[Objective] Over 7 million people die from bacterial infections every year due to the inappropriate or untimely use of antibiotics. Antimicrobial susceptibility test (AST) is a routine method guiding antibiotic therapy in clinical practice, while the existing methods have limitations as they require at least 16–20 h to get the results. We aimed to develop a novel rapid AST method based on bacterial morphology analysis, which can improve the diagnosis efficiency and provide timely treatment for the patients with bacterial infections, especially for urgent cases. [Methods] We developed an in-situ time-lapsed imaging (ISM-TLI) technique for microcolonies by combining bacterial incubation, in vitro antibiotic treatment, microscopic imaging, and image processing algorithm. The combination of the imaging device with the algorithm enabled the tracing of the morphological changes of colonies treated with different concentrations of antibiotics in-situ and the rapid obtainment of AST results. [Results] This technique worked effectively for both Gram-positive and Gram-negative bacteria and could determine the minimum inhibitory concentration (MIC) within 2 h, with the results in agreement with those obtained with the clinical gold standard method. [Conclusion] Our rapid AST technique significantly reduced the turn-around-time for obtaining the AST profile and had great potential for AST of a wide range of other strains.

[Objective] To explore the effects of CbrB from the carbon catabolite repression system on the biocontrol performance of Pseudomonas protegens Pf-5. [Methods] The mutant Pf5274 with the markerless deletion of the coding region of cbrB was constructed by a double-crossover recombination event based on pJQ200SK. Moreover, the complementary strain of cbrB and the control strains were constructed by the plasmid complementation method based on pBBR1Am. Lastly, the effects of CbrB on the growth, biofilm formation, motility, antifungal activity, and pyoluteorin synthesis of Pf-5 were analyzed by the measurement of OD₆₀₀, crystal violet staining, agar plate culture, plate confrontation method, and pltL'-'lacZ fusion report strains, respectively. [Results] CbrB greatly slowed the growth of Pf-5 in the natural medium LB, but greatly sped up the growth of Pf-5 in the basic medium M9-glucose. In addition, CbrB significantly promoted the motility but inhibited the biofilm formation, antifungal activity, and pyoluteorin synthesis of Pf-5. [Conclusion] CbrB plays a role in regulating the growth, biofilm formation, motility, antifungal activity, and pyoluteorin synthesis of P. protegens Pf-5, thus regulating the biocontrol performance of this strain. This study provides a theoretical basis for the biocontrol capabilities of strains by genetic engineering and lays a foundation for probing into the biosynthesis of pyoluteorin.

[Objective] Monensin is a polyether antibiotic produced by Streptomyces cinnamonensis. To enhance the production of monensin by microbial fermentation, we employed metabolic engineering to strengthen the synthesis pathway of the key precursor methylmalonyl-CoA in S. cinnamonensis 2110. [Methods] Firstly, crotonyl-CoA reductase (CCR) was overexpressed to strengthen the acetoacetyl-CoA pathway. Subsequently, methylmalonyl-CoA mutase (MCM) was overexpressed to improve the succinyl-CoA pathway. Finally, an engineered strain with tandem overexpression of CCR and MCM was constructed and evaluated for the fermentation performance. [Results] The overexpression of CCR increased the strain biomass and monensin titer by 10.4% and 19.0%, respectively, after 10 days of shake-flask fermentation. The overexpression of MCM increased the monensin titer by 9.9%, whereas it did not increase the strain biomass after 10 days of shake-flask fermentation. The tandem overexpression of CCR and MCM increased the biomass and monensin titer by 9.4% and 26.8%, respectively, after 10 days of shake-flask fermentation. In a 5 L bioreactor, the engineered strain 2110-CCR-MCM reached the highest biomass of 54.6 g/L and monensin titer of 11.3 kU/mL, which increased by 12.7% and 36.2%, respectively, compared with those of the starting strain 2110. [Conclusion] CCR and MCM mediated the key metabolic pathway of monensin biosynthesis in S. cinnamonensis, and the overexpression of CCR and MCM was highly favorable for monensin synthesis. This study provides technical reference for the engineering of strains with high yields of other polyketides.

[Objective] This study explored the application potential of symbiotic fungi and bacteria of Odontotermes formosanus in the degradation of wheat (Triticum aestivum L.) straw resources, aiming to provide a theoretical basis and supplement strain resources for the industrialization of straw biodegradation. [Methods] The lignocellulose-degrading fungi and bacteria were isolated from O. formosanus and screened by Congo red staining and the agar plate supplemented with carboxymethyl cellulose sodium (CMC-Na), and the enzyme production of the strains screened out was examined. Liquid fermentation at room temperature was carried out to evaluate the degradation effects of different strains and strain combinations on wheat straw. Fourier transform infrared spectroscopy (FTIR), X-ray crystal diffraction (XRD), and scanning electron microscopy (SEM) were employed to analyze the physical and chemical properties of wheat straw before and after degradation. [Results] Five species of fungi and three species of bacteria were isolated from the termite combs, including two species of Penicillium, one species of Purpureocillium, one species of Aspergillus, one species of Eutypella, one species of Bacillus, one species of Escherichia, and one species of Stenotrophomonas. Four efficient degrading strains were screened out and identified as Penicillium citrinum, Aspergillus nomiae, Bacillus subtilis, and Escherichia coli. In the liquid fermentation at room temperature, the combination of the four strains had the strongest comprehensive degradation performance, with the degradation rates of 24.35%, 47.24%, 35.75%, and 32.72% for dry matter, cellulose, hemicellulose, and lignin, respectively, in 12 days. The degradation destroyed the chemical bonds, the intermolecular force, and the composite structure of lignocellulose in wheat straw, and decreased the crystallinity of cellulose from 37.40% to 32.97%. [Conclusion] The combination of P. citrinum, A. nomiae, B. subtilis, and E. coli isolated from the combs of O. formosanus had a good degradation effect on wheat straw, demonstrating the application potential in the industrialization of straw biodegradation.

