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The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether c-di-GMP participates in regulating nitrate assimilation is unclear. Here, we found that NasT, an antiterminator involved in nitrate assimilation in Pseudomonas putida, specifically bound c-di-GMP. NasT was essential for expressing the nirBD operon encoding nitrite reductase during nitrate assimilation. High-level c-di-GMP inhibited the binding of NasT to the leading RNA of nirBD operon (NalA), thus attenuating the antitermination function of NasT, resulting in decreased nirBD expression and nitrite reductase activity, which in turn led to increased nitrite accumulation in cells and its export. Molecular docking and point mutation assays revealed five residues in NasT (R70, Q72, D123, K127 and R140) involved in c-di-GMP-binding, of which R140 was essential for both c-di-GMP-binding and NalA-binding. Three diguanylate cyclases (c-di-GMP synthetases) were found to interact with NasT and inhibited nirBD expression, including WspR, PP_2557, and PP_4405. Besides, the c-di-GMP-binding ability of NasT was conserved in the other three representative Pseudomonas species, including P. aeruginosa, P. fluorescens and P. syringae. Our findings provide new insights into nitrate assimilation regulation by revealing the mechanism by which c-di-GMP inhibits nitrate assimilation via NasT., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase and degraded by c-di-GMP-specific phosphodiesterase. The genome of Pseudomonas putida contains dozens of genes encoding diguanylate cyclase/phosphodiesterase, but the phenotypical-genotypical correlation and functional mechanism of these genes are largely unknown. Herein, we characterize the function and mechanism of a P. putida phosphodiesterase named DibA. DibA consists of a PAS domain, a GGDEF domain, and an EAL domain. The EAL domain is active and confers DibA phosphodiesterase activity. The GGDEF domain is inactive, but it promotes the phosphodiesterase activity of the EAL domain via binding GTP. Regarding phenotypic regulation, DibA modulates the cell surface adhesin LapA level in a c-di-GMP receptor LapD-dependent manner, thereby inhibiting biofilm formation. Moreover, DibA interacts and colocalizes with LapD in the cell membrane, and the interaction between DibA and LapD promotes the PDE activity of DibA. Besides, except for interacting with DibA and LapD itself, LapD is found to interact with 11 different potential diguanylate cyclases/phosphodiesterases in P. putida, including the conserved phosphodiesterase BifA. Overall, our findings demonstrate the functional mechanism by which DibA regulates biofilm formation and expand the understanding of the LapD-mediated c-di-GMP signaling network in P. putida., (© 2023 John Wiley & Sons Ltd.)

Type VI secretion systems (T6SS) are specific antibacterial weapons employed by diverse bacteria to protect themselves from competitors. Pseudomonas putida KT2440 possesses a functional T6SS (K1-T6SS) and exhibits antibacterial activity towards a broad range of bacteria. Here we found that the Wsp signal transduction system regulated K1-T6SS expression via synthesizing the second messenger cyclic di-GMP (c-di-GMP), thus mediating antibacterial activity in P. putida. High-level c-di-GMP produced by Wsp system repressed the transcription of K1-T6SS genes in structural operon and vgrG1 operon. Transcriptional regulator FleQ and ATPase FleN functioned as repressors in the Wsp system-modulated K1-T6SS transcription. However, FleQ and FleN functioned as activators in biofilm formation, and Wsp system promoted biofilm formation largely in a FleQ/FleN-dependent manner. Furthermore, FleQ-FleN complex bound directly to the promoter of K1-T6SS structural operon in vitro, and c-di-GMP promoted the binding. Besides, P. putida biofilm cells showed higher c-di-GMP levels and lower antibacterial activity than planktonic cells. Overall, our findings reveal a mechanism by which Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN, and demonstrate the relationship between plankton/biofilm lifestyles and antibacterial activity in P. putida., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

