Rice is one of the most widely cultivated and highest-yielding crops worldwide, and its yield stability is closely linked to global food security. As a typical mycorrhizal crop, rice exhibits enhanced growth and stress tolerance after colonization of arbuscular mycorrhizal fungi (AMF). Rhizosphere bacteria, a major component of the rhizosphere microbiome, interact synergistically with AMF, collectively contributing to enhanced nutrient acquisition and improved rice performance. This review summarizes recent advances in the interactions between AMF and rhizosphere bacteria. We highlight that their synergistic interactions regulate rice growth, nutrient acquisition, and stress tolerance, which contribute to the ecosystem stability and biodiversity of the rhizosphere. However, most available studies on the interactions between AMF and rhizosphere bacteria have been conducted under controlled conditions, which limits their applicability to the complex natural field environments. Therefore, this review proposes several suggestions for future research. First, utilize multi-omics technologies such as metagenomics and isotope tracing techniques to elucidate the molecular mechanisms underlying the AMF-bacterial interaction. Second, in practical applications, long-term field positioning trials should be conducted to screen out superior microbial agents that meet the requirements. Finally, optimize regulatory conditions to achieve large-scale propagation of AMF and overcome the bottleneck in AMF field propagation provides theoretical support for promoting the practical application of this technology in agricultural production.

Dimorphic prosthecate bacteria (DPB) are a group of prokaryotes that reproduce through asymmetric division, producing two morphologically and functionally highly differentiated daughter cells. This unique lifestyle strategy endows them with a competitive advantage in nutrient-poor environments and makes them ideal models for studying bacterial developmental regulation, morphological evolution, and ecological adaptation. This review systematically summarizes the research progress in DPB in terms of their taxonomy, molecular mechanisms of lifestyle regulation, extensive ecological distribution, and environmental adaptation strategies. Additionally, it discusses the application potential of DPB in environmental remediation and biotechnology. Finally, this review makes an outlook on future research directions, aiming to provide a reference for deeply understanding the biological characteristics of these bacteria and expanding their application value.

Ciliates of the family Balantidiidae widely parasitize the digestive systems of various invertebrates and vertebrates. Among them, Balantioides coli with cosmopolitan distribution is a major zoonotic pathogen. For a long time, due to the similar morphology of trophozoites of Balantidiidae ciliates from various hosts and the influences of various growing stages and host intestinal microenvironments on morphological characteristics, there have been some inconsistencies and confusions in their classification and nomenclature in literature. Recently, significant progress has been achieved in the study of the phylogenetic relationship and genetic diversity of Balantidiidae ciliates, providing new evidences for their taxonomic reconstruction. On the basis of a systematic review of literature and our findings, this article summarizes the taxonomic status and genetic diversity of Balantidiidae ciliates from different hosts in terms of the morphological characteristics, encystation ability, host specificity, and phylogenetic relationships. The existing evidence supports the division of the family Balantidiidae into two genera Balantidium and Balantioides and indicates that the ciliate infecting humans and various mammals should be classified as Balantioides coli. The related findings and understanding help clarify the taxonomy and nomenclature system of Balantidiidae ciliates and provide an important reference for studying the biological characteristics of Balantidiidae ciliates as well as the diagnosis and control of balantiosis in humans and animals.

