oxygenases found to be present in the cytoplasm (7, 14). In strain C5pp, the enzymes involved in carbaryl degradation are organized into three putative operons, “upper” (carbaryl to salicylate), “middle” (salicylate to gentisate), and “lower” (gentisate to central carbon pathway intermediates) (15). The “upper” pathway genes encoding CH and 1NH were reported to be a part of an integron, while the “middle” and “lower” pathways were present as two distinct class I composite transposons. These findings suggest that in strain C5pp, the carbaryl degradation capability must have been acquired via horizontal gene transfer. Genes encoding CH and 1NH with novel features have probably evolved under the positive selection pressure of carbaryl (15). Thus, translocating CH to the periplasm and evolution of a new cytoplasmic enzyme like 1NH might be a successful strategy to regulate the concentration of 1-naphthol, thereby reducing its toxicity to the isolates. This seems to be an essential adaptation process for the organism to survive in a carbaryl-contaminated environment.

FIG 5
FIG 5

Proposed compartmentalization of the carbaryl degradation pathway in Pseudomonas sp. strains C5pp and C7. The kinetic constants were determined for the purified CH and 1NH from Pseudomonas sp. strain C5pp (15; this study). Carbaryl is proposed to be transported into the periplasmic space through the nonspecific outer membrane porin(s) and/or diffusion across the outer membrane. The transport of 1-naphthol across the inner membrane is proposed to be through diffusion and/or partitioning. Cytoplasmic enzymes such as 1NH, 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase, salicylate 5-hydroxyalse, gentisate dioxygenase, etc. (14; this study), are involved in the metabolism of 1-naphthol to central carbon metabolism in the cytosol.

MATERIALS AND METHODS

Bioinformatics analysis.The draft genome sequence of Pseudomonas sp. strain C5pp is available at GenBank with accession number JWLN00000000.1 (15). The amino acid sequence of CH encoded by mcbA (accession no. AMK37578.1) was analyzed by TMHMM (19), Phobius (20), PrediSi (21), SignalP 4.1 (22), YRC Philius (23), and PRED-TAT (24) to predict Tmd, Sp, and the enzyme location.

Cultures and growth conditions.The bacterial isolates used in this study, are Pseudomonas sp. strain C5pp (MCC 2562) and Pseudomonas sp. strain C7 (MCC 3207), have been deposited in the Microbial Culture Collection, Pune, India (MCC). Pseudomonas sp. strains C5pp and C7 were grown on minimal salt medium (MSM) (7) supplemented aseptically with 0.1% (wt/vol) carbaryl in baffled Erlenmeyer flasks at 30°C and 200 rpm. Escherichia coli strains DH5α and BL21(DE3) used in this study were grown in lysogeny broth (LB) (42) with or without antibiotic (kanamycin, 40 μg · ml−1).

Preparation of periplasmic and cytoplasmic fractions.The periplasmic protein fraction was prepared as described previously (29). Briefly, C5pp and C7 cells were grown in MSM supplemented with carbaryl or glucose (0.1%, wt/vol) until the late log phase (optical density at 540 nm [OD540] = 1.0) (see Fig. S7 in the supplemental material), harvested by centrifugation (7,000 × g, 10 min, 4°C), and washed twice with potassium phosphate (K-PO4) buffer (20 mM, pH 7.5). The cells (1 g cells in 5 ml buffer) were resuspended in the K-PO4 buffer (20 mM, pH 7.5) containing MgCl2 (0.2 M) and incubated in a shaking water bath at 35°C for 10 min, followed by incubation on ice for 10 min. This “cold-osmotic shock” treatment was repeated two more times to release proteins from the periplasmic space without breaking/lysing the cells. The cell suspension was centrifuged (20,000 × g, 20 min, 4°C), and the supernatant obtained was termed the periplasmic fraction. The cell pellet obtained after osmotic shock treatment was washed once with buffer A (50 mM K-PO4, pH 7.5), resuspended in buffer A (1 g cells in 5 ml buffer), and lysed by sonication on ice (four cycles with 4-min intervals, each cycle consisting of 15 pulses of 1 s with 1-s intervals; output, 11 W; GE130 Ultrasonic processor). The cell homogenate was centrifuged at 30,000 × g for 30 min, and the clear pale brown supernatant obtained was referred to as the cytoplasmic fraction.

