Enhanced denitrification and p-nitrophenol removal performance via hydrophilic sponge carriers fixed with dual-bacterial: Optimization, performance, and enhancement mechanism.

Effective treatment of industrial wastewater containing complex pollutants, such as nitrate (NO3--N) and organic pollutants, remains a significant challenge to date. Here, a strain Nocardioides sp. ZS2 with denitrification and degradation of p-nitrophenol (PNP) was isolated and its culture conditions were optimized by kinetic analysis. Hydrophilic sponge carriers were prepared using polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), and chitosan (CS) to construct bioreactors. Furthermore, to further enhance the PNP degradation and denitrification performance of bioreactors, Pseudomonas stutzeri GF2 with denitrification capability was introduced. The results revealed that the removal efficiencies of PNP and NO3--N reached 97.9 % and 91.9 %, respectively, when hydraulic retention time (HRT) of 6 h, C/N of 2.0, and pH of 6.5. The bioreactor exhibited stable denitrification performance even with fluctuations in the influent PNP concentration. The potential functional prediction results revealed that the abundance of amino acids, fatty acids, and carbohydrates increased as the influent C/N decreased, reflecting a tendency of the microbial community to adjust carbon source utilization to maintain cell growth, metabolic balance, and resist adverse C/N environments. This research provides new insights into the effective removal of organic pollutants and NO3--N in wastewater treatment.

Evaluation of aerobic granulation performance bioaugmented with the auto-aggregating bacterium Pseudomonas stutzeri strain XL-2 with heterotrophic nitrification-aerobic denitrification capacity.

In this study, the possibility of an auto-aggregating bacterium Pseudomonas strain XL-2 with heterotrophic nitrification-aerobic denitrification capacity for improving granulation and nitrogen removal was evaluated. The results showed that the supplementation of strain XL-2 promoted granulation, making R1 (experimental group with strain XL-2) dominated by granules at 14 d, which was 12 days earlier than R2 (control group without strain XL-2). This was attributed to the promotion of extracellular polymeric substances (EPS) secretion, particularly proteins by adding strain XL-2, thereby improving the hydrophobicity of sludge and altering the proteins secondary structures to facilitate aggregation. Meanwhile, adding strain XL-2 improved simultaneous nitrification and denitrification efficiency of R1. Microbial community analysis indicated that strain XL-2 successfully proliferated in aerobic granule sludge and might induce the enrichment of genera such as Flavobacterium and Paracoccus that were favorable for EPS secretion and denitrification, jointly promoting granulation and enhancing nitrogen removal efficiency.

Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501.

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCE: Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Enhancing nitrogen removal performance using immobilized aerobic denitrifying bacteria by modified polyvinyl alcohol/sodium alginate (PVA/SA).

Aerobic denitrification has emerged as a promising and efficient method for nitrogen removal from wastewater. However, the direct application of aerobic denitrifying bacteria has faced challenges such as low nitrogen removal efficiency, bacterial loss, and poor stability. To address these issues, this study developed a novel microbial particle carrier using NaHCO3-modified polyvinyl alcohol (PVA)/sodium alginate (SA) gel (NaHCO3-PVA/SA). This carrier exhibits several advantageous properties, including excellent mass transfer efficiency, favorable biocompatibility, convenient film formation, abundant biomass, and exceptional pollutant treatment capacity. The carrier was modified with 0.3% NaHCO3, 8.0% PVA, and 1.0% SA, resulting in a remarkable 3.4-fold increase in the average pore diameter and a 12.8% improvement in mass transfer efficiency. This carrier was utilized to immobilize the aerobic denitrifying bacterium Stutzerimonas stutzeri W-2 to enhance nitrogen removal (NaHCO3-PVA/SA@W-2), resulting in a NO3--N removal efficiency of 99.06%, which was 21.39% higher than that without modification. Compared with the non-immobilized W-2, the degradation efficiency was improved by 43.70%. After five reuses, the NO3--N and TN removal rates remained at 99% and 93.01%, respectively. These results provide a solid foundation for the industrial application of the modified carrier as an effective tool for nitrogen removal in large-scale wastewater treatment processes.

Inoculation with Stutzerimonas stutzeri strains decreases N2O emissions from vegetable soil by altering microbial community composition and diversity.