Neopestalotiopsis cubana Maharachch. is the primary pathogen responsible for leaf spot and fruit ring rot in guava (Psidium guajava L.). The ADP-ribosylation factor-like protein 8 (Arl8) belonging to the ADP-ribosylation factor (Arf) superfamily is associated with lysosomal processes including localization, trafficking, and fusion. The function of Arl8 in phytopathogenic fungi remains poorly understood. Objective To elucidate the function of Arl8 in N. cubana. Methods An Arl8 gene was identified from the genome of N. cubana and designated as NecArl8. A NecArl8 knockout mutant (ΔNecArl8) and a complemented strain (ΔNecArl8-C) were generated via clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9, (CRISPR/Cas9)-mediated genome editing. These strains were subsequently analyzed through phenotypic experiments and RNA-seq. Results ΔNecArl8 showed a reduction of 34.93% in sporulation capacity and significant increases of 5.74% and 7.39% in inhibition rates by salt and osmotic stresses, respectively. In addition, this mutant exhibited weakened pathogenicity, as indicated by a reduction of 5.74 mm in the average spot diameter. In contrast, the mutant displayed significantly enhanced resistance to oxidative stress, which showed an inhibition rate of -19.22%. After long-term culture, a large number of vacuolated cells were observed in the mutant. RNA-seq analysis was performed on ΔNecArl8 and the wild-type strain to identify differentially expressed genes. Transcriptomic analysis showed that 2 330 genes were significantly down-regulated and 1 355 genes were significantly up-regulated. Gene set enrichment analysis further revealed that transmembrane transport proteins were broadly suppressed, and the expression levels of 16 fungal-type vacuolar-related proteins were significantly down-regulated, suggesting that the structure and functions of fungal-type vacuoles were seriously damaged. The reverse transcription quantitative PCR (RT-qPCR) results showed the expression patterns of 9 randomly selected transmembrane transport genes were consistent with those obtained by transcriptome sequencing, indicating that the transcriptome data were highly reliable. Conclusion NecArl8 may affect the pathogenicity of N. cubana on guava by regulating function of transmembrane transport. This study lays a theoretical foundation for exploring the pathogenic mechanism of N. cubana and developing new control strategies.

Objective To overcome the limitations such as high costs and restricted substrate utilization of mono-culture fermentation, bacterium-alga co-culture based on resource complementarity offers a promising new avenue for ectoine production. This study investigated the co-culture conditions of Dunaliella pseudosalina ZBY-1 and Halomonas campaniensis XH26 and the variations in ectoine yield, aiming to elucidate the metabolic regulation mechanism of ectoine biosynthesis in the co-culture system. Methods Strains XH26 and ZBY-1 were co-cultured at different inoculation ratios (1/0, 1/5, 1/10, 1/15, and 1/20) to screen the ratio yielding the highest ectoine production. Targeted metabolomics analysis was performed on the co-culture group [H group (H)], the bacterial control group [XH26 group (X)], and the algal control group [ZBY-1 group (D)] to identify significant differential metabolites. Results The highest ectoine yield was achieved at a bacterium-to-alga ratio of 1:15, while the pigment content of the algal strain was lower than that of the control group. Metabolomics analysis identified 15 (H vs. D), 16 (H vs. X), and 16 (X vs. D) significant differential metabolites, including L-alanine, L-asparagine, L-aspartic acid, L-phenylalanine, malic acid, and pyruvic acid. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis revealed that alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, and arginine biosynthesis were the significantly altered metabolic pathways. Conclusion The co-culture system exhibited an asymmetric pattern characterized by bacterial proliferation and algal inhibition. The co-culture system significantly activated the central carbon metabolic network of the bacteria. Notably, aspartic acid and glutamic acid were significantly accumulated in cells, serving as the direct carbon skeleton and amino donor, respectively, to directly promote the efficient synthesis of ectoine.

Objective To clone and express a gene encoding the β-xylosidase from Enterobacter cloacae GX-3, a putative member of the glycoside hydrolase family 3, systematically characterize the recombinant enzyme, and improve the xylose tolerance by molecular engineering of key amino acid residues involved in xylose binding. Methods On the basis of the whole-genome sequencing data of E. cloacae GX-3, primers were designed to amplify the β-xylosidase gene annotated as GH3. The target gene was amplified by PCR and cloned into the pQE30 expression vector, and the resulting recombinant plasmid was transformed into Escherichia coli M15 for induced expression. The recombinant enzyme was purified by nickel-affinity chromatography, and its enzymatic properties were studied. Site-directed mutagenesis was conducted on amino acid residues associated with xylose tolerance. Results The β-xylosidase gene belonging to the GH3 family was successfully cloned from E. cloacae GX-3 and heterologously expressed in E. coli M15. Substrate specificity analysis revealed that the recombinant enzyme EXYL was a multifunctional enzyme exhibiting β-xylosidase, β-glucosidase, and α-L-arabinofuranosidase activities. EXYL showed the optimal performance with the substrate of pNPX and at pH 5.5 and 45 ℃. The K_m and V_max values of this enzyme were (0.73±0.06) mmol/L and (130.00±6.85) μmol/(mg·min), respectively. The inhibition constant (K_i) for xylose was (51.95±2.36) mmol/L. When EXYL acted on xylooligosaccharides (X3-X5), the main products were xylose and xylobiose, each accounting for approximately 50% of the yield. Site-directed mutagenesis of xylose tolerance-related residues yielded positive mutants W138C and W138A, which showed 2.38-fold and 1.83-fold improvements in xylose tolerance, respectively. Conclusion This study provides insights into the multifunctional activities of β-xylosidases and offers new strategies for enhancing the xylose tolerance of β-xylosidases in the GH3 family.

