Productivity of Pseudomonas putida TISTR 1522 in polyhydroxyalkanoates (PHAs) production from saponified palm oil.

Polyhydroxyalkanoates (PHAs) have attracted attention as an environmentally degradable bioplastic which potentially replaces synthetic polymers used in a wide range of industries. One of most promising microorganisms for the production of PHAs is Pseudomonas putida. In this study, we purpose to develop sustainable processes to convert abundant palm oil available in local market to high value PHAs and optimize PHAs production by Pseudomonas putida TISTR 1522 from saponified palm oil. We found that the highest yield of PHAs production (0.95 g/L, 40.15%) was obtained in culture medium supplemented with 1% (w/v) fatty acid salt by P. putida TISTR 1522 after 24-h cultivation. The intracellular PHAs were located in granules inside the cells, which fluoresced bright yellow by staining with Nile red. The physical appearance of intracellular PHAs investigated by transmission electron microscope (TEM) revealed that PHAs accumulate in granules, about 3-10 granules per cell. These granules are white and roundish-shaped with 0.3-0.5-µm diameter. The 1H NMR spectrum represented the typical characters of medium-chain length-PHAs. This variation of all parameters was successfully demonstrated a good intracellular PHAs accumulation in P. putida TISTR 1522 by fatty acid salt utilization.

Parameter identification for gompertz and logistic dynamic equations.

In this paper, we generalize and compare Gompertz and Logistic dynamic equations in order to describe the growth patterns of bacteria and tumor. First of all, we introduce two types of Gompertz equations, where the first type 4-paramater and 3-parameter Gompertz curves do not include the logarithm of the number of individuals, and then we derive 4-parameter and 3-parameter Logistic equations. We notice that Logistic curves are better in modeling bacteria whereas the growth pattern of tumor is described better by Gompertz curves. Increasing the number of parameters of Logistic curves give favorable results for bacteria while decreasing the number of parameters of Gompertz curves for tumor improves the curve fitting. Moreover, our results overshadow some of the existing results in the literature.

Role of bacterial cell surface sulfhydryl sites in cadmium detoxification by Pseudomonas putida.

Understanding bacterial metal detoxification systems is crucial for determining the environmental impacts of metal pollution and for developing advanced bioremediation and water disinfection strategies. Here, we explore the role of cell surface sulfhydryl sites in bacterial detoxification of Cd, using Pseudomonas putida with surface sulfhydryl sites mostly on its EPS molecules as a model organism. Our results show that 5 and 20 ppm Cd in LB growth medium affects the lag phase of P. putida, but not the overall extent of cell growth at stationary phase, indicating that P. putida can detoxify Cd at these concentrations. EXAFS analysis of Cd bound to biomass from the different growth stages indicates that Cd binds to both sulfhydryl and non-sulfhydryl sites, but that the importance of Cd-sulfhydryl binding increases from early exponential to stationary phase. Cell growth is positively correlated to the measured sulfhydryl concentration on different biomass samples, but is independent of the measured non-sulfhydryl binding site concentration on the cell surfaces. Taken together, our results demonstrate that the sulfhydryl binding sites on EPS molecules can play an important role in binding and detoxifying toxic metals, significantly decreasing the bioavailability of the metal by sequestering it away from the bacterial cells.

Synergistic effect of Pseudomonas putida II-2 and Achromobacter sp. QC36 for the effective biodegradation of the herbicide quinclorac.

Quinclorac (QNC) is an effective but environmentally persistent herbicide commonly used in rice production. However, few studies have investigated its environmental behavior and degradation. In the present study, we carried out microbial cultures in the presence of QNC to observe changes in soil microbiota and to identify species capable of QNC degradation by using high-throughput sequencing of the 16S rRNA. Pseudomonas was the dominant genus, and Pseudomonas putida II-2 and other species were found to be capable of mineralizing QNC as a source of carbon and energy. However, this degradation rate was slow, only reaching 51.5 ± 1.6% for 7 days at 30 °C on QNC + minimal salt medium. Achromobacter sp. QC36 co-metabolized QNC when rice straw was added into the mineral salt medium containing QNC, and a mixed culture of both strains could mineralize approximately 92% of the 50 mg/L QNC after 5 days of cultivation in the presence of rice straw, at 25-35 °C and pH 6.0-8.0. Non-phytotoxicity of tobacco after degradation of QNC by mixed strains was evidenced in a pot experiment. These results suggest that this mixed culture may be useful in QNC bioremediation and can be used as a bio-formulation for agro-economical and industrial application.