Inoculation with plant growth-promoting rhizobacteria (PGPR) strains has promoted plant growth and decreased nitrous oxide (N2O) emissions from agricultural soils simultaneously. However, limited PGPR strains can mitigate N2O emissions from agricultural soils, and the microbial ecological mechanisms underlying N2O mitigation after inoculation are poorly understood. In greenhouse pot experiments, the effects of inoculation with Stutzerimonas stutzeri NRCB010 and NRCB025 on tomato growth and N2O emissions were investigated in two vegetable agricultural soils with contrasting textures. Inoculation with NRCB010 and NRCB025 significantly promoted tomato growth in both soils. Moreover, inoculation with NRCB010 decreased the N2O emissions from the fine- and coarse-textured soils by 38.7% and 52.2%, respectively, and inoculation with NRCB025 decreased the N2O emissions from the coarse-textured soil by 76.6%. Inoculation with NRCB010 and NRCB025 decreased N2O emissions mainly by altering soil microbial community composition and the abundance of nitrogen-cycle functional genes. The N2O-mitigating effect might be partially explained by a decrease in the (amoA + amoB)/(nosZI + nosZII) and (nirS + nirK)/(nosZI + nosZII) ratios, respectively. Soil pH and organic matter were key variables that explain the variation in abundance of N-cycle functional genes and subsequent N2O emission. Moreover, the N2O-mitigating effect varied depending on soil textures and individual strain after inoculation. This study provides insights into developing biofertilizers with plant growth-promoting and N2O-mitigating effects. IMPORTANCE: Plant growth-promoting rhizobacteria (PGPR) have been applied to mitigate nitrous oxide (N2O) emissions from agricultural soils, but the microbial ecological mechanisms underlying N2O mitigation are poorly understood. That is why only limited PGPR strains can mitigate N2O emissions from agricultural soils. Therefore, it is of substantial significance to reveal soil ecological mechanisms of PGPR strains to achieve efficient and reliable N2O-mitigating effect after inoculation. Inoculation with Stutzerimonas stutzeri strains decreased N2O emissions from two soils with contrasting textures probably by altering soil microbial community composition and gene abundance involved in nitrification and denitrification. Our findings provide detailed insight into soil ecological mechanisms of PGPR strains to mitigate N2O emissions from vegetable agricultural soils.

Mechanisms of nanoscale zero-valent iron mediating aerobic denitrification in Pseudomonas stutzeri by promoting electron transfer and gene expression.

Aerobic denitrification and its mechanism by P. stutzeri was investigated in the presence of nanoscale zero-valent iron (nZVI). The removal of nitrate and ammonia was accelerated and the nitrite nitrogen accumulation was reduced by nZVI. The particle size and dosage of nZVI were key factors for enhancing aerobic denitrification. nZVI reduced the negative effects of low carbon/nitrogen, heavy metals, surfactants and salts to aerobic denitrification. nZVI and its dissolved irons were adsorbed into the bacteria cells, enhancing the transfer of electrons from nicotinamide adenine dinucleotide (NADH) to nitrate reductase. Moreover, the activities of NADH-ubiquinone reductase involved in the respiratory system, and the denitrifying enzymes were increased. The expression of denitrifying enzyme genes napA and nirS, as well as the iron metabolism gene fur, were promoted in the presence of nZVI. This work provides a strategy for enhancing the biological denitrification of wastewater using the bio-stimulation of nanomaterials.

Effects of silver nanoparticles and various forms of silver on nitrogen removal by the denitrifier Pseudomonas stutzeri and their toxicity mechanisms.

