Metabolic and Microbial Profiling of Soil Microbial Community under Per- and Polyfluoroalkyl Substance (PFAS) Stress.

Per- and polyfluoroalkyl substances (PFAS) represent significant stress to organisms and are known to disrupt microbial community structure and function. Nevertheless, a detailed knowledge of the soil microbial community responding to PFAS stress at the metabolism level is required. Here we integrated UPLC-HRMS-based metabolomics data with 16S rRNA and ITS amplicon data across soil samples collected adjacent to a fluoropolymer production facility to directly identify the biochemical intermediates in microbial metabolic pathways and the interactions with microbial community structure under PFAS stress. A strong correlation between metabolite and microbial diversity was observed, which demonstrated significant variations in soil metabolite profiles and microbial community structures along with the sampling locations relative to the facility. Certain key metabolites were identified in the metabolite-PFAS co-occurrence network, functioning on microbial metabolisms including lipid metabolism, amino acid metabolism, and secondary metabolite biosynthesis. These results provide novel insights into the impacts of PFAS contamination on soil metabolomes and microbiomes. We suggest that soil metabolomics is an informative and useful tool that could be applied to reinforce the chemical evidence on the disruption of microbial ecological traits.

Microfluidics as an Emerging Platform for Exploring Soil Environmental Processes: A Critical Review.

Investigating environmental processes, especially those occurring in soils, calls for innovative and multidisciplinary technologies that can provide insights at the microscale. The heterogeneity, opacity, and dynamics make the soil a "black box" where interactions and processes are elusive. Recently, microfluidics has emerged as a powerful research platform and experimental tool which can create artificial soil micromodels, enabling exploring soil processes on a chip. Micro/nanofabricated microfluidic devices can mimic some of the key features of soil with highly controlled physical and chemical microenvironments at the scale of pores, aggregates, and microbes. The combination of various techniques makes microfluidics an integrated approach for observation, reaction, analysis, and characterization. In this review, we systematically summarize the emerging applications of microfluidic soil platforms, from investigating soil interfacial processes and soil microbial processes to soil analysis and high-throughput screening. We highlight how innovative microfluidic devices are used to provide new insights into soil processes, mechanisms, and effects at the microscale, which contribute to an integrated interrogation of the soil systems across different scales. Critical discussions of the practical limitations of microfluidic soil platforms and perspectives of future research directions are summarized. We envisage that microfluidics will represent the technological advances toward microscopic, controllable, and in situ soil research.

Biochar Effectively Inhibits the Horizontal Transfer of Antibiotic Resistance Genes via Restraining the Energy Supply for Conjugative Plasmid Transfer.

Horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) through plasmid-mediated conjugation poses a major threat to global public health. Biochar, a widely used environmental remediation material, has remarkable impacts on the fate of ARGs. However, although biochar was reported being able to inhibit the HGT of ARGs via conjugation and transformation, little is known about the intracellular process that mediates the inhibition effects. On the other hand, as typical natural organic matter, fulvic acid is a common environmental influencer, and how it interferes with the effect of biochar on the HGT of ARGs is unknown. Therefore, this study investigated the effects on the conjugative transfer of ARGs between Escherichia coli MG1655 and E. coli HB101 carrying plasmid RP4, with biochars pyrolyzed at three temperatures and with the corresponding biochars coating with fulvic acid. Results showed that biochar with higher pyrolyzed temperature had a more substantial inhibitory effect on the conjugative transfer of the RP4 plasmid. The inhibitory effect of biochar was mainly attributed to (i) down-regulation of plasmid transfer gene expression, including the formation of conjugative transfer channel and plasmid replication, due to restrained adenosine triphosphate (ATP) energy supply and (ii) decreased cell membrane permeability. Conversely, the fulvic acid coating diminished this inhibition effect of biochar, mainly by providing more ATP and strengthening intracellular reactive oxygen species (ROS) defense. Our findings shed light on the intracellular process that mediates the effects of biochar on the conjugative transfer of ARGs, which would provide support for using biochar to reduce the spread of ARGs.

The aggregate distribution of Pseudomonas aeruginosa on biochar facilitates quorum sensing and biofilm formation.

