Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarS_cyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarS_cyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarS_cyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarS_cyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.
Humans ; Histidine Kinase/metabolism* ; Fusobacterium nucleatum/metabolism* ; Automobiles ; Protein Kinases/genetics* ; Escherichia coli/metabolism* ; Colorectal Neoplasms

The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound β-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. β-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that β-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of β-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined β-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.
Humans ; Tigecycline/pharmacology* ; Escherichia coli/metabolism* ; Reactive Oxygen Species/therapeutic use* ; Plasmids ; Anti-Bacterial Agents/metabolism* ; Escherichia coli Infections/microbiology* ; Bacteria/genetics* ; Microbial Sensitivity Tests

The evaluation of the bioavailability of pollutants in soil is crucial to accurately assess the pollution risk, and whole-cell biosensor is one of the important tools for such evaluation. This study aimed to develop a novel whole-cell biosensor for the detection of methyl parathion in soil using. First, a whole-cell biosensor was constructed by the screened methyl parathion hydrolase mpd gene, the existing specific induction element pobR, and the pUC19 plasmid skeleton. Then, the detection method of methyl parathion in soil extracts was established using 96-well microtiter plate as carrier and five whole-cell biosensors as indicator. The method was applied in the detection of methyl parathion in tested and field soil extracts. Taking E. coli DH5α/pMP-AmilCP with the best detection performance as an example, this biosensor had a detection limit of 6.21-6.66 µg/L and a linear range of 10-10 000 µg/L for methyl parathion in four soil extracts. E. coli DH5α/pMP-RFP and E. coli DH5α/pMP-AmilCP methods have good detection performance for the analysis of methyl parathion in soil extract samples. This biosensor method can help to quickly assess the bioavailability of methyl parathion in soil, and thus help to understand the risk of soil pollution caused by organophosphorus pesticide methyl parathion.
Methyl Parathion/analysis* ; Pesticides/analysis* ; Organophosphorus Compounds ; Escherichia coli/genetics* ; Soil ; Farms ; Biosensing Techniques

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni²⁺-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.
Animals ; Rabbits ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Moths/genetics* ; Blotting, Western ; Larva/genetics* ; Isoantibodies/metabolism* ; Antibody Specificity

Objective: To investigate the antimicrobial resistance of food-borne diarrheagenic Escherichia coli (DEC) and the prevalence of mcr genes that mediates mobile colistin resistance in parts of China, 2020. Methods: For 91 DEC isolates recovered from food sources collected from Fujian province, Hebei province, Inner Mongolia Autonomous Region and Shanghai city in 2020, Vitek2 Compact biochemical identification and antimicrobial susceptibility testing platform was used for the detection of antimicrobial susceptibility testing (AST) against to 18 kinds of antimicrobial compounds belonging to 9 categories, and multi-polymerase chain reaction (mPCR) was used to detect the mcr-1-mcr-9 genes, then a further AST, whole genome sequencing (WGS) and bioinformatics analysis were platformed for these DEC isolates which were PCR positive for mcr genes. Results: Seventy in 91 isolates showed different antimicrobial resistance levels to the drugs tested with a resistance rate of 76.92%. The isolates showed the highest antimicrobial resistance rates to ampicillin (69.23%, 63/91) and trimethoprim-sulfamethoxazole (59.34%, 54/91), respectively. The multiple drug-resistant rate was 47.25% (43/91). Two mcr-1 gene and ESBL (extended-spectrum beta-lactamase) positive EAEC (enteroaggregative Escherichia coli) strains were detected. One of them was identified as serotype of O11:H6, which showed a resistance profile to 25 tested drugs referring to 10 classes, and 38 drug resistance genes were predicted by genome analysis. The other one was O16:H48 serotype, which was resistant to 21 tested drugs belonging to 7 classes and carried a new variant of mcr-1 gene (mcr-1.35). Conclusion: An overall high-level antimicrobial resistance was found among foodborne DEC isolates recovered from parts of China in 2020, and so was the MDR (multi-drug resistance) condition. MDR strains carrying multiple resistance genes such as mcr-1 gene were detected, and a new variant of mcr-1 gene was also found. It is necessary to continue with a dynamic monitoring on DEC contamination and an ongoing research into antimicrobial resistance mechanisms.
Humans ; Colistin/pharmacology* ; Anti-Bacterial Agents/pharmacology* ; Escherichia coli Infections/epidemiology* ; Escherichia coli Proteins/genetics* ; Drug Resistance, Bacterial/genetics* ; China/epidemiology* ; Escherichia coli ; Plasmids/genetics* ; Microbial Sensitivity Tests