Silver nanoparticles (AgNPs) are widely used in daily life and industry because of their excellent antibacterial properties. AgNPs can exist in wastewater in various forms, such as Ag+, Ag2SO4, Ag2CO3, Ag2S, Ag2O, and AgCl. To assess the potential environmental risk of AgNPs and various forms of Ag, their toxic effects were investigated using the common denitrifier species Pseudomonas stutzeri (P. stutzeri). The inhibitory effect of AgNPs and various forms of Ag on P. stutzeri growth and its denitrification performance occurred in a concentration-dependent manner. The denitrification efficiency of P. stutzeri decreased from 95%∼97% to 89∼95%, 74∼95%, and 56∼85% under low, medium, and high exposure doses, respectively, of AgNPs and various forms of Ag. The changes in cell membrane morphology and increases in lactate dehydrogenase (LDH) release indicated that AgNPs and various forms of Ag damaged the cell membrane of P. stutzeri. Oxidative stress caused by excessive accumulation of reactive oxygen species (ROS) increased superoxide dismutase (SOD) and catalase (CAT) activities and decreased glutathione (GSH) levels. Overall, this study will help elucidate the impact of AgNPs and their transformation products on nitrogen removal efficiency in wastewater biological treatment systems.

Genetic and Functional Characterization of a Salicylate 1-monooxygenase Located on an Integrative and Conjugative Element (ICE) in Pseudomonas stutzeri AJR13.

Pseudomonas stutzeri strain AJR13 was isolated for growth on the related compounds biphenyl (BPH) and diphenylmethane (DPM). The BPH and DPM degradative pathway genes are present on an integrative and conjugative element (ICE) in the chromosome. Examination of the genome sequence of AJR13 revealed a gene encoding a salicylate 1-monooxygenase (salA) associated with the ICE even though AJR13 did not grow on salicylate. Transfer of the ICE to the well-studied Pseudomonas putida KT2440 resulted in a KT2440 strain that could grow on salicylate. Knockout mutagenesis of the salA gene on the ICE in KT2440 eliminated the ability to grow on salicylate. Complementation of the knockout with the cloned salA gene restored growth on salicylate. Transfer of the cloned salA gene under control of the lac promoter to KT2440 resulted in a strain that could grow on salicylate. Heterologous expression of the salA gene in E. coli BL21 DE3 resulted in the production of catechol from salicylate, confirming that it is indeed a salicylate 1-monooxygenase. Interestingly, transfer of the cloned salA gene under control of the lac promoter to AJR13 resulted in a strain that could now grow on salicylate, suggesting that gene expression for the downstream catechol pathway is intact.

Metal oxide nanoparticles inhibit nitrogen fixation and rhizosphere colonization by inducing ROS in associative nitrogen-fixing bacteria Pseudomonas stutzeri A1501.

The potential effects of engineered metal oxide nanoparticles (MONPs) on bacterial nitrogen fixation are of great concern. Herein, the impact and mechanism of the increasing-used MONPs, including TiO2, Al2O3, and ZnO nanoparticles (TiO2NP, Al2O3NP, and ZnONP, respectively), on nitrogenase activity was studied at the concentrations ranging from 0 to 10 mg L-1 using associative rhizosphere nitrogen-fixing bacteria Pseudomonas stutzeri A1501. Nitrogen fixation capacity was inhibited by MONPs in an increasing degree of TiO2NP < Al2O3NP < ZnONP. Realtime qPCR analysis showed that the expressions of nitrogenase synthesis-related genes, including nifA and nifH, were inhibited significantly when MONPs were added. MONPs could cause the explosion of intracellular ROS, and ROS not only changed the permeability of the membrane but also inhibited the expression of nifA and biofilm formation on the root surface. The repressed nifA gene could inhibit transcriptional activation of nif-specific genes, and ROS reduced the biofilm formation on the root surface which had a negative effect on resisting environmental stress. This study demonstrated that MONPs, including TiO2NP, Al2O3NP, and ZnONP, inhibited bacterial biofilm formation and nitrogen fixation in the rice rhizosphere, which might have a negative effect on the nitrogen cycle in bacteria-rice system.

Cometabolic degradation of pyrene with phenanthrene as substrate: assisted by halophilic Pseudomonas stutzeri DJP1.

At present, cometabolic degradation is an extensive method for the biological removal of high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) in the marine environment. However, due to the refractory to degradation and high toxicity, there are few studies on pyrene (PYR) cometabolic degradation with phenanthrene (PHE) as substrate. In this study, a Pseudomonas stutzeri DJP1 strain isolated from sediments was used in the cometabolic system of PHE and PYR. The biomass and the activity of key enzymes such as dehydrogenase and catechol 12 dioxygenase of strain were improved, but the enhancement of biotoxicity resulted in the inhibition of cometabolism simultaneously. Seven metabolites were identified respectively in PYR, PHE degradation cultures. It was speculated that the cometabolism of PHE and PYR had a common phthalic acid pathway, and the degradation pathway of PHE was included in the downstream pathway of PYR. The functional genes such as PhdF, NidD and CatA involved in DJP1 degradation were revealed by Genome analysis. This study provides a reference for the biodegradation of PYR and PHE in real marine environment.

