Objective To identify the species and investigate the diversity of 120 Burkholderia cepacia complex (Bcc) strains isolated from industrial products and their production environments between 2022 and 2023. Additionally, the whole genome of a novel sequence type (ST) strain, Burkholderia aenigmatica ST2120, was analyzed to assess its virulence and pathogenicity. Methods Multilocus sequence typing (MLST) was employed to assign sequence types (STs) of Bcc strains. Multilocus sequence analysis (MLSA) was conducted for phylogenetic analysis and species identification of novel ST Bcc strains. Whole genome sequencing of ST2120 was performed on the Nanopore platform, followed by genome assembly, gene prediction, functional annotation, and prediction of biosynthetic gene clusters (BGCs) for secondary metabolites. Results Among the 120 Bcc strains, seven species (B. aenigmatica, B. cenocepacia, B. cepacia, B. contaminans, B. vietnamiensis, B. stabilis, and B. multivorans) and 38 STs were identified. Twenty-two novel alleles and 20 new STs were discovered. The novel ST strains were predominantly identified as B. aenigmatica and B. vietnamiensis. B. aenigmatica accounted for 55% of Bcc strains associated with industrial contamination, representing the most prevalent species within the industrial contamination-related Bcc. The genome (8 909 914 bp, G+C content: 65.73%) of B. aenigmatica ST2120 comprised 8 192 protein-coding genes, and the genome data were deposited in NCBI under the accession number CP184468-CP184476. Genomic analysis predicted siderophore-related BGCs for secondary metabolites (e.g., ornibactin C8 and chromobactin), five efflux pump-associated antibiotic resistance genes, and virulence genes linked to secretion systems, host adhesion/invasion, immune modulation, and quorum sensing. Conclusion B. aenigmatica has emerged as a predominant Bcc species in industrial contamination. The genome of B. aenigmatica ST2120 contains comprehensive virulence genes, indicating significant pathogenicity.

Objective To explore the functions of type XI histidine kinase genes in Botrytis cinerea, thereby establishing a foundation for further elucidation of the molecular mechanisms involving histidine kinase-related genes in the growth, development, and pathogenic processes of plant pathogenic fungi. Methods Through knockout vector construction, ATMT transformation, and PCR/qPCR validation, we obtained the mutant ΔBcHK91. The colony growth, sclerotium formation, conidial production and morphology, conidial germination rate, appressorium and infection cushion formation rates, pathogenicity, cell wall/membrane integrity assays and transcriptome profiling were conducted to evaluate the effect of BcHK91 knockout on B. cinerea strain B05.10, thereby elucidating the biological function of this gene. Results Two BcHK91 knockout mutants were successfully obtained and designated as ΔBcHK91-A and ΔBcHK91-B. The phenotypic analysis revealed that the knockout of BcHK91 reduced the conidial length, conidial production, conidial germination rate, appressorium formation rate, infection cushion formation rate, and pathogenicity. Moreover, the mutant formed no sclerotium and showed increased sensitivityto Congo red and SDS compared with the wild-type strain B05.10 and the ectopic insertion strain ET. To reveal the transcription mechanisms of BcHK91, we compared the transcriptomes of B05.10 and ΔBcHK91 by RNA-seq. A total of 1 533 differentially expressed genes (DEGs) were predicted in ΔBcHK91, including 1 017 genes with up-regulated expression and 516 genes with down-regulated expression. Gene ontology (GO) and clusters of orthologous groups (COG) annotation showed that the DEGs were mainly involved in cell process and metabolic process of biological processes, cellular anatomical entity and intracellular of cellular components, and catalytic activity, binding, and transporter activity of molecular functions. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the DEGs were mainly involved in carbohydrate transport and metabolism, starch and sucrose metabolism, and mitogen-activated protein kinase (MAPK) cascade response and other physiological metabolic pathways related tothe phenotype and pathogenicity of ΔBcHK91. In silico analysis of the DEGs suggested that 17 DEGs were related to the growth and development, oxidative stress, cell wall biosynthesis, cell membrane integrity, sclerotial initials, and pathogenicity. The results of qPCR demonstrated that the expression levels of 13 genes matched the trends observed in RNA-seq data, confirming the high reliability of the transcriptome analysis. Conclusion In B. cinerea, BcHK91 plays critical roles in asexual development, environmental stress responses, and pathogenicity.