Enzyme assays.The activity of CH was monitored spectrophotometrically (Lambda 35; PerkinElmer, USA) by measuring the rate of increase in the absorbance at 322 nm due to the formation of 1-naphthol (ε322 = 2,200 M−1 · cm−1) (7). The reaction mixture (1 ml) contained buffer A, carbaryl (100 μM), and an appropriate amount of the extract/enzyme. Gentisate 1,2-dioxygenase (GDO), an aromatic ring-cleaving enzyme, was used as a cytoplasmic marker to check the intactness of cells during cold-osmotic shock treatment (GDO was predicted to be a cytoplasmic enzyme by PSORTb 3.0 [30]). The activity of GDO was monitored spectrophotometrically by measuring the increase in the absorbance at 330 nm due to the formation of maleylpyruvate (ε330 = 13,000 M−1 · cm−1) (43). The reaction mixture contained buffer A, gentisate (100 μM), and an appropriate amount of the extract. Protein estimation was carried out by the Bradford method using bovine serum albumin (BSA) as the standard (44). The specific activities are expressed as micromoles · minute−1 · mg of protein−1.

To determine the mode of hydrolysis by CH from C7 and rCH, the substrate disappearance or product formation was monitored by recording the time-dependent spectral changes observed during the enzyme reaction using various substrates, such as carbaryl, 1-naphthylacetate, and p-nitrophenylacetate (all containing an ester linkage) and 1-naphthalene acetamide (containing an amide linkage). The activities were calculated by monitoring the amount of product formed (1-naphthol for 1-naphthylacetate and 1-naphthalene acetamide or p-nitrophenol [ε405 = 18,000 M−1 cm−1; 45] for p-nitrophenylacetate), and the specific activities are reported as micromoles · minute−1 · mg of protein−1.

Purification of CH from strains C5pp and C7.Wild-type CH was partially purified from the periplasmic fraction prepared from strain C5pp and C7 cells grown on MSM supplemented with carbaryl (0.1%, wt/vol). The periplasmic fraction was prepared as described above, dialyzed against buffer A to remove MgCl2, and loaded onto Q-Sepharose ion-exchange column (43 by 6 mm; bed volume, 4.9 ml) preequilibrated with buffer A (flow rate, 30 ml · h−1). The column was washed with buffer A (10 column volumes), and the unbound and wash fractions (3 ml) were collected. CH activity was seen in the unbound fractions. Active fractions were pooled, glycerol and (NH4)2SO4 were added to final concentrations of 5% (vol/vol) and 50% (wt/vol), respectively, and the solution was stirred for 30 min and centrifuged at 30,000 × g for 30 min. The supernatant (containing CH) was loaded onto a phenyl-Sepharose CL-4B (42.5 by 6 mm; bed volume, 4.8 ml) hydrophobic chromatography column preequilibrated with (NH4)2SO4 (50%, wt/vol) in buffer A containing 5% (vol/vol) glycerol. The column was washed with the same buffer (10 column volumes), and bound CH was eluted using a simultaneous increasing gradient of ethylene glycol (0 to 60%, vol/vol) and decreasing gradient of (NH4)2SO4 (50 to 0%, wt/vol; flow rate, 30 ml · h−1; fraction size, 2 ml) in buffer A. Active fractions were pooled and dialyzed against buffer B (10 mM K-PO4 [pH 7.0], 5% [vol/vol] glycerol) and applied to hydroxyapatite matrix (18 by 6 mm; bed volume, 2 ml; flow rate 30 ml · h−1) equilibrated with buffer B. The column was washed with buffer B (10 column volumes), and the bound enzyme was eluted using an increasing linear gradient of K-PO4 buffer (pH 7.0; 10 to 400 mM; fraction size, 1.0 ml). For C7, the active fractions were pooled and concentrated to ∼1.5 ml using Centricon (membrane cutoff, 30 kDa; Millipore, USA). The concentrated enzyme was loaded onto Sephacryl S-200HR column (10 by 900 mm; bed volume, 80 ml; void volume, 37 ml) equilibrated in buffer A. Fractions (1 ml) were collected (flow rate, 4 ml · h−1) and assayed for activity. The purity of the enzyme was assessed by SDS-PAGE (12%, wt/vol) (46). Active and pure fractions were pooled, concentrated, and used for kinetic characterization. The partially purified CH from C5pp was resolved by SDS-PAGE (12%, wt/vol) and transferred onto a polyvinylidene difluoride (PVDF) membrane (0.45 μm; Pall, USA). The protein band was excised and sequenced by automated Edman degradation at Monash Biomedical Proteomics Facility, Clayton, through Biotrance Exim Pvt. Ltd., India.

