Cyclic di-GMP inhibits nitrate assimilation by impairing the antitermination function of NasT in Pseudomonas putida.

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 phosphodiesterase DibA interacts with the c-di-GMP receptor LapD and specifically regulates biofilm in Pseudomonas putida.

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.

Portable Mass Spectrometry for On-site Detection of Hazardous Volatile Organic Compounds via Robotic Extractive Sampling.

Various hazardous volatile organic compounds (VOCs) are frequently released into environments during accidental events that cause many hazards to ecosystems and humans. Therefore, rapid, sensitive, and on-site detection of hazardous VOCs is crucial to understand their compositions, characteristics, and distributions in complex environments. However, manual handling of hazardous VOCs remains a challenging task, because of the inaccessible environments and health risk. In this work, we designed a quadruped robotic sampler to reach different complex environments for capturing trace hazardous VOCs using a needle trap device (NTD) by remote manipulation. The captured samples were rapidly identified by portable mass spectrometry (MS) within minutes. Rapid detection of various hazardous VOCs including toxicants, chemical warfare agents, and burning materials from different environments was successfully achieved using this robot-MS system. On-site detection of 83 typical hazardous VOCs was examined. Acceptable analytical performances including low detection limits (at subng/mL level), good reproducibility (relative standard deviation (RSD) < 20%, n = 6), excellent quantitative ability (R2 > 0.99), and detection speed (within minutes) were also obtained. Our results show that the robot-MS system has excellent performance including safety, controllability, applicability, and robustness under dangerous chemical conditions.

Organo-organic interactions dominantly drive soil organic carbon accrual.

Organo-mineral interactions have been regarded as the primary mechanism for the stabilization of soil organic carbon (SOC) over decadal to millennial timescales, and the capacity for soil carbon (C) storage has commonly been assessed based on soil mineralogical attributes, particularly mineral surface availability. However, it remains contentious whether soil C sequestration is exclusively governed by mineral vacancies, making it challenging to accurately predict SOC dynamics. Here, through a 400-day incubation experiment using 13 C-labeled organic materials in two contrasting soils (i.e., Mollisol and Ultisol), we show that despite the unsaturation of mineral surfaces in both soils, the newly incorporated C predominantly adheres to "dirty" mineral surfaces coated with native organic matter (OM), demonstrating the crucial role of organo-organic interactions in exogenous C sequestration. Such interactions lead to multilayered C accumulation that is not constrained by mineral vacancies, a process distinct from direct organo-mineral contacts. The coverage of native OM by new C, representing the degree of organo-organic interactions, is noticeably larger in Ultisol (~14.2%) than in Mollisol (~5.8%), amounting to the net retention of exogenous C in Ultisol by 0.2-1.3 g kg-1 and in Mollisol by 0.1-1.0 g kg-1 . Additionally, organo-organic interactions are primarily mediated by polysaccharide-rich microbial necromass. Further evidence indicates that iron oxides can selectively preserve polysaccharide compounds, thereby promoting the organo-organic interactions. Overall, our findings provide direct empirical evidence for an overlooked but critically important pathway of C accumulation, challenging the prevailing "C saturation" concept that emphasizes the overriding role of mineral vacancies. It is estimated that, through organo-organic interactions, global Mollisols and Ultisols might sequester ~0.1-1.0 and ~0.3-1.7 Pg C per year, respectively, corresponding to the neutralization of ca. 0.5%-3.0% of soil C emissions or 5%-30% of fossil fuel combustion globally.

Protists regulate microbially mediated organic carbon turnover in soil aggregates.