Myosmine, also known as 3-(3,4-dihydro-2H-pyrrol-5-yl) pyridine, is a tobacco alkaloid found not only in tobacco but also in various foods, fruits, and vegetables. It serves as one of the precursors for the formation of the carcinogenic tobacco-specific nitrosamine N′-nitrosonornicotine, posing a potential threat to human health. Objective To screen bacterial strains capable of degrading myosmine and preliminarily explore the pathways and mechanisms underlying myosmine degradation. Methods We used myosmine as the sole carbon source to enrich and isolate the myosmine-degrading bacterial strain from tobacco-growing soil. Taxonomic identification of this myosmine-degrading strain was achieved by a combination of morphological observation, physiological and biochemical testing, and molecular analysis. The myosmine degradation products by this strain were analyzed by HPLC and UHPLC-MS/MS. The degradation genes were predicted by BLAST comparison. Results A strain G-2 capable of degrading myosmine was successfully isolated. The strain was identified as a member of Shinella, designated Shinella sp. G-2. HPLC and UHPLC-MS/MS identified five degradation products. Genomic analysis showed that strain G-2 possessed a homologous gene cluster of a variant of the pyridine and pyrrolidine pathway (VPP) gene cluster. Conclusion In this study, a strain Shinella sp. G-2 with the ability to degrade myosmine was isolated. Strain G-2 might use enzymes in the VPP pathway to degrade myosmine through a metabolic pathway similar to the VPP pathway.

Objective The probiotic Escherichia coli Nissle 1917 (ECN) is engineered by synthetic biology to construct a tumor-targeting strain capable of colonizing the tumor tissue, converting glucose and metabolic waste ammonia in the tumor microenvironment into the photosensitizer precursor 5-aminolevulinic acid (5-ALA) and the immunomodulatory amino acid arginine, while synergizing with immune checkpoint inhibitors for enhanced antitumor efficacy. Methods The genes hemA^M, hemL, and argA were co-expressed in ECN, and thyA was knocked out via the λ-Red homologous recombination system to improve the tumor-targeting specificity. Shake-flask fermentation experiments, UV spectrophotometry, and HPLC were employed to quantify 5-ALA and arginine production. The antitumor effects of the engineered ECN were systematically evaluated by in vitro cellular assays and a murine colorectal cancer model. Results The engineered strain achieved 5-ALA and arginine yields of (173.00±11.46) mg/L and (1.70±0.09) g/L, which represented 8.2-fold and 20-fold increases, respectively, over that of wild-type ECN (P<0.000 1). The deletion of thyA enabled selective proliferation of the strain in tumor cells (HCT116 and CT26), with a two-fold increase in OD₆₀₀ compared with that in normal Vero cells (P<0.000 1), confirming enhanced tumor targeting. Both in vitro and in vivo experiments demonstrated sustained synthesis of 5-ALA and arginine in tumors. Compared with wild-type ECN, the engineered strain induced 2.7-fold and 1.9-fold increases in CD8⁺ and CD4⁺ T-cell infiltration (P<0.000 1), alongside 1.7-fold and 2.4-fold elevations in IL-6 and TNF-α secretion (P<0.000 1), respectively. The engineered strain combined with the anti-PD-L1 therapy achieved a tumor volume inhibition rate of 77.6% (P<0.000 1). Conclusion This study establishes a metabolically and immunologically dual-functional ECN platform that synergizes localized delivery of photodynamic therapy precursors, arginine-mediated immunometabolic reprogramming, and immune checkpoint blockade, providing a novel solution for the combined therapy against solid tumors. The engineered system offers a groundbreaking strategy for precise tumor microenvironment modulation, advancing the research on targeted cancer therapeutics.

Objective As biocatalysts, halohydrin dehalogenases can catalyze both cyclization and ring-opening reactions and are widely used in the synthesis of chiral epoxides and other compounds. The eco-friendly and efficient preparation of halohydrin dehalogenases is thus of great significance. In this study, we optimized the N-terminus of the coding sequence of the halohydrin dehalogenase HheC based on the mRNA secondary structure to achieve the efficient expression of this enzyme and then applied this enzyme in the synthesis of chiral epichlorohydrin. Methods The mRNA prediction tools was used to predict the secondary structure and thermodynamic properties of 5′mRNA. To reduce the stability of the mRNA secondary structure and increase folding free energy (ΔG), we designed the 5′mRNA sequence without changing the amino acid sequence. Furthermore, we characterized the expression efficiency and catalytic performance of this enzyme. Results The HheC mutant was obtained via the design of the 5′mRNA sequence, with the protein level increasing from 16.71% to 33.39% and the relative activity towards 1,3-dichloro-2-propanol increasing by three folds. Conclusion The optimization based on the secondary structure of 5′mRNA improves the expression level of HheC and enhances the synthesis efficiency of the target product, laying a foundation for constructing the route of enzymatic catalytic synthesis of chiral epichlorohydrin.

