[Objective] To compare the metabolism and transcription between the probiotic Escherichia coli Nissle 1917 (EcN) and the model strains, thus providing a reference for the engineering and promoting the application of the food-safe strain EcN. [Methods] The genome and transcriptome were compared between EcN and model strains BL21(DE3) and W3110 by software, and plasmids were constructed to verify the differences. EcN-derived microcin was expressed in BL21(DE3) and the antibacterial effect of microcin was verified. [Results] A total of 904 differentially coding genes were identified. The differences in carbon source absorption and utilization of different strains were verified by experiments with different carbon sources as substrates. The expression of the promoter P_flic confirmed the differences in transcription among different strains. The recombinant strain of microcin showed an increase of 30.3% in the inhibition rate after 12 h of culture. [Conclusion] This study clarifies the metabolic characteristics of EcN and confirms the differences in transcription between EcN and model strains. Moreover, this study provides ideas for the development of microcin as a narrow-spectrum therapeutic drug to inhibit intestinal pathogens and reduce intestinal bacterial blooms.

[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.

The spread of antibiotic resistance has aroused global concern. The development of technologies for detecting antibiotic resistance genes (ARGs) is essential for curbing the migration and spread of ARGs from the environment to plants/animals and human populations. This paper describes the development timeline of existing nucleic acid detection technologies and their first applications to the detection of ARGs and summarizes their detection principles, advantages and disadvantages, and development potential. Furthermore, this paper prospects that isothermal amplification combined with CRISPR/Cas might be the core technology for the development of in-situ rapid detection methods. By reviewing the development history of each technology, this paper aims to give insights into the development and applications of technologies for detecting ARGs and provide technical support for the research and control of antibiotic resistance transmission.

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] The protein SakA encoded by sakA is a member of the mitogen-activated protein kinase (MAPK) family in Aspergillus niger. However, little is known about the roles of SakA in A. niger. In this study, we constructed the A. niger strains with knockout of sakA to investigate the roles of this gene. [Methods] The Agrobacterium-mediated method was utilized to construct ΔsakA strains from A. niger RAF106 (the wild type, WT). The growth and spore production of ΔsakA and WT were observed on three different media. The sensitivity of ΔsakA and WT to different stress conditions was studied. The intracellular and extracellular levels of amylase, pectinase, and cellulase were compared between ΔsakA and WT. Real-time quantitative polymerase chain reaction (qRT-PCR) was employed to determine the relative transcript levels of the genes associated with spore production, amylase, pectinase, cellulase, and hyperosmotic regulation. [Results] Three ΔsakA strains were successfully obtained and verified by PCR and qRT-PCR. The ΔsakA strains had slow growth, delayed spore production, and delayed conidiophore differentiation compared with WT. The ΔsakA strains showcased slower colony growth than WT under the stress conditions of 0.6 mol/L KCl, 0.8 mol/L NaCl, and 1.2 mol/L NaCl. Compared with WT, the knockout of sakA increased the extracellular amylase production by 20.68%–21.43% and decreased the intracellular amylase production by 19.18%–20.26%, decreased the extracellular pectinase production by 36.71%–38.30% and increased the intracellular pectinase production by 35.68%–36.53%, decreased the extracellular cellulase production by 28.04%–33.82% and increased the intracellular cellulase production by 15.28%–18.19%. Compared with WT, the knockout of sakA down-regulated the transcript levels of spore production-related genes (fluG, sfgA, flbA, flbB, flbD, laeA, brlA, abaA, vosA, stuA, and velB) by 8.53%–90.87%. Furthermore, it down-regulated the transcript levels of amylase-related genes (amyC, amyD, amyE, amyF, amyG, and amyH) and the transcription factor (amyR) by 8.87%–87.50%, the pectinase-related genes (aglB, lacA, pexB, pecA, pecC, pecB, endA, endC, and poly) by 23.23%–84.01%, the cellulase-related genes (xlnR, chbA, chbB, and eglB) by 3.75%–81.02%, and the hyperosmotic regulation-related genes (ena1, ena2, sho1, nik1, ypdl, pkA, and hAD) by 5.27%–94.36%. [Conclusion] The sakA gene of A. niger positively regulates spore production and is essential for spore production. The knockout of sakA affects the spore production of A. niger. Furthermore, SakA plays a crucial role in the synthesis and secretion of amylase, pectinase, and cellulase as well as osmotic stress response.

