Galactomannan oligosaccharides (GMOS), composed of 2–10 mannose units linked with β-1, 4 glycosidic bond as the main chain and galactose linked with α-1, 6 glycosidic bond as the side chain, are crucial for probiotic food synthesis due to their ability to promote the growth and activity of beneficial intestinal microbiota, enhance the host immune system, and improve nutrient digestion. GMOS is usually obtained by hydrolyzing plants such as locust bean gum and guar gum with mannanase. β-mannanase ManA from Alkaliphilic Bacillus sp. N16-5 can hydrolyze β-1, 4 glycosidic bond of galactomannan. In this study, an immobilization system was employed utilizing polyhydroxyalkanoate (PHA) biopolymers, which naturally have an affinity mainly mediated by hydrophobic interaction for PhaP protein. Fusion protein combining ManA with PhaP from Aeromonas hydrophila, was subsequently immobilized on PHA support to form a multi-enzyme complex, facilitating the hydrolysis of locust bean gum to generate GMOS. This immobilized enzyme enhances enzyme stability and reusability, can be reused up to 32 times while maintaining ~ 80% of its activity, offering substantial cost savings through in-situ enzyme and product separation. Additionally, the different PHA forms were developed to hydrolyze locust bean gum to produce GMOS, such as nano PHA particles, PHA electrospun materials, while these preliminary investigations show promise, further research is needed to optimize their performance and practical application.

Wild-type Bacillus strains have significant industrial and medical value, but their effective utilization often requires strain improvement. The CRISPR/Cas9 system has become the primary tool for genome editing, allowing precise introduction of desired mutations at specific chromosomal locations. However, the practical application of CRISPR/Cas9 in most wild-type Bacillus strains remains challenging due to cellular toxicity resulting from Cas9/sgRNA activity. Therefore, controlling Cas9 toxicity is essential for the widespread application of the CRISPR/Cas9 system in wild-type Bacillus strains. We employed AcrIIA4, an anti-CRISPR protein that inhibits the Cas9/sgRNA ribonucleoprotein complex from interacting with DNA, to mitigate Cas9/sgRNA-mediated toxicity, thereby enabling CRISPR/Cas9-based genome editing in wild-type strains. The newly constructed CRISPR/anti-CRISPR (CAC) plasmids harbor both cas9 and acrIIA4 genes controlled by the Pspac and Pxyl promoters, respectively, along with the repressor genes lacI and xylR. This design allows precise control of Cas9 activity through inducers. Xylose, which induces AcrIIA4 expression, effectively alleviated Cas9/sgRNA-mediated toxicity during transformation. Under xylose induction, the CAC plasmid led to a remarkable 139-fold increase in the transformation efficiency of wild-type Bacillus subtilis compared to a plasmid lacking anti-CRISPR. Meanwhile, IPTG induction promoted Cas9 expression, facilitating efficient genome editing. Upon IPTG induction, the genome editing efficiency in wild-type B. subtilis increased from 0 to 95.8% in transformants carrying the CAC plasmid. Importantly, our findings extend beyond B. subtilis, revealing that the anti-CRISPR protein dramatically enhanced transformation and genome editing efficiencies in Bacillus pumilus. Moreover, we demonstrated that the CAC system successfully enabled the generation of spo0A mutants in Bacillus mojavensis, Bacillus tequilensis, and Paenibacillus polymyxa. Conclusions In this study, we developed a CAC system that utilizes the anti-CRISPR protein AcrIIA4 to reduce Cas9/sgRNA-mediated toxicity in Bacillus strains. This system enables precise control of AcrIIA4 and Cas9 expression through inducers, significantly enhancing the efficiency of transformation and genome editing in wild-type Bacillus strains. Therefore, the CAC system stands as a powerful tool to facilitate genome editing in diverse wild-type Bacillus species.

Alterations in gut microbiota have been linked to chronic kidney disease (CKD), but large-scale studies and mechanistic insights are limited. Here we analyzed gut metagenome data from 1,550 older individuals (aged 65–93 years) with comprehensive kidney function measurements. Segatella copri was positively associated with kidney function through microbial ammonia metabolism-related pathways and the asnA gene, which encodes an ammonia-assimilating enzyme. These associations were replicated in two external studies. In mice, ammonia supplementation increased serum levels of creatinine and blood urea nitrogen, accelerating CKD progression. In vitro cultures of S. copri or asnA-overexpressing Escherichia coli reduced ammonia concentrations, which was markedly attenuated in asnA-knockout S. copri. Gavage of either S. copri or asnA-overexpressing E. coli, but not asnA-knockout S. copri, mitigated ammonia-induced CKD progression in mice. These findings highlight the role of gut microbial ammonia metabolism in CKD pathogenesis and underscore the therapeutic potential of microbial-based interventions. Using human data and mice models, it was revealed that assimilation of ammonia by a gut microbe prevents its harmful accumulation that can impair kidney function.

Transgenic plants are essential for both basic and applied plant biology. Recently, fluorescent and colorimetric markers were developed to enable nondestructive identification of transformed seeds and accelerate the generation of transgenic plant lines. Yet, transformation often results in the integration of multiple copies of transgenes in the plant genome. Multiple transgene copies can lead to transgene silencing and complicate the analysis of transgenic plants by requiring researcher to track multiple T-DNA loci in future generations. Thus, to simplify analysis of transgenic lines, plant researchers typically screen transformed plants for lines where the T-DNA inserted in a single locus — an analysis that involves laborious manual counting of fluorescent and non-fluorescent seeds for screenable markers.  To expedite T-DNA segregation analysis, we developed SeedSeg, an image analysis tool that uses a segmentation algorithm to count the number of transformed and wild-type seeds in an image. SeedSeg runs a chi-squared test to determine the number of T-DNA loci. Parameters can be adjusted to optimize for different brightness intensities and seed sizes. By automating the seed counting process, SeedSeg reduces the manual labor associated with identifying transgenic lines containing a single T-DNA locus. SeedSeg is adaptable to different seed sizes and visual transgene markers, making it a versatile tool for accelerating plant research.

Understanding the relationships between bacteria, their ecological and genomic traits, and their environment, is important to elucidate microbial community dynamics and their roles in ecosystem functioning. Here, we examined the relationships between soil properties and bacterial traits within highly managed agricultural soil systems subjected to arable crop rotations or management as permanent grass. We assessed the bacterial communities using metabarcoding and assigned each amplicon trait scores for rRNA copy number, genome size, and GC content, which are classically associated with potential growth rates and specialisation. We also calculated the niche breadth trait of each amplicon as a measure of social ubiquity within the examined samples. Within this soil system, we demonstrated that pH was the primary driver of bacterial traits. The weighted mean trait scores of the samples revealed that bacterial communities associated with soils at lower pH (<7) tended to have larger genomes (potential plasticity), have more rRNA (higher growth rate potential), and are more ubiquitous (have less niche specialisation) than the bacterial communities from higher pH soils. Our findings highlight not only the association between pH and bacterial community composition but also the importance of pH in driving community functionality by directly influencing genomic and niche traits.