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The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment. New discoveries will be greatly facilitated by the ability to screen many natural and engineered microbes in a combinatorial manner against large numbers of fluorinated compounds simultaneously. Here, we describe a low-volume, high-throughput screening method to determine defluorination capacity of microbes and their enzymes. The method is based on selective binding of fluoride to a lanthanum chelate complex that gives a purple-colored product. It was miniaturized to determine biodefluorination in 96-well microtiter plates by visual inspection or robotic handling and spectrophotometry. Chemicals commonly used in microbiological studies were examined to define usable buffers and reagents. Base-catalyzed, purified enzyme and whole-cell defluorination reactions were demonstrated with fluoroatrazine and showed correspondence between the microtiter assay and a fluoride electrode. For discovering new defluorination reactions and mechanisms, a chemical library of 63 fluorinated compounds was screened

The exopolysaccharide (EPS) Pea is essential for wrinkly colony morphology, pellicle formation, and robust biofilm production in Pseudomonas putida. The second messenger cyclic diguanylate monophosphate (c-di-GMP) induces wrinkly colony morphology in P. putida through an unknown mechanism(s). Herein, we found that c-di-GMP modulates wrinkly colony morphology via the regulation of expression of eppA (PP_5586), a small individually transcribed gene of 177 bp, and this gene was adjacent to the upstream region of the pea cluster. Phenotype observation revealed that eppA was essential for Pea-dependent phenotypes. The deletion of eppA led to a smooth colony morphology and impaired biofilm, which was analogous to the phenotypes with loss of the entire pea operon. eppA expression was positively regulated by c-di-GMP via the transcriptional effector FleQ, and eppA was essential for the c-di-GMP-induced wrinkly colony morphology. Structure prediction results implied that EppA had two transmembrane regions, and Western blotting revealed that EppA was located on the cell membrane. Transcriptomic analysis indicated that EppA had no significant effect on the transcriptomic profile of P. putida. A bacterial two-hybrid (BTH) assay suggested that there was no direct interaction between EppA and the proteins in the pea cluster and adjacent operons. Overall, these findings reveal that EppA is essential for Pea-dependent phenotypes and that c-di-GMP modulates Pea-dependent phenotypes via regulation of eppA expression in P. putida. IMPORTANCE Microbe-secreted EPSs are high-molecular-weight polysaccharides that have the potential to be used as industrially important biomaterials. The EPS Pea in P. putida is essential for wrinkly colony morphology and pellicle formation. Here, we identified a function-unknown protein, EppA, which was also essential for Pea-dependent wrinkly colony morphology and pellicle formation, and EppA was probably involved in Pea secretion. Meanwhile, our results indicated that the second messenger c-di-GMP positively regulated the expression of EppA, resulting in Pea-dependent wrinkly colony morphology. Our results reveal the relationship of c-di-GMP, EppA, and Pea-dependent phenotypes and provide a possible pathway to construct genetically engineered strains for high Pea production.

Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield., Pseudomonas putida S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida S12 carries several gene clusters involved in solvent tolerance, and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent tolerance of this strain. Whole-genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% [vol/vol]). We identified mutations in an RND efflux pump regulator, arpR, that resulted in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β′, a global two-component regulatory system (GacA/GacS), and a putative AraC family transcriptional regulator, Afr. Transcriptomic analysis further revealed a constitutive downregulation of energy-consuming activities in ALE-derived strains, such as flagellar assembly, FoF1 ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of the solvent tolerance trait in P. putida S12 lacking its megaplasmid. IMPORTANCE Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of Pseudomonas putida; however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida S12 strain to regain its solvent tolerance. Through these adapted strains, we began to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in P. putida. This work sheds light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB–Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields. IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

Sustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during the bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates, or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant Pseudomonas putida S12 for molecular adaptation to an organic solvent. In addition to the solvent extrusion pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to short-term tolerance in moderate solvent concentrations, as well as to the stability of pTTS12. These two gene clusters were successfully expressed in non-solvent-tolerant strains of P. putida and Escherichia coli strains to confer and enhance solvent tolerance., Pseudomonas putida S12 is highly tolerant of organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies revealed that P. putida S12 contains the single-copy 583-kbp megaplasmid pTTS12. pTTS12 carries several important operons and gene clusters facilitating P. putida S12 survival and growth in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major determinant of solvent tolerance on the megaplasmid pTTS12. In addition, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA, respectively) encoded on pTTS12 which contributes to the solvent tolerance phenotype and is important for conferring stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with the slvAT gene pair created a solvent tolerance phenotype in non-solvent-tolerant strains, such as P. putida KT2440, Escherichia coli TG1, and E. coli BL21(DE3). IMPORTANCE Sustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during the bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates, or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant Pseudomonas putida S12 for molecular adaptation to an organic solvent. In addition to the solvent extrusion pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to short-term tolerance in moderate solvent concentrations, as well as to the stability of pTTS12. These two gene clusters were successfully expressed in non-solvent-tolerant strains of P. putida and Escherichia coli strains to confer and enhance solvent tolerance.