[Objective] To study the morphological and physiological characteristics of hybrids compared with their parents and contribute to research on the mechanisms of speciation and evolution. [Methods] Sonneratia×hainanensis, a natural hybrid of the mangrove plants Sonneratia alba and S. ovata, usually presents hybrid weakness than its parents. In this study, Illumina high-throughput sequencing was employed to compare the rhizosphere microbiomes (including bacteria and fungi) between the hybrid and its parents, on the basis of which the reason for hybrid weakness was explored. [Results] The principal coordinate analysis (PCoA) revealed no significant difference in the rhizosphere bacterial or fungal community structure between the hybrid and its parents. However, the rhizosphere microbiome of the hybrid was different from that of the female parent S. alba with strong survival ability but similar to that of the male parent S. ovata. The rhizosphere bacteria belonged to 388 genera, 320 families of 76 phyla. The dominant phylum Pseudomonadota had the relative abundance above 41.00% in the rhizosphere of the three plant species, reaching 55.33% in the hybrid, which was higher than that in the parents. At the genus level, 18 common genera including Desulfococcus (3.23%) and Rhodoplanes (0.94%) in all the three mangrove plants showed the relative abundance of 15.77%. Among them, 8 salt-tolerant genera such as Mariprofundus showed decreased relative abundance in the hybrid, which may affect the salt tolerance. The rhizosphere fungi were dominated by Ascomycota and Basidiomycota with the relative abundance of 41.89% and 4.53%, respectively, which was significantly lower than that in the parents. Moreover, the predominant fungal genera were different in the three mangrove plants. Functional annotation of prokaryotic taxa (FAPROTAX) predicted that the mangrove prokaryotes were involved in sulfur metabolism and nitrogen metabolism. Although the hybrid had higher Shannon and Simpson indexes of rhizosphere bacteria than S. alba, some dominant taxa such as B-42 (unclassified Trueperaceae), Mariprofundus, and Sulfurimonas participating in the nitrogen cycle were not inherited by the hybrid. The soil total nitrogen (TN) and total phosphorus (TP) of the hybrid was significantly lower than that of S. alba. TN was significantly positively correlated with the relative abundance of Mariprofundus, B-42, Aspergillus, and Rhodotorula, which, however, demonstrated decreased relative abundance in the rhizosphere of the hybrid. [Conclusion] The results help to understand the mechanisms of hybrid weakness in Sonneratia×hainanensis.

Potato scab caused by Streptomyces scabies has become a bottleneck threatening the sustainable development of the potato industry. Using biocontrol strains to combat bacterial infections is considered an ideal approach. [Objective] We screened the phosphorus-solubilizing antagonistic strains of S. scabies and investigated their effects on the prevention and control of potato scab, aiming to provide candidate strains for the development of composite functional bacterial agents. [Methods] The target strains were screened by plate confrontation and phosphorus-solubilizing tests. Their taxonomic status was determined by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequencing. Pot and field experiments were carried out to measure the inhibitory effects of the target strains on S. scabies, and the relationship between phosphorus-solubilizing function and antagonistic effect was analyzed. [Results] Four antagonistic strains BN4-4, BN4-5, BN5-2, and YN17-2, were screened out. Among them, BN4-4 and BN5-2 showed strong ability to degrade inorganic phosphorus. BN4-4 and BN4-5 were identified as Bacillus atrophaeus, while BN5-2 and YN17-2 as Bacillus subtilis. The pot experiments showed that the relative control effects of the four strains were 71.35%, 38.70%, 62.18%, and 36.22%, respectively. In the field experiments, BN4-4 and BN4-5 showed the control effects of 69.07% and 56.20%, respectively. The four strains exerted inhibitory effects on four plant pathogens: Alternaria solani, Fusarium oxysporum, Rhizoctonia solani, and Verticillium dahliae. The four strains showed tolerance to pH 1.0–13.0, NaCl concentration of 1%–13%, and high temperatures (up to 80 ℃). Furthermore, they were insensitive to commonly used fungicides such as avermectin, zhongshengmycin, thiophanate-methyl, kasugamycin, and carbendazim. All the four stains exhibited the ability to metabolize and produce indole-3-acetic acid (IAA), which has been shown to promote the growth and increase the yield of plants. Notably, strains BN4-4 and BN5-2 capable of solubilizing phosphorus exhibited stronger inhibitory effects on S. scabies than the non-phosphorus-solubilizing strains. [Conclusion] B. atrophaeus BN4-4 with phosphorus-solubilizing function demonstrated promising preventive effects against S. scabies. This strain demonstrated broad-spectrum disease resistance, thermal stability, salinity tolerance, plant growth-promoting effect, and colonizing ability, serving as a potential functional strain for preventing and controlling potato scab and other soil-borne diseases in crops.