[Objective] To investigate the fungal diversity and community dynamics in the roots and root zone soil of the invasive species Spathoglottis plicata at different development stages and mine potential fungal resources. [Methods] The developmental period of S. plicata was divided into the protocorm, pre-seedling, middle-seedling, late-seedling, adult, and flowering stages. Fungal community composition and diversity in the roots and root zone soil were analyzed by QIIME 2 and other software. Fungal strains were isolated by the tissue culture method from the protocorms and seedlings of S. plicata, and the phylogenetic relationship of the strains was analyzed. [Results] The dominant fungi in the roots and root zone soil of S. plicata were Tulasnellaceae and Onygenaceae, respectively. The fungal community composition of S. plicata varied across different developmental stages. Tulasnellaceae was the most prevalent from the protocorm to middle-seedling stage, while Nectriaceae and Trichocomaceae became dominant during the late-seedling stage and adult stage. At the flowering stage, Ceratobasidiaceae presented high relative abundance. A total of 101 fungal strains were isolated from the protocorms and seedling roots of S. plicata, belonging to 13 families, in which strains of Tulasnellaceae accounted for 35.65%, being dominant. [Conclusion] The present study elucidated the fungal community composition and diversity dynamics in the roots and root zone soil of S. plicata at different development stages. The findings offer a theoretical foundation for comprehensively understanding of the intricate relationship between orchids and symbiotic fungi and facilitating the selection of optimal habitats for the conservation of rare and endangered orchid species.

L-threonine is one of the eight essential amino acids that cannot be synthesized by the human body and must be taken from food. It is an important component of protein synthesis and is widely used in food, feed, medicine and other fields. At present, Escherichia coli can achieve a high threonine yield in fermentation, being the main bacterium used for industrial production of threonine. With the development of metabolic engineering, the modification of strains is no longer limited to mutagenesis, and the directed modification of strains greatly improves the production of L-threonine, facilitating the development of the L-threonine industry. This paper introduces the physicochemical properties and synthesis pathway of L-threonine and reviews the achievements in improving L-threonine production by metabolic engineering, aiming to enrich the knowledge about the modification of Escherichia coli for efficient synthesis of threonine.

[Objective] To mine the differentially expressed genes (DEGs) of Kocuria rhizophila DC2201 exposed to clindamycin hydrochloride at 0.5 minimum inhibitory concentration (MIC) and reveal the response mechanism of Kocuria rhizophila DC2201 to clindamycin hydrochloride. [Methods] With the Kocuria rhizophila DC2201 cells cultured in LB liquid medium as the control, Illumina HiSeq platform was used for paired-end sequencing to determine the gene expression of Kocuria rhizophila DC2201 cells exposed to clindamycin hydrochloride at 0.5 MIC. Real-time fluorescence quantitative PCR was then conducted for validation. [Results] A total of 1 202 significantly DEGs were screened out from Kocuria rhizophila DC2201 under the stress of clindamycin hydrochloride, including 604 significantly up-regulated genes and 598 significantly down-regulated genes. After gene ontology (GO) annotation, 1 041 significantly DEGs were annotated into 35 GO terms of molecular function (MF), cell composition (CC), and biological process (BP). The Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis predicted 16 significantly DEGs related to DNA repair, 43 significantly DEGs related to ribosomal synthesis, 28 DEGs associated with ATP-binding cassette (ABC) transporters, 77 significantly DEGs associated with the pentose phosphate pathway, glycolysis, tricarboxylic acid (TCA) cycle, starch and sucrose, pyruvate, butyrate and other carbohydrate metabolisms, and 5 significantly DEGs related to peptidoglycan synthesis. [Conclusion] Kocuria rhizophila DC2201 exposed to clindamycin hydrochloride adopts a global response mechanism. It increases the efflux of clindamycin hydrochloride by up-regulating the gene expression of major facilitator superfamily (MFS) transporters in the multidrug resistance (MDR) family. By enhancing DNA repair and RNA metabolism pathways, the strain ensures the genomic stability and normal RNA function. In addition, it enhances the ribosome synthesis pathway to compensate for the protein synthesis barrier caused by the binding of clindamycin hydrochloride with the 50S ribosome. Furthermore, the strain reduces the absorption and transportation of carbohydrates to restrain the energy metabolisms pathways, thus slowing down the growth and reducing the energy demand. Correspondingly, the cell wall stability of Kocuria rhizophila DC2201 is also affected.