Valorization of lignocellulose (nonedible plant matter) is of key interest for the sustainable production of chemicals from renewable resources. Lignin, one of the main constituents of lignocellulose, is a heterogeneous aromatic biopolymer that can be chemically depolymerized into a heterogeneous mixture of aromatic building blocks; those can be further converted by certain microbes into value-added aromatic chemicals, e.g., the flavoring agent vanillin. We previously isolated a Pseudomonas sp. strain with the (for the genus) unusual trait of vanillyl alcohol production during growth on vanillin. Whole-genome sequencing of the isolate led to the identification of a vanillin reductase candidate gene whose deletion in a recombinant vanillin-accumulating P. putida strain almost completely alleviated the undesired vanillyl alcohol by-product yield. These results represent an important step toward biotechnological production of vanillin from lignin using bacterial cell factories., Microbial degradation of lignin and its related aromatic compounds has great potential for the sustainable production of chemicals and bioremediation of contaminated soils. We previously isolated Pseudomonas sp. strain 9.1 from historical waste deposits (forming so-called fiber banks) released from pulp and paper mills along the Baltic Sea coast. The strain accumulated vanillyl alcohol during growth on vanillin, and while reported in other microbes, this phenotype is less common in wild-type pseudomonads. As the reduction of vanillin to vanillyl alcohol is an undesired trait in Pseudomonas strains engineered to accumulate vanillin, connecting the strain 9.1 phenotype with a genotype would increase the fundamental understanding and genetic engineering potential of microbial vanillin metabolism. The genome of Pseudomonas sp. 9.1 was sequenced and assembled. Annotation identified oxidoreductases with homology to Saccharomyces cerevisiae alcohol dehydrogenase ScADH6p, known to reduce vanillin to vanillyl alcohol, in both the 9.1 genome and the model strain Pseudomonas putida KT2440. Recombinant expression of the Pseudomonas sp. 9.1 FEZ21_09870 and P. putida KT2440 PP_2426 (calA) genes in Escherichia coli revealed that these open reading frames encode aldehyde reductases that convert vanillin to vanillyl alcohol, and that P. putida KT2440 PP_3839 encodes a coniferyl alcohol dehydrogenase that oxidizes coniferyl alcohol to coniferyl aldehyde (i.e., the function previously assigned to calA). The deletion of PP_2426 in P. putida GN442 engineered to accumulate vanillin resulted in a decrease in by-product (vanillyl alcohol) yield from 17% to ∼1%. Based on these results, we propose the reannotation of PP_2426 and FEZ21_09870 as areA and PP_3839 as calA-II. IMPORTANCE Valorization of lignocellulose (nonedible plant matter) is of key interest for the sustainable production of chemicals from renewable resources. Lignin, one of the main constituents of lignocellulose, is a heterogeneous aromatic biopolymer that can be chemically depolymerized into a heterogeneous mixture of aromatic building blocks; those can be further converted by certain microbes into value-added aromatic chemicals, e.g., the flavoring agent vanillin. We previously isolated a Pseudomonas sp. strain with the (for the genus) unusual trait of vanillyl alcohol production during growth on vanillin. Whole-genome sequencing of the isolate led to the identification of a vanillin reductase candidate gene whose deletion in a recombinant vanillin-accumulating P. putida strain almost completely alleviated the undesired vanillyl alcohol by-product yield. These results represent an important step toward biotechnological production of vanillin from lignin using bacterial cell factories.

Caffeine and other methylxanthines are stimulant molecules found in formulated beverages, including sodas and energy drinks, and in brewed beverages, such as coffee and teas. Previously, we developed a bioassay for caffeine that involves monitoring the growth of a ΔguaB mutant of Escherichia coli defective in de novo guanine biosynthesis. When supplemented with a plasmid expressing the genes for an N-demethylation pathway from Pseudomonas putida CBB5, these bacteria demethylate caffeine (1,3,7-trimethylxanthine) and other methylxanthines into xanthine, which is then converted into guanine to support cell growth. A major limitation of this bioassay was that it could only measure the total concentration of all methylxanthines in a mixture. Therefore, it could not be used to measure the caffeine content of beverages like teas, which contain substantial quantities of multiple methylxanthines. To overcome this limitation, we created seven new plasmids containing all subsets of the three demethylase genes (ndmA, ndmB, and ndmC). We show that strains of ΔguaBE. coli containing each plasmid are able to demethylate specific subsets of methylxanthines and that they can be used to determine the concentrations of individual methylxanthines in complex mixtures containing multiple methylxanthines, including coffee doped with an additional methylxanthine. While validating this assay, we also discovered an unexpected demethylation event at the 1-methyl position when NdmB and NdmC were expressed in the absence of NdmA. The improved cell-based bioassay is inexpensive, is easy to use, and gives results comparable to standard high-performance liquid chromatography methods for measuring methylxanthine concentrations. IMPORTANCE Caffeine (1,3,7-trimethylxanthine) is the dominant neurostimulant found in coffee, teas, sodas, and energy drinks. Measuring the amount of caffeine and other methylxanthines in these beverages is important for quality assurance and safety in food science. Methylxanthines are also used in medicine and as performance-enhancing drugs, two contexts in which accurately determining their concentrations in bodily fluids is important. Liquid chromatography is the standard method for measuring methylxanthine concentrations in a sample, but it requires specialized equipment and expertise. We improved a previous bioassay that links E. coli growth to methylxanthine demethylation so that it can now be used to determine the amounts of individual methylxanthines in complex mixtures or beverages, such as coffee.