Construction, expression, and localization of GFP fusion protein.Predicted Tmd and Sp regions of CH from strain C5pp were fused with green fluorescent protein (GFP) at the N terminus. The linker region (6 amino acids long, GAAGGG) was placed between Tmd+Sp and GFP so as to allow the proper folding of the expressed GFP. The primers used to construct GFP fusion proteins are listed in Table 3. The gene coding for GFP was amplified from pET28-EGFP by using primers GFP_FP and GFP_RP (Table 3). The Tmd+Sp, Tmd, and Sp regions were amplified from the genomic DNA of strain C5pp by using primers listed in Table 3. Amplified products were restriction digested and ligated to form (Tmd+Sp)-GFP, Tmd-GFP, and Sp-GFP cassettes, which were cloned into the pET-28a(+) vector at either the NheI and BamHI or the NdeI and BamHI sites to give GFP fusion constructs. All constructs were transformed independently into E. coli BL21(DE3) and grown in LB medium containing kanamycin (40 μg · ml−1). Randomly selected transformant colonies were grown in LB medium containing kanamycin at 37°C to an OD600 of 0.6. Cultures were precooled on ice for 45 min and induced with IPTG (isopropyl-β-d-thiogalactopyranoside) (500 μM) for 16 h at 25°C on a rotary shaker (200 rpm). The cells were harvested at 10,000 × g for 2 min at 4°C and resuspended in phosphate-buffered saline (pH 7.4). Cells were fixed onto a microscopic glass slide with paraformaldehyde (4%, wt/vol) and imaged under a confocal microscope with a 488-nm excitation filter (Zeiss FV100-IX81; Olympus America).

View this table:
TABLE 3

Primers used in the study

Cloning and overexpression of various CH constructs in E. coli.The construct carrying the full-length mcbA gene in pET-28a(+) encoding rCH was used in this study (15). rCH was expressed in E. coli BL21 as described previously (15). A deletion mutant of mcbA lacking Tmd, rCHΔ(Tmd)a, was constructed in the pET-28a(+) vector at the NdeI and XhoI sites by cloning the PCR product obtained using primers Δ(Tmd)CHa_FP and Δ(Tmd)CHa_RP (Table 3) and genomic DNA of strain C5pp as a template (see Fig. S2A in the supplemental material). Deletion mutants lacking both Tmd and Sp were constructed as follows: (i) rCHΔ(Tmd+Sp)a was constructed in the pET-28a(+) vector at the NdeI and XhoI sites by cloning a PCR product obtained using primers Δ(Tmd+Sp)CHa_FP and Δ(Tmd+Sp)CHa_RP (Table 3) and genomic DNA of strain C5pp as the template, which possessed a His6 tag at the N terminus, and (ii) rCHΔ(Tmd+Sp)b was constructed in pET-41a(+) at the NdeI and XhoI sites by cloning a PCR product obtained using primers Δ(Tmd+Sp)CHb_FP and Δ(Tmd+Sp)CHb_RP (Table 3), which possessed a His tag at the C terminus of the polypeptide.