[Objective] To explore the effects of cell walls of Saccharomyces cerevisiae on the intestinal microbiota in finishing bulls by 16S rDNA and ITS sequencing. [Methods] A total of 40 simmental crossbred finishing bulls weighing about 550 kg were randomized into 4 groups, with 10 bulls in each group. The control group was fed with a basic diet, and 5, 10, and 15 g cell walls of S. cerevisiae were added to the diet of each bull per day in trial 1, 2, and 3 groups, respectively. The preliminary trial and trial lasted for 10 days and 94 days, respectively. Intestinal feces were collected 7 days before the end of the trial. [Results] 16S rDNA: (1) The Chao and ACE indices in the trial 3 group were higher than those in other groups (P < 0.05); (2) Firmicutes and Bacteroidota were the dominant phyla, and Prevotella_9, Faecalibacterium, Succinivibrio, Bacteroides, and Bifidobacterium were the dominant genera; (3) The linear discriminant analysis effect size (LEfSe) revealed one differential species (LDA≥4.0, P < 0.05) playing an important role in the trial 2 group. ITS: (1) There was no significant difference in the alpha or beta diversity among groups (P > 0.05); (2) Ascomycota with the relative abundance above 50.00% was the dominant phylum. Penicillium, unidentified_Ascomycota_sp., Aspergillus, Orpinomyces, and Eurotium were the dominant genera; (3) LEfSe revealed 8 differential species (LDA≥3.0, P < 0.05), which included 3, 3, and 2 differential species playing an important role in the control, trial 2, and trial 3 groups, respectively. [Conclusion] Under conditions of this study, adding 10–15 g/d cell walls of S. cerevisiae in the basic diet increased the richness of intestinal microbiota and the relative abundance of beneficial bacteria Provetella_9, Tolypocladium, and Torulaspora, which were conducive to improve intestinal microecological environment of finishing bulls.

Escherichia coli, a facultative anaerobic, flagellated, Gram-negative rod bacterium commonly parasitic in the intestines of humans and animals, is one of the common zoonotic pathogens. E. coli is easy to form biofilms, which are special aggregates formed by bacterial cells attached to each other and encased with self-produced extracellular matrix. Biofilm formation is a major reason for the difficulty in curing bacterial infectious diseases in clinical practice. It not only helps bacteria evade the host defense system but also reduces or prevents drugs from working, thereby inducing biofilm-associated infections (BAIs). This review introduces the molecular mechanism of E. coli biofilm formation from the perspective of the gene regulatory system and related regulatory proteins and summarizes the strategies for the prevention and treatment of BAIs, providing references for finding appropriate drug targets and preventing BAIs.

[Objective] Considering the important role of signal peptides in the secretory expression of heterologous proteins, we devised an automated high-throughput platform for the automatic screening of signal peptides, aiming to explore the effects of different signal peptides in Bacillus subtilis on the expression of heterologous proteins. [Methods] First, using the Escherichia coli-B. subtilis shuttle vectors pHP13 and pMA5 as the skeleton, we amplified the cell division B lethal gene (ccdB) and then ligated it to the middle of the promoter and the target gene to build the signal peptide screening vector. With the genomic DNA of B. subtilis 168 as the template, 173 signal peptides were amplified. An automated platform was established for the expression and screening of heterologous proteins in B. subtilis. Furthermore, the recombinant strains of heterologous proteins containing different signal peptides were constructed, and the effects of different signal peptides on the secretory expression of heterologous proteins were investigated. [Results] Five signal peptides (RpmG, AspB, CitH, LytF, and YkwD) showed strong abilities to induce the export of GFP from B. subtilis. Among them, RpmG had the strongest ability to induce the export of GFP, and the extracellular GFP fluorescence of the recombinant strain increased by 236% compared with that of the control strain. In addition, 41 signal peptides were not compatible with pullulanase (PulA), while the two signal peptides RpmG and AspB showed strong abilities to export PulA. The highest PulA activity of 116 U/mL was detected from the recombinant strain carrying the signal peptide RpmG, and the extracellular enzyme activity was 52 U/mL. The secretion rate of the PulA recombinant strain carrying the signal peptide AspB reached 74%, which was 68% higher than that of the control strain. [Conclusion] We developed an automated platform for high-throughput screening of the heterologous protein signal peptides in B. subtilis and obtained the signal peptides capable of improving the secretory expression of GFP and PulA. This automated platform allowed the parallel processing of a considerable number of samples, which simplified the repetitive manual laboratory work. This platform outperformed manual operation in terms of both time consumption and cost. The advantage of the automated high-throughput platform will be more significant with the increase in sample size. In summary, the established automatic high-throughput screening platform not only accelerates the screening process of signal peptides of heterologous proteins, but also provides new technical support for the modification and iteration of industrial strains of other value-added proteases.