Potential high-risk release sources of thallium and arsenic from surrounding rocks of a typical thallium and arsenic mining area in southwest China.

Abundant naturally and anthropogenically exposed surrounding rocks (NESRs and AESRs) in mining areas may pose persistent threats as sources of potentially toxic elements (PTEs), but this has been historically overlooked, especially for thallium (Tl) and arsenic (As). Here, the release risks of Tl and As from both NESRs and AESRs in a typical TlAs sulfide mining area were investigated. In a single leaching process, AESRs released 10.4 % of total Tl (157 µg L-1) and 32.5 % of total As (4089 µg L-1), 2-3 orders of magnitude higher than NESRs. Prolonged multiple leaching tests revealed notable and long-term risks of release of Tl and As from AESRs, associated with oxidation and dissolution of iron/sulfur-bearing minerals. Substantial release of PTEs was linked to the transformation/degradation of the -OH functional group and extensive dissolution of secondary sulfate minerals in AESRs. Ultrafiltration and STEM-EDS indicate that 18.4 % of water-extracted As released from AESRs existed as natural nanoparticles consisting of iron/sulfur-bearing minerals. This study highlights the high risks of Tl and As release from anthropogenically exposed surrounding rocks and the importance of nanoparticles in PTE transport, and provides insights into the control of PTEs in mining areas.

Metagenomic analysis of a thermophilic bacterial consortium and its use in the bioremediation of a petroleum-contaminated soil.

Biodegradation is difficult at high temperatures due to the limited capacity of microorganisms to survive and function outside their optimum temperature range. Here, a thermophilic petroleum-degrading consortium was enriched from compost at a temperature of 55 °C. 16S rDNA and metagenomic techniques were used to analyze the composition of the consortium and the mechanisms of degradation. The consortium degraded 17000 mg total petroleum hydrocarbons (TPHs) L-1 with a degradation efficiency of 81.5% in 14 days. The consortium utilized a range of substrates such as n-hexadecane, n-docosane, naphthalene and pyrene and grew well over a wide range of pH (4-10) and salinity (0-90 g L-1). The hydrocarbon-degrading extremophilic consortium contained, inter alia, (relative abundance >1%) Caldibacillus, Geobacillus, Mycolicibacterium, Bacillus, Chelatococcus, and Aeribacillus spp. Metagenomic analysis was conducted to discover the degradation and environmental tolerance functional genes of the consortium. Two alkane hydroxylase genes, alkB and ladA, were found. A microcosm study shows that the consortium promoted the bioremediation of soil TPHs. The results indicate that the consortium may be a good candidate for the high-temperature bioremediation of petroleum-contaminated soils.

Cadmium toxicity to and accumulation in a soil collembolan (Folsomia candida): major factors and prediction using a back-propagation neural network model.

Accurate prediction of cadmium (Cd) ecotoxicity to and accumulation in soil biota is important in soil health. However, very limited information on Cd ecotoxicity on naturally contaminated soils. Herein, we investigated soil Cd ecotoxicity using Folsomia candida, a standard single-species test animal, in 28 naturally Cd-contaminated soils, and the back-propagation neural network (BPNN) model was used to predict Cd ecotoxicity to and accumulation in F. candida. Soil total Cd and pH were the primary soil properties affecting Cd toxicity. However, soil pH was the main factor when the total Cd concentration was < 3 mg kg-1. Interestingly, correlation analysis and the K-spiked test confirmed nutrient potassium (K) was essential for Cd accumulation, highlighting the significance of studying K in Cd accumulation. The BPNN model showed greater prediction accuracy of collembolan survival rate (R2 = 0.797), reproduction inhibitory rate (R2 = 0.827), body Cd concentration (R2 = 0.961), and Cd bioaccumulation factor (R2 = 0.964) than multiple linear regression models. Then the developed BPNN model was used to predict Cd ecological risks in 57 soils in southern China. Compared to multiple linear regression models, the BPNN models can better identify high-risk regions. This study highlights the potential of BPNN as a novel and rapid tool for the evaluation and monitoring of Cd ecotoxicity in naturally contaminated soils.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.

Differences in the activities of six soil enzymes in response to cadmium contamination of paddy soils in high geological background areas.