Two-component systems (TCSs) are predominant means by which bacteria sense and respond to environment signals. Genome of Pseudomonas putida contains dozens of putative TCS-encoding genes, but phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterized function and transcriptional regulation of a conserved P. putida TCS, named TarR-TarS. TarS (PP_0769) encodes a potential histidine kinase, and tarR (PP_0768) encodes a potential response regulator. Protein-protein interaction assay and phosphorylation assay confirmed that TarR-TarS was a functional TCS. Growth assay under antibiotics revealed that TarR-TarS positively regulated bacterial resistance to multiple antibiotics. Pull-down assay revealed that TarR directly interacted with PP_0800 (a hypothetical protein) and GroEL (the chaperonin). GroEL played a positive role in antibiotic resistance, while PP_0800 seemed to have no effect on antibiotic resistance. The regulator FleQ indirectly activated tarR-tarS transcription. However, the second messenger c-di-GMP antagonized FleQ activation to inhibit tarR-tarS transcription. The sigma factor FliA directly activated tarR-tarS transcription via a consensus motif. These findings reveal function and transcriptional regulation of TarR-TarS, and enrich knowledge regarding the relationship between c-di-GMP and antibiotic susceptibility in P. putida., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Cyclic diguanylate (c-di-GMP) is a broadly conserved bacterial signalling molecule that modulates diverse cellular processes, such as biofilm formation, colony morphology and swimming motility. The intracellular level of c-di-GMP is controlled by diguanylate cyclases (DGCs) with GGDEF domain and phosphodiesterases (PDEs) with either EAL or HD-GYP domain. Pseudomonas putida KT2440 has a large group of genes on its genome encoding proteins with GGDEF/EAL/HD-GYP domains. However, phenotypic-genotypic correlation and c-di-GMP metabolism of these genes were largely unknown. Herein, by systematically constructing deletion mutants/overexpression strains of the 42 predicted c-di-GMP metabolism-related genes and analysing the phenotypes, we preliminarily revealed the role of each gene in biofilm formation, colony morphology and swimming motility. Subsequent results from protein sequence alignments and cellular c-di-GMP assessment indicated that 25 out of the 42 genes were likely to encode DGCs, nine genes were predicted to encode PDEs, four genes encoded bifunctional enzymes and the other four genes encoded enzymatically inactive proteins. This study offers a basic understanding of the roles of these 42 genes and can serve as a toolkit for investigators to further elucidate the functions of these GGDEF and EAL/HD-GYP domain-containing proteins., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents that would damage cells or even cause cell death. Bacteria have developed defensive systems, including induction of stress-sensing proteins and detoxification enzymes, to handle oxidative stress. Cyclic diguanylate (c-di-GMP) is a ubiquitous intracellular bacterial second messenger that coordinates diverse aspects of bacterial growth and behavior. In this study, we revealed a mechanism by which c-di-GMP regulated bacterial oxidative stress resistance in Pseudomonas putida KT2440. High c-di-GMP level was found to enhance bacterial resistance towards hydrogen peroxide. Transcription assay showed that expression of two oxidative stress resistance genes, fpr-1 and katE, was promoted under high c-di-GMP level. Deletion of fpr-1 and katE both decreased bacterial tolerance to hydrogen peroxide and weakened the effect of c-di-GMP on oxidative stress resistance. The promoted expression of fpr-1 under high c-di-GMP level was caused by increased cellular ROS via a transcriptional regulator FinR. We further demonstrated that the influence of high c-di-GMP on cellular ROS depend on the existence of FleQ, a transcriptional regulatory c-di-GMP effector. Besides, the regulation of katE by c-di-GMP was also FleQ dependent in an indirect way. Our results proved a connection between c-di-GMP and oxidative stress resistance and revealed a mechanism by which c-di-GMP regulated expression of fpr-1 and katE in P. putida KT2440.

In Pseudomonas putida KT2440, the exopolysaccharide Pea is associated with biofilm stability and pellicle formation; however, little is known about its regulatory pathway. In this study, we identified that the gene cluster pea was transcribed from 25 bp upstream of the operon and the stationary phase alternative sigma factor RpoS regulated the transcription of pea. When RpoS was absent, another sigma factor, likely the housekeeping sigma factor RpoD, could also mediate pea transcription but at a low level. The function of Pea polysaccharide was further confirmed to be necessary for full production of biofilm, formation of pellicle and c-di-GMP-dependent wrinkly colony morphology. Additionally, evidences were provided to demonstrate that the transcriptional regulator AmrZ was a negative regulator for pea expression. DNase I footprinting studies verified that AmrZ bound directly to the site overlapping the pea promoter, which might interfere with the binding of RNA polymerase to the promoter and resulted in inhibition of transcription initiation., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

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 ., (Copyright © 2018 American Society for Microbiology.)