Au nanoparticle-decorated aragonite microdumbbells for enhanced antibacterial and anticancer activities.

The present work reports the very first hydrothermal synthesis of 100% triclinic phase pure aragonite (A1) with microdumbbell microstructural architecture and Au Nanoparticle-decorated (AuNP-decorated) aragonites (A2, A3 and A4) with spherical, pentagonal/hexagonal and agglomerated AuNP-decorated microdumbbells having triclinic aragonite phase as the major and cubic AuNPs as the minor phase. Even in dark the AuNP-decorated aragonites (especially A2) show efficacies as high 90% against gram-negative e.g., Pseudomonas putida (P. putida) bacteria. Further the AuNP-decorated aragonites (A3) show anti-biofilm capability of as high as about 20% against P. putida. Most importantly the AuNP-decorated aragonites (A3) offer anti-cancer efficacy of as high as 53% while those of A1, A2, and A4 are e.g., 26%, 46% and 37%, respectively. For the very first time, based on detailed investigations, the mechanisms behind such advance antibiofilm and anticancer activities are linked to the generation of excess labile toxic reactive oxygen species (ROS). Thus, these materials show enormous potential as futuristic, multi-functional biomaterials for anti-bacterial, anti-biofilm and anti-cancer applications.

Benzoate transport in Pseudomonas putida CSV86.

Pseudomonas putida strain CSV86 metabolizes variety of aromatic compounds as the sole carbon source. Genome analysis revealed the presence of genes encoding putative transporters for benzoate, p-hydroxybenzoate, phenylacetate, p-hydroxyphenylacetate and vanillate. Bioinformatic analysis revealed that benzoate transport and metabolism genes are clustered at the ben locus as benK-catA-benE-benF. Protein topology prediction suggests that BenK (aromatic acid-H+ symporter of major facilitator superfamily) has 12 transmembrane α-helices with the conserved motif LADRXGRKX in loop 2, while BenE (benzoate-H+ symporter protein) has 11 predicted transmembrane α-helices. benF and catA encode benzoate specific porin, OprD and catechol 1,2-dioxygenase, respectively. Biochemical studies suggest that benzoate was transported by an inducible and active process. Inhibition (90%-100%) in the presence of dinitrophenol suggests that the energy for the transport process is derived from the proton motive force. The maximum rate of benzoate transport was 484 pmole min-1 mg-1 cells with an affinity constant, Kmof 4.5 µM. Transcriptional analysis of the benzoate and glucose-grown cells showed inducible expression of benF, benK and benE, suggesting that besides outer membrane porin, both inner membrane transporters probably contribute for the benzoate transport in P. putida strain CSV86.

Production of Polyhydroxyalkanoates from Sludge Palm Oil Using Pseudomonas putida S12.

Polyhydroxyalkanoates (PHAs) are biodegradable plastics produced by bacteria, but their use in diverse applications is prohibited by high production costs. To reduce these costs, the conversion by Pseudomonas strains of P HAs from crude s ludge p alm oil ( SPO) a s an inexpensive renewable raw material was tested. Pseudomonas putida S12 was found to produce the highest yield (~41%) of elastomeric medium-chain-length (MCL)-PHAs from SPO. The MCL-PHA characteristics were analyzed by gas-chromatography/mass spectrometry, gel permeation chromatography, and differential scanning calorimetry. These findings may contribute to more widespread use of PHAs by reducing PHA production costs.

Numerical modelling of biophysicochemical effects on multispecies reactive transport in porous media involving Pseudomonas putida for potential microbial enhanced oil recovery application.

pH and resident time of injected slug plays a critical role in characterizing the reservoir for potential microbial enhanced oil recovery (MEOR) application. To investigate MEOR processes, a multispecies (microbes-nutrients) reactive transport model in porous media was developed by coupling kinetic and transport model. The present work differs from earlier works by explicitly determining parametric values required for kinetic model by experimental investigations using Pseudomonas putida at different pH conditions and subsequently performing sensitivity analysis of pH, resident time and water saturation on concentrations of microbes, nutrients and biosurfactant within reservoir. The results suggest that nutrient utilization and biosurfactant production are found to be maximum at pH 8 and 7.5 respectively. It is also found that the sucrose and biosurfactant concentrations are highly sensitive to pH rather than reservoir microbial concentration, while at larger resident time and water saturation, the microbial and nutrient concentrations were lesser due to enhanced dispersion.

Effects of even and odd number fatty acids cofeeding on PHA production and composition in Pseudomonas putida Bet001 isolated from palm oil mill effluent.