A cell’s innate fluorescence signature is an assemblage of fluorescence signals emitted by diverse biomolecules within a cell. It is known that the innate fluoresce signature reflects various cellular properties and physiological statuses; thus, they can serve as a rich source of information in cell characterization as well as cell identification. However, conventional techniques focus on the analysis of the innate fluorescence signatures at the population level but not at the single-cell level and thus necessitate a clonal culture. In the present study, we developed a technique to analyze the innate fluorescence signature of a single microbial cell. Using this novel method, we found that even very similarly shaped cells differ noticeably in their autofluorescence features, and the innate fluorescence signature changes dynamically with growth phases. We also demonstrated that the different cell types can be classified accurately within a mixed population under a microscope at the resolution of a single cell, depending solely on the innate fluorescence signature information. We suggest that single-cell autofluoresce signature analysis is a promising tool to directly assess the taxonomic or physiological heterogeneity within a microbial population, without cell tagging., Here we analyzed the innate fluorescence signature of the single microbial cell, within both clonal and mixed populations of microorganisms. We found that even very similarly shaped cells differ noticeably in their autofluorescence features and that the innate fluorescence signatures change dynamically with growth phases. We demonstrated that machine learning models can be trained with a data set of single-cell innate fluorescence signatures to annotate cells according to their phenotypes and physiological status, for example, distinguishing a wild-type Aspergillus nidulans cell from its nitrogen metabolism mutant counterpart and log-phase cells from stationary-phase cells of Pseudomonas putida. We developed a minimally invasive method (confocal reflection microscopy-assisted single-cell innate fluorescence [CRIF] analysis) to optically extract and catalog the innate cellular fluorescence signatures of each of the individual live microbial cells in a three-dimensional space. This technique represents a step forward from traditional techniques which analyze the innate fluorescence signatures at the population level and necessitate a clonal culture. Since the fluorescence signature is an innate property of a cell, our technique allows the prediction of the types or physiological status of intact and tag-free single cells, within a cell population distributed in a three-dimensional space. Our study presents a blueprint for a streamlined cell analysis where one can directly assess the potential phenotype of each single cell in a heterogenous population by its autofluorescence signature under a microscope, without cell tagging. IMPORTANCE A cell’s innate fluorescence signature is an assemblage of fluorescence signals emitted by diverse biomolecules within a cell. It is known that the innate fluoresce signature reflects various cellular properties and physiological statuses; thus, they can serve as a rich source of information in cell characterization as well as cell identification. However, conventional techniques focus on the analysis of the innate fluorescence signatures at the population level but not at the single-cell level and thus necessitate a clonal culture. In the present study, we developed a technique to analyze the innate fluorescence signature of a single microbial cell. Using this novel method, we found that even very similarly shaped cells differ noticeably in their autofluorescence features, and the innate fluorescence signature changes dynamically with growth phases. We also demonstrated that the different cell types can be classified accurately within a mixed population under a microscope at the resolution of a single cell, depending solely on the innate fluorescence signature information. We suggest that single-cell autofluoresce signature analysis is a promising tool to directly assess the taxonomic or physiological heterogeneity within a microbial population, without cell tagging.

Conjugative transfer of bacterial plasmids to recipient cells is often mediated by type IV secretion machinery. Experimental investigations into the minimal gene sets required for efficient conjugative transfer suggest that such gene sets are variable, depending on plasmids. We have been analyzing the conjugative transfer of Pseudomonas-derived and IncP-9 plasmids, NAH7 and pWW0, whose conjugation systems belong to the MPF(T) type. Our deletion analysis and synthetic biology analysis in this study showed that these plasmids require previously uncharacterized genes, mpfK (formerly orf34) and its functional homolog, kikA, respectively, for their efficient conjugative transfer. MpfK was localized in periplasm and had four cysteine residues whose intramolecular or intermolecular disulfide bond formation was suggested to be important for efficient conjugative transfer. The mpfK homologs were specifically carried by many MPF(T)-type plasmids, including non-IncP-9 plasmids, such as R388 and R751. Intriguingly, the mpfK homologs from the two non-IncP-9 plasmids were not required for conjugation of their plasmids, but were able to complement efficiently the transfer defect of the NAH7 mpfK mutant. Our results suggested the importance of the mpfK homologs for conjugative transfer of MPF(T)-type plasmids. IMPORTANCE IncP-9 plasmids are important mobile genetic elements for the degradation of various aromatic hydrocarbons. Elucidation of conjugative transfer of such plasmids is expected to greatly contribute to our understanding of its role in the bioremediation of polluted environments. The present study mainly focused on the conjugation system of NAH7, a well-studied and naphthalene-catabolic IncP-9 plasmid. Our analysis showed that the NAH7 conjugation system uniquely requires, in addition to the conserved components of the type IV secretion system (T4SS), a previously uncharacterized periplasmic protein, MpfK, for successful conjugation. Our findings collectively revealed a unique type of T4SS-associated conjugation system in the IncP-9 plasmids.

We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His 6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1 S )-2α-hydroxy-1,8-cineole or (1 R )-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida . Compared to P450 cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii , these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.

Soil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme. While GDH-dependent phosphate solubilization has been observed in numerous bacteria, little is known concerning the mechanism by which this process is regulated. Here we use the model rhizosphere-dwelling bacterium Pseudomonas putida KT2440 to explore GDH activity and PQQ synthesis, as well as gene expression of the GDH-encoding gene ( gcd ) and PQQ biosynthesis genes ( pqq operon) while under different growth conditions. We also use reverse transcription-PCR to identify transcripts from the pqq operon to more accurately map the operon structure. GDH specific activity and PQQ levels vary according to growth condition, with the highest levels of both occurring when glucose is used as the sole carbon source and under conditions of low soluble phosphate. Under these conditions, however, PQQ levels limit in vitro phosphate solubilization. GDH specific activity data correlate well with gcd gene expression data, and the levels of expression of the pqqF and pqqB genes mirror the levels of PQQ synthesized, suggesting that one or both of these genes may serve to modulate PQQ levels according to the growth conditions. The pqq gene cluster ( pqqFABCDEG ) encodes at least two independent transcripts, and expression of the pqqF gene appears to be under the control of an independent promoter and terminator. IMPORTANCE Plant growth promotion can be enhanced by soil- and rhizosphere-dwelling bacteria by a number of different methods. One method is by promoting nutrient acquisition from soil. Phosphorus is an essential nutrient that plants obtain through soil, but in many cases it is locked up in forms that are not available for plant uptake. Bacteria such as the model bacterium Pseudomonas putida KT2440 can solubilize insoluble soil phosphates by secreting gluconic acid. This chemical is produced from glucose by the activity of the bacterial enzyme glucose dehydrogenase, which requires a coenzyme called PQQ. Here we have studied how the glucose dehydrogenase enzyme and the PQQ coenzyme are regulated according to differences in bacterial growth conditions. We determined that glucose dehydrogenase activity and PQQ production are optimal under conditions when the bacterium is grown with glucose as the sole carbon source and under conditions of low soluble phosphate.