Though abundant studies have targeted the characterization of heavy metal adsorption by either clay minerals or bacteria, to date, minimal literature exists which specifically assesses bacteria-clay mineral interactions in the context of metal immobilization. The adsorption of Pb onto montmorillonite, Pseudomonas putida, and their 1:1, 2:1, 6:1 and 12:1 mass ratio composites were investigated by using a combination of atomic force microscope (AFM), X-ray diffraction (XRD), surface complexation modeling (SCM), Pb-L III edge extended X-ray absorption fine structure (EXAFS) spectroscopy and isothermal titration calorimetry (ITC). The SCM and EXAFS demonstrated that Pb ions coordinate with phosphoryl and carboxyl functional groups on bacteria at low and high concentrations, respectively. The ITC analysis found adverse enthalpy values for Pb adsorption to permanent (-2.91 kJ/mol) and variable charge sites (6.93 kJ/mol) on montmorillonite. The ternary bridging model, EXAFS and ITC provide molecular and thermodynamic evidences for the formation of enthalpy driven (-4.74 kJ/mol) ternary complex (>AlO-Pb-PO 4 ) in the composites. The proportion for the bridging structures increased at pH > 5 and high bacterial mass ratios. The formation of ternary complex did not result in the enhanced adsorption of Pb on the composites, but promoted the allocation of Pb on the mineral fraction. The results obtained from SCM, EXAFS and ITC may provide an essential assumption for predicting the speciation and fate of Pb in soils and associated environments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Surface complexation modeling, isothermal titration calorimetry, and batch adsorption were employed to characterize the adsorption of Cu onto montmorillonite, Pseudomonas putida X4, and their composites at mass ratios of 2:1, 6:1, and 12:1, respectively. Different enthalpy values were found for Cu adsorption to permanent (-6.43kJ/mol) and variable charge sites (8.51kJ/mol) on montmorillonite. The component additivity (CA) method was used to predict the adsorption of Cu on the composites by combining end member models for montmorillonite and P. putida. A reduced adsorption was observed at pH<5.5 due to physical blocking between montmorillonite and P. putida. By contrast, the enhanced binding at high pH levels was ascribed to the formation of bridging structures among the clay mineral-Cu-bacteria complexes. These deviations in the CA method were corrected by adding reactions of >RCOOH…XNa and >RCOOCuOHXNa. The increased adsorption of the composites confirmed the decrease of permanent negative charge sites and the formation of ternary complexes. The results help elucidate the effect of Cu adsorption onto clay minerals-bacteria composites. The newly developed "CA-site masking-bridging" model can be used to predict Cu speciation in systems in which the active interaction of bacteria and clay minerals occurs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The global regulatory molecule (p)ppGpp is synthesized under limited nutrition conditions and involves in many cellular processes in bacteria. (p)ppGpp has been reported to affect biofilm formation in several bacterial species. Here, we found that deletion of (p)ppGpp synthase genes of Pseudomonas putida KT2440 led to enhanced biofilm formation in polystyrene microtitre plates. Besides, the pellicle of this mutant formed at the air-liquid interface lost the robust structure and became frail. The biofilm formation and its structure are mainly determined by exopolysaccharides (EPSs) and adhesins. Transcriptional analysis of four EPS operons designated as pea, peb, alg and bcs and two adhesin genes nominated as lapA and lapF showed that the deletion of (p)ppGpp synthase genes increased the expression of peb, bcs and lapA but repressed the expression of pea and lapF. Furthermore, expression of the regulation factor FleQ was significantly augmented in (p)ppGpp-synthase mutants while the expression of sigma factor RpoS was reduced. Since FleQ and RpoS play important roles in regulating expression of EPS and adhesin genes, (p)ppGpp may mediate the synthesis of biofilm matrix via influencing these regulators to control the biofilm formation and pellicle structure., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Bacteria-phyllosilicate complexes are commonly found in natural environments and are capable of immobilizing trace metals. However, the molecular binding mechanisms of heavy metals to these complex aggregates still remain poorly understood. This study investigated Cd adsorption on Gram-positive Bacillus subtilis, Gram-negative Pseudomonas putida and their binary mixtures with montmorillonite using surface complexation model, Cd K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and isothermal titration calorimetry (ITC). We have shown that larger amounts of Cd are adsorbed by B. subtilis than by P. putida at pH<∼6, and Cd sorption that binding to phosphate groups plays a more important role in P. putida than in B. subtilis. This remind us that we should consider the microbe species when predict the biochemical behavior of trace metals in microbe-bearing environments. The observed Cd adsorption on the binary bacteria-clay composites was more than that predicted based on the component additivity approach. When taking bacteria-clay (1:1 mass ratio) as a representative example, an approximately 68%:32% metal distribution between the bacterial and mineral fraction was found. Both the EXAFS and ITC fits showed that the binding stoichiometry for Cd-carboxyl/phosphate was smaller in the binary mixtures than that in pure bacteria. We proposed that the significant deviations were possibly due to the physical-chemical interaction between the composite fractions that might reduce the agglomeration of the clay grains, increase the negative surface charges, and provide additional bridging of metals ions between bacterial cells and clays., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