The biosynthesis of medium-chain-length poly-3-hydroxyalkanoates by Pseudomonas putida Bet001 cultivated on mixed carbon sources was investigated. The mixed carbon sources consisted of heptanoic acid (HA) and oleic acid (OA). A relatively low PHA content at 1.2% (w/w) and 11.4% (w/w) was obtained when HA or OA was used as the sole carbon source. When these fatty acids were supplied as a mixture, PHA content increased threefold. Interestingly, the mixture-derived PHA composed of both odd and even monomer units, namely. 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate and no unsaturated monomer was detected. It is hypothesized that the even-numbered monomers were derived primarily from OA, whereas the odd-numbered monomer was derived from HA. This also points out to an efficient and yet distinct fatty acids metabolism that fed the PHA biosynthesis machinery of this particular microorganism. PHA obtained was elastomeric because melting temperature (Tm ) and crystallinity were absent. It showed good thermal stability with degradation temperature (Td ) ranging from 275.96 to 283.05 °C.

Bypasses in intracellular glucose metabolism in iron-limited Pseudomonas putida.

Decreased biomass growth in iron (Fe)-limited Pseudomonas is generally attributed to downregulated expression of Fe-requiring proteins accompanied by an increase in siderophore biosynthesis. Here, we applied a stable isotope-assisted metabolomics approach to explore the underlying carbon metabolism in glucose-grown Pseudomonas putida KT2440. Compared to Fe-replete cells, Fe-limited cells exhibited a sixfold reduction in growth rate but the glucose uptake rate was only halved, implying an imbalance between glucose uptake and biomass growth. This imbalance could not be explained by carbon loss via siderophore production, which accounted for only 10% of the carbon-equivalent glucose uptake. In lieu of the classic glycolytic pathway, the Entner-Doudoroff (ED) pathway in Pseudomonas is the principal route for glucose catabolism following glucose oxidation to gluconate. Remarkably, gluconate secretion represented 44% of the glucose uptake in Fe-limited cells but only 2% in Fe-replete cells. Metabolic (13) C flux analysis and intracellular metabolite levels under Fe limitation indicated a decrease in carbon fluxes through the ED pathway and through Fe-containing metabolic enzymes. The secreted siderophore was found to promote dissolution of Fe-bearing minerals to a greater extent than the high extracellular gluconate. In sum, bypasses in the Fe-limited glucose metabolism were achieved to promote Fe availability via siderophore secretion and to reroute excess carbon influx via enhanced gluconate secretion.

Impact of nanoscale zero valent iron on bacteria is growth phase dependent.

The toxic effect of nanoscale zero valent iron (nZVI) particles on bacteria from different growth phases was studied. Four bacterial strains namely Escherichia coli strains JM109 and BW25113, and Pseudomonas putida strains KT2440 and F1 were experimented. The growth curves of these strains were determined. Bacterial cells were harvested based on the predetermined time points, and exposed to nZVI. Cell viability was determined by the plate count method. Bacterial cells in lag and stationary phases showed higher resistance to nZVI for all four bacterial strains, whereas cells in exponential and decline phases were less resistant to nZVI and were rapidly inactivated when exposed to nZVI. Bacterial inactivation increased with the concentration of nZVI. Furthermore, less than 14% bacterial inactivation was observed when bacterial cells were exposed to the filtrate of nZVI suspension suggesting that the physical interaction between nZVI and cell is necessary for bacterial inactivation.

Stability of the GraA Antitoxin Depends on Growth Phase, ATP Level, and Global Regulator MexT.

UNLABELLED: Bacterial type II toxin-antitoxin systems consist of a potentially poisonous toxin and an antitoxin that inactivates the toxic protein by binding to it. Most of the toxins regulate stress survival, but their activation depends on the stability of the antitoxin that has to be degraded in order for the toxin to be able to attack its cellular targets. The degradation of antitoxins is usually rapid and carried out by ATP-dependent protease Lon or Clp, which is activated under stress conditions. The graTA system of Pseudomonas putida encodes the toxin GraT, which can affect the growth rate and stress tolerance of bacteria but is under most conditions inactivated by the unusually stable antitoxin GraA. Here, we aimed to describe the stability features of the antitoxin GraA by analyzing its degradation rate in total cell lysates of P. putida. We show that the degradation rate of GraA depends on the growth phase of bacteria being fastest in the transition from exponential to stationary phase. In accordance with this, higher ATP levels were shown to stabilize GraA. Differently from other antitoxins, the main cellular proteases Lon and Clp are not involved in GraA stability. Instead, GraA seems to be degraded through a unique pathway involving an endoprotease that cleaves the antitoxin into two unequal parts. We also identified the global transcriptional regulator MexT as a factor for destabilization of GraA, which indicates that the degradation of GraA may be induced by conditions similar to those that activate MexT. IMPORTANCE: Toxin-antitoxin (TA) modules are widespread in bacterial chromosomes and have important roles in stress tolerance. As activation of a type II toxin is triggered by proteolytic degradation of the antitoxin, knowledge about the regulation of the antitoxin stability is critical for understanding the activation of a particular TA module. Here, we report on the unusual degradation pathway of the antitoxin GraA of the recently characterized GraTA system. While GraA is uncommonly stable in the exponential and late-stationary phases, its degradation increases in the transition phase. The degradation pathway of GraA involves neither Lon nor Clp, which usually targets antitoxins, but rather an unknown endoprotease and the global regulator MexT, suggesting a new type of regulation of antitoxin stability.

Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste.

Treatment of solid and liquid tobacco wastes with high nicotine content remains a longstanding challenge. Here, we explored an environmentally friendly approach to replace tobacco waste disposal with resource recovery by genetically engineering Pseudomonas putida. The biosynthesis of 3-succinoyl-pyridine (SP), a precursor in the production of hypotensive agents, from the tobacco waste was developed using whole cells of the engineered Pseudomonas strain, S16dspm. Under optimal conditions in fed-batch biotransformation, the final concentrations of product SP reached 9.8 g/L and 8.9 g/L from aqueous nicotine solution and crude suspension of the tobacco waste, respectively. In addition, the crystal compound SP produced from aqueous nicotine of the tobacco waste in batch biotransformation was of high purity and its isolation yield on nicotine was 54.2%. This study shows a promising route for processing environmental wastes as raw materials in order to produce valuable compounds.

Pseudomonas aeruginosa displays an altered phenotype in vitro when grown in the presence of mannitol.

D-mannitol has been approved in dry powder formulation as an effective antimucolytic agent in patients with cystic fibrosis. What is not known is the effect of adding a metabolisable sugar on the biology of chronic bacterial pathogens in the CF lung. Therefore, a series of simple in vitro experiments were performed to examine the effect of adding D-mannitol on the phenotype of the CF respiratory pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia. Clinical isolates (n = 86) consisting of P. aeruginosa (n = 51), B. cenocepacia (n = 26), P. putida (n = 4), Stenotrophomonas maltophila (n = 3) and Pseudomonas spp. (n = 2) were examined by supplementing basal nutrient agar with varying concentrations of D-mannitol (0-20% [w/v]) and subsequently examining for any change in microbial phenotype. The effect of supplementation with mannitol was four-fold, namely i) To increase the proliferation and increase in cell density of all CF organisms examined, with an optimal concentration of 2-4% (w/v) D-mannitol. No such increase in cell proliferation was observed when mannitol was substituted with sodium chloride. ii) Enhanced pigment production was observed in 2/51 (3.9%) of the P. aeruginosa isolates examined, in one of the P. putida isolates, and in 3/26 (11.5%) of the B. cenocepacia isolates examined. iii). When examined at 4.0% (w/v) supplementation with mannitol, 11/51 (21.6%) P. aeruginosa isolates and 3/26 (11.5%) B. cenocepacia isolates were seen to exhibit the altered adhesion phenotype. iv). With respect to the altered mucoid phenotype, 5/51 (9.8%) P. aeruginosa produced this phenotype when grown at 4% mannitol. Mucoid production was greatest at 4%, was poor at 10% and absent at 20% (w/v) mannitol. The altered mucoid phenotype was not observed in the B. cenocepacia isolates or any of the other clinical taxa examined. Due consideration therefore needs to be given, where there is altered physiology within the small airways, leading to a potentially altered biological state of the colonising microorganisms in novel inhaled pharmaceutical interventions in CF, particularly those, which are not designated as antimicrobial agents.

Biocompatibility of low molecular weight polymers for two-phase partitioning bioreactors.