Objective To screen the biocontrol bacterial isolates with antagonistic activity against root rot pathogens of Panax notoginseng from the rhizosphere of Bletilla striata in a P. notoginseng-B. striata rotation system, identify the isolates, and evaluate their control effects, thereby providing a scientific basis for the application of P. notoginseng-B. striata rotation in alleviating continuous cropping obstacles and screening biocontrol strains from the rhizosphere of the plants for rotation rather than from that of target plants in the plant rotation system for controlling soil-borne diseases in the field. Methods The dilution plate method was employed to isolate culturable microorganisms from the rhizosphere soil of B. striata. The antagonistic activities of the isolates against root rot pathogens of P. notoginseng were evaluated via the dual culture assay. Molecular identification was performed based on the 16S rRNA gene sequence. In pot experiments, antagonistic bacteria were inoculated, and then the ability of antagonistic bacteria to control root rot was evaluated based on the root rot incidence of P. notoginseng. Results The rotation with B. striata significantly reduced the incidence and disease index of P. notoginseng root rot compared with continuous cropping. A total of 200 bacterial strains were isolated from the rhizosphere soil of B. striata, from which 25 strains exhibiting antagonistic activities against the root rot pathogens including Ilyonectria destructans RS6, Fusarium solani F3, and Fusarium oxysporum Z5 of P. notoginseng were screened out. The isolation efficiency of antagonistic bacteria was 12.5%. The 25 strains were identified as 12 species belonging to 5 genera, demonstrating rich diversity. There were 14 strains of Bacillus sp., including 4 strains of B. subtilis, 2 strains of B. velezensis, 6 strains of B. amyloliquefaciens, 1 strain of B. cereus, and 1 strain of B. toyonensis. There were 5 strains of Acinetobacter, including 2 strains of A. johnsonii, 2 strains of A. junii, and 1 strain of A. pittii. There were 4 strains of Pseudomonas, including 3 strains of P. putida and 1 strain of P. fulva. In addition, 1 strain of Enterobacter asburiae and 1 strain of Aeromonas caviae with antagonistic activities were isolated. Four strains (B. amyloliquefaciens BJ1, B. subtilis BJ7, B. amyloliquefaciens BJ8, and A. johnsonii YB10) antagonistic to all the three pathogens were applied to continuous cropping soil. They significantly reduced the root rot incidence and enhanced the fresh weight of P. notoginseng. Conclusion The rhizosphere soil of B. striata in rotation with P. notoginseng harbors diverse biocontrol strains against the root rot pathogens of P. notoginseng. This study lays a theoretical foundation for the rotation of P. notoginseng with B. striata to alleviate continuous cropping obstacles.

Objective The mucosal microbiota of large yellow croaker (Larimichthys crocea) plays a vital role in host health and environmental adaptation. However, differences in the mucosal microbiota composition between wild and cultured populations of large yellow croaker, as well as their assembly mechanisms, remain unclear. This study compared the microbiota composition and characteristics in the skin, gill, and intestine of wild and cultured large yellow croaker, aiming to elucidate the impacts of aquaculture environments on host-microbiota interactions. Methods We employed 16S rRNA gene high-throughput sequencing to analyze the microbiota characteristics in the skin, gill, intestine, and surrounding seawater of wild and cultured large yellow croaker. Key bacterial strains were isolated and characterized. Results Significant divergence of mucosal microbiota was observed between cultured and wild populations. (1) Cultured fish exhibited homogenized mucosal microbiota across the skin, gill, and intestine, whereas wild fish maintained strong tissue-specific microbial signatures. (2) Cultured fish had more unique taxa in the skin (156 vs. 93) and gill (171 vs. 50) but fewer unique taxa in the intestine (118 vs. 253) than wild counterparts. (3) LEfSe revealed enrichment of potential opportunistic pathogens (e.g., Vibrio and Photobacterium) in the skin/gill of wild fish, while myxobacteria (e.g., Haliangium) were specifically enriched in the intestine of cultured fish. (4) Co-occurrence networks demonstrated predominantly cooperative interactions in both groups, yet wild fish microbiota displayed stronger competitive relationships. (5) Lysinibacillus sp. isolated from the mucosal tissue of large yellow croaker demonstrated cross-tissue colonization in cultured populations, exhibiting broad-spectrum antibiotic susceptibility and potential probiotic properties. Conclusion The aquaculture environment significantly reshaped the mucosal microbiota composition, diversity, and interaction networks in large yellow croaker, resulting in reduced microbiota stability and impaired metabolic processes, immune competence, and environmental adaptability of the host. These findings provide critical insights for refining aquaculture practices, developing probiotic resources, and mitigating microbiota dysbiosis in large yellow croaker.