East Yunnan province in southwest China is a region with elevated natural abundance (high geological background levels) of Cd due to high metal (loid) contents in the soils. Enzyme activities are useful indicators of metal (loid) toxicity in contaminated soils and whether Cd inhibits enzyme activities in paddy soils in high geological background areas is of considerable public concern. A pot experiment combined with field investigation was conducted to assess the effects of Cd on six soil enzymes that are essential to the cycling of C, N, and P in soils. Inhibitory effects of Cd fractions on enzyme activities were assessed using ecological dose-response models. The impact of soil properties on the inhibition of sensitive soil enzymes by Cd were assessed using linear and structural equation models. Cadmium was enriched in the paddy soils with 72.2 % of soil samples from high geological background areas exceeding the Chinese threshold values (GB 15618-2018) of Cd. Enzyme responses to Cd contamination varied markedly with a negative response by catalase but a positive response by invertase. Urease, ß-glucosidase, and alkaline phosphatase activities were stimulated at low Cd concentrations and inhibited at high concentrations. The average inhibition ratios of ß-glucosidase, urease, and catalase in high Cd levels were 19.9, 38.9, and 51.9%, respectively. Ecological dose-response models indicate that catalase and urease were the most Cd-sensitive of the enzymes studied and were suitable indicators of soil quality in high geological background areas. Structural equation modeling (SEM) indicates that soil properties influenced sensitive enzymes through various pathways, indicating that soil properties were factors determining Cd inhibition of enzyme activities. This suggests that Cd concentrations and soil physicochemical properties under a range of environmental conditions should be considered in addressing soil Cd pollution.

Climate and mineral accretion as drivers of mineral-associated and particulate organic matter accumulation in tidal wetland soils.

Tidal wetlands sequester vast amounts of organic carbon (OC) and enhance soil accretion. The conservation and restoration of these ecosystems is becoming increasingly geared toward "blue" carbon sequestration while obtaining additional benefits, such as buffering sea-level rise and enhancing biodiversity. However, the assessments of blue carbon sequestration focus primarily on bulk SOC inventories and often neglect OC fractions and their drivers; this limits our understanding of the mechanisms controlling OC storage and opportunities to enhance blue carbon sinks. Here, we determined mineral-associated and particulate organic matter (MAOM and POM, respectively) in 99 surface soils and 40 soil cores collected from Chinese mangrove and saltmarsh habitats across a broad range of climates and accretion rates and showed how previously unrecognized mechanisms of climate and mineral accretion regulated MAOM and POM accumulation in tidal wetlands. MAOM concentrations (8.0 ± 5.7 g C kg-1 ) (±standard deviation) were significantly higher than POM concentrations (4.2 ± 5.7 g C kg-1 ) across the different soil depths and habitats. MAOM contributed over 51.6 ± 24.9% and 78.9 ± 19.0% to OC in mangrove and saltmarsh soils, respectively; both exhibited lower autochthonous contributions but higher contributions from terrestrial or marine sources than POM, which was derived primarily from autochthonous sources. Increased input of plant-derived organic matter along the increased temperature and precipitation gradients significantly enriched the POM concentrations. In contrast, the MAOM concentrations depended on climate, which controlled the mineral reactivity and mineral-OC interactions, and on regional sedimentary processes that could redistribute the reactive minerals. Mineral accretion diluted the POM concentrations and potentially enhanced the MAOM concentrations depending on mineral composition and whether the mineral accretion benefited plant productivity. Therefore, management strategies should comprehensively consider regional climate while regulating sediment supply and mineral abundance with engineering solutions to tap the OC sink potential of tidal wetlands.

Structural and functional diversity of type IV secretion systems.

Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host-pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.

Root-associated bacterial microbiome shaped by root selective effects benefits phytostabilization by Athyrium wardii (Hook.).