Two phase partitioning bioreactors (TPPBs) improve the efficiency of fermentative processes by limiting the exposure of microorganisms to toxic solutes by sequestering them into a non-aqueous phase (NAP). A potential limitation of this technology, when using immiscible organic solvents as the NAP, is the cytoxicity that these materials may exert on the microbes. An improved TPPB configuration is one in which polymeric NAPs are used to replace organic solvents in order to take advantage of their low cost, improved handling qualities, and biocompatibility. A recent study has shown that low molecular weight polymers may confer improved solute uptake relative to high molecular weight polymers (i.e., have higher partition coefficients), but it is unknown whether sufficiently low molecular weight polymers may inhibit cell growth. This study has investigated the biocompatibility of a range of low molecular weight polymers, and compared trends in biocompatibility to the well-established "critical log P" concept. This was achieved by determining the biocompatibility of polypropylene glycol polymers over a molecular weight (MW) range of 425-4,000 to Saccharomyces cerevisiae and Pseudomonas putida, two organisms which have been previously used in TPPB systems. The lower MW polymers were shown to have lower average log P values, and showed more cytotoxicity than polymers of the same structure but with higher molecular weight. Since polymers are generally polydisperse (i.e., polymer samples contain a distribution of MWs), removal of the lower MW fractions via water washing was found to result in improved polymer biocompatibility. These results suggest that the critical log P concept remains useful for describing the toxicity of polymeric substances of different MWs, although it is complicated by the presence of the low MW fractions in the polymers arising from polydispersity.

[The role of mineral phosphorus compounds in naphthalene biodegradation by Pseudomonas putida].

The effect of phosphate concentration in the culture medium on the growth and naphthalene degradation by Pseudomonas putida BS 3701 was studied. The limiting concentration of phosphate was 0.4 mM and 0.1 mM under cultivation in media with naphthalene and glucose, respectively The phosphate deficiency correlated with a decrease in the activities of naphthalene dioxygenase and salicylate hydroxylase and with salicylate accumulation in the culture medium. We suggest that this fact indicates the impaired regulation of gene expression of "upper" and "lower" pathways of naphthalene oxidation. Under naphthalene degradation, the cells accumulated three times more inorganic polyphosphates as compared with the consumption of glucose. The involvement of polyphosphates in the regulation of naphthalene metabolism has been considered.

Dynamics of suspended and attached aerobic toluene degraders in small-scale flow-through sediment systems under growth and starvation conditions.

The microbially mediated reactions, that are responsible for field-scale natural attenuation of organic pollutants, are governed by the concurrent presence of a degrading microbial community, suitable energy and carbon sources, electron acceptors, as well as nutrients. The temporal lack of one of these essential components for microbial activity, arising from transient environmental conditions, might potentially impair in situ biodegradation. This study presents results of small scale flow-through experiments aimed at ascertaining the effects of substrate-starvation periods on the aerobic degradation of toluene by Pseudomonas putida F1. During the course of the experiments, concentrations of attached and mobile bacteria, as well as toluene and oxygen were monitored. Results from a fitted reactive-transport model, along with the observed profiles, show the ability of attached cells to survive substrate-starvation periods of up to four months and suggest a highly dynamic exchange between attached and mobile cells under growth conditions and negligible cell detachment under substrate-starvation conditions. Upon reinstatement of toluene, it was readily degraded without a significant lag period, even after a starvation period of 130 days. Our experimental and modeling results strongly suggest that aerobic biodegradation of BTEX-hydrocarbons at contaminated field sites is not hampered by intermittent starvation periods of up to four months.

Genome reduction boosts heterologous gene expression in Pseudomonas putida.

BACKGROUND: The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome). RESULTS: In this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440. CONCLUSIONS: The two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.

Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity.

This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1-3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.

Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis.

Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain isolated from the tobacco rhizosphere. We successfully performed a comprehensive whole-genome analysis of nicotine metabolism-associated genes by Tn5 transposon mutagenesis in P. putida J5. A total of 18 mutants with unique insertions screened from 16,324 Tn5-transformants failed to use nicotine as the sole carbon source. Flanking sequences of the Tn5 transposon were cloned with a shotgun method from all of the nicotine-growth-deficient mutants. The potentially essential products of mutated gene were classified as follows: oxidoreductases, protein and metal transport systems, proteases and peptidases, transcriptional and translational regulators, and unknown proteins. Bioinformatic analysis of the Tn5 insertion sites indicated that the nicotine metabolic genes were separated and widely distributed in the genome. One of the mutants, M2022, was a Tn5 insert into a gene encoding a homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in Arthrobacter nicotinovorans. Genetic and biochemical analysis confirmed that three open reading frames (ORFs) from an approximately 13-kb fragment recovered from the mutant M2022 were responsible for the transformation of nicotine to 3-succinoyl-pyridine via pseudooxynicotine and 3-succinoyl semialdehyde-pyridine, the first three steps of nicotine degradation. Further research on these mutants and the Tn5-inserted genes will help us characterize nicotine metabolic processes in P. putida J5.