These constructs were transformed in E. coli BL21(DE3) and grown in LB broth containing kanamycin (40 μg · ml−1) at 37°C. Cultures were precooled on ice for 45 min and induced. Cells with rCH were induced with IPTG (100 μM) for 16 h at 16°C on a rotary shaker. Cells harboring rCHΔ(Tmd)a were induced with IPTG (10 μM) for 16 h at 20°C. Cells carrying constructs rCHΔ(Tmd+Sp) in pET-28a(+) and rCHΔ(Tmd+Sp) in pET-41a were induced with IPTG (100 μM) for 16 h at 20°C and IPTG (100 μM) for 6 h at 30°C, respectively.

Purification of rCHΔ(Tmd)a.The protein was purified from the cell extract of IPTG-induced E. coli cells carrying rCHΔ(Tmd)a using an Ni-NTA matrix. Briefly, cells were resuspended in buffer C (10 mM K-PO4, pH 7.5) (1g cells in 10 ml buffer) and lysed by sonication (10 cycles with 5-min intervals; each cycle, 15 pulses; output, 11 W; GE130 Ultrasonic processor) on ice. The cell homogenate was centrifuged at 30,000 × g for 30 min, and the supernatant obtained was referred to as the cell extract or soluble fraction. The cell extract (∼45 mg protein) was loaded onto an Ni-NTA matrix (80 by 6 mm; bed volume, 9 ml) preequilibrated with buffer C and washed with buffer C (10 column volumes). Bound CH was eluted using an increasing linear gradient of imidazole (0 to 120 mM; flow rate, 30 ml · h−1; fraction size, 1 ml) in buffer C. Fractions were analyzed by SDS-PAGE. Active and pure fractions were pooled, dialyzed against buffer C, concentrated, and used for kinetic characterization.

Determination of kinetic constants, substrate specificity and mode of hydrolysis.The kinetic constants were determined by measuring the initial reaction rates for CH using various concentration of carbaryl (2.5 to 1,000 μM). The amount of enzyme used per assay was 0.5 μg for C5pp and rCHΔ(Tmd)a and 1 μg for C7. The data were fitted using the Michaelis-Menten equation, v = Vmax[S]/(Km + [S]), where v and Vmax are the initial and maximum velocities, [S] is the substrate concentration, and Km is the Michaelis-Menten constant.

Statistical analysis.For all experiments, the means and standard deviations were calculated using values obtained from triplicates of at least three independent experiments, unless otherwise specified.

ACKNOWLEDGMENTS

K. thanks IIT-B for a teaching assistantship. V.D.T. acknowledges CSIR, Government of India, for a senior research fellowship. M.V. thanks IIT-B for an IRCC internship. P.S.P. acknowledges a research grant from Board of Research in Nuclear Sciences, Government of India.

We thank N. S. Punekar, Department of Bioscience and Bioengineering, IIT-Bombay, for providing plasmid pET28-EGFP. We acknowledge the Confocal Laser Scanning Microscope Facility at the Department of Biosciences and Bioengineering and IRCC, IIT-Bombay.

FOOTNOTES

    • Received 23 September 2017.
    • Accepted 24 October 2017.
    • Accepted manuscript posted online 27 October 2017.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02115-17.

