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 As a zoonotic pathogen, Proteus mirabilis poses a serious challenge to public health due to its multi-antibiotic resistance and the synergistic effect of virulence genes. To characterize the antibiotic resistance transmission of bacteria in the food chain in Zhejiang Province, this study systematically monitored the antibiotic resistance phenotypes and genes of isolates from cattle slaughterhouses and farmers’ markets, and analyzed the distribution differences of antibiotic resistance genes (ARGs), virulence genes (VGs), and mobile genetic elements (MGEs). Methods A total of 384 samples (feces, carcasses, environment, etc.) were collected from cattle slaughterhouses and farmers’ markets, and the strains were identified by 16S rRNA gene sequencing. Twenty ARGs and 10 VGs were detected by the K-B disc diffusion method and PCR, and the ARGs and VGs carried by P. mirabilis were analyzed. The ARG clusters were analyzed by sequencing of integron gene cassettes, and the co-occurrence network of ARGs, VGs, and MGEs was constructed. Subsequently, conjugative transfer experiments were carried out to explore the horizontal transmission potential of ARGs. Results A total of 101 strains of P. mirabilis were isolated, with the total isolation rate of 26.30%. The isolation rate of strains from slaughterhouses (33.85%) was significantly higher than that from farmers’ markets (18.75%). The resistance rates to ceftriaxone sodium, amoxicillin, and erythromycin were all over 90.00%. Among the ARGs, bla_TEM (89.09%), sul1 (77.71%), and tetA (63.29%) had the highest detection rates, and the distribution of ARGs in slaughterhouses was more complex. The VGs fliL (92.08%) and zapA (80.20%) were highly expressed in the isolates, which suggested potential pathogenicity. The detection rate of integrons in slaughterhouses was significantly higher than that in farmers’ markets, and PCR amplification results showed that there were a variety of ARGs, including aminoglycoside and trimethoprim resistance genes. Co-occurrence network analysis showed that ARGs, VGs, and MGEs had significantly positive correlations, and type I integron (intI1) was the hub gene. Conjugative transfer experiments confirmed that bla_TEM could be transmitted across species via horizontal transmission. Conclusion Compared with farmers’ markets, slaughterhouses are key nodes in the spread of antibiotic resistance due to the antibiotic exposure pressure, high organism density, and rich mobile components. The findings emphasize the importance of strengthening antibiotic management and monitoring the transmission chain of ARGs, providing a scientific basis for the prevention and control of antibiotic resistance under the framework of “One Health”.

The cyclic oligonucleotide (CO)-based anti-phage signaling system (CBASS), an innate immune system widely distributed in bacteria, is composed of oligonucleotide cyclases cGAS/DncV-like nucleotidyltransferases (CD-NTases), CD-NTase-associated protein (Cap), and accessory proteins. When bacteria are infected by phages, CD-NTases generate COs to amplify signals. Subsequently, effectors are activated by COs, inducing cell death through multiple mechanisms such as damaging cell membranes, degrading DNA, and depleting essential metabolites. Accessory proteins are responsible for regulating the CBASS, ultimately inhibiting phage infection. This review introduces the composition and classification of CBASS and further discusses the process by which CD-NTases recognize and bind to phage RNA to activate the synthesis of the second messenger CO. Effectors encoded by Cap effector genes mediate cell killing by binding to COs, while accessory proteins encoded by Cap auxiliary genes are involved in regulating the activity of CBASS. In addition, the immune evasion of phages from CBASS is also discussed. This review helps to understand the detailed mechanisms and biological significance of the interactions between phages and their host bacteria from the perspective of CBASS.

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.

There are a large number of non-rhizobia endophytes in addition to rhizobia in leguminous root nodules, which are of great significance for promoting plant growth and improving the bacterial abundance in root nodules. Some NREs can not only help rhizobia expand their host range but also participate in the leguminous plant-rhizobia symbiotic nodulation process. In this review, we systematically summarize the genetic diversity, classification, and functions of NREs, specifically describe the pathways of NREs entering rhizobia, the interaction mechanism between NREs and rhizobia, and the diversity of NREs in soil ecosystems, and discuss the application potential and future research directions of NREs. Furthermore, this article summarizes the current research progress in leguminous plant-rhizobia-NREs interactions and explores the methods of improving crop productivity and health through interactions mediated by nodule microecology, aiming to provide theoretical support for sustainable agricultural development.

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.