Biochar when applied into soil, together with soil clay minerals, may provide habitats for soil microbes and shift soil microbial community structure. Although several mechanisms have been proposed to explain the effects of biochar on microbial community, the impact of biochar on quorum sensing (QS) and QS-regulated behavior is poorly understood. In this study, we compared the effects of biochar and three common soil minerals (i.e., montmorillonite, kaolinite, and goethite) on QS and biofilm formation. Pseudomonas aeruginosa PAO1 with complete QS systems was selected as a model organism. Our results showed that biochar and goethite effectively promoted microbial QS and biofilm formation, while montmorillonite and kaolinite posed no significant effect. Live/Dead staining, SEM and density-dependent QS activity indicated that biochar was beneficial to cell viability maintenance and cell aggregations, which improved the efficiency of intercellular communications through QS. QS mutant strain experiments confirmed that biochar enhanced PAO1 biofilm formation by promoting QS. Goethite promoted biofilm formation with a different mechanism that cell debris induced by iron ions and positive charge on goethite surface provided raw materials for bacterial biofilm formation. Our findings provide evidence that the presence of biochar can enhance QS and biofilm formation through a feedforward loop of the QS system. This contributes to better understand biochar-mediated microbial cell to cell communications through QS.

Contribution of enrofloxacin and Cu2+ to the antibiotic resistance of bacterial community in a river biofilm.

Pollutants discharged from wastewater are the main cause of the spread of antibiotic resistance in river biofilms. There is controversy regarding the primary contribution of environmental selectors such as antibiotics and heavy metals to the development of antibiotic resistance in bacterial communities. Here, this study compared the effect of environmental safety concentration Cu2+ and enrofloxacin (ENR) on the evolution of antibiotic resistance by examining phenotypic characteristics and genotypic profiles of bacterial communities in a river biofilm, and then distinguished the major determinants from a comprehensive perspective. The pollution induced community tolerance in ENR-treated group was significantly higher than that in Cu2+-treated group (at concentration levels of 100 and 1000 µg/L). Metagenomic sequencing results showed that ENR significantly increased the number and total abundance of antibiotic resistance genes (ARGs), but there was no significant change in the Cu2+- treated group. Compared with Cu2+, ENR was the major selective agent in driving the change of taxonomic composition because the taxonomic composition in ENR was the most different from the original biofilm. Comparing and analyzing the prokaryotic composition, the phylum of Proteobacteria was enriched in both ENR and Cu2+ treated groups. Among them, Acidovorax and Bosea showed resistance to both pollutants. Linking taxonomic composition to ARGs revealed that the main potential hosts of fluoroquinolone resistance genes were Comamonas, Sphingopyxis, Bradyrhizobium, Afipia, Rhodopseudomonas, Luteimonas and Hoeflea. The co-occurrence of ARGs and metal resistance genes (MRGs) showed that the multidrug efflux pump was the key mechanism connecting MRGs and ARGs. Network analysis also revealed that the reason of Cu2+ selected for fluoroquinolones resistant bacterial communities was the coexistence of multidrug efflux gene and MRGs. Our research emphasizes the importance of antibiotics in promoting the development of antibiotic resistant bacterial communities from the perspective of changes in community structure and resistome in river biofilms.

Conditional quorum-sensing induction of a cyanide-insensitive terminal oxidase stabilizes cooperating populations of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic pathogen of humans, uses quorum sensing (QS) to regulate the production of extracellular products that can benefit all members of the population. P. aeruginosa can police QS-deficient cheaters by producing hydrogen cyanide, which is also QS regulated; however, the mechanism by which cooperators selectively protect themselves from the toxicity of cyanide remained unresolved. Here, we show that a cyanide-insensitive terminal oxidase encoded by cioAB provides resistance to cyanide, but only in QS-proficient strains. QS-deficient cheaters do not activate cioAB transcription. QS-mediated regulation of cioAB expression depends on production of both cyanide by cooperators (which is QS regulated) and reactive oxygen species (ROS) from cheaters (which is not QS regulated). This type of regulatory system allows cooperating populations to respond, via ROS, to the presence of cheaters, and might allow them to defer the substantial metabolic cost of policing until cheaters are present in the population.

Changes to tetracyclines and tetracycline resistance genes in arable soils after single and multiple applications of manure containing tetracyclines.