Soil protists, the major predator of bacteria and fungi, shape the taxonomic and functional structure of soil microbiome via trophic regulation. However, how trophic interactions between protists and their prey influence microbially mediated soil organic carbon turnover remains largely unknown. Here, we investigated the protistan communities and microbial trophic interactions across different aggregates-size fractions in agricultural soil with long-term fertilization regimes. Our results showed that aggregate sizes significantly influenced the protistan community and microbial hierarchical interactions. Bacterivores were the predominant protistan functional group and were more abundant in macroaggregates and silt + clay than in microaggregates, while omnivores showed an opposite distribution pattern. Furthermore, partial least square path modeling revealed positive impacts of omnivores on the C-decomposition genes and soil organic matter (SOM) contents, while bacterivores displayed negative impacts. Microbial trophic interactions were intensive in macroaggregates and silt + clay but were restricted in microaggregates, as indicated by the intensity of protistan-bacterial associations and network complexity and connectivity. Cercozoan taxa were consistently identified as the keystone species in SOM degradation-related ecological clusters in macroaggregates and silt + clay, indicating the critical roles of protists in SOM degradation by regulating bacterial and fungal taxa. Chemical fertilization had a positive effect on soil C sequestration through suppressing SOM degradation-related ecological clusters in macroaggregate and silt + clay. Conversely, the associations between the trophic interactions and SOM contents were decoupled in microaggregates, suggesting limited microbial contributions to SOM turnovers. Our study demonstrates the importance of protists-driven trophic interactions on soil C cycling in agricultural ecosystems.

Human genetic associations of the airway microbiome in chronic obstructive pulmonary disease.

Little is known about the relationships between human genetics and the airway microbiome. Deeply sequenced airway metagenomics, by simultaneously characterizing the microbiome and host genetics, provide a unique opportunity to assess the microbiome-host genetic associations. Here we performed a co-profiling of microbiome and host genetics with the identification of over 5 million single nucleotide polymorphisms (SNPs) through deep metagenomic sequencing in sputum of 99 chronic obstructive pulmonary disease (COPD) and 36 healthy individuals. Host genetic variation was the most significant factor associated with the microbiome except for geography and disease status, with its top 5 principal components accounting for 12.11% of the microbiome variability. Within COPD individuals, 113 SNPs mapped to candidate genes reported as genetically associated with COPD exhibited associations with 29 microbial species and 48 functional modules (P < 1 × 10-5), where Streptococcus salivarius exhibits the strongest association to SNP rs6917641 in TBC1D32 (P = 9.54 × 10-8). Integration of concurrent host transcriptomic data identified correlations between the expression of host genes and their genetically-linked microbiome features, including NUDT1, MAD1L1 and Veillonella parvula, TTLL9 and Stenotrophomonas maltophilia, and LTA4H and Haemophilus influenzae. Mendelian randomization analyses revealed a potential causal link between PARK7 expression and microbial type III secretion system, and a genetically-mediated association between COPD and increased relative abundance of airway Streptococcus intermedius. These results suggest a previously underappreciated role of host genetics in shaping the airway microbiome and provide fresh hypotheses for genetic-based host-microbiome interactions in COPD.

Trade-off between Pore-Throat Structure and Mineral Composition in Modulating the Stability of Soil Organic Carbon.

The preservation of soil organic carbon (OC) is an effective way to decelerate the emission of CO2 emission. However, the coregulation of pore structure and mineral composition in OC stabilization remains elusive. We employed the in situ nondestructive oxidation of OC by low-temperature ashing (LTA) combined with near edge X-ray absorption fine structure (NEXAFS), high-resolution microtomography (µ-CT), field emission electron probe microanalysis (FE-EPMA) with C-free embedding, and novel Cosine similarity measurement to investigate the C retention in different aggregate fractions of contrasting soils. Pore structure and minerals contributed equally (ca. 50%) to OC accumulation in macroaggregates, while chemical protection played a leading role in C retention with 53.4%-59.2% of residual C associated with minerals in microaggregates. Phyllosilicates were discovered to be more prominent than Fe (hydr)oxides in C stabilization. The proportion of phyllosilicates-associated C (52.0%-61.9%) was higher than that bound with Fe (hydr)oxides (45.6%-55.3%) in all aggregate fractions tested. This study disentangled quantitatively for the first time a trade-off between physical and chemical protection of OC varying with aggregate size and the different contributions of minerals to OC preservation. Incorporating pore structure and mineral composition into C modeling would optimize the C models and improve the soil C content prediction.

