Microorganisms are key players in global cycling of nitrogen (N) and carbon (C), controlling their availability and fluxes, including the emissions of the powerful greenhouse gases nitrous oxide (N2O) and methane (CH4). Standard sequencing methods often reveal only a limited fraction of their diversity, because of their low relative abundance, the insufficient sequencing depth of traditional metagenomes of complex communities, and limitations in coverage of PCR-based assays. Here, we developed and tested a targeted metagenomics approach based on probe capture and hybridization to simultaneously characterize the diversity of multiple key metabolic genes involved in inorganic N and CH4 cycling. We designed comprehensive probe libraries for each of the 14 target marker genes comprising 264 111 unique probes. In validation experiments with the mock communities, targeted metagenomics yielded gene profiles similar to the original communities. Only GC content had a small effect on probe efficiency, as low GC targets were less efficiently detected than those with high GC, within the mock communities. Furthermore, the relative abundances of the marker genes obtained using targeted or traditional shotgun metagenomics were significantly correlated. In addition, using archaeal amoA genes as a case-study, targeted metagenomics identified substantially higher taxonomic diversity and a larger number of sequence reads per sample, yielding diversity estimates 28 or 1.24 times higher than shotgun metagenomics or amplicon sequencing, respectively. Our results show that targeted metagenomics complements current approaches to characterize key microbial populations and functional guilds in biogeochemical cycles in different ecosystems, enabling more detailed, simultaneous characterization of multiple functional genes.

Pseudomonas fluorescens is a Gram-negative bacterium with a remarkable metabolic and physiological versatility that enables it to adapt and colonize diverse ecological niches, including the human small intestine. While serotonin is primarily found in high concentrations in gut tissue, its levels in the lumen can be elevated in conditions such as celiac disease, where P. fluorescens is also found in increased abundance. The potential effects of serotonin on P. fluorescens in such contexts remain unclear. We demonstrate that P. fluorescens metabolizes serotonin primarily into 5-hydroxyindole-3-acetic acid (5-HIAA) and, to a lesser extent, into 5-hydroxytryptophol and N-acetylserotonin. Gene expression analysis revealed significant changes in oxidative stress-related pathways over time, and proteomic analysis confirmed the shifts seen particularly in amino acid catabolic pathways. Serotonin metabolism also enhanced bacterial resistance to oxidative stress, suggesting a protective role. The findings reveal a novel mechanism by which serotonin modulates the metabolism and stress responses of P. fluorescens. This study provides insight into how P. fluorescens adapts to serotonin-rich environments, such as in celiac disease, and may inform future research on microbial interactions with host-derived metabolites in disease contexts.

Most biofilm research has centered on a few motile model organisms that form thick biofilms. During dispersal, motile bacteria actively escape through motility; however, non-motile species likely rely on the modulation of adhesive interactions to regulate detachment. In this study we show that the non-motile soil organism Paracoccus denitrificans maintains thin biofilms through constitutive quorum sensing (QS). We show that a LuxI/LuxR-type QS system actively suppresses cell-to-cell adhesion to maintain a thin biofilm architecture, while maintaining a basal level of cell-surface adhesion. We identified a polysaccharide biosynthesis gene, pxm, which is essential for cellular aggregation. Disruption of QS leads to thick biofilm formation with necrotic cores, mediated by Pxm-dependent production. Using microfluidic chambers, we examined interactions between Pxm and BapA adhesion factors under varying spatial confinement. Our findings reveal that QS-dependent suppression of adhesion factors enables P. denitrificans to form thin, loosely connected biofilms that promote nutrient access and dispersal, providing an ecological advantage in spatially confined environments such as soil or aquatic sediments.