REFERENCES

  1. 1.↵
    1. Klaassen CD
    . 2008. Casarett and Doull's toxicology: the basic science of poisons. McGraw-Hill, Health Professions Division, New York, NY.
  2. 2.↵
    1. Sud RK,
    2. Sud AK,
    3. Gupta KG
    . 1972. Degradation of sevin (1-naphthyl-N-methyl carbamate) by Achromobacter sp. Arch Mikrobiol 87:353–358. doi:10.1007/BF00409134.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Mulbry WW,
    2. Eaton RW
    . 1991. Purification and characterization of the N-methylcarbamate hydrolase from Pseudomonas strain CRL-OK. Appl Environ Microbiol 57:3679–3682.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Hayatsu M,
    2. Nagata T
    . 1993. Purification and characterization of carbaryl hydrolase from Blastobacter sp. strain M501. Appl Environ Microbiol 59:2121–2125.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    1. Hayatsu M,
    2. Hirano M,
    3. Nagata T
    . 1999. Involvement of two plasmids in the degradation of carbaryl by Arthrobacter sp. strain RC 100. Appl Environ Microbiol 65:1015–1019.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Hashimoto M,
    2. Fukui M,
    3. Hayano K,
    4. Hayatsu M
    . 2002. Nucleotide sequence and genetic structure of a novel carbaryl hydrolase gene (cehA) from Rhizobium sp. strain AC100. Appl Environ Microbiol 68:1220–1227. doi:10.1128/AEM.68.3.1220-1227.2002.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Swetha VP,
    2. Phale PS
    . 2005. Metabolism of carbaryl via 1,2-dihydroxynaphthalene by soil isolates Pseudomonas sp. strains C4, C5, and C6. Appl Environ Microbiol 71:5951–5956. doi:10.1128/AEM.71.10.5951-5956.2005.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Seo JS,
    2. Keum YS,
    3. Li QX
    . 2013. Metabolomic and proteomic insights into carbaryl catabolism by Burkholderia sp. C3 and degradation of ten N-methylcarbamates. Biodegradation 24:795–811. doi:10.1007/s10532-013-9629-2.
    OpenUrlCrossRefPubMed
  9. 9.↵
    1. Trivedi VD,
    2. Bharadwaj A,
    3. Varunjikar MS,
    4. Singha AK,
    5. Upadhyay P,
    6. Gautam K,
    7. Phale PS
    . 2017. Insights into metabolism and sodium chloride adaptability of carbaryl degrading halotolerant Pseudomonas sp. strain C7. Arch Microbiol doi:10.1007/s00203-017-1363-4.
    OpenUrlCrossRef
  10. 10.↵
    1. Horvat CM,
    2. Wolfenden RV
    . 2005. A persistent pesticide residue and the unusual catalytic proficiency of a dehalogenating enzyme. Proc Natl Acad Sci U S A 102:16199–16202. doi:10.1073/pnas.0508176102.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Dong YJ,
    2. Bartlam M,
    3. Sun L,
    4. Zhou YF,
    5. Zhang ZP,
    6. Zhang CG,
    7. Rao Z,
    8. Zhang XE
    . 2005. Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3. J Mol Biol 353:655–663. doi:10.1016/j.jmb.2005.08.057.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    1. Wu PC,
    2. Liu YH,
    3. Wang ZY,
    4. Zhang XY,
    5. Li H,
    6. Liang WQ,
    7. Luo N,
    8. Hu JM,
    9. Lu JQ,
    10. Luan TG,
    11. Cao LX
    . 2006. Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112. J Agric Food Chem 54:836–842. doi:10.1021/jf052691u.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Wang BZ,
    2. Guo P,
    3. Hang BJ,
    4. Li L,
    5. He J,
    6. Li SP
    . 2009. Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from Sphingobium sp. strain JZ-1 and characterization of the gene product. Appl Environ Microbiol 75:5496–5500. doi:10.1128/AEM.01298-09.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Singh R,
    2. Trivedi VD,
    3. Phale PS
    . 2013. Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6. Arch Microbiol 195:521–535. doi:10.1007/s00203-013-0903-9.
    OpenUrlCrossRef
  15. 15.↵
    1. Trivedi VD,
    2. Jangir PK,
    3. Sharma R,
    4. Phale PS
    . 2016. Insights into functional and evolutionary analysis of carbaryl metabolic pathway from Pseudomonas sp. strain C5pp. Sci Rep 6:38430. doi:10.1038/srep38430.
    OpenUrlCrossRef
  16. 16.↵
    1. Hayatsu M,
    2. Mizutani A,