Ore improver additions alter livestock manure compost ecosystem C:N:P stoichiometry.

Deciphering the pivotal components of nutrient metabolism in compost is of paramount importance. To this end, ecoenzymatic stoichiometry, enzyme vector modeling, and statistical analysis were employed to explore the impact of exogenous ore improver on nutrient changes throughout the livestock composting process. The total phosphorus increased from 12.86 to 18.72 g kg-1, accompanied by a marked neutralized pH with ore improver, resulting in the Carbon-, nitrogen-, and phosphorus-related enzyme activities decreases. However, the potential C:P and N:P acquisition activities represented by ln(ßG + CB): ln(ALP) and ln(NAG): ln(ALP), were increased with ore improver addition. Based on the ecoenzymatic stoiometry theory, these changes reflect a decreasing trend in the relative P/N limitation, with pH and total phosphorus as the decisive factors. Our study showed that the practical employment of eco stoichiometry could benefit the manure composting process. Moreover, we should also consider the ecological effects from pH for the waste material utilization in sustainable agriculture.

Portable Mass Spectrometry Approach Combined with Machine Learning for Onsite Field Detection of Huanglongbing Disease.

Huanglongbing (HLB) is one of the most serious citrus diseases in the world. Rapid, onsite, and accurate field detection of HLB is a challenging task in analytical science for a long time. Herein, we have developed a novel HLB detection method that combines headspace solid phase microextraction with portable gas chromatography-mass spectrometry (PGC-MS) approach for onsite field detection of volatile metabolites of citrus leaves. Detectability and characteristics of HLB-affected metabolites from leaves were validated, and the important biomarkers were verified by authentic compounds. A machine learning approach based on random forest algorithm is established to model the volatile metabolites from healthy, symptomatic, and asymptomatic citrus leaves. In this work, a total of 147 citrus leaf samples were analyzed. Analytical performances of this newly developed method were investigated by in-field detection of various volatile metabolites. Results demonstrated limits of detection and quantification of 0.04-0.12 and 0.17-0.44 ng/mL for different metabolites, respectively. Linear calibration curves of various metabolites were established over a concentration dynamic range of at least three orders (R2 > 0.96). Good reproducibility was obtained for intraday (3.0-17.5%, n = 6) and interday precision (8.7-18.2%, n = 7). This new HLB field detection method provides a rapid detection with 6 min for each sample via a simple optimized procedure, including onsite sampling, PGC-MS analysis, and data process and provides a high accuracy (93.3%) for simultaneous identification of healthy, symptomatic, and asymptomatic trees. These data support the use of this new method for reliable field detection of HLB. Furthermore, metabolic pathways of HLB-affected metabolites were also proposed. Overall, our results not only provide a rapid and onsite field HLB detection method but also provide valuable information for understanding metabolic change of HLB infection.

The newest Oxford Nanopore R10.4.1 full-length 16S rRNA sequencing enables the accurate resolution of species-level microbial community profiling.