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity.

BACKGROUND: Biological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166. RESULTS: Twenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain. CONCLUSIONS: The endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Heterogeneous distribution of carbofuran shaped by Pseudomonas stutzeri biofilms enhances biodegradation of agrochemicals.

Biodegradation, harnessing the metabolic versatility of microorganisms to reduce agrochemical contaminations, is commonly studied with enriched planktonic cells but overlooking the dominant lifestyle of microorganisms is to form biofilms, which compromises the efficiency of biodegradation in natural environment. Here, we employed a carbofuran-degrading bacterium Pseudomonas stutzeri PS21 to investigate how the bacterial biofilms formed and responded to agrochemicals. First, the PS21 biofilms formed with a core of bacterial cells enclosing with extracellular polymeric substances (EPSs), and the biofilms were active and resilient when exposed to carbofuran (up to 50 mg L-1). The formation was regulated by the second messenger bis-(3'-5')-cyclic di-guanosine monophosphate signaling, which strengthened the structural resistance and metabolic basis of biofilms to remain the degrading efficiency as comparable as the planktonic cells. Second, carbofuran distributed heterogeneously in the near-biofilm microenvironment via the covalent adsorption of biofilms, which provided a spontaneous force that enhanced the combination of carbofuran with biofilms to maintain high degrading activity. Additionally, we elucidated the biodegradation was driven by the integrated metabolic system of biofilms involving the extracellular enzymes located in the EPSs. This study exhibited the structural and metabolic advantages of microbial biofilms, highlighting the attractive potentials of exploring biofilm-based strategies to facilitate the in-situ bioremediation of organic contaminations.

Biodegradation of asphaltenes by an indigenous bioemulsifier-producing Pseudomonas stutzeri YWX-1 from shale oil in the Ordos Basin: Biochemical characterization and complete genome analysis.

Crude oil pollution is environmentally ubiquitous and has become a global public concern about its impact on human health. Asphaltenes are the key components of heavy crude oil (HCO) that are underutilized due to their high viscosity and density, and yet, the associated information about biodegradation is extremely limited in the literature. In the present study, an indigenous bacterium with effective asphaltene-degrading activity was isolated from oil shale and identified as Pseudomonas stutzeri by a polyphasic taxonomic approach, named YWX-1. Supplemented with 75 g L-1 heavy crude oil as the sole carbon source for growth in basic mineral salts liquid medium (MSM), strain YWX-1 was able to remove 49% of asphaletene fractions within 14 days, when it was cultivated with an initial inoculation size of 1%. During the degradation process, the bioemulsifier produced by strain YWX-1 could emulsify HCO obviously into particles, as well as it had the ability to solubilize asphaletenes. The bioemulsifier was identified to be a mixture of polysaccharide and protein through Fourier transform infrared spectroscopy (FT-IR). The genome of strain YWX-1 contains one circular chromosome of 4488441 bp with 63.98% GC content and 4145 protein coding genes without any plasmid. Further genome annotation indicated that strain YWX-1 possesses a serial of genes involved in bio-emulsification and asphaltenes biodegradation. This work suggested that P. stutzeri YWX-1 could be a promising species for bioremediation of HCO and its genome analysis provided insight into the molecular basis of asphaltene biodegradation and bioemulsifier production.

Pseudomonas stutzeri PM101005 inhaled with atmospheric particulate matter induces lung damage through inflammatory responses.