The remarkable metal resistance of many microorganisms is related to the presence of multiple metal resistance operons. Pseudomonas putida KT2440 can be considered a model for these microorganisms since its arsenic resistance is due to the action of proteins encoded by the two paralogous arsenic resistance operons ARS1 and ARS2. Both operons contain the genes encoding the transcriptional regulators ArsR1 and ArsR2 that control operon expression. We show here that purified ArsR1 and ArsR2 bind the trivalent salt of arsenic (arsenite) with similar affinities (~30 μM), whereas no binding is observed for the pentavalent salt (arsenate). Furthermore, trivalent salts of bismuth and antimony showed binding to both paralogues. The positions of cysteines, found to bind arsenic in other homologues, indicate that ArsR1 and ArsR2 employ different modes of arsenite recognition. Both paralogues are dimeric and possess significant thermal stability. Both proteins were used to construct whole-cell, lacZ -based biosensors. Whereas responses to bismuth were negligible, significant responses were observed for arsenite, arsenate, and antimony. Biosensors based on the P. putida arsB1 arsB2 arsenic efflux pump double mutant were significantly more sensitive than biosensors based on the wild-type strain. This sensitivity enhancement by pump mutation may be a convenient strategy for the construction of other biosensors. A frequent limitation found for other arsenic biosensors was their elevated background signal and interference by inorganic phosphate. The constructed biosensors show no interference by inorganic phosphate, are characterized by a very low background signal, and were found to be suitable to analyze environmental samples. IMPORTANCE Arsenic is at the top of the priority list of hazardous compounds issued by the U.S. Agency for Toxic Substances and Disease. The reason for the stunning arsenic resistance of many microorganisms is the existence of paralogous arsenic resistance operons. Pseudomonas putida KT2440 is a model organism for such bacteria, and their duplicated ars operons and in particular their ArsR transcription regulators have been studied in depth by in vivo approaches. Here we present an analysis of both purified ArsR paralogues by different biophysical techniques, and data obtained provide valuable insight into their structure and function. Particularly insightful was the comparison of ArsR effector profiles determined by in vitro and in vivo experimentation. We also report the use of both paralogues to construct robust and highly sensitive arsenic biosensors. Our finding that the deletion of both arsenic efflux pumps significantly increases biosensor sensitivity is of general relevance in the biosensor field.

The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA , were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the Δ fleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes. IMPORTANCE The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis in Pseudomonas aeruginosa . Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation.

Fungal mycelia serve as effective dispersal networks for bacteria in water-unsaturated environments, thereby allowing bacteria to maintain important functions, such as biodegradation. However, poor knowledge exists on the effects of dispersal networks at various osmotic (Ψ o ) and matric (Ψ m ) potentials, which contribute to the water potential mainly in terrestrial soil environments. Here we studied the effects of artificial mycelium-like dispersal networks on bacterial dispersal dynamics and subsequent effects on growth and benzoate biodegradation at ΔΨ o and ΔΨ m values between 0 and −1.5 MPa. In a multiple-microcosm approach, we used a green fluorescent protein (GFP)-tagged derivative of the soil bacterium Pseudomonas putida KT2440 as a model organism and sodium benzoate as a representative of polar aromatic contaminants. We found that decreasing ΔΨ o and ΔΨ m values slowed bacterial dispersal in the system, leading to decelerated growth and benzoate degradation. In contrast, dispersal networks facilitated bacterial movement at ΔΨ o and ΔΨ m values between 0 and −0.5 MPa and thus improved the absolute biodegradation performance by up to 52 and 119% for ΔΨ o and ΔΨ m , respectively. This strong functional interrelationship was further emphasized by a high positive correlation between population dispersal, population growth, and degradation. We propose that dispersal networks may sustain the functionality of microbial ecosystems at low osmotic and matric potentials.

Many studies on phage biology are based on isolation methods that may inadvertently select for narrow-host-range phages. Consequently, broad-host-range phages, whose ecological significance is largely unexplored, are consistently overlooked. To enhance research on such polyvalent phages, we developed two sequential multihost isolation methods and tested both culture-dependent and culture-independent phage libraries for broad infectivity. Lytic phages isolated from activated sludge were capable of interspecies or even interorder infectivity without a significant reduction in the efficiency of plating (0.45 to 1.15). Two polyvalent phages (PX1 of thePodoviridaefamily and PEf1 of theSiphoviridaefamily) were characterized in terms of adsorption rate (3.54 × 10−10to 8.53 × 10−10ml/min), latent time (40 to 55 min), and burst size (45 to 99 PFU/cell), using different hosts. These phages were enriched with a nonpathogenic host (Pseudomonas putidaF1 orEscherichia coliK-12) and subsequently used to infect model problematic bacteria. By using a multiplicity of infection of 10 in bacterial challenge tests, >60% lethality was observed forPseudomonas aeruginosarelative to uninfected controls. The corresponding lethality forPseudomonas syringaewas ∼50%. Overall, this work suggests that polyvalent phages may be readily isolated from the environment by using different sequential hosts, and this approach should facilitate the study of their ecological significance as well as enable novel applications.

The Pseudomonas genus contains a metabolically versatile group of organisms that are known to occupy numerous ecological niches, including the rhizosphere and endosphere of many plants. Their diversity influences the phylogenetic diversity and heterogeneity of these communities. On the basis of average amino acid identity, comparative genome analysis of >1,000 Pseudomonas genomes, including 21 Pseudomonas strains isolated from the roots of native Populus deltoides (eastern cottonwood) trees resulted in consistent and robust genomic clusters with phylogenetic homogeneity. All Pseudomonas aeruginosa genomes clustered together, and these were clearly distinct from other Pseudomonas species groups on the basis of pangenome and core genome analyses. In contrast, the genomes of Pseudomonas fluorescens were organized into 20 distinct genomic clusters, representing enormous diversity and heterogeneity. Most of our 21 Populus -associated isolates formed three distinct subgroups within the major P. fluorescens group, supported by pathway profile analysis, while two isolates were more closely related to Pseudomonas chlororaphis and Pseudomonas putida . Genes specific to Populus -associated subgroups were identified. Genes specific to subgroup 1 include several sensory systems that act in two-component signal transduction, a TonB-dependent receptor, and a phosphorelay sensor. Genes specific to subgroup 2 contain hypothetical genes, and genes specific to subgroup 3 were annotated with hydrolase activity. This study justifies the need to sequence multiple isolates, especially from P. fluorescens , which displays the most genetic variation, in order to study functional capabilities from a pangenomic perspective. This information will prove useful when choosing Pseudomonas strains for use to promote growth and increase disease resistance in plants.

Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames ( vppA S and vppA L [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5α and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.

2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02′ in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase.

( R )-Specific enoyl-coenzyme A (enoyl-CoA) hydratases (PhaJs) are capable of supplying monomers from fatty acid β-oxidation to polyhydroxyalkanoate (PHA) biosynthesis. PhaJ1 Pp from Pseudomonas putida showed broader substrate specificity than did PhaJ1 Pa from Pseudomonas aeruginosa , despite sharing 67% amino acid sequence identity. In this study, the substrate specificity characteristics of two Pseudomonas PhaJ1 enzymes were investigated by site-directed mutagenesis, chimeragenesis, X-ray crystallographic analysis, and homology modeling. In PhaJ1 Pp , the replacement of valine with isoleucine at position 72 resulted in an increased preference for enoyl-coenzyme A (CoA) elements with shorter chain lengths. Conversely, at the same position in PhaJ1 Pa , the replacement of isoleucine with valine resulted in an increased preference for enoyl-CoAs with longer chain lengths. These changes suggest a narrowing and broadening in the substrate specificity range of the PhaJ1 Pp and PhaJ1 Pa mutants, respectively. However, the substrate specificity remains broader in PhaJ1 Pp than in PhaJ1 Pa . Additionally, three chimeric PhaJ1 enzymes, composed from PhaJ1 Pp and PhaJ1 Pa , all showed significant hydratase activity, and their substrate preferences were within the range exhibited by the parental PhaJ1 enzymes. The crystal structure of PhaJ1 Pa was determined at a resolution of 1.7 Å, and subsequent homology modeling of PhaJ1 Pp revealed that in the acyl-chain binding pocket, the amino acid at position 72 was the only difference between the two structures. These results indicate that the chain-length specificity of PhaJ1 is determined mainly by the bulkiness of the amino acid residue at position 72, but that other factors, such as structural fluctuations, also affect specificity.

Many proteobacteria harbor FinR homologues in their genomes as putative LysR-type proteins; however, the function of FinR is poorly studied except in the induction of fpr-1 under superoxide stress conditions in Pseudomonas putida and Pseudomonas aeruginosa. Here, by analyzing the influence of finR deletion on the transcriptomic profile of P. putida KT2440 through RNA sequencing and real-time quantitative PCR (RT-qPCR), we found 11 operons that are potentially regulated by FinR. Among them, the expression of nicC and nicX operons, which were reported to be responsible for the aerobic degradation of nicotinic acid (NA), was significantly decreased in the finR mutant, and complementation with intact finR restored the expression of the two operons. The results of bacterial NA utilization demonstrated that the deletion of finR impaired bacterial growth in minimal medium supplemented with NA/6HNA (6-hydroxynicotinic acid) as the sole carbon source and that complementation with intact finR restored the growth of the mutant strain. The expression of nicC and nicX operons was previously revealed to be repressed by the NicR repressor and induced by NA/6HNA. Our transcriptional assay revealed that the deletion of finR weakened the induction of nicC and nicX by NA/6HNA. Meanwhile, the deletion of finR largely decreased the effect of nicR deletion on the expression of nicC and nicX operons. These results suggest that finR plays a positive role and cooperates with NicR in the regulation of nicC and nicX operons. In vitro experiments showed that both FinR and NicR bound to nicX and nicC promoter regions directly. The results of this study deepened our knowledge of FinR function and nicotinic acid degradation in P. putida. IMPORTANCE This study analyzed the influence of finR deletion on the transcriptomic profile of Pseudomonas putida KT2440. The FinR regulator is widely distributed but poorly studied in diverse proteobacteria. Here, we found 11 operons that potentially are regulated by FinR in KT2440. We further demonstrated that FinR played a positive role and cooperated with the NicR repressor in bacterial nicotinic acid (NA) degradation via regulating the expression of nicC and nicX operons. Furthermore, a transcriptomic analysis also indicated a potentially negative role of FinR in the expression of the hut cluster involved in bacterial histidine utilization. The work deepened our knowledge of FinR function and nicotinic acid degradation in P. putida.

Alkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) from Mycobacterium chubuense strain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved in Mycobacterium smegmatis mc2155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C2 to C4), and their activity was much lower on liquid alkenes (C5 to C8). Despite intensive efforts to express the complete EtnABCD enzyme in Escherichia coli, this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 μmol cytochrome c reduced/min/mg protein). Cloning the EtnABCD gene cluster into Pseudomonas putida KT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression of cpn60 chaperonins from either Mycobacterium spp. or E. coli Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels.IMPORTANCE Alkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.

Many proteobacteria harbor FinR homologues in their genomes as putative LysR-type proteins; however, the function of FinR is poorly studied except in the induction of fpr-1 under superoxide stress conditions in Pseudomonas putida and Pseudomonas aeruginosa. Here, by analyzing the influence of finR deletion on the transcriptomic profile of P. putida KT2440 through RNA sequencing and real-time quantitative PCR (RT-qPCR), we found 11 operons that are potentially regulated by FinR. Among them, the expression of nicC and nicX operons, which were reported to be responsible for the aerobic degradation of nicotinic acid (NA), was significantly decreased in the finR mutant, and complementation with intact finR restored the expression of the two operons. The results of bacterial NA utilization demonstrated that the deletion of finR impaired bacterial growth in minimal medium supplemented with NA/6HNA (6-hydroxynicotinic acid) as the sole carbon source and that complementation with intact finR restored the growth of the mutant strain. The expression of nicC and nicX operons was previously revealed to be repressed by the NicR repressor and induced by NA/6HNA. Our transcriptional assay revealed that the deletion of finR weakened the induction of nicC and nicX by NA/6HNA. Meanwhile, the deletion of finR largely decreased the effect of nicR deletion on the expression of nicC and nicX operons. These results suggest that finR plays a positive role and cooperates with NicR in the regulation of nicC and nicX operons. In vitro experiments showed that both FinR and NicR bound to nicX and nicC promoter regions directly. The results of this study deepened our knowledge of FinR function and nicotinic acid degradation in P. putida. IMPORTANCE This study analyzed the influence of finR deletion on the transcriptomic profile of Pseudomonas putida KT2440. The FinR regulator is widely distributed but poorly studied in diverse proteobacteria. Here, we found 11 operons that potentially are regulated by FinR in KT2440. We further demonstrated that FinR played a positive role and cooperated with the NicR repressor in bacterial nicotinic acid (NA) degradation via regulating the expression of nicC and nicX operons. Furthermore, a transcriptomic analysis also indicated a potentially negative role of FinR in the expression of the hut cluster involved in bacterial histidine utilization. The work deepened our knowledge of FinR function and nicotinic acid degradation in P. putida.