The long-read amplicon provides a species-level solution for the community. With the improvement of nanopore flowcells, the accuracy of Oxford Nanopore Technologies (ONT) R10.4.1 has been substantially enhanced, with an average of approximately 99%. To evaluate its effectiveness on amplicons, three types of microbiomes were analyzed by 16S ribosomal RNA (hereinafter referred to as "16S") amplicon sequencing using Novaseq, Pacbio sequel II, and Nanopore PromethION platforms (R9.4.1 and R10.4.1) in the current study. We showed the error rate, recall, precision, and bias index in the mock sample. The error rate of ONT R10.4.1 was greatly reduced, with a better recall in the case of the synthetic community. Meanwhile, in different types of environmental samples, ONT R10.4.1 analysis resulted in a composition similar to Pacbio data. We found that classification tools and databases influence ONT data. Based on these results, we conclude that the ONT R10.4.1 16S amplicon can also be used for application in environmental samples. IMPORTANCE The long-read amplicon supplies the community with a species-level solution. Due to the high error rate of nanopore sequencing early on, it has not been frequently used in 16S studies. Oxford Nanopore Technologies (ONT) introduced the R10.4.1 flowcell with Q20+ reagent to achieve more than 99% accuracy as sequencing technology advanced. However, there has been no published study on the performance of commercial PromethION sequencers with R10.4.1 flowcells on 16S sequencing or on the impact of accuracy improvement on taxonomy (R9.4.1 to R10.4.1) using 16S ONT data. In this study, three types of microbiomes were investigated by 16S ribosomal RNA (rRNA) amplicon sequencing using Novaseq, Pacbio sequel II, and Nanopore PromethION platforms (R9.4.1 and R10.4.1). In the mock sample, we displayed the error rate, recall, precision, and bias index. We observed that the error rate in ONT R10.4.1 is significantly lower, especially when deletions are involved. First and foremost, R10.4.1 and Pacific Bioscience platforms reveal a similar microbiome in environmental samples. This study shows that the R10.4.1 full-length 16S rRNA sequences allow for species identification of environmental microbiota.

Microbial autotrophy explains large-scale soil CO2 fixation.

Microbial communities play critical roles in fixing carbon from the atmosphere and fixing it in the soils. However, the large-scale variations and drivers of these microbial communities remain poorly understood. Here, we conducted a large-scale survey across China and found that soil autotrophic organisms are critical for explaining CO2 fluxes from the atmosphere to soils. In particular, we showed that large-scale variations in CO2 fixation rates are highly correlated to those in autotrophic bacteria and phototrophic protists. Paddy soils, supporting a larger proportion of obligate bacterial and protist autotrophs, display four-fold of CO2 fixation rates over upland and forest soils. Precipitation and pH, together with key ecological clusters of autotrophic microbes, also played important roles in controlling CO2 fixation. Our work provides a novel quantification on the contribution of terrestrial autotrophic microbes to soil CO2 fixation processes at a large scale, with implications for global carbon regulation under climate change.

Climatic seasonality challenges the stability of microbial-driven deep soil carbon accumulation across China.

Microbial residues contribute to the long-term stabilization of carbon in the entire soil profile, helping to regulate the climate of the planet; however, how sensitive these residues are to climatic seasonality remains virtually unknown, especially for deep soils across environmental gradients. Here, we investigated the changes of microbial residues along soil profiles (0-100 cm) from 44 typical ecosystems with a wide range of climates (~3100 km transects across China). Our results showed that microbial residues account for a larger portion of soil carbon in deeper (60-100 cm) vs. shallower (0-30 and 30-60 cm) soils. Moreover, we find that climate especially challenges the accumulation of microbial residues in deep soils, while soil properties and climate share their roles in controlling the residue accumulation in surface soils. Climatic seasonality, including positive correlations with summer precipitation and maximum monthly precipitation, as well as negative correlations with temperature annual range, are important factors explaining microbial residue accumulation in deep soils across China. In particular, summer precipitation is the key regulator of microbial-driven carbon stability in deep soils, which has 37.2% of relative independent effects on deep-soil microbial residue accumulation. Our work provides novel insights into the importance of climatic seasonality in driving the stabilization of microbial residues in deep soils, challenging the idea that deep soils as long-term carbon reservoirs can buffer climate change.

Dichotomous Role of Humic Substances in Modulating Transformation of Antibiotic Resistance Genes in Mineral Systems.