FleN generally functions as an antagonist of FleQ in regulating flagellar genes and biofilm matrix related genes in Pseudomonas aeruginosa. Here, we found that in Pseudomonas putida KT2440, FleN and FleQ play a synergistic role in regulating two biofilm matrix coding operons, lapA and bcs. FleN deletion decreased the transcription of lapA and increased the transcription of bcs operon, and the same trend was observed in fleQ deletion mutant before. In vitro experiments showed that FleN promoted the binding of FleQ to the lapA/bcs promoter DNA especially in the presence of ATP. Both phenotype observation and transcription analysis showed that, similar to fleQ deletion, fleN deletion significantly weaken the effect of high c-di-GMP level on biofilm formation, surface winkle phenotype and expression of lapA and bcs operons. Mutagenesis of the putative ATP binding motif in FleN K21Q revealed that FleN ATPase activity played an essential role in the regulation of flagellar number and swimming motility but was not critical for biofilm formation. Our results revealed that FleN was not an antagonist of FleQ but a synergistic factor of FleQ in regulating the two biofilm matrix coding operons in P. putida KT2440., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Flagella-mediated motility is an important capability of many bacteria to survive in nutrient-depleted and harsh environments. Decreasing the intracellular cyclic di-GMP (c-di-GMP) level by overexpression of phosphodiesterase BifA promotes flagellar-mediated motility and induces planktonic lifestyle in Pseudomonas. The mechanism that regulates expression of bifA gene was poorly studied. Here we showed that expression of BifA was partly controlled by flagellar sigma factor FliA (σ 28 ) in Pseudomonas putidaKT2440. FliA deletion led to an approximately twofold decrease in transcription of bifA. 5' race assay revealed two transcription start points in bifA promoter region, with the putative σ 70 and σ 28 promoter sequences upstream, respectively. Point mutation in σ 28 promoter region reduced transcriptional activity of the promoter in wild-type KT2440, but showed no influence on that in fliA deletion mutant. FliA overexpression decreased the intracellular c-di-GMP level in a BifA-dependent way, suggesting that FliA was able to modulate the intracellular c-di-GMP level and BifA function was required for the modulation. Besides, FliA overexpression enhanced swimming ability of wild-type strain, while made no difference to the bifA mutant. Our results suggest that FliA acts as a negative regulator to modulate the c-di-GMP level via controlling transcription of bifA to facilitate swimming motility., (© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that regulates diverse cellular processes, is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). GcbA is a well conserved DGC among Pseudomonas species, and has been reported to influence biofilm formation and flagellar motility in Pseudomonas fluorescens and Pseudomonas aeruginosa. Here we confirm the function of GcbA in Pseudomonas putida and reveal that expression of GcbA is regulated by FleQ in response to c-di-GMP. GcbA deletion impaired initial biofilm formation and enhanced swimming motility, but showed no influence on biofilm maturation in Pseudomonas putida. Deletion of the c-di-GMP effector FleQ led to a significant decrease in transcription of gcbA. Moreover, reducing c-di-GMP levels promoted gcbA transcription in a FleQ dependent way, while enhancing c-di-GMP levels abolished the promotion. In in vitro experiments we found that FleQ bound to gcbA promoter DNA and the binding was inhibited by c-di-GMP. Besides, FleN, an anti-activator of FleQ, and the sigma factor RpoN also participated in transcription of gcbA. Our finding expands the complexity of FleQ-dependent regulation and reveals a self-regulation function of c-di-GMP by regulating GcbA expression via FleQ., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The characteristics and mechanisms of competitive adsorption of trace metals on bacteria-associated clay mineral composites have never been studied, despite their being among the most common organic-mineral complexes in geological systems. Herein, competitive adsorption of Pb and Cd on Pseudomonas putida-montmorillonite composite was investigated through adsorption-desorption experiment, isothermal titration calorimetry (ITC), and synchrotron micro X-ray fluorescence (μ-XRF). From the experiment, stronger competition was observed on clay mineral than on bacteria-clay composite because more non-specific sites accounted for heavy metal adsorption on clay mineral surface at the studied pH 5. Both competing heavy metals tended to react with bacterial fractions in the composite, which was verified by the higher correlation of Cd (and Pb) with Zn (R 2  = 0.41) elemental distribution than with Si (R 2  = 0.10). ITC results showed that competitive adsorption exhibited a lower entropy change (ΔS) at the metal-sorbent interfaces compared with single-metal adsorption, revealing that Cd and Pb are bound to the same types of adsorption sites on the sorbent. The competitive effect on bacteria-clay composite was found to be helpful for a better understanding on the fixation, remobilization and subsequent migration of heavy metals in multi-metal contaminated environments., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A multifunctional Pseudomonas putida X3 strain was successfully engineered by introducing methyl parathion (MP)-degrading gene and enhanced green fluorescent protein (EGFP) gene in P. putida X4 (CCTCC: 209319). In liquid cultures, the engineered X3 strain utilized MP as sole carbon source for growth and degraded 100 mg L(-1) of MP within 24 h; however, this strain did not further metabolize p-nitrophenol (PNP), an intermediate metabolite of MP. No discrepancy in minimum inhibitory concentrations (MICs) to cadmium (Cd), copper (Cu), zinc (Zn), and cobalt (Co) was observed between the engineered X3 strain and its host strain. The inoculated X3 strain accelerated MP degradation in different polluted soil microcosms with 100 mg MP kg(-1) dry soil and/or 5 mg Cd kg(-1) dry soil; MP was completely eliminated within 40 h. However, the presence of Cd in the early stage of remediation slightly delayed MP degradation. The application of X3 strain in Cd-contaminated soil strongly affected the distribution of Cd fractions and immobilized Cd by reducing bioavailable Cd concentrations with lower soluble/exchangeable Cd and organic-bound Cd. The inoculated X3 strain also colonized and proliferated in various contaminated microcosms. Our results suggested that the engineered X3 strain is a potential bioremediation agent showing competitive advantage in complex contaminated environments.