In this study, we developed a method that provides profiles of community-level surface dispersal from environmental samples under controlled hydration conditions and enables us to isolate and uncover the diversity of the fastest bacterial dispersers. The method expands on the porous surface model (PSM), previously used to monitor the dispersal of individual bacterial strains in liquid films at the surface of a porous ceramic disc. The novel procedure targets complex communities and captures the dispersed bacteria on a solid medium for growth and detection. The method was first validated by distinguishing motile Pseudomonas putida and Flavobacterium johnsoniae strains from their nonmotile mutants. Applying the method to soil and lake water bacterial communities showed that community-scale dispersal declined as conditions became drier. However, for both communities, dispersal was detected even under low-hydration conditions (matric potential, −3.1 kPa) previously proven too dry for P. putida strain KT2440 motility. We were then able to specifically recover and characterize the fastest dispersers from the inoculated communities. For both soil and lake samples, 16S rRNA gene amplicon sequencing revealed that the fastest dispersers were substantially less diverse than the total communities. The dispersing fraction of the soil microbial community was dominated by Pseudomonas species cells, which increased in abundance under low-hydration conditions, while the dispersing fraction of the lake community was dominated by Aeromonas species cells and, under wet conditions (−0.5 kPa), also by Exiguobacterium species cells. The results gained in this study bring us a step closer to assessing the dispersal ability within complex communities under environmentally relevant conditions. IMPORTANCE Dispersal is a key process of bacterial community assembly, and yet, very few attempts have been made to assess bacterial dispersal at the community level, as the focus has previously been on pure-culture studies. A crucial factor for dispersal in habitats where hydration conditions vary, such as soils, is the thickness of the liquid films surrounding solid surfaces, but little is known about how the ability to disperse in such films varies within bacterial communities. Therefore, we developed a method to profile community dispersal and identify fast dispersers on a rough surface resembling soil surfaces. Our results suggest that within the motile fraction of a bacterial community, only a minority of the bacterial types are able to disperse in the thinnest liquid films. During dry periods, these efficient dispersers can gain a significant fitness advantage through their ability to colonize new habitats ahead of the rest of the community.

Medium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A from Marinobacter aquaeolei VT8 (CYP153A M.aq ; the strain is also known as Marinobacter hydrocarbonoclasticus VT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153A M.aq is limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkR M.aq is functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153A M.aq operon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) from Pseudomonas putida GPo1. Using a recombinant Escherichia coli strain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane–1-dodecanol substrate mixture. This study reports a very efficient method of producing C 12 /C 14 alkanols and C 12 1,12-alkanediol by whole-cell biotransformation. IMPORTANCE To produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.

Chemoreceptors are at the beginnings of chemosensory signaling cascades that mediate chemotaxis. Most bacterial chemoreceptors are functionally unannotated and are characterized by a diversity in the structure of their ligand binding domains (LBDs). The data available indicate that there are two major chemoreceptor families at the functional level, namely, those that respond to amino acids or to Krebs cycle intermediates. Since pseudomonads show chemotaxis to many different compounds and possess different types of chemoreceptors, they are model organisms to establish relationships between chemoreceptor structure and function. Here, we identify PP2861 (termed McpP) of Pseudomonas putida KT2440 as a chemoreceptor with a novel ligand profile. We show that the recombinant McpP LBD recognizes acetate, pyruvate, propionate, and l -lactate, with K D (equilibrium dissociation constant) values ranging from 34 to 107 μM. Deletion of the mcpP gene resulted in a dramatic reduction in chemotaxis toward these ligands, and complementation restored a native-like phenotype, indicating that McpP is the major chemoreceptor for these compounds. McpP has a CACHE-type LBD, and we present data indicating that CACHE-containing chemoreceptors of other species also mediate taxis to C 2 and C 3 carboxylic acids. In addition, the LBD of NbaY of Pseudomonas fluorescens , an McpP homologue mediating chemotaxis to 2-nitrobenzoate, bound neither nitrobenzoates nor the McpP ligands. This work provides further insight into receptor structure-function relationships and will be helpful to annotate chemoreceptors of other bacteria.

Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr , pnd , and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr , whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than 2-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of lignin-derived aromatics. IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related compound, 4-hydroxybenzoate. This optimized strain can now be used as the basis for the characterization of novel pathways.