Widespread antibiotic resistance genes (ARGs) have emerged as a focus of attention for public health. Transformation is essential for ARGs dissemination in soils and associated environments; however, the mechanisms of how soil components contribute to the transformation of ARGs remain elusive. Here we demonstrate that three representative mineral-humic acid (HA) composites exert contrasting influence on the transformation of plasmid-borne ARGs in Bacillus subtilis. Mineral surface-bound HA facilitated transformation in kaolinite and montmorillonite systems, while an inhibitory effect of HA was observed for goethite. The elevated transformation by HA-coated kaolinite was mainly attributed to the enhanced activity of competence-stimulating factor (CSF), while increased transformation by montmorillonite-HA composites was assigned to the weakened adsorption affinity of DNA and enhanced gene expression induced by flagella-driven cell motility. In goethite system, HA played an overriding role in suppressing transformation via alleviation of cell membrane damage. The results obtained offer insights into the divergent mechanisms of humic substances in modulating bacterial transformation by soil minerals. Our findings would help for a better understanding on the fate of ARGs in soil systems and provide potentials for the utilization of soil components, particularly organic matter, to mitigate the spread of ARGs in a range of settings.

Core Species Derived from Multispecies Interactions Facilitate the Immobilization of Cadmium.

Microbial consortia have opened new avenues for heavy-metal remediation. However, the limited understanding of the overall effect of interspecific interactions on remediation efficacy hinders its application. Here, the effects of multispecies growth and biofilm formation on Cd immobilization were explored from direct and multiple interactions through random combinations of two or three rhizosphere bacteria. In monocultures, Cd stress resulted in an average decrease in planktonic biomass of 26%, but through cooperation, the decrease was attenuated in dual (21%) and triple cultures (13%), possibly involving an increase in surface polysaccharides. More than 65% of the co-cultures exhibited induction of biofilm formation under Cd stress, which further enhanced the role of biofilms in Cd immobilization. Notably, excellent biofilm-forming ability or extensive social induction makes Pseudomonas putida and Brevundimonas diminuta stand out in multispecies biofilm formation and Cd immobilization. These two core species significantly increase the colonization of soil microorganisms on rice roots compared to the control, resulting in a 40% decrease in Cd uptake by rice. Our study enhances the understanding of bacterial interactions under Cd stress and provides a novel strategy for adjusting beneficial soil consortia for heavy-metal remediation.

Phagotrophic Protists Modulate Copper Resistance of the Bacterial Community in Soil.

Protist predation is a crucial biotic driver modulating bacterial populations and functional traits. Previous studies using pure cultures have demonstrated that bacteria with copper (Cu) resistance exhibited fitness advantages over Cu-sensitive bacteria under the pressure of protist predation. However, the impact of diverse natural communities of protist grazers on bacterial Cu resistance in natural environments remains unknown. Here, we characterized the communities of phagotrophic protists in long-term Cu-contaminated soils and deciphered their potential ecological impacts on bacterial Cu resistance. Long-term field Cu pollution increased the relative abundances of most of the phagotrophic lineages in Cercozoa and Amoebozoa but reduced the relative abundance of Ciliophora. After accounting for soil properties and Cu pollution, phagotrophs were consistently identified as the most important predictor of the Cu-resistant (CuR) bacterial community. Phagotrophs positively contributed to the abundance of a Cu resistance gene (copA) through influencing the cumulative relative abundance of Cu-resistant and -sensitive ecological clusters. Microcosm experiments further confirmed the promotion effect of protist predation on bacterial Cu resistance. Our results indicate that the selection by protist predation can have a strong impact on the CuR bacterial community, which broadens our understanding of the ecological function of soil phagotrophic protists.

Rice-crayfish co-culture increases microbial necromass' contribution to the soil nitrogen pool.

The rice-crayfish co-culture (RC) is a putative sustainable agricultural system. However, studies on the ecological effects of long-term RC systems were still lacking. Here, we compare enzymatic stoichiometry, microbial necromass, and microbial community between the RC and rice monoculture systems (RM). Soil enzymatic stoichiometry analysis showed that after transformation from RM to RC for about three years, ammonium nitrogen (NH4+-N) availability increased to depress relative N-acquiring enzyme production, especially for leucine aminopeptidase. The contents of microbial necromass increased approximately onefold in the RC system, making microbial necromass' contribution to the soil nitrogen (N) reach up to 46.72%. Elevation in NH4+ decreased N-acquiring enzyme, and a relatively more effective C acquisition likely benefited microbial necromass retention and production in the RC system. This study highlights that the rice-crayfish co-culture could modify the N pool of the surface paddy soil.