The role of different czcRS genes in metal resistance and the cross-link between czcRS and czcCBA in Pseudomonas putida X4 were studied to advance understanding of the mechanisms by which P. putida copes with metal stress. Similar to P. putida KT2440, two complete czcRS1 and czcRS2 two-component systems, as well as a czcR3 without the corresponding sensing component were amplified in P. putida X4. The histidine kinase genes czcS1 and czcS2 were inactivated and fused to lacZ by homologous recombination. The lacZ fusion assay revealed that Cd2+ and Zn2+ caused a decrease in the transcription of czcRS1, whereas Cd2+ treatment enhanced the transcription of czcRS2. The mutation of different czcRSs showed that all czcRSs are necessary to facilitate full metal resistance in P. putida X4. A putative gene just downstream of czcR3 is related to metal ion resistance, and its transcription was activated by Zn2+. Data from quantitative real-time polymerase chain reaction (qRT-PCR) strongly suggested that czcRSs regulate the expression of czcCBA, and a cross-link exists between different czcRSs.

Monkey metallothionein α domain tandem repeats (4mMTα), which exhibit high cadmium affinity, have been displayed for the first time on the surface of a bacterium using ice nucleation protein N-domain (inaXN) protein from the Xanthomonas campestris pv (ACCC-10049) as an anchoring motif. The shuttle vector pIME, which codes for INAXN-4mMTα-EGFP fusion, was constructed and used to target 4mMTα and EGFP on the surface of Pseudomonas putida X4 (CCTCC-209319). The surface location of the INAXN-4mMTα-EGFP fusion was further verified by western blot analysis and immunofluorescence microscopy. The growth of X4 showed resistance to cadmium presence. The presence of surface-exposed 4mMTα on the engineered strains was four times higher than that of the wild-type X4. The Cd²⁺ accumulation by X4/pIME was not only four times greater than that of the original host bacterial cells but was also remarkably unaffected by the presence of Cu²⁺ and Zn²⁺. Moreover, the surface-engineered strains could effectively bind Cd²⁺ under a wide range of pH levels, from 4 to 7. P. putida X4/pIME with surface-expressed 4mMTα-EGFP had twice the cadmium binding capacity as well as 1.4 times the fluorescence as the cytoplasmic 4mMTa-EGFP. These results suggest that P. putida X4 expressing 4mMTα-EGFP with the INAXN anchor motif on the surface would be a useful tool for the remediation and biodetection of environmental cadmium contaminants.