    3. Hashimoto M,
    4. Sato K,
    5. Hayano K
    . 2001. Purification and characterization of carbaryl hydrolase from Arthrobacter sp. RC100. FEMS Microbiol Lett 201:99–103. doi:10.1111/j.1574-6968.2001.tb10739.x.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    1. Chapalmadugu S,
    2. Chaudhry GR
    . 1993. Isolation of a constitutively expressed enzyme for hydrolysis of carbaryl in Pseudomonas aeruginosa. J Bacteriol 175:6711–6716. doi:10.1128/jb.175.20.6711-6716.1993.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Öztürk B,
    2. Ghequire M,
    3. Nguyen TP,
    4. De Mot R,
    5. Wattiez R,
    6. Springael D
    . 2016. Expanded insecticide catabolic activity gained by a single nucleotide substitution. Environ Microbiol 18:4878–4887. doi:10.1111/1462-2920.13409.
    OpenUrlCrossRef
  19. 19.↵
    1. Krogh A,
    2. Larsson B,
    3. von Heijne G,
    4. Sonnhammer EL
    . 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580. doi:10.1006/jmbi.2000.4315.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    1. Käll L,
    2. Krogh A,
    3. Sonnhammer EL
    . 2004. A combined transmembrane topology and signal peptide prediction method. J Mol Biol 338:1027–1036. doi:10.1016/j.jmb.2004.03.016.
    OpenUrlCrossRefPubMedWeb of Science
  21. 21.↵
    1. Hiller K,
    2. Grote A,
    3. Scheer M,
    4. Münch R,
    5. Jahn D
    . 2004. PrediSi: prediction of signal peptides and their cleavage positions. Nucleic Acids Res 32:W375–W379. doi:10.1093/nar/gkh378.
    OpenUrlCrossRefPubMedWeb of Science
  22. 22.↵
    1. Petersen TN,
    2. Brunak S,
    3. von Heijne G,
    4. Nielsen H
    . 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. doi:10.1038/nmeth.1701.
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    1. Reynolds SM,
    2. Käll L,
    3. Riffle ME,
    4. Bilmes JA,
    5. Noble WS
    . 2008. Transmembrane topology and signal peptide prediction using dynamic bayesian networks. PLoS Comput Biol 4:1–14. doi:10.1371/journal.pcbi.1000213.
    OpenUrlCrossRef
  24. 24.↵
    1. Bagos PG,
    2. Nikolaou EP,
    3. Liakopoulous TD,
    4. Tsirigos KD
    . 2010. Combined prediction of Tat and Sec signal peptides with hidden markov models. Bioinformatics 26:2811–2817. doi:10.1093/bioinformatics/btq530.
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    1. Nossal NG,
    2. Heppel LA
    . 1966. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241:3055–3062.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    1. Wood PM
    . 1978. Periplasmic location of the terminal reductase in nitrite respiration. FEBS Lett 92:214–218. doi:10.1016/0014-5793(78)80757-1.
    OpenUrlCrossRefPubMedWeb of Science
  27. 27.↵
    1. Robles-Price A,
    2. Wong TY,
    3. Sletta H,
    4. Valla S,
    5. Schiller NL
    . 2004. AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa. J Bacteriol 186:7369–7377. doi:10.1128/JB.186.21.7369-7377.2004.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Jensch T,
    2. Fricke B
    . 1997. Localization of alanyl aminopeptidase and leucyl aminopeptidase in cells of Pseudomonas aeruginosa by application of different methods for periplasm release. J Basic Microbiol 37:115–128. doi:10.1002/jobm.3620370206.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Basu A,
    2. Shrivastava R,
    3. Basu B,
    4. Apte SK,
    5. Phale PS
    . 2007. Modulation of glucose transport causes preferential utilization of aromatic compounds in Pseudomonas putida CSV86. J Bacteriol 189:7556–7562. doi:10.1128/JB.01235-07.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Yu NY,
    2. Wagner JR,
    3. Laird MR,
    4. Melli G,
    5. Rey S,
    6. Lo R,
    7. Dao P,
    8. Sahinalp SC,
    9. Ester M,
    10. Foster LJ,
    11. Brinkman FS
    . 2010. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608–1615. doi:10.1093/bioinformatics/btq249.
    OpenUrlCrossRefPubMedWeb of Science
  31. 31.↵
    1. Gupta RC
    . 2006. Toxicology of organophosphate and carbamate compounds. Academic Press Inc., New York, NY.
  32. 32.↵