Microbial bioremediation is a promising approach for the removal of polycyclic aromatic hydrocarbon (PAH) contaminants. Many degraders of PAHs possess efflux pump genes in their genomes; however, their specific roles in the degradation of PAHs have not been clearly elucidated. In this study, two efflux pumps, TtgABC and SrpABC, were systematically investigated to determine their functions in a PAH-degrading Pseudomonas putida strain B6-2 (DSM 28064). The disruption of genes ttgABC or srpABC resulted in a defect in organic solvent tolerance. TtgABC was found to contribute to antibiotic resistance; SrpABC only contributed to antibiotic resistance under an artificial overproduced condition. Moreover, a mutant strain without srpABC did not maintain its activity in long-term biphenyl (BP) degradation, which correlated with the loss of cell viability. The expression of SrpABC was significantly upregulated in the course of BP degradation. BP, 2-hydroxybiphenyl, 3-hydroxybiphenyl, and 2,3-dihydroxybiphenyl (2,3-DHBP) were revealed to be the inducers of srpABC . 2,3-DHBP was verified to be a substrate of pump SrpABC; SrpABC can enhance the tolerance to 2,3-DHBP by pumping it out. The mutant strain B6-2Δ srpS prolonged BP degradation with the increase of srpABC expression. These results suggest that the pump SrpABC of strain B6-2 plays a positive role in BP biodegradation by pumping out metabolized toxic substances such as 2,3-DHBP. This study provides insights into the versatile physiological functions of the widely distributed efflux pumps in the biodegradation of PAHs. IMPORTANCE Polycyclic aromatic hydrocarbons (PAHs) are notorious for their recalcitrance to degradation in the environment. A high frequency of the occurrence of the efflux pump genes was observed in the genomes of effective PAH degraders; however, their specific roles in the degradation of PAHs are still obscure. The significance of our study is in the identification of the function and mechanism of the efflux pump SrpABC of Pseudomonas putida strain B6-2 (DSM 28064) in the biphenyl degradation process. SrpABC is crucial for releasing the toxicity caused by intermediates that are unavoidably produced in PAH degradation, which enables an understanding of how cells maintain the intracellular balance of materials. The findings from this study provide a new perspective on PAH recalcitrance and shed light on enhancing PAH degradation by genetic engineering.

The effective elimination of xenobiotic pollutants from the environment can be achieved by efficient degradation by microorganisms even in the presence of sugars or organic acids. Soil isolate Pseudomonas putida CSV86 displays a unique ability to utilize aromatic compounds prior to glucose. The draft genome and transcription analyses revealed that glucose uptake and benzoate transport and metabolism genes are clustered at the glc and ben loci, respectively, as two distinct operons. When grown on glucose plus benzoate, CSV86 displayed significantly higher expression of the ben locus in the first log phase and of the glc locus in the second log phase. Kinetics of substrate uptake and metabolism matched the transcription profiles. The inability of succinate to suppress benzoate transport and metabolism resulted in coutilization of succinate and benzoate. When challenged with succinate or benzoate, glucose-grown cells showed rapid reduction in glc locus transcription, glucose transport, and metabolic activity, with succinate being more effective at the functional level. Benzoate and succinate failed to interact with or inhibit the activities of glucose transport components or metabolic enzymes. The data suggest that succinate and benzoate suppress glucose transport and metabolism at the transcription level, enabling P. putida CSV86 to preferentially metabolize benzoate. This strain thus has the potential to be an ideal host to engineer diverse metabolic pathways for efficient bioremediation. IMPORTANCE Pseudomonas strains play an important role in carbon cycling in the environment and display a hierarchy in carbon utilization: organic acids first, followed by glucose, and aromatic substrates last. This limits their exploitation for bioremediation. This study demonstrates the substrate-dependent modulation of ben and glc operons in Pseudomonas putida CSV86, wherein benzoate suppresses glucose transport and metabolism at the transcription level, leading to preferential utilization of benzoate over glucose. Interestingly, succinate and benzoate are cometabolized. These properties are unique to this strain compared to other pseudomonads and open up avenues to unravel novel regulatory processes. Strain CSV86 can serve as an ideal host to engineer and facilitate efficient removal of recalcitrant pollutants even in the presence of simpler carbon sources.

Phenazine-1-carboxylic acid, the main component of shenqinmycin, is widely used in southern China for the prevention of rice sheath blight. However, the fate of phenazine-1-carboxylic acid in soil remains uncertain. Sphingomonas wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources for growth. In this study, dioxygenase-encoding genes, pcaA1A2 , were found using transcriptome analysis to be highly upregulated upon phenazine-1-carboxylic acid biodegradation. PcaA1 shares 68% amino acid sequence identity with the large oxygenase subunit of anthranilate 1,2-dioxygenase from Rhodococcus maanshanensis DSM 44675. The dioxygenase was coexpressed in Escherichia coli with its adjacent reductase-encoding gene, pcaA3 , and ferredoxin-encoding gene, pcaA4 , and showed phenazine-1-carboxylic acid consumption. The dioxygenase-, ferredoxin-, and reductase-encoding genes were expressed in Pseudomonas putida KT2440 or E. coli BL21, and the three recombinant proteins were purified. A phenazine-1-carboxylic acid conversion capability occurred in vitro only when all three components were present. However, P. putida KT2440 transformed with pcaA1A2 obtained phenazine-1-carboxylic acid degradation ability, suggesting that phenazine-1-carboxylic acid 1,2-dioxygenase has low specificities for its ferredoxin and reductase. This was verified by replacing PcaA3 with RedA2 in the in vitro enzyme assay. High-performance liquid chromatography–mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analysis showed that phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation, indicating that PcaA1A2A3A4 constitutes the initial phenazine-1-carboxylic acid 1,2-dioxygenase. This study fills a gap in our understanding of the biodegradation of phenazine-1-carboxylic acid and illustrates a new dioxygenase for decarboxylation. IMPORTANCE Phenazine-1-carboxylic acid is widely used in southern China as a key fungicide to prevent rice sheath blight. However, the degradation characteristics of phenazine-1-carboxylic acid and the environmental consequences of the long-term application are not clear. S. wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources. In this study, a three-component dioxygenase, PcaA1A2A3A4, was determined to be the initial dioxygenase for phenazine-1-carboxylic acid degradation in S. wittichii DP58. Phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation. This finding may help us discover the pathway for phenazine-1-carboxylic acid degradation.

Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization.