The root-associated microbiome assembly substantially promotes (hyper)accumulator plant growth and metal accumulation and is influenced by multiple factors, especially host species and environmental stress. Athyrium wardii (Hook.) is a phytostabilizer that grows in lead (Pb)-zinc (Zn) mine tailings and shows high root Pb accumulation. However, there remains little information on the assembly of the root-associated microbiome of A. wardii and its role in phytostabilization. A field study investigated the structural and functional variation in the root-associated bacterial microbiome of Athyrium wardii (Hook.) exposed to different levels of contamination in Pb-Zn mine tailings. The root compartment dominated the variation in the root-associated bacterial microbiome but the levels of contaminants showed less impact. Bacterial co-occurrence was enhanced in the rhizosphere soil and rhizoplane but tended to be much simpler in the endosphere in terms of network complexity and connectivity. This indicates that the microbial community assembly of A. wardii was non-random and shaped by root selective effects. Proteobacteria, Chloroflexi, Actinobacteria, Cyanobacteria, and Acidobacteriota were generally the dominant bacterial phyla. The genera Crossiella and Bradyrhizobium were enriched in the rhizosphere and cyanobacterial genera were enriched in the endosphere, demonstrating substantial advantages to plant survival and adaptation in the harsh mine environment. Functional categories involved in amino acid and carbohydrate metabolism were abundant in the rhizosphere soil, thus contributing to metal solubility and bioavailability in the rhizosphere. Membrane transporters, especially ATP-binding cassette transporters, were enriched in the endosphere, indicating a potential role in metal tolerance and transportation in A. wardii. The study shows substantial variation in the structure and function of microbiomes colonizing different compartments, with the rhizosphere and endophytic microbiota potentially involved in plant metal tolerance and accumulation during phytostabilization.

Does phytoextraction with Sedum plumbizincicola increase cadmium leaching from polluted agricultural soil?

Sedum plumbizincicola is a cadmium (Cd) and zinc hyperaccumulator that can activate Cd by rhizosphere acidification. However, there is little understanding of the Cd leaching risk from polluted soil during phytoextraction process. Here, pot and column experiments were conducted to monitor soil Cd leaching characteristics under different rainfall simulation conditions during S. plumbizincicola phytoextraction. Soil Cd leaching increased significantly with increasing simulated rainfall intensity. Compared with normal rainfall (NR), weak rainfall (WR) resulted in a 34.3% decrease in Cd uptake by S. plumbizincicola and also led to a 68.7% decline in Cd leaching. In contrast, Cd leaching under heavy rainfall (HR) was 2.12 times that of NR in the presence of S. plumbizincicola. After two successive growing periods, phytoextraction resulted in a 53.5-66.4% decline in the amount of soil Cd leached compared with controls in which S. plumbizincicola was absent. Even compared with maize cropping as a control, S. plumbizincicola did not instigate a significant increase in Cd leaching. The contribution of Cd leaching loss to the decline in soil total Cd concentration was negligible after phytoextraction in the pot experiment. Overall, the results contribute to our understanding of soil Cd leaching risk by phytoextraction with S. plumbizincicola.

Repeated phytoextraction by hyperaccumulator Sedum plumbizincicola is an important remediation technology to remove Cd from contaminated soils. At the same time, Sedum plumbizincicola can also activate soil Cd by rhizosphere acidification. However, studies on the leaching risk of soil activated Cd during the phytoextraction process are very few. This study looked at the effects of Sedum plumbizincicola growth on soil Cd leaching with the changes in rainfall simulation and plant type. Results showed that repeated phytoextraction with Sedum plumbizincicola did not increase Cd leaching from contaminated soil.

Potential mobilization of water-dispersible colloidal thallium and arsenic in contaminated soils and sediments in mining areas of southwest China.

Water-dispersible colloids (WDCs) are vital for trace element migration, but there is limited information about the abundance, size distribution and elemental composition of WDC-bound thallium (Tl) and arsenic (As) in mining-contaminated soils and sediments solutions. Here, we investigated the potential mobilization of WDC-bound Tl and As in soils and sediments in a typical Tl/As-contaminated area. Ultrafiltration results revealed on average > 60% of Tl and As in soil solution (< 220 nm) coexisted in colloidal form whereas Tl and As in sediment solution primarily existed in the truly dissolved state (< 10 kDa) due to increased acidity. Using AF4-UV-ICP-MS and STEM-EDS, we identified Fe-bearing WDCs in association with aluminosilicate minerals and organic matter were main carriers of Tl and As. SAED further verified jarosite nanoparticles were important components of soil WDC, directly participating in the migration of Tl and As. Notably, high pollution levels and solution pH promoted the release of Tl/As-containing WDCs. This study provides quantitative and visual insights into the distribution of Tl and As in WDC, highlighting the important roles of Fe-bearing WDC, soil solution pH and pollution level in the potential mobilization of Tl and As in contaminated soils and sediments.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate - TraD and TraD - T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.

Dynamics of trace element enrichment in blue carbon ecosystems in relation to anthropogenic activities.