Atmospheric particulate matter (PM) contains a mixture of chemical and biological elements that pose threat to human health by increasing susceptibility to respiratory diseases. Although the identification of the microorganisms composing the PM has been assessed, their immunological impacts are still questionable. Here, we examined the mechanisms responsible for the pathogenicity of Pseudomonas stutzeri PM101005 (PMPS), a bacterium isolated from fine dust, in lung epithelial cells, alveolar cells, and macrophages. Relative to its comparative strain Pseudomonas stutzeri (PS), infections with PMPS induced higher production of inflammatory cytokines and chemokines, mediated by the activation of NF-κB and MAPK signaling pathways. Additionally, with three-dimensional (3D) airway spheroids which mimic the human bronchial epithelium, we confirmed that PMPS infections lead to relatively higher induction of pro-inflammatory cytokines than PM infections. Consistent results were observed in murine models as the infections with PMPS provoked greater inflammatory responses than the infections with PS. These PMPS-induced responses were mediated by the signaling pathways of the Toll-like receptors (TLRs), which regulated PMPS infection and played an important role in the expression of the antibiotic peptide ß-defensin 3 (BD3) that suppressed PMPS proliferation. Moreover, PM pretreatment enhanced inflammatory responses and tissue damage of PMPS, while reducing BD3 expression. Overall, these results indicate that PM-isolated PMPS induce TLR-mediated inflammatory responses in lung tissues, and contributes to the understanding of the etiology of PM-induced respiratory damage.

Nitrogen Removal Characteristics of a Marine Denitrifying Pseudomonas stutzeri BBW831 and a Simplified Strategy for Improving the Denitrification Performance Under Stressful Conditions.

A marine aerobic denitrifying bacterium was isolated and identified as Pseudomonas stutzeri BBW831 from the seabed silt of Beibu Gulf in China. According to the genome analysis, P. stutzeri BBW831 possessed a total of 14 genes (narG, narH, narI, narJ, napA, napB, nirB, nirD, nirS, norB, norC, norD, norQ, and nosZ) responsible for fully functional enzymes (nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase) involved in the complete aerobic denitrification pathway, suggesting that it had the potential for reducing nitrate to the final N2. Denitrification results showed that P. stutzeri BBW831 exhibited efficient nitrogen removal characteristics. Within 12 h, the NO3--N removal efficiency and rate reached 94.64% and 13.09 mg·L-1·h-1 under 166.10 ± 3.75 mg/L NO3--N as the sole nitrogen source, and removal efficiency of the mixed nitrogen (50.50 ± 0.55, 62.28 ± 0.74, and 64.26 ± 0.90 mg/L of initial NH4+-N, NO3--N, and NO2--N, respectively) was nearly 100%. Furthermore, a simplified strategy, by augmenting the inoculation biomass, was developed for promoting the nitrogen removal performance under high levels of NO2--N and salinity. As a result, the removal efficiency of the initial NO2--N up to approximately 130 mg/L reached 99.46% within 8 h, and the NO3--N removal efficiency achieved at 59.46% under the NaCl concentration even up to 50 g/L. The C/N ratio of 10 with organic acid salt such as trisodium citrate and sodium acetate as the carbon source was most conducive for cell growth and nitrogen removal by P. stutzeri BBW831, respectively. In conclusion, the marine P. stutzeri BBW831 contained the functional genes responsible for a complete aerobic denitrification pathway (NO3--N → NO2--N → NO → N2O → N2), and had great potential for the practical treatment of high-salinity nitrogenous mariculture wastewater.

Biogenic ferrihydrite-humin coprecipitate as an electron donor for the enhancement of microbial denitrification by Pseudomonas stutzeri.

Nitrate pollution of groundwater has become an increasingly serious environmental problem that poses a great threat to aquatic ecosystems and to human health. Previous studies have shown that solid-phase humin (HM) can act as an additional electron donor to support microbial denitrification in the bioremediation of nitrate-contaminated groundwater where electron donor is deficient. However, the electron-donating capacities of HMs vary widely. In this study, we introduced ferrihydrite and prepared ferrihydrite-humin (Fh-HM) coprecipitates via biotic means to strengthen their electron-donating capacities. The spectroscopic results showed that the crystal phase of Fh did not change after coprecipitation with HM in the presence of Shewanella oneidensis MR-1, and iron may have complexed with the organic groups of HM. The Fh-HM coprecipitate prepared with an optimal initial Fh-HM mass ratio of 14:1 enhanced the microbial denitrification of Pseudomonas stutzeri with an electron-donating capacity 2.4-fold higher than that of HM alone, and the enhancement was not caused by greater bacterial growth. The alginate bead embedding assay indicated that the oxidation pathway of Fh-HM coprecipitate was mainly through direct contact between P. stutzeri and the coprecipitate. Further analyses suggested that quinone and organic-complexed Fe were the main electron-donating fractions of the coprecipitate. The results of the column experiments demonstrated that the column filled with Fh-HM-coated quartz sand exhibited a higher denitrification rate than the one filled with quartz sand, indicating its potential for practical applications.