Objective To screen out a strain with the ability to degrade penicillin G (PENG) and identify the key enzymes involved in PENG catabolism, providing strain and gene resources for the biological treatment of penicillin waste. Methods Bacterial strains capable of utilizing penicillin G potassium (PGK) as the sole carbon source were screened by enrichment culture. Key enzymes involved in the catabolism of PGK were identified by genome and transcriptome analyses, and their evolutionary origins were examined. The key enzymes were expressed and purified, and their kinetics were analyzed. The physiological roles of the key genes in bacterial growth on PGK were revealed by gene knockout and complementation. Results The obtained strain Delftia sp. PG-8 can degrade PGK and utilize it as the sole carbon source for growth. The strain showed the best performance in PENG degradation and growth at pH 7.0, 35 ℃, and 10.00 mmol/L PGK. PgkA catalyzed the rapid degradation of PGK, with K_m=(99.19±19.45) μmol/L and k_cat/K_m=(1.96±0.55)×10⁵ L/(mol·s). Compared with the functionally characterized β-lactamases, PgkA had a unique evolutionary origin. PgkB also had the ability to catalyze the transformation of PGK, while its substrate affinity was only 1/5 that of PgkA, in addition to the lower catalytic efficiency. The degradation and utilization of PGK for growth by strains PG-8-ΔpgkA and PG-8-ΔpgkB were significantly reduced, with PG-8-ΔpgkA showing a more pronounced decline. Although PG-8-ΔpgkAB, in which both pgkA and pgkB were knocked out, still degraded a certain amount of substrate, it was almost unable to use PGK as the sole carbon source for growth. Conclusion PG-8 is the first strain of Delftia capable of using PGK as the sole carbon source for growth. Both pgkA and pgkB play important physiological roles during PG-8 growth on PGK, with pgkA playing a dominant role.

Objective Bacteria of Pseudomonas are the main cause of food-borne and clinical infections. At present, mass spectrometry and 16S rRNA gene are widely used to identify Pseudomonas, while the facilities relied on are expensive and require cumbersome operation. Internationally, the numerical identification products require manual single-sample loading procedures, which are cumbersome. This study aims to develop a numerical identification kit for Pseudomonas with high accuracy and simple operation. Methods On the basis of the existing biochemical reaction data of Pseudomonas, we developed a numerical identification model via the branching diagram method and designed and optimized 11 biochemical matrix formulas for microquantization. We then used the kit to identify the standard strains and isolates of Pseudomonas and compared the results with those from mass spectrometry and PCR to evaluate the performance of the kit. Results A numerical identification kit was developed, and it was capable of identifying 10 species of Pseudomonas with just one sample addition. The accuracy rate of the kit in the identification for 5 standard strains and 135 isolates reached 97.04%, and the accuracy rate in actual samples was 97.74%. The biochemical test was stable and reproducible, and the identification cost (25 CNY/sample) of the kit was only 10% of that (240 CNY/sample) of the comparable product from bioMérieux (France). Conclusion The numerical identification kit developed in this study for Pseudomonas has simple operation, a low price, and high accuracy, which can be used in clinical diagnosis and food detection.

Legionnaires’ disease, a serious pulmonary infection caused by Legionella, requires early diagnosis for effective treatment and favorable outcomes. Laboratory diagnostic methods primarily encompass isolation and culture, antigen detection, nucleic acid detection, and serological testing. Although isolation and culture are considered the gold standard, it is characterized by low sensitivity and extended culture duration. Antigen detection offers simple and fast operation but necessitates improved specificity. Nucleic acid detection, although being highly sensitive, has complex operation and high costs. Serological testing provides limited value for early diagnosis. Urine antigen detection, extensively employed globally, is limited to the detection of Legionella pneumophila serogroup I. Novel nucleic acid detection technologies, including digital PCR, isothermal amplification, and next-generation sequencing, present promising applications. Future research should aim to develop more streamlined, rapid, and sensitive detection methods to enhance the early diagnostic rate of Legionnaires’ disease.