Blue carbon ecosystems (BCEs), located at the land-sea interface, provide critical ecological services including the buffering of anthropogenic pollutants. Understanding the interactions between trace element (TE) loads in BCEs and socioeconomic development is imperative to informing management plans to address pollution issues. However, the identification of anthropogenic TE pollution in BCEs remains uncertain due to the complex geochemical and depositional processes and asynchronous socioeconomic development along continental coastlines. Here, priority-controlled TE (As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn) concentrations in the mangrove, saltmarsh and seagrass soils and plant tissues along the coastline of China were investigated while taking bare flat and upland soils as corresponding references. We demonstrate that blue carbon (BC) soils accumulated markedly higher concentrations of anthropogenic TEs than the reference soils, mainly due to the effective trapping of fine-grained particles and higher binding capacities. We identify the time course of TE changes over the last 100 years which shows increasing anthropogenic TE accumulation resulting from military activities (1930-1950) and the growth of industrial and agricultural activities (1950-1980), then reaching a maximum after national economic reform (1980-2000). Since the 2000s, decreases in TE discharges driven by socioeconomic reform and strengthened environmental regulations have led to a widespread reversal of anthropogenic TE concentrations in BC soils. Based on the current TE flux we estimate that BCEs can filter over 27.3-100 % of the TEs emitted in industrial wastewaters from Chinese coastal provinces annually. However, the uptake of these TEs by plants can be substantially reduced through various mechanisms offered by edaphic properties such as organic carbon, clay, and sulfur contents. Therefore, enhancing TE filtering while preventing TEs from entering food webs through the conservation and restoration of BCEs will greatly aid in achieving the sustainable development goal of the coastal zone under intensified anthropogenic activities.

Optimizing cropping systems to close the gap between economic profitability and environmental health.

Supporting food security while maintaining ecosystem sustainability is one of the most important global challenges for humanity. Optimization of cropping systems is expected to promote the ecosystem services of agroecosystems. Yet, how and why cropping system influences the trade-offs between economic profitability and multiple ecosystem services remain poorly understood. We investigate the influence of six cropping systems on trade-offs between economic profitability and multiple ecosystem services after considering 36 agricultural ecosystem properties using field experiment data from 2020 to 2022. We show that designing cropping system is a critical tool to closing the gap between ecosystem sustainability and commercial profitability. Cropping system with three harvests within 2 yr had higher performance in overall ecosystem multiple services through enhancement of supporting, regulating, and economic performance without compromising provisioning compared with four other systems. These systems diminished the trade-off among multiple services, resulting in a 'win-win' situation for economics and multiple services. By contrast, the monoculture and double cropping systems lead to a strong trade-off between pairwise services including ecosystem health and profitability. Our work illustrates the substantial potential of rotation systems with three harvests within 2 yr in enforcing ecosystem services and closing the trade-offs among multiple agricultural ecosystem services.

Metagenomics reveals mechanism of pyrene degradation by an enriched bacterial consortium from a coking site contaminated with PAHs.

A bacterial consortium, termed WPB, was obtained from polycyclic aromatic hydrocarbons (PAHs) contaminated soil from a coking site. The consortium effectively degraded 100 mg L-1 pyrene by 94.8 % within 12 days. WPB was also able to degrade phenanthrene (98.3 %) and benzo[a]pyrene (24.6 %) in 12 days, while the individual isolates showed no PAHs degrading ability. Paracoccus sp. dominated the bacterial consortium (65.0-86.2 %) throughout the degradation process. Metagenomic sequencing reveals the proportion of sequences with xenobiotics biodegradation and metabolism increased throughout the degradation process indicating the great potential of WPB to degrade pollutants. The annotation of genes by metagenomic analysis help reconstruct the degradation pathways ("phthalate pathway" and "naphthalene degradation") and reveal how different bacteria contribute to the degradation process. Mycobacterium gilvum was found to carry nidAB genes that catalyze the first step of high-molecular-weight (HMW) PAHs in the degradation process despite Mycobacterium gilvum accounting for only 0.005-0.06 %. In addition, genomes of Paracoccus denitrificans and some other genera affiliated with Devosia, Pusillimonas caeni and Eoetvoesia caeni were successfully recovered and were found to carry genes responsible for the degradation of the intermediates of pyrene. These results enable further understanding of the metabolic patterns of pyrene-degrading consortia and provide direction for further cultivation and discovery of key players in complex microbial consortia.

Phytoextraction of highly cadmium-polluted agricultural soil by Sedum plumbizincicola: An eight-hectare field study.