The nitric oxide synthase gene from Bacillus subtilis was heterologously expressed in Pseudomonas putida using a broad-host expression vector. Both the performance of the nitric oxide-specific fluorescent probe and the quantitative assessment of the nitric oxide end products demonstrated the generation of nitric oxide. The endogenous nitric oxide increased the motility of P. putida and decreased the capacity of P. putida and other multispecies bacteria to develop biofilms. On the practical side, endogenous nitric oxide offers an advantage in generating continuous, controllable levels of nitric oxide, which suggests a new method to use nitric oxide in the control of biofilms.

Equilibrium adsorption along with scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and isothermal titration calorimetry (ITC) techniques were employed to investigate the adhesion of Pseudomonas putida on goethite. The adhesion isotherm revealed the high affinity of P. putida for goethite. The SEM analysis also showed a tight association between bacteria and mineral particles. Larger amounts of adhesion of bacteria on goethite were observed at pH lower than the isoelectric point (IEP) of goethite. The bacterial adhesion increased with increasing concentration of K(+). The calorimetric results demonstrated that the P. putida-goethite adhesion was an exothermic process. The adhesion enthalpy increased with increasing pH and concentrations of electrolyte. The increase of the negative enthalpy with increment of temperature indicated that the bacteria-goethite adhesion was an enthalpy-driven process. Electrostatic interactions and hydrogen bonding were considered to contribute mainly to the adhesion of bacterial adhesion on goethite. The data obtained in this study would provide valuable information for a better understanding of the mechanisms of mineral-microorganism interactions in soil and associated environments.

In order to have a better understanding of the interactions of heavy metals with bacteria and minerals in soil and associated environments, isothermal titration calorimetry (ITC), potentiometric titration and equilibrium sorption experiments were conducted to investigate the adsorption behavior of Cu(II) by Bacillus thuringiensis, Pseudomonas putida and their composites with minerals. The interaction of montmorillonite with bacteria increased the reactive sites and resulted in greater adsorption for Cu(II) on their composites, while decreased adsorption sites and capacities for Cu(II) were observed on goethite-bacteria composites. A gram-positive bacterium B. thuringiensis played a more important role than a gram-negative bacterium P. putida in determining the properties of the bacteria-minerals interfaces. The enthalpy changes (DeltaH(ads)) from endothermic (6.14 kJ mol(-1)) to slightly exothermic (-0.78 kJ mol(-1)) suggested that Cu(II) is complexed with the anionic oxygen ligands on the surface of bacteria-mineral composites. Large entropies (32.96-58.89 J mol(-1) K(-1)) of Cu(II) adsorption onto bacteria-mineral composites demonstrated the formation of inner-sphere complexes in the presence of bacteria. The thermodynamic data implied that Cu(II) mainly bound to the carboxyl and phosphoryl groups as inner-sphere complexes on bacteria and mineral-bacteria composites., (Copyright 2010 Elsevier B.V. All rights reserved.)

Equilibrium adsorption experiments, isothermal titration calorimetry and potentiometric titration techniques were employed to investigate the adsorption of Cu(II) by extracellular polymeric substances (EPS) extracted from Pseudomonas putida X4, minerals (montmorillonite and goethite) and their composites. Compared with predicted values of Cu(II) adsorption on composites, the measured values of Cu(II) on EPS-montmorillonite composite increased, however, those on EPS-goethite composite decreased. Potentiometric titration results also showed that more surface sites were observed on EPS-montmorillonite composite and less reactive sites were found on EPS-goethite composite. The adsorption of Cu(II) on EPS molecules and their composites with minerals was an endothermic reaction, while that on minerals was exothermic. The positive values of enthalpy change (Delta H) and entropy change (DeltaS) for Cu(II) adsorption on EPS and mineral-EPS composites indicated that Cu(II) mainly interacts with carboxyl and phosphoryl groups as inner-sphere complexes on EPS molecules and their composites with minerals., ((c) 2010 Elsevier Ltd. All rights reserved.)