Salinity-triggered homogeneous selection constrains the microbial function and stability in lakes.

Climate change and anthropogenic exploitation have led to the gradual salinization of inland waters worldwide. However, the impacts of this process on the prokaryotic plankton communities and their role in biogeochemical cycles in the inland lake are poorly known. Here, we take a space-for-time substitution approach, using 16S rRNA gene amplicon sequencing and metagenomic sequencing. We analyzed the prokaryotic plankton communities of 11 lakes in northwest China, with average water salinities ranging from 0.002 to 14.370%. The results demonstrated that, among the various environmental parameters, salinity was the most important driver of prokaryotic plankton ß-diversity (Mantel test, r = 0.53, P < 0.001). (1) Under low salinity, prokaryotic planktons were assembled by stochastic processes and employed diverse halotolerant strategies, including the synthesis and uptake of compatible solutes and extrusion of Na+ or Li+ in exchange for H+. Under elevated salinity pressure, strong homogeneous selection meant that only planktonic prokaryotes showing an energetically favorable halotolerant strategy employing an Mnh-type Na+/H+ antiporter remained. (2) The decreasing taxonomic diversity caused by intense environmental filtering in high-salinity lakes impaired functional diversity related to substance metabolism. The prokaryotes enhanced the TCA cycle, carbon fixation, and low-energy-consumption amino acid biosynthesis in high-salinity lakes. (3) Elevated salinity pressure decreased the negative:positive cohesion and the modularity of the molecular ecology networks for the planktonic prokaryotes, indicating a precarious microbial network. Our findings provide new insights into plankton ecology and are helpful for the protecting of the biodiversity and function of inland lakes against the background of salinization. KEY POINTS: • Increased salinity enhances homogeneous selection in the microbial assembly. • Elevated salinity decreases the microbial co-occurrence networks stability. • High salinity damages the microbial function diversity.

Elevated temperature induces contrasting transformation of exogenous copper to soil solution and solid phases in an arable soil.

Heavy metal contamination of soils has been a global environmental issue over the past decades, threatening food security and human health. Understanding the migration and transformation of heavy metals in soils is critical for restoring an impaired environment and developing sustainable agriculture, particularly in the face of global warming. However, little effort has been devoted to investigating the impact of elevated temperatures on the migration and distribution of exogenous heavy metals in soils. This study experimented with a 180-day incubation at 15 °C, 30 °C, and 45 °C with an arable soil (Alfisol) of Huang-Huai-Hai River Basin, China, which was initially spiked with copper (Cu). A comparison of the results revealed that the percentage of soil water-soluble Cu doubled at 45 °C compared with 15 °C. The percentage of protein-like substances in dissolved organic matter (DOM) was the highest at 45 °C, suggesting that proteinaceous components play a more significant role in controlling the dissolution of Cu into DOM. Moreover, by sequential extraction and micro-X-ray fluorescence (µ-XRF), Cu was facilitatively transformed from exchangeable, and specifically adsorbed fractions, to iron (Fe)/manganese (Mn) oxides bound species by 7.75%23.63% with the elevation of temperature from 15 °C to 45 °C. The conversion of Cu speciation is attributed to the significant release of organic carbon from Fe/Mn oxides, especially the Mn oxide components, which are available for Cu binding. The findings of this work will provide an in-depth understanding of the fate of Cu in soils, which is fundamental for the risk assessment and remediation of Cu-polluted soils in the Huang-Huai-Hai River Basin under the context of global warming.

Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN complex in Pseudomonas putida.

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.

Second Messenger c-di-GMP Modulates Exopolysaccharide Pea-Dependent Phenotypes via Regulation of eppA Expression in Pseudomonas putida.

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.