Phytoextraction with Sedum plumbizincicola is an in-situ, environmentally friendly and highly efficient remediation technique for slightly Cd-polluted soils but it remains a challenge to remediate highly Cd-polluted soils under field conditions. Here, an 8-ha field experiment was conducted to evaluate the feasibility of repeated phytoextraction by S. plumbizincicola of a highly Cd-polluted acid agricultural soil (pH 5.61, [Cd] 2.58 mg kg-1) in Yunnan province, southwest China. Mean shoot dry biomass production, Cd concentration and Cd uptake were 1.95 t ha-1, 170 mg kg-1 and 339 g ha-1 at the first harvest, and 0.91 t ha-1, 172 mg kg-1 and 142 g ha-1 at the second harvest. After two seasons of phytoextraction, soil total and CaCl2-extractable Cd concentrations decreased from 2.58 ± 0.69 to 1.53 ± 0.43 mg kg-1 and 0.22 ± 0.12 to 0.14 ± 0.07 mg kg-1, respectively. Stepwise multiple linear regression analysis shows that the shoot Cd concentration and uptake of S. plumbizincicola were positively related to soil CaCl2-extractable Cd concentrations, especially in the first crop. A negative relationship indicates that soil organic matter content played an important role in soil Cd availability and shoot Cd concentration in the first crop. In addition, the rhizosphere effect on soil CaCl2-extractable Cd concentration was negatively correlated with soil pH in the first crop. The accuracy of the calculation of soil Cd phytoextraction efficiency at field scale depends on all of the following factors being considered: shoot Cd uptake, cropping pattern, standardized sampling points, and the leaching and surface runoff of Cd. Phytoextraction with S. plumbizincicola is a feasible technique for efficient Cd removal from highly polluted soils and wide variation in soil properties can influence phytoextraction efficiency at the field scale.

Ligand-displaying Escherichia coli cells and minicells for programmable delivery of toxic payloads via type IV secretion systems.

IMPORTANCE: The rapid emergence of drug-resistant bacteria and current low rate of antibiotic discovery emphasize the urgent need for alternative antibacterial strategies. We engineered Escherichia coli to conjugatively transfer plasmids to specific E. coli and Pseudomonas aeruginosa recipient cells through the surface display of cognate nanobody/antigen (Nb/Ag) pairs. We further engineered mobilizable plasmids to carry CRISPR/Cas9 systems (pCrispr) for the selective killing of recipient cells harboring CRISPR/Cas9 target sequences. In the assembled programmed delivery system (PDS), Nb-displaying E. coli donors with different conjugation systems and mobilizable pCrispr plasmids suppressed the growth of Ag-displaying recipient cells to significantly greater extents than unpaired recipients. We also showed that anucleate minicells armed with conjugation machines and pCrispr plasmids were highly effective in killing E. coli recipients. Together, our findings suggest that bacteria or minicells armed with PDSs may prove highly effective as an adjunct or alternative to antibiotics for antimicrobial intervention.

Ligand-Displaying E. coli Cells and Minicells for Programmable Delivery of Toxic Payloads via Type IV Secretion Systems.

Bacterial type IV secretion systems (T4SSs) are highly versatile macromolecular translocators and offer great potential for deployment as delivery systems for therapeutic intervention. One major T4SS subfamily, the conjugation machines, are well-adapted for delivery of DNA cargoes of interest to other bacteria or eukaryotic cells, but generally exhibit modest transfer frequencies and lack specificity for target cells. Here, we tested the efficacy of a surface-displayed nanobody/antigen (Nb/Ag) pairing system to enhance the conjugative transfer of IncN (pKM101), IncF (F/pOX38), or IncP (RP4) plasmids, or of mobilizable plasmids including those encoding CRISPR/Cas9 systems (pCrispr), to targeted recipient cells. Escherichia coli donors displaying Nb's transferred plasmids to E. coli and Pseudomonas aeruginosa recipients displaying the cognate Ag's at significantly higher frequencies than to recipients lacking Ag's. Nb/Ag pairing functionally substituted for the surface adhesin activities of F-encoded TraN and pKM101-encoded Pep, although not conjugative pili or VirB5-like adhesins. Nb/Ag pairing further elevated the killing effects accompanying delivery of pCrispr plasmids to E. coli and P. aeruginosa transconjugants bearing CRISPR/Cas9 target sequences. Finally, we determined that anucleate E. coli minicells, which are clinically safer delivery vectors than intact cells, transferred self-transmissible and mobilizable plasmids to E. coli and P. aeruginosa cells. Minicell-mediated mobilization of pCrispr plasmids to E. coli recipients elicited significant killing of transconjugants, although Nb/Ag pairing did not enhance conjugation frequencies or killing. Together, our findings establish the potential for deployment of bacteria or minicells as Programmed Delivery Systems (PDSs) for suppression of targeted bacterial species in infection settings. IMPORTANCE: The rapid emergence of drug-resistant bacteria and current low rate of antibiotic discovery emphasize an urgent need for alternative antibacterial strategies. We engineered Escherichia coli to conjugatively transfer plasmids to specific E. coli and Pseudomonas aeruginosa recipient cells through surface display of cognate nanobody/antigen (Nb/Ag) pairs. We further engineered mobilizable plasmids to carry CRISPR/Cas9 systems (pCrispr) for selective killing of recipient cells harboring CRISPR/Cas9 target sequences. In the assembled Programmed Delivery System (PDS), Nb-displaying E. coli donors with different conjugation systems and mobilizable pCrispr plasmids suppressed growth of Ag-displaying recipient cells to significantly greater extents than unpaired recipients. We also showed that anucleate minicells armed with conjugation machines and pCrispr plasmids were highly effective in killing of E. coli recipients. Together, our findings suggest that bacteria or minicells armed with PDSs may prove highly effective as an adjunct or alternative to antibiotics for antimicrobial intervention.