The influence of manure containing tetracyclines (TCs) on the prevalence of antibiotic resistance genes in soils remains poorly understood. Here, three different TCs (oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC)) were mixed respectively with unpolluted manure to fertilize arable soil. The soil received either a single application of 0 µg kg-1, 300 µg kg-1 (TC and CTC), or 700 µg kg-1 (OTC) or multiple applications every 14 days for 140 days. Four tetracycline resistance genes (TRGs), including tet(A), tet(L), tet(M), and tet(Q), were monitored. Although the abundances of the four TRGs in the single application treatment initially increased rapidly, they decreased over time and were significantly lower than those of the repeated treatments after day 112. All additions of TCs stopped on day 140, but we continued to assess the long-term accumulation of TRGs. Most of the TRGs were detected even after the TC-containing manures had not been applied for more than 15 months. The abundance of the TRGs after ceasing fertilization with the TC-containing manures was higher in the repeated application treatments than in the single application treatments. Therefore, more attention should be paid to repeated applications of antibiotic-containing manure to arable soils.

A Metabolic Trade-Off Modulates Policing of Social Cheaters in Populations of Pseudomonas aeruginosa.

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of public goods such as the secreted protease elastase. P. aeruginosa requires the LasI-LasR QS circuit to induce elastase and enable growth on casein as the sole carbon and energy source. The LasI-LasR system also induces a second QS circuit, the RhlI-RhlR system. During growth on casein, LasR-mutant social cheaters emerge, and this can lead to a population collapse. In a minimal medium containing ammonium sulfate as a nitrogen source, populations do not collapse, and cheaters and cooperators reach a stable equilibrium; however, without ammonium sulfate, cheaters overtake the cooperators and populations collapse. We show that ammonium sulfate enhances the activity of the RhlI-RhlR system in casein medium and this leads to increased production of cyanide, which serves to control levels of cheaters. This enhancement of cyanide production occurs because of a trade-off in the metabolism of glycine: exogenous ammonium ion inhibits the transformation of glycine to 5,10-methylenetetrahydrofolate through a reduction in the expression of the glycine cleavage genes gcvP1 and gcvP2, thereby increasing the availability of glycine as a substrate for RhlR-regulated hydrogen cyanide synthesis. Thus, environmental ammonia enhances cyanide production and stabilizes QS in populations of P. aeruginosa.

A common evolutionary pathway for maintaining quorum sensing in Pseudomonas aeruginosa.

In the bacterium Pseudomonas aeruginosa, the synthesis and secretion of extracellular protease is a typical cooperative behavior regulated by quorum sensing. However, this type of cooperative behavior is easily exploited by other individuals who do not synthesize public goods, which is known as the "tragedy of the commons". Here P. aeruginosa was inoculated into casein media with different nitrogen salts added. In casein broth, protease (a type of public good) is necessary for bacterial growth. After 30 days of sequential transfer, some groups propagated stably and avoided "tragedy of the commons". The evolved cooperators who continued to synthesize protease were isolated from these stable groups. By comparing the characteristics of quorum sensing in these cooperators, an identical evolutionary pattern was found. A variety of cooperative behaviors regulated by quorum sensing, such as the synthesis and secretion of protease and signals, were significantly reduced during the process of evolution. Such reductions improved the efficiency of cooperation, helping to prevent cheating. In addition, the production of pyocyanin, which is regulated by the RhlIR system, increased during the process of evolution, possibly due to its role in stabilizing the cooperation. This study contributes towards our understanding of the evolution of quorum sensing of P. aeruginosa.

Effects of low-level engineered nanoparticles on the quorum sensing of Pseudomonas aeruginosa PAO1.

The toxicity of engineered nanoparticles (ENPs) on bacteria has aroused much interest. However, few studies have focused on the effects of low-level ENPs on bacterial group behaviors that are regulated by quorum sensing (QS). Herein, we investigated the effects of nine ENPs (Ag, Fe, ZnO, TiO2, SiO2, Fe2O3, single-wall carbon nanotubes (SWCNTs), graphene oxide (GO), and C60) on QS in Pseudomonas aeruginosa PAOl. An ENP concentration of 100 µg L-1 did not impair bacterial growth. However, concentrations of 100 µg L-1 of Ag and GO ENPs induced significant increases in 3OC12-HSL in the culture and significantly promoted protease production and biofilm formation of PAO1. C4-HSL synthase and its transcription factors were less sensitive to 100 µg L-1 Ag and GO ENPs compared with 3OC12-HSL. Fe ENPs induced a significant increase in the 3OC12-HSL concentration, similar to Ag and GO ENPs. However, Fe ENPs did not induce any significant increase in protease production or biofilm formation. Different size distributions, chemical compositions, and aggregation states of the ENPs had different effects on bacterial QS. These whole circuit indicators could clarify the effects of ENPs on bacterial QS. This study furthers our understanding of the effects of low-level ENPs on bacterial social behaviors.