Cysteine-Mediated Extracellular Electron Transfer of Lysinibacillus varians GY32.

Microbial extracellular electron transfer (EET) is essential in many natural and engineering processes. Compared with the versatile EET pathways of Gram-negative bacteria, the EET of Gram-positive bacteria has been studied much less and is mainly limited to the flavin-mediated pathway. Here, we investigate the EET pathway of a Gram-positive filamentous bacterium Lysinibacillus varians GY32. Strain GY32 has a wide electron donor spectrum (including lactate, acetate, formate, and some amino acids) in electrode respiration. Transcriptomic, proteomic, and electrochemical analyses show that the electrode respiration of GY32 mainly depends on electron mediators, and c-type cytochromes may be involved in its respiration. Fluorescent sensor and electrochemical analyses demonstrate that strain GY32 can secrete cysteine and flavins. Cysteine added shortly after inoculation into microbial fuel cells accelerated EET, showing cysteine is a new endogenous electron mediator of Gram-positive bacteria, which provides novel information to understand the EET networks in natural environments. IMPORTANCE Extracellular electron transport (EET) is a key driving force in biogeochemical element cycles and microbial chemical-electrical-optical energy conversion on the Earth. Gram-positive bacteria are ubiquitous and even dominant in EET-enriched environments. However, attention and knowledge of their EET pathways are largely lacking. Gram-positive bacterium Lysinibacillus varians GY32 has extremely long cells (>1 mm) and conductive nanowires, promising a unique and enormous role in the microenvironments where it lives. Its capability to secrete cysteine renders it not only an EET pathway to respire and survive, but also an electrochemical strategy to connect and shape the ambient microbial community at a millimeter scale. Moreover, its incapability of using flavins as an electron mediator suggests that the common electron mediator is species-dependent. Therefore, our results are important to understanding the EET networks in natural and engineering processes.

Function-Oriented Graphene Quantum Dots Probe for Single Cell in situ Sorting of Active Microorganisms in Environmental Samples.

Functional microorganisms play a vital role in removing environmental pollutants because of their diverse metabolic capability. Herein, a function-oriented fluorescence resonance energy transfer (FRET)-based graphene quantum dots (GQDs-M) probe was developed for the specific identification and accurate sorting of azo-degrading functional bacteria in the original location of environmental samples for large-scale culturing. First, nitrogen-doped GQDs (GQDs-N) were synthesized using a bottom-up strategy. Then, a GQDs-M probe was synthesized based on bonding FRET-based GQDs-N to an azo dye, methyl red, and the quenched fluorescence was recovered upon cleavage of the azo bond. Bioimaging confirmed the specific recognition capability of GQDs-M upon incubation with the target bacteria or environmental samples. It is suggested that the estimation of environmental functional microbial populations based on bioimaging will be a new method for rapid preliminary assessment of environmental pollution levels. In combination with a visual single-cell sorter, the target bacteria in the environmental samples could be intuitively screened at the single-cell level in 17 bacterial strains, including the positive control Shewanella decolorationis S12, and were isolated from environmental samples. All of these showed an azo degradation function, indicating the high accuracy of the single-cell sorting strategy using the GQDs-M. Furthermore, among the bacteria isolated, two strains of Bacillus pacificus and Bacillus wiedmannii showed double and triple degradation efficiency for methyl red compared to the positive control (strain S12). This strategy will have good application prospects for finding new species or high-activity species of specific functional bacteria.

Visualizing and Isolating Iron-Reducing Microorganisms at the Single-Cell Level.

Iron-reducing microorganisms (FeRM) play key roles in many natural and engineering processes. Visualizing and isolating FeRM from multispecies samples are essential to understand the in situ location and geochemical role of FeRM. Here, we visualized FeRM by a "turn-on" Fe2+-specific fluorescent chemodosimeter (FSFC) with high sensitivity, selectivity, and stability. This FSFC could selectively identify and locate active FeRM from either pure culture, coculture of different bacteria, or sediment-containing samples. Fluorescent intensity of the FSFC could be used as an indicator of Fe2+ concentration in bacterial cultures. By combining the use of the FSFC with that of a single-cell sorter, we obtained three FSFC-labeled cells from an enriched consortium, and all of them were subsequently shown to be capable of iron reduction; two unlabeled cells were shown to have no iron-reducing capability, further confirming the feasibility of the FSFC.IMPORTANCE Visualization and isolation of FeRM from samples containing multiple species are commonly needed by researchers from different disciplines, such as environmental microbiology, environmental sciences, and geochemistry. However, no available method has been reported. In this study, we provide a method to visualize FeRM and evaluate their activity even at the single-cell level. When this approach is combined with use of a single-cell sorter, FeRM can also be isolated from samples containing multiple species. This method can be used as a powerful tool to uncover the in situ or ex situ role of FeRM and their interactions with ambient microbes or chemicals.

FRET-based fluorescent nanoprobe platform for sorting of active microorganisms by functional properties.

Fluorescence-activated cell sorting (FACS) has rarely been applied to screening of microorganisms because of poor detection resolution, which is compromised by poor stability, toxicity, or interference from background fluorescence of the fluorescence sensors used. Here, a fluorescence-based rapid high-throughput cell sorting method was first developed using a fluorescence resonance energy transfer (FRET) fluorescent nanoprobe NP-RA, which was constructed by coating a silica nanoparticle with Rhodamine B and methyl-red (an azo dye). Rhodamine B (inner layer) is the FRET donor and methyl-red (outer layer) is the acceptor. This ready-to-use NP-RA is non-fluorescent, but fluoresces once the outer layer is degraded by microorganisms. In our experiment, NP-RA was ultrasensitive to model strain Shewanella decolorationis S12, showing a broad detection range from 8.0 cfu/mL to 8.7 × 108 cfu/mL under confocal laser scanning microscopy, and from 1.1 × 107 to 9.36 × 108 cfu/mL under a fluorometer. In addition, NP-RA bioimaging can clearly identify other azo-respiring cells in the microbial community, including Bosea thiooxidans DSM 9653 and Lysinibacillus pakistanensis NCCP-54. Furthermore, the fluorescent probe NP-RA is compatible with downstream FACS so that azo-respiring cells can be rapidly sorted out directly from an artificial microbial community. To our knowledge, no fluorescent nanoprobe has yet been designed for tracking and sorting azo-respiration functional microorganisms.