Impact of lignin constituents on the bacterial community and polycyclic aromatic hydrocarbon co-metabolism in an agricultural soil.

Lignin is a complex biopolymer comprising phenolic monomers with different degrees of methoxylation and may potentially enhance the degradation of soil pollutants such as polycyclic aromatic hydrocarbons (PAHs) through co-metabolism. However, the contribution of lignin constituents, including phenolic and methoxy subunits, to PAH biodegradation remains unclear. Here, p-hydroxybenzoate (pHBA), vanillate and methanol were selected to simulate phenolic units and methoxy groups of lignin. Soil microcosms receiving these compounds were established to evaluate their regulation on the bacterial community and PAH co-metabolism. There were different effects of different components on the biodegradation of a four-ring PAH, benzo(a)anthracene (BaA), as characterized using an isotopic tracer. Only vanillate significantly stimulated BaA mineralization to CO2, with pHBA and methanol leading to no appreciable change in the allocation of BaA in soil compartments. The lignin constituents had differential impacts on the soil bacterial community, with substantial enrichment of methylotrophs occurring in methanol-supplemented microcosms. Both vanillate and pHBA selected several aromatic degraders. Vanillate caused additional enrichment of methylotrophs, suggesting structure-dependent stimulation of bacterial functional guilds by lignin monomers. Compared with its constituents, lignin produced more extensive responses in terms of bacterial diversity and composition and the fate of BaA. However, it was difficult to link BaA co-metabolism to any specific bacterial taxa in the presence of lignin or its subunits. The results indicate that the co-metabolism effects of lignin may not be directly associated with phenolic or methoxy metabolism but with its regulation of the soil microbiome.

Endogenous biohydrogen from a rhizobium-legume association drives microbial biodegradation of polychlorinated biphenyl in contaminated soil.

Endogenous hydrogen (H2) is produced through rhizobium-legume associations in terrestrial ecosystems worldwide through dinitrogen fixation. In turn, this gas may alter rhizosphere microbial community structure and modulate biogeochemical cycles. However, very little is understood about the role that this H2 leaking to the rhizosphere plays in shaping the persistent organic pollutants degrading microbes in contaminated soils. Here, we combined DNA-stable isotope probing (DNA-SIP) with metagenomics to explore how endogenous H2 from the symbiotic rhizobium-alfalfa association drives the microbial biodegradation of tetrachlorobiphenyl PCB 77 in a contaminated soil. The results showed that PCB77 biodegradation efficiency increased significantly in soils treated with endogenous H2. Based on metagenomes of 13C-enriched DNA fractions, endogenous H2 selected bacteria harboring PCB degradation genes. Functional gene annotation allowed the reconstruction of several complete pathways for PCB catabolism, with different taxa conducting successive metabolic steps of PCB metabolism. The enrichment through endogenous H2 of hydrogenotrophic Pseudomonas and Magnetospirillum encoding biphenyl oxidation genes drove PCB biodegradation. This study proves that endogenous H2 is a significant energy source for active PCB-degrading communities and suggests that elevated H2 can influence the microbial ecology and biogeochemistry of the legume rhizosphere.