Objective Bacillomycin L, a cyclic lipopeptide antibiotic produced by Bacillus velezensis Bs916, has been demonstrated to possess strong antifungal activity. However, its low yield has become a critical bottleneck limiting its large-scale application. This study aims to identify the negative transcriptional regulators involved in bacillomycin L biosynthesis and, based on this, adopt a dual strategy combining genetic engineering and fermentation process optimization to promote its large-scale production. Methods Homologous recombination was employed to construct single (ΔresD, ΔabrB) and double (ΔresDΔabrB) knockout strains. The regulatory characteristics were elucidated by HPLC, inhibition zone measurement, and RT-qPCR. Furthermore, EMSA and DNase I footprinting assays were conducted to investigate the binding activity and identify the specific binding sites of these transcription factors with the flanking sequences of the transcription initiation site of the Bac gene cluster. Finally, fermentation process optimization was performed in a bioreactor to further enhance the production of bacillomycin L. Results HPLC results demonstrated that the bacillomycin L yields of all the mutant strains cultured in the LB media significantly increased by 5.8, 11.3, and 12.0 folds compared with that of the wild-type strain. The antagonistic activities of the mutants against plant pathogenic fungi exhibited corresponding increases. RT-qPCR results further confirmed that both ResD and AbrB acted as negative regulators of bacillomycin L biosynthesis. EMSA experiments revealed that both ResD and AbrB possessed strong binding activities with their target sequences. DNase I footprinting assays further elucidated that AbrB exhibited a propensity for binding to A+T-rich gene fragments and displayed extensive DNA-binding capabilities, directly interacting with the promoter region, 5′ UTR, and coding regions of the Bac gene cluster. Unfortunately, the specific binding site of ResD remained to be identified. Under fermentation conditions, the genetically engineered strain ΔresDΔabrB achieved gram-per-liter level production of bacillomycin L in an optimized glucose-mineral salts medium. This result greatly promoted the large-scale production of bacillomycin L. Conclusion Adopting a dual strategy that combines genetic engineering with fermentation process optimization effectively breaks the bottleneck of low yields for Bacillus-derived antimicrobial peptides. This study provides a reference for the mass production of other microbial secondary metabolites.

Objective To achieve high-level expression and purification of the recombinant 9 kDa and 15 kDa chicken NK-lysin proteins (designated as cNKL9 and cNKL15) and evaluate their in vitro antibacterial and immunomodulatory activities. Methods The target genes—cNKL9 and cNKL15—were individually cloned into the pPIC9K vector to construct recombinant expression plasmids for heterologous expression in Pichia pastoris. After confirming the correct expression of the recombinant proteins via SDS-PAGE and Western blotting, we used nickel affinity chromatography to obtain highly purified recombinant cNKL9 and cNKL15 proteins. The in vitro antibacterial activitiesof recombinant proteins against Salmonella typhimurium and Escherichia coli O157:H7 were assessed by colony forming unit (CFU) assays. We measured the mRNA levels by RT-qPCR and analyzed the transcriptional changes of relevant cytokines after applying the recombinant proteins to the chicken macrophage cell line HD11. High-level expression of the recombinant proteins was achieved in a 15 L fermenter for scale-up culture via optimization of the high-density fermentation process. Results The P. pastoris expression plasmids pPIC9K-cNKL9 andpPIC9K-cNKL15 were successfully constructed. SDS-PAGE and Western blotting results revealed specific bands of recombinant cNKL9 and cNKL15 proteins in the culture supernatant, confirming the successful secretory expression of the target proteins. Antibacterial activity assay demonstrated that the recombinant cNKL9 protein exhibited significant inhibitory activities against both S. typhimurium and E. coli O157:H7, whereas the recombinant cNKL15 protein showed no obvious inhibitory effects on either pathogenic strain. RT-qPCR analysis showed that the recombinant cNKL15 protein significantly upregulated the transcription levels of cytokines such as CCL4 and CCL20 in HD11 cells, while the recombinant cNKL9 protein did not exhibit immunomodulatory activity. Furthermore, in the 15 L fermenter, the heterologous expression yields of cNKL9 and cNKL15 reached 0.64 g/L and 0.53 g/L, respectively. Conclusion For the first time, we achieved high-level expression of recombinant cNKL9 and cNKL15 proteins in the P. pastoris system. Specifically, cNKL9 exhibited inhibitory activities against S. typhimurium and E. coli O157:H7, while cNKL15 exerted immunomodulatory effects by upregulating the mRNA levels of cytokines in the chicken macrophage cell line HD11. These findings provide experimental evidence for the development of cNKL9 and cNKL15 as antibacterial or immunomodulatory agents, indicating their considerable application potential.