The stringent response represses translation and is activated when cells enter the stationary phase and intracellular amino acid levels drop. Bacillus subtilis, a well-known industrial enzyme production organism, secretes most enzymes during the stationary phase. However, translation pausing can affect protein folding and production. Here, we examined whether the stringent response affects ribosome pausing in B. subtilis. To investigate this, genome-wide ribosome profiling was performed using a stringent response mutant that overexpressed the α-amylase AmyM. Blocking the stringent response did increase overall protein synthesis, however AmyM production was reduced. Ribosome profiling revealed that this was not caused by a reduction in translation. In fact, absence of the stringent response did not markedly influence ribosome pausing. Late into the stationary phase an increased ribosome pausing at tryptophan codons was observed, suggesting a depletion of tryptophan. A strong suppression of tryptophan biosynthesis and acquisition genes, under control of the trp RNA-binding attenuation protein TRAP, likely accounts for this. Why this occurs is unclear since TRAP does not belong to the stringent response regulon of B. subtilis. Finally, the genome-wide ribosome profiles revealed several operon genes with unusually low translation activities, illustrating the importance of translation initiation as a regulatory element of expression.

Previous high-throughput evaluations of CRISPR activities for a large number of target and guide RNA sequences were based on measuring insertion–deletion frequencies rather than cleavage efficiencies. Here we develop two high-throughput in vitro methods, Cut-seq1 and Cut-seq2, to evaluate Cas9 cleavage efficiency for tens of thousands, or even hundreds of thousands, of guide RNA–target pairs. These methods reveal low correlations between in vitro cleavage efficiencies and insertion–deletion frequencies in cells, yet high concordances in PAM compatibility. Using the resulting large datasets of in vitro cleavage efficiencies, we develop DeepCut, a set of deep learning models that can identify optimized single-guide RNAs that can selectively cleave specific sequences, even in the presence of similar noise sequences. Using these optimized single-guide RNAs, we develop a method, CLOVE-seq (which stands for cleavage for large-scale optimized variant enrichment sequencing), to enrich rare variants in a multiplexed manner by Cas9-mediated specific cleavage of noise or rare variant sequences. Our methods can enhance the understanding of CRISPR nuclease activities and could be used to detect a large number of rare variants in various biomedical contexts. Cut-seq1, Cut-seq2, DeepCut and CLOVE-seq methods find optimized single-guide RNAs to detect a large number of rare variants in cells.

The world is witnessing a serious challenge of poverty which is reflected as hidden hunger and simultaneously overpopulation puts a grave burden on our ecosystem. In order to overcome these serious issues, the sustainable development goals (SDGs) serve a very significant role. Concurrently, the contribution of microorganisms in achieving the SDGs cannot be overlooked. Bacillus species as a probiotic has led to the development of various commercial fermented products that are available in the market. Additionally, the formulated products have been clinically validated for their health benefits. Bacillus species have also been reported to serve as bio-preservatives and biocontrol agents because they aid in preserving and maintaining the final food product as well as post-harvest goods. The utilization of Bacillus spp. as probiotics has significantly contributed to the overall well-being of animals. Moreover, many different species of Bacillus have been reported to be utilized as bio-cementing agents. All the aforementioned roles of Bacillus species contribute to addressing zero hunger (SDG 2), good health and well-being (SDG 3), and industry, infrastructure, and innovation (SDG 9).

Reprogramming the specificity of proteases toward alternative target sequences could enable an array of exciting applications, ranging from proteome editing to therapeutic interventions. Here we report an in vivo life–death selection system for protease reprogramming using the toxic N-terminal domain of gasdermin D (GD-N) protein as a selection marker. The approach is a modular system that can be used to cover the protease mutational diversity in the billions through only a few cycles of directed evolution. By inserting the desired cleavage sequence into the loop region of the GD-N protein, which is toxic to host bacteria cells, the system selects for efficient substrate cleavage—rendering GD-N nontoxic—by enrichment of bacteria in liquid culture. Using the tobacco etch virus protease (TEVp) and corresponding substrate sequence as a model, we demonstrated that our platform could select and enrich an efficient protease variant millionfold after a single round of selection. We also evolve TEVp to cut sequences on target proteins with known pathological roles. An in vivo selection system based on the toxicity regulation of the N-terminal domain of gasdermin D and evolution of tobacco etch virus protease (TEVp) has been developed. The method enables development of a TEVp capable of precise cleavage of specific sequences on target proteins.