Adsorption and desorption of salmon sperm DNA on bacteria (Bacillus thuringiensis, Pseudomonas putida), two different colloidal fractions (organic and inorganic clay) from an Alfisol, minerals (montmorillonite, kaolinite and goethite) and colloid-bacteria composites were studied. Similar adsorption capacity and affinity of DNA were observed on two bacterial cells. However, the two bacterial strains played different roles in affecting the adsorption of DNA on the composites of soil colloidal particles with bacteria. The introduction of B. thuringiensis in soil colloids and minerals systems dramatically promoted DNA adsorption on colloidal particles especially organic clay, while P. putida decreased the adsorption of DNA on kaolinite and goethite. Electrostatic force and ligand exchange are regarded to be the major driving forces involved in the adsorption of DNA on bacterial cells, montmorillonite, soil colloids and goethite. Presence of bacteria enhanced the proportion of DNA adsorption on soil colloidal particles by electrostatic force and depressed that by ligand exchange process. Information obtained in this study is of fundamental significance for the understanding of the ultimate fate of extracellular DNA in soil systems.

Bacterial adhesion to minerals in soils and sediments is of fundamental importance in mineral weathering and formation, soil aggregate stability, organic matter degradation and the fate of pollutants. Bacterial surface properties are considered to govern adhesion, and these properties likely change as a function of bacterial growth phase. However, the effect of growth stage on bacterial adhesion to clay minerals remains unclear. This work examined the influence of growth phase on the adhesion of Pseudomonas putida to kaolinite-coated coverslips. Fluorescence microscopy, together with a bacterial viability stain, was used to directly quantify surface cell density and viability of adhered P. putida . In situ attenuated total reflectance Fourier transform infrared spectroscopy was applied to yield molecular information about the characteristics of the bacteria, and the adsorption and desorption kinetics. Stationary-phase cells exhibited a higher adsorption density on kaolinite surfaces than mid-exponential-phase cells under static deposition conditions. Compared with the mid-exponential-phase cells, the stationary-phase cells displayed higher saturation coverage, and we fitted the results using a pseudo-first-order kinetics equation. The greater extent of adhesion of the stationary-phase cells was probably due to their smaller cell size and less negative surface charges compared with the cells from other growth stages, which resulted in deeper secondary energy minima and lower energy barriers for adhesion. The results from this study suggest that growth phase may strongly influence cell mobility and biofilm formation in aqueous geochemical environments. [ABSTRACT FROM AUTHOR]

Substantial interactions of microbes with soil particles present fundamental influences on microbial activities relevant to a series of biogeochemical processes. However, how soil surface-active particles modulate microbial metabolism has received scant attention. The extent to which composition of soil colloids alter the metabolism is not well addressed. This work examined the impacts of soil colloids and minerals on the metabolic activity ofPseudomonas putidausing microcalorimetry and carbon utilization. The results showed that montmorillonite remarkably improved metabolic activity ofP. putida, whereas kaolinite, goethite and soil colloids significantly inhibited the activity. Humus may weaken the inhibition of soil colloids on bacterial metabolism via interfacial interaction rather than nutrient supplements. Soils bearing higher amount of kaolinite and iron oxide may have greater depression on bacterial activity. The thermodynamic method provides different and complementary information to that from other techniques in characterizing microbial activities. The quantity and affinity for the adhesion of bacteria onto soil components together with the detoxification of metabolites were assigned to the modifications of bacterial activities. [ABSTRACT FROM PUBLISHER]

Purpose: Bacterial adhesion to soil particles is fundamentally important in mineral weathering, organic matter degradation, heavy metal transformation, and fate of pollutants. However, the adhesion mechanism between bacteria and soil colloids under continuous flow systems in the natural environments remains unknown. Materials and methods: The kinetics of Pseudomonas putida cellular adsorption and desorption on Red soil colloid films under controlled flow systems were examined using in situ attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Derjaguin-Landau-Verwey-Overbeek (DLVO) and non-DLVO interactions were employed to elucidate the cellular adsorption and desorption kinetics. Results and discussion: In situ ATR-FTIR spectroscopy can be used effectively to investigate the kinetics of bacterial adhesion to a soil colloid deposit. Surface proteins may be involved in the bacterial adhesion to soil colloids. The adsorption followed pseudo-first-order kinetic equation. High adsorption rate constant and great saturation coverage of adsorbed bacteria were found at high ionic strengths in dynamic systems. Conclusions: P. putida bacterial cellular adsorption on the soil colloid deposit was irreversible in a wide range of ionic strengths under controlled flow systems. The less reversible adhesion was probably attributed to the DLVO predicted deep secondary energy minima together with non-DLVO factors including polymer bridging, local charge heterogeneities, surface roughness, and Lewis acid-base interactions. [ABSTRACT FROM AUTHOR]