[Objective] Ergothioneine, a rare natural amino acid, is a powerful antioxidant with important physiological functions in the body. It has been widely used in the fields of food, medicine, and cosmetics. However, extracting ergothioneine from mushrooms and chemical synthesis suffer from low yields and high costs. This study aims to use metabolic engineering approaches to improve the yield of ergothioneine in Actinoplanes sp. HS. [Methods] Firstly, we locked onto the genes potentially involved in ergothioneine synthesis in Actinoplanes sp. HS by bioinformatics analysis. Then, we identified the functions of these genes by heterologous expression in Escherichia coli BL21(DE3). Finally, the identified functional genes were combined and overexpressed in Actinoplanes sp. HS, and the yields of ergothioneine in the mutant strains were measured. By adding different concentrations of precursors to the fermentation medium, we investigated the impact of precursor concentration on the yield of ergothioneine. [Results] The enzymes encoded by BC03-04016, BC03-04015, BC03-04014, and BC03-04013 in Actinoplanes sp. HS could synthesize the ergothioneine precursor hercynine-cysteine-sulphoxide (HER-Cys-Sul). The enzymes encoded by BC03-04046 and BC03-04917 had the function of cleaving carbon-sulfur bonds, which could catalyze the formation of the final product ergothioneine from HER-Cys-Sul. The mutant strains YC313 and YC314 obtained through overexpression of genes in ergothioneine synthesis showed ergothioneine yields of 125 mg/L and 108 mg/L, respectively, which were 2.9 times and 2.5 times that of the wild-type strain Actinoplanes sp. HS. Additional supplementation of 0.35 g/L methionine in the fermentation medium increased the ergothioneine yield of YC313 by 24% compared with that in the original medium, and the addition of 10 g/L soybean meal resulted in a 19% increase in the ergothioneine yield. [Conclusion] In this study, we identified the genes and their functions in ergothioneine synthesis in Actinoplanes sp. HS and obtained two strains with high yields of ergothioneine through metabolic engineering. Furthermore, we investigated the impact of precursor supply on the yield of ergothioneine, which provided strategic support for the production of ergothioneine by Actinoplanes sp.

[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.

[Objective] To investigate the changes of pathogenicity and DNA methylation levels and patterns of Ralstonia solanacearum strains with different pathogenicity during consecutive subculture. [Methods] R. solanacearum strains with different pathogenicity were consecutively subcultured for 50 passages. The pathogenicity of different strains was determined by the attenuated index (AI) method and the pot experiments. Methylation sensitive amplification polymorphism (MSAP) analysis was performed to profile the DNA methylation levels of different strains. Moreover, the relative expression levels of genes related to methylases and demethylases were determined by real-time fluorescent quantitative PCR (qRT-PCR). [Results] After 50 passages, both of the virulent strain FJAT15304 and the intermediate strain FJAT445 evolved into avirulent strains, while the avirulent strain FJAT15249 remained to be avirulent. Compared with F₁ strains, FJAT15304.F₅₀ and FJAT445.F₅₀ showed the total methylation rates increasing by 7.82% and 38.22%, respectively. However, both of FJAT15249.F₁ and FJAT15249.F₅₀ had the total methylation rate of 33.33%. Full methylation was the main pattern in the virulent and intermediate strains, while hemi-methylation was the main pattern in all the avirulent strains. Compared with F₁ strains, strains FJAT15304.F₅₀ and FJAT445.F₅₀ showed up-regulated expression of three methylase-related genes dam, dcm, and ftsZ and down-regulated expression of demethylase-related gene alkB, which suggested that the change of DNA methylation might play a key role in the debilitation of pathogenicity. [Conclusion] The pathogenicity of R. solanacearum attenuates during the consecutive subculture, which might be related to the level of DNA methylation. The findings provide a scientific basis for the application of avirulent strains in the biocontrol of bacterial wilt.