PHA stimulated denitrification through regulation of preferential cofactor provision and intracellular carbon metabolism at different dissolved oxygen levels by Pseudomonas stutzeri.

Denitrification, a typical biological process mediated by complex environmental factors, i.e., carbon sources and dissolved oxygen (DO), has attracted great attention due to its contribution to the control of eutrophication and the biochemical cycling of nitrogen. However, the effects of carbon source on electron distribution and enzyme expression for enhanced denitrification under competition of electron acceptors (DO and nitrate) remain unclear. Here, we profile the carbon metabolic pathway of polyhydroxybutyrate (PHB) and glucose (Glu) at high and low DO levels (50% and 10% saturated DO, respectively). It was found that PHB enhanced the growth of Pseudomonas stutzeri (model denitrifying bacterium) and improved the specific nitrogen removal rate (SNRR) at all DO levels. The functional proteins had a better affinity for the cofactor nicotinamide-adenine dinucleotide (NADH) than for nicotinamide adenine dinucleotide phosphate (NADPH); thus, more electrons were involved in nitrogen reduction and intracellular PHB production in the PHB groups than in the Glu groups. Furthermore, the expression difference of enzymes in glucose and PHB metabolism was demonstrated by metaproteomic and target protein analysis, implying that PHB-driven intracellular carbon accumulation could optimize the intracellular electron allocation and correspondingly promote nitrogen metabolism. Our work integrated the mechanisms of intracellular carbon metabolism with preferences for electron transfer pathways in denitrification, providing a new perspective on how the selective parameters regulated microbial functions involved in denitrification.

Production of Membrane Proteins in Pseudomonas stutzeri.

Functional and structural studies on membrane proteins are often hampered by insufficient yields, misfolding and aggregation during the production and purification process. Escherichia coli is the most commonly used expression host for the production of recombinant prokaryotic integral membrane proteins. However, in many cases expression hosts other than E. coli are more appropriate for certain target proteins. Here, we report a convenient, systematically developed expression system using the γ-proteobacterium Pseudomonas stutzeri as an alternative production host for over-expression of integral membrane proteins. P. stutzeri can be easily and inexpensively cultured in large quantities. The Pseudomonas expression vectors are designed for inducible expression of affinity-tagged fusion proteins controlled by the PBAD promoter. This chapter provides detailed protocols of the different steps required to successfully produce and isolate recombinant membrane proteins with high yields in P. stutzeri.

Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation.

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the medium, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylation state of proteins when heterologously expressed, since their biochemical and physiological properties can be dependent on post-translational modification.

Insight rifampicin-resistant (rpoB) mutation in Pseudomonas stutzeri leads to enhance the biosynthesis of secondary metabolites to survive against harsh environments.

In this study, a wild-type and five distinct rifampicin-resistant (Rifr) rpoB mutants of Pseudomonas stutzeri (i.e., Q518R, D521Y, D521V, H531R and I614T) ability were investigated against harsh environments (particularly nutritional complexity). Among these, the robust Rifr phenotype of P. Stutzeri was associated only with base replacements of the amino deposits. The use of carboxylic and amino acids significantly increased in various Rifr mutants than that of wild type of P. stutzeri. The assimilation of carbon and nitrogen (N) sources of Rifr mutants' confirmed that the organism maintains the adaptation in nutritionally complex environments. Acetylene reduction assay at different times also found the variability for N-fixation in all strains. Among them, the highest nitrogenase activity was determined in mutant 'D521V'. The assimilation of carbon and nitrogen sources of P. stutzeri and its Rifr mutants ensures that the organism maintains the adaptability in nutritionally complex environments through fixing more nitrogen.