Several bacteria use the plant hormone indole-3-acetic acid (IAA) as a sole carbon and energy source. A cluster of genes (named iac ) encoding IAA degradation has been reported in Pseudomonas putida 1290, but the functions of these genes are not completely understood. The plant-growth-promoting rhizobacterium Paraburkholderia phytofirmans PsJN harbors iac gene homologues in its genome, but with a different gene organization and context than those of P. putida 1290. The iac gene functions enable P. phytofirmans to use IAA as a sole carbon and energy source. Employing a heterologous expression system approach, P. phytofirmans iac genes with previously undescribed functions were associated with specific biochemical steps. In addition, two uncharacterized genes, previously unreported in P. putida and found to be related to major facilitator and tautomerase superfamilies, are involved in removal of an IAA metabolite called dioxindole-3-acetate. Similar to the case in strain 1290, IAA degradation proceeds through catechol as intermediate, which is subsequently degraded by ortho -ring cleavage. A putative two-component regulatory system and a LysR-type regulator, which apparently respond to IAA and dioxindole-3-acetate, respectively, are involved in iac gene regulation in P. phytofirmans . These results provide new insights about unknown gene functions and complex regulatory mechanisms in IAA bacterial catabolism. IMPORTANCE This study describes indole-3-acetic acid (auxin phytohormone) degradation in the well-known betaproteobacterium P. phytofirmans PsJN and comprises a complete description of genes, some of them with previously unreported functions, and the general basis of their gene regulation. This work contributes to the understanding of how beneficial bacteria interact with plants, helping them to grow and/or to resist environmental stresses, through a complex set of molecular signals, in this case through degradation of a highly relevant plant hormone.

NAH7 and pWW0 from gammaproteobacterial Pseudomonas putida strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are well-characterized, experimental investigation of their conjugation systems remains at a primitive level. To clarify these conjugation systems, in this study, we investigated the NAH7-encoded conjugation system by (i) analyzing the origin of its conjugative transfer ( oriT )-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT ( oriT N ) region was located within a 430-bp fragment, and the strand-specific nicking ( nic ) site and its upstream sequences that were important for efficient conjugation in the oriT N region were identified. Unlike many other relaxases, the NAH7 relaxase exhibited unique features in its ability to catalyze, in a conjugation-independent manner, the site-specific intramolecular recombination between two copies of the oriT N region, between two copies of the pWW0 oriT ( oriT W ) region (which is clearly different from the oriT N region), and between the oriT N and oriT W regions. The pWW0 relaxase, which is also clearly different from the NAH7 relaxase, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriT N -containing plasmid. Such a plasmid was, in the presence of the NAH7Δ nic derivative, conjugatively transferable to alphaproteobacterial and betaproteobacterial strains in which the NAH7 replication machinery is nonfunctional, indicating that the NAH7 conjugation system has a broader host range than its replication system. IMPORTANCE Various studies have strongly suggested an important contribution of conjugative transfer of catabolic plasmids to the rapid and wide dissemination of the plasmid-loaded degradation genes to microbial populations. Degradation genes on such plasmids are often loaded on transposons, which can be inserted into the genomes of the recipient bacterial strains where the transferred plasmids cannot replicate. The aim was to advance detailed molecular knowledge of the determinants of host range for plasmids. This aim is expected to be easily and comprehensively achieved using an experimental strategy in which the oriT region is connected with a plasmid that has a broad host range of replication. Using such a strategy in this study, we showed that (i) the NAH7 oriT -relaxase system has unique properties that are significantly different from other well-studied systems and (ii) the host range of the NAH7 conjugation system is broader than previously thought.

What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacterium Pseudomonas putida with glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re)definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathways sensu stricto are not a part of central carbon metabolism in P. putida , as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation.

1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene ( dhaA31 ) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon.

Pseudomonas putida is a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome of P. putida reveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, β-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes were ttgA , ttgB , and ttgC , which code for the major multidrug efflux pump TtgABC in P. putida KT2440. Investigation of an ECF-10 and ttgA double-knockout strain and a ttgABC -overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance in P. putida KT2440.

The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods. Pseudomonas putida , a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylum Proteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla Actinobacteria , Bacteroidetes , and Firmicutes were detected only by the culture-independent method. Members of the genus Pseudomonas (class Gammaproteobacteria ) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas Delftia species (class Betaproteobacteria ) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containing Delftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable Delftia strains as recipients, fluorescence in situ hybridization detected pCAR1-containing Delftia cells, suggesting that Delftia was a “transient” host of pCAR1.

On the basis of bioinformatic evidence, we suspected that proteins with a CYTH (CyaB thiamine triphosphatase) domain and/or a CHAD (conserved histidine α-helical domain) motif might represent polyphosphate (polyP) granule-associated proteins. We found no evidence of polyP targeting by proteins with CYTH domains. In contrast, two CHAD motif-containing proteins from Ralstonia eutropha H16 (A0104 and B1017) that were expressed as fusions with enhanced yellow fluorescent protein (eYFP) colocalized with polyP granules. While the expression of B1017 was not detectable, the A0104 protein was specifically identified in an isolated polyP granule fraction by proteome analysis. Moreover, eYFP fusions with the CHAD motif-containing proteins MGMSRV2-1987 from Magnetospirillum gryphiswaldense and PP2307 from Pseudomonas putida also colocalized with polyP granules in a transspecies-specific manner. These data indicated that CHAD-containing proteins are generally attached to polyP granules. Together with the findings from four previously polyP-attached proteins (polyP kinases), the results of this study raised the number of polyP-associated proteins in R. eutropha to six. We suggest designating polyP granule-bound proteins with CHAD motifs as phosins (phosphate), analogous to phasins and oleosins that are specifically bound to the surface of polyhydroxyalkanoate (PHA) granules in PHA-accumulating bacteria and to oil droplets in oil seed plants, respectively.

Tunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of the Pseudomonas putida F1 cym / cmt system. The resulting promoter, termed P Q5 , responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in other Alphaproteobacteria , such as the model organisms Caulobacter crescentus , Paracoccus denitrificans , and Methylobacterium extorquens . In the noninduced state, expression from P Q5 is low enough to allow gene depletion analysis, as demonstrated with the essential gene phyP of Sphingomonas sp. strain Fr1. A set of P Q5 -based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.

Pseudomonas sp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designated nox ) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans . The nox gene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteria Escherichia coli DH5α (DH-nox) and Pseudomonas putida KT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that the nox gene is expressed in both DH5α and KT2440, suggesting that additional factors required for nicotine degradation are present in a Pseudomonas strain(s), but not in E. coli . The mutant of strain HZN6 with nox disrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that the nox gene is responsible for the first step of nicotine degradation. The ( RS )-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.