Sulfate-reducing bacteria (SRB) with unique reductive capabilities enable simultaneous sulfate reduction and heavy metal removal, demonstrating potential in heavy metal pollution remediation. Objective To isolate efficient SRB strains from marine sediments and investigate their reductive characteristics and application prospects in Cr(VI) contamination remediation. Methods We enriched and screened out an efficient SRB strain and systematically analyzed its sulfate reduction efficiency, Cr(VI) removal efficiency, and metabolic responses under environmental stressors (such as pH, sulfate concentration, and heavy metal concentration). This strain was then used to synthesize biological iron sulfide composite, the physicochemical characteristics of which were then investigated by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). Furthermore, the feasibility of applying the biological iron sulfide composite in the remediation of Cr(VI)-contaminated environments was explored. Results Strain S5 was identified as Desulfovibrio sp. with GenBank accession number OR140726. Its protein concentration and sulfate reduction followed an S-shaped curve. This strain exhibited a certain degree of acid tolerance, with the OD₆₀₀ and sulfate reduction rate reaching 0.16±0.01 and (83.71±1.49)% at pH 5.0, respectively. The strain exhibited sulfate reduction capability in the presence of 0.5-1.3 g/L sulfate, achieving a maximum reduction rate of (92.27±1.20)%. In the presence of 10-30 mg/L Cr(VI), strain S5 demonstrated efficient Cr(VI) removal. However, when the Cr(VI) concentration was higher than 30 mg/L, the Cr(VI) removal rate of this strain decreased significantly. The biological iron sulfide composite prepared based on strain S5 was porous, amorphous, and rich in functional groups such as C=O, N-H, and Fe-S, and its Cr(VI) removal rates were above 85% and did not differ significantly when exposed to Cr(VI) at high concentrations. Conclusion Desulfovibrio sp. S5 is a strain with high efficiency of sulfate reduction and Cr(VI) removal, and the biological iron sulfide composite prepared with it can overcome the limitation of higher Cr(VI) concentration and maintain high Cr(VI) removal rate, which has obvious advantages in the remediation of Cr(VI) pollution. The results of this study can provide a scientific basis for the application of SRB in the bioremediation of Cr(VI)-polluted environments.

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.

Objective To study the protective effect of baicalin on mice infected by porcine extraintestinal pathogenic Escherichia coli (ExPEC) PCN033 strain and explore the underlying mechanism. Methods The mouse infection model and Western blotting were employed to determine the clinical features, survival rate, bacterial loads in different tissue samples, pathological changes, and expression levels of P65, IκBα, NLRP3, ASC, and Caspase-1 of mice in the infection group and the baicalin treatment group. Results After baicalin treatment, the mental state of mice in the baicalin treatment group was obviously better than that in the infection group. The survival rate of mice in the baicalin treatment group was higher than that in the infection group, and the colonization ability of PCN033 in the blood, brain, and lung of mice in the baicalin treatment group was lower than that in the infection group. Further studies showed that baicalin inhibited the PCN033 infection-induced activation of phosphorylation of P65 and IκBα and down-regulated the expression levels of NLRP3, ASC, and Caspase-1 in mice. Conclusion Baicalin alleviated the inflammatory response caused by porcine ExPEC infection by inhibiting NF-κB signaling pathway and blocking the activation of NLRP3 inflammasome, thereby reducing the clinical symptoms and tissue damage in mice. It is suggested that baicalin may be a potential drug to prevent and treat porcine ExPEC infection by regulating the inflammatory response.

Objective To breed Bifidobacterium adolescentis strains that can adapt to the gastrointestinal environment of felines and have strong intestinal colonization capabilities. Methods B. adolescentis was isolated from the feces of felines with long and regular life spans. The original strain QC-Y (with the life span extension rate reaching 33.85%) was selected through biomass assessments and mouse life span experiments and it was identified as B. adolescentis. After radiation-induced mutation, QC-Y-09 was screened out by the biomass assessment, gastrointestinal tolerance domestication, and evaluation. Results QC-Y-09 showed the biomass 55.667 times and the tolerance score 5.66 times that of QC-Y. Moreover, the tolerance of the strain to the feline gastrointestinal environment showed good genetic stability. The survival rates of the 10th generation of QC-Y-09 in the artificial gastric juice, intestinal juice, anaerobic, and micro-aerobic environments were 18.80%, 41.60%, 93.26%, and 48.39%, respectively, which were 62.67%, 108.53%, 97.92%, and 94.40% of those of the original generation. The intestinal colonization test showed that the colonization ability of QC-Y-09 in felines was significantly stronger than that of QC-Y and human-derived B. adolescentis. Seven days after the feeding of the microbial inoculum was stopped, the viable count of B. adolescentis in the feline feces of the QC-Y-09 group still reached 4.37 lg CFU/g, which was 1.74 lg CFU/g and 3.02 lg CFU/g, respectively, higher than those of the QC-Y group and the human-derived B. adolescentis group. In addition, QC-Y-09 had a good relieving effect on the feline food change stress, reducing the feline food change stress rate by 85.71%. The results of SNP analysis showed that QC-Y-09 was significantly different from QC-Y at the gene level, and the differentially expressed genes were mainly enriched in the ribosome structure and aminoacyl-tRNA biosynthesis pathway. Conclusion Feline-derived B. adolescentis QC-Y-09 bred in this study can effectively adapt to the gastrointestinal environment and colonize the intestines of felines. This study provides both theoretical and practical bases for the application of B. adolescentis in functional food for felines.