Objective To investigate the effects of ferric uptake regulator (Fur) on iron homeostasis, oxidative stress response, and virulence in Vibrio parahaemolyticus. Methods BLASTp and multiple sequence alignment analyses were employed to analyze the conservation of Fur proteins in several Gram-negative bacteria. A fur gene deletion mutant (Δfur) and a complementation strain (CΔfur) were constructed. The growth of the wild-type (WT), Δfur, and CΔfur was compared under normal (iron-replete), iron-excess, iron-restricted, and oxidative stress conditions. Inductively coupled plasma mass spectrometry was employed to measure intracellular metal content in each strain. A zebrafish survival assay and a competition infection assay were performed to assess the impact of Fur on the virulence of V. parahaemolyticus. RNA sequencing was conducted to identify the genes regulated by Fur. Results The Fur proteins were highly conserved among several Gram-negative bacteria. The deletion of fur attenuated the growth of V. parahaemolyticus under normal (iron-replete) and iron-excess conditions, and reduced its sensitivity to iron restriction. The intracellular iron content in Δfur was significantly lower than that in the WT and CΔfur strains. Fur regulated the oxidative stress response in V. parahaemolyticus. Fur played no significant role in the virulence of V. parahaemolyticus in the zebrafish model. The expression of multiple genes related to iron uptake, iron storage, and type Ⅲ secretion system 1 (T3SS1) was significantly upregulated, whereas genes encoding iron-containing proteins and type Ⅵ secretion system 2 (T6SS2) components were significantly downregulated in Δfur. Conclusion Fur regulates iron homeostasis and oxidative stress response in V. parahaemolyticus, but does not affect its virulence in zebrafish.

Objective To clarify the role and molecular mechanism of the host protein solute carrier family 25 member 6 (SLC25A6) during fowl adenovirus serotype-4 (FAdV-4) infection, thus providing a theoretical basis for elucidating the pathogenic mechanism of FAdV-4 and developing novel prevention and control strategies. Methods First, we confirmed that infection of LMH cells with FAdV-4 at different time points and multiplicities of infection (MOI) resulted in obvious cytopathic effects (CPE). Second, on the basis of the host protein SLC25A6 identified in previous screening, Western blotting was employed to examine the effect of FAdV-4 on the expression of endogenous SLC25A6 in cells. Subsequently, transfection experiments were performed to regulate the expression of SLC25A6 (overexpression or interference with endogenous expression). RT-qPCR and Western blotting were employed to analyze the effect of SLC25A6 on FAdV-4 replication from the aspects of mRNA level, protein level, and viral titer. Finally, co-immunoprecipitation (Co-IP) was employed to verify the interaction between SLC25A6 and the core capsid protein Hexon of FAdV-4. Results FAdV-4 significantly inhibited the expression of endogenous SLC25A6 in cells. The overexpression of SLC25A6 markedly inhibited FAdV-4 replication, while interference with endogenous SLC25A6 promoted viral replication. SLC25A6 could directly interact with the Hexon protein of FAdV-4. Conclusion The host protein SLC25A6 inhibits FAdV-4 replication through its interaction with the viral Hexon protein. The results provide a theoretical basis for further elucidating the pathogenic mechanism of FAdV-4 and developing novel prevention and control strategies.