The inducibility and specificity of different czcRS operons in Pseudomonas putida X4 were studied by lacZ gene fusions. The data of β-glycosidase activity confirmed that the czcR3 promoter responded quantitatively to zinc. A zinc-specific biosensor, P. putida X4 (pczcR3GFP), was constructed by fusing a promoterless enhanced green fluorescent protein (egfp) gene with the czcR3 promoter in the chromosome of P. putida X4. In water extracts of four different soils amended with zinc, the reporter strain detected about 90% of the zinc content of the samples. Both the bioavailability assessment and the sequential extraction analysis demonstrated that the immobilization of zinc was highly dependent on the physico-chemical properties of soils. The results also showed that the lability of zinc decreased over time. It is concluded that the biosensor constitutes an alternative system for the convenient evaluation of zinc toxicity in the environment. [Copyright &y& Elsevier]

Mineral-organic interfacial reactions strongly influence the adsorption, distribution and bioavailability of metal cations in soil systems. The molecular binding mechanisms and distribution of Cd onto goethite, humic acid, Pseudomonas putida cells, and their composites at different mass ratios were studied through the combination of bulk adsorption coupled with EXAFS, ITC and SCM. In binary and ternary composites, the energetics of the overall adsorption of Cd was dominated by the entropy of Cd adsorption onto the organic fraction. The formation of a type-B HA bridging complex >FeOH−HACOOCdOH enhanced Cd adsorption by 10−30% at low Cd concentrations, and more than 93.5% of the adsorbed Cd was bound onto HA fraction. In ternary systems, the component additivity over-estimated Cd adsorption onto bacteria by ~21.8%, likely due to site blocking effects. Models involving the masking of phosphoryl sites and HA bridging reactions can simulate the distribution of Cd in the composites. Our modelling suggests that HA is the main scavenger of Cd under a range of environmental conditions, and that bacteria become important in affecting the distribution of Cd under lower pH settings. This study demonstrates the impact of iron oxide−HA−bacteria interactions on the fate and distribution of Cd in soils and associated environments. ga1 • Adsorption of Cd was driven by entropy increment on organic fraction in composites. • Composite SCMs for Cd adsorption were established based on EXAFS and ITC. • Formation of type-B HA bridging complex enhanced Cd adsorption. • Masking of phosphoryl and HA bridging reduced allocation of Cd onto bacteria. • HA is the main scavenger of Cd under a range of environmental conditions. [ABSTRACT FROM AUTHOR]

The accurate modeling of heavy metal adsorption in complex systems is fundamental for risk assessments in soils and associated environments. Bacteria-iron (hydr)oxide associations in soils and sediments play a critical role in heavy metal immobilization. The reduced adsorption of heavy metals on these composites have been widely reported using the component additivity (CA) method. However, there is a lack of a mechanism model to account for these deviations. In this study, we established models for Cd adsorption on goethite- Pseudomonas putida composites at 1:1 and 5:1 mass ratios. Cadmium adsorption on the 5:1 composite was consistent with the additivity method. However, the CA method over predicted Cd adsorption by approximately 8% on the 1:1 composite at high Cd concentration. The deviation was corrected by adding the site blockage reactions between P. putida and goethite. Both CA and “CA-site masking” models for Cd adsorption onto the composites were in line with the ITC data. These results indicate that CA method in simulating Cd adsorption on bacteria-iron oxides composites is limited to low bacterial and Cd concentrations. Therefore the interfacial complexation reactions that occur between iron (hydr)oxides and bacteria should be taken into account when high concentrations of bacteria and heavy metals are present. [ABSTRACT FROM AUTHOR]

Abstract: In situ immobilization of heavy metals via physical, chemical, and biological processes presents practical remediation techniques for contaminated agricultural soils. In the current study, plant yield and cadmium (Cd) uptake by pakchoi (Brassica chinensis L.) in relation to soil Cd fractionations were investigated to evaluate the remediating effect of EGFP-tagged Pseudomonas putida X4 in Cd-contaminated soil. The soil treated with various levels of Cd (0–10 mg kg−1 soil) was inoculated with P. putida X4::gfp at an initial cell density of 107. Bacterial application transformed 7.5%–24.8% of soluble/exchangeable Cd mainly to the organic-bound fraction. Cd uptake decreased in pakchoi shoots by 10.0%–62.0% and pakchoi roots by 8.1%–60.1% after microbial remediation. The test bacterium was able to rapidly colonize the Cd-contaminated habitats and could persist at relatively stable cell densities during the whole pot experiments. This low cost and eco-friendly remediation method is very effective in the restoration of Cd-contaminated soils. [Copyright &y& Elsevier]