    1. Oliver DB
    . 1996. Periplasm, p 88–103. In Neidhardt FC, Curtiss R, III, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (ed), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, DC.
  33. 33.↵
    1. Modak A,
    2. Bhaumik P,
    3. Phale PS
    . 2014. Periplasmic glucose-binding protein from Pseudomonas putida CSV86—identification of the glucose-binding pocket by homology-model-guided site-specific mutagenesis. FEBS J 281:365–375. doi:10.1111/febs.12607.
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Nagata Y,
    2. Futamura A,
    3. Miyauchi K,
    4. Takagi M
    . 1999. Two different types of dehalogenases, LinA and LinB, involved in gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26 are localized in the periplasmic space without molecular processing. J Bacteriol 181:5409–5413.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Natale P,
    2. Bruser T,
    3. Driessen JMA
    . 2008. Sec and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms. Biochim Biophys Acta 1778:1735–1756. doi:10.1016/j.bbamem.2007.07.015.
    OpenUrlCrossRefPubMedWeb of Science
  36. 36.↵
    1. Simone D,
    2. Bay DC,
    3. Leach T,
    4. Turner RJ
    . 2013. Diversity and evolution of bacterial twin arginine translocase protein, TatC, reveals a protein secretion system that is evolving to fit its environmental niche. PLoS One 8:e78742. doi:10.1371/journal.pone.0078742.
    OpenUrlCrossRef
  37. 37.↵
    1. Cai X,
    2. Wang W,
    3. Lin L,
    4. He D,
    5. Huang G,
    6. Shen Y,
    7. Wei W,
    8. Wei D
    . 2017. Autotransporter domain-dependent enzymatic analysis of a novel extremely thermostable carboxylesterase with high biodegradability towards pyrethroid pesticides. Sci Rep 7:3461. doi:10.1038/s41598-017-03561-8.
    OpenUrlCrossRef
  38. 38.↵
    1. Yan QX,
    2. Wang YX,
    3. Li SP,
    4. Li WJ,
    5. Hong Q
    . 2010. Sphingobium qiguonii sp. nov., a carbaryl-degrading bacterium isolated from a wastewater treatment system. Int J Syst Evol Microbiol 60:2724–2728. doi:10.1099/ijs.0.020362-0.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Hamada M,
    2. Matar A,
    3. Bashir A
    . 2015. Carbaryl degradation by bacterial isolates from a soil ecosystem of the Gaza Strip. Braz J Microbiol 46:1087–1091. doi:10.1590/S1517-838246420150177.
    OpenUrlCrossRef
  40. 40.↵
    1. Janardhan Garikipati SVB,
    2. Peeples TL
    . 2015. Solvent resistance pumps of Pseudomonas putida S12: applications in 1-naphthol production and biocatalyst engineering. J Biotechnol 210:91–99. doi:10.1016/j.jbiotec.2015.06.419.
    OpenUrlCrossRef
  41. 41.↵
    1. Trivedi VD,
    2. Majhi P,
    3. Phale PS
    . 2014. Kinetic and spectroscopic characterization of 1-naphthol 2-hydroxylase from Pseudomonas sp. strain C5. Appl Biochem Biotechnol 172:3964–3977. doi:10.1007/s12010-014-0815-4.
    OpenUrlCrossRef
  42. 42.↵
    1. Sezonov G,
    2. Joseleau-Petit D,
    3. D'Ari R
    . 2007. Escherichia coli physiology in Luria-Bertani broth. J Bacteriol 189:8746–8749. doi:10.1128/JB.01368-07.
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Suárez M,
    2. Ferrer E,
    3. Martin M
    . 1996. Purification and biochemical characterization of gentisate 1,2-dioxygenase from Klebsiella pneumoniae M5a1. FEMS Microbiol Lett 143:89–95.
    OpenUrlCrossRefPubMed
  44. 44.↵
    1. Bradford MM
    . 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3.
    OpenUrlCrossRefPubMedWeb of Science
  45. 45.↵
    1. Zhang YZ,
    2. Vanetten RL
    . 1991. Pre-steady state and steady state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart. J Biol Chem 266:1516–1525.
    OpenUrlAbstract/FREE Full Text
  46. 46.↵
    1. Laemmli UK
    . 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0.
    OpenUrlCrossRefPubMedWeb of Science
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KEYWORDS

inducible carbaryl hydrolase
periplasmic localization
signal peptide analysis
kinetic characterization
Pseudomonas

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