The α-amino acid ester acyltransferase (SAET) from Sphingobacterium siyangensis is one of the enzymes with the highest catalytic ability for the biosynthesis of l-alanyl-l-glutamine (Ala-Gln) with unprotected l-alanine methylester and l-glutamine. To improve the catalytic performance of SAET, a one-step method was used to rapidly prepare the immobilized cells (SAET@ZIF-8) in the aqueous system. The engineered Escherichia coli (E. coli) expressing SAET was encapsulated into the imidazole framework structure of metal organic zeolite (ZIF-8). Subsequently, the obtained SAET@ZIF-8 was characterized, and the catalytic activity, reusability and storage stability were also investigated. Results showed that the morphology of the prepared SAET@ZIF-8 nanoparticles was basically the same as that of the standard ZIF-8 materials reported in literature, and the introduction of cells did not significantly change the morphology of ZIF-8. After repeated use for 7 times, SAET@ZIF-8 could still retain 67% of the initial catalytic activity. Maintained at room temperature for 4 days, 50% of the original catalytic activity of SAET@ZIF-8 could be retained, indicating that SAET@ZIF-8 has good stability for reuse and storage. When used in the biosynthesis of Ala-Gln, the final concentration of Ala-Gln reached 62.83 mmol/L (13.65 g/L) after 30 min, the yield reached 0.455 g/(L·min), and the conversion rate relative to glutamine was 62.83%. All these results suggested that the preparation of SAET@ZIF-8 is an efficient strategy for the biosynthesis of Ala-Gln.
Escherichia coli/genetics* ; Glutamine ; Zeolites/chemistry* ; Amino Acids

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S₁₀₅, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD₆₀₀, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S₁₀₅, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.
Rhamnose/pharmacology* ; Plasmids/genetics* ; Pseudomonas aeruginosa ; Escherichia coli/metabolism*

At present, the research of biological living materials mainly focuses on applications in vitro, such as using a single bacterial strain to produce biofilm and water plastics. However, due to the small volume of a single strain, it is easy to escape when used in vivo, resulting in poor retention. In order to solve this problem, this study used the surface display system (Neae) of Escherichia coli to display SpyTag and SpyCatcher on the surface of two strains, respectively, and constructed a double bacteria "lock-key" type biological living material production system. Through this force, the two strains are cross-linked in situ to form a grid-like aggregate, which can stay in the intestinal tract for a longer time. The in vitro experiment results showed that the two strains would deposit after mixing for several minutes. In addition, confocal imaging and microfluidic platform results further proved the adhesion effect of the dual bacteria system in the flow state. Finally, in order to verify the feasibility of the dual bacteria system in vivo, mice were orally administrated by bacteria A (p15A-Neae-SpyTag/sfGFP) and bacteria B (p15A-Neae-SpyCatcher/mCherry) for three consecutive days, and then intestinal tissues were collected for frozen section staining. The in vivo results showed that the two bacteria system could be more detained in the intestinal tract of mice compared with the non-combined strains, which laid a foundation for further application of biological living materials in vivo.
Animals ; Mice ; Bacteria ; Microorganisms, Genetically-Modified ; Escherichia coli/genetics*

The construction of efficient and stable Lactobacillus expression vector is critical for strain improvement and development of customized strains. In this study, four endogenous plasmids were isolated from Lacticaseibacillus paracasei ZY-1 and subjected to functional analysis. The Escherichia coli-Lactobacillus shuttle vectors pLPZ3N and pLPZ4N were constructed by combining the replicon rep from pLPZ3 or pLPZ4, the chloramphenicol acetyltransferase gene cat from pNZ5319 and the replicon ori from pUC19. Moreover, the expression vectors pLPZ3E and pLPZ4E with the promoter P_ldh3 of lactic acid dehydrogenase and the mCherry red fluorescent protein as a reporter gene were obtained. The size of pLPZ3 and pLPZ4 were 6 289 bp and 5 087 bp, respectively, and its GC content, 40.94% and 39.51%, were similar. Both shuttle vectors were successfully transformed into Lacticaseibacillus, and the transformation efficiency of pLPZ4N (5.23×10²-8.93×10² CFU/μg) was slightly higher than that of pLPZ3N. Furthermore, the mCherry fluorescent protein was successfully expressed after transforming the expression plasmids pLPZ3E and pLPZ4E into L. paracasei S-NB. The β-galactosidase activity of the recombinant strain obtained from the plasmid pLPZ4E-lacG constructed with P_ldh3 as promoter was higher than that of the wild-type strain. The construction of shuttle vectors and expression vectors provide novel molecular tools for the genetic engineering of Lacticaseibacillus strains.
Lacticaseibacillus ; Lacticaseibacillus paracasei ; Plasmids/genetics* ; Genetic Vectors/genetics* ; Lactobacillus/genetics* ; Escherichia coli/genetics*

OBJECTIVE: To explore the genotyping characteristics of human fecal Escherichia coli( E. coli) and the relationships between antibiotic resistance genes (ARGs) and multidrug resistance (MDR) of E. coli in Miyun District, Beijing, an area with high incidence of infectious diarrheal cases but no related data. METHODS: Over a period of 3 years, 94 E. coli strains were isolated from fecal samples collected from Miyun District Hospital, a surveillance hospital of the National Pathogen Identification Network. The antibiotic susceptibility of the isolates was determined by the broth microdilution method. ARGs, multilocus sequence typing (MLST), and polymorphism trees were analyzed using whole-genome sequencing data (WGS). RESULTS: This study revealed that 68.09% of the isolates had MDR, prevalent and distributed in different clades, with a relatively high rate and low pathogenicity. There was no difference in MDR between the diarrheal (49/70) and healthy groups (15/24). CONCLUSION We developed a random forest (RF) prediction model of TEM.1 + baeR + mphA + mphB + QnrS1 + AAC.3-IId to identify MDR status, highlighting its potential for early resistance identification. The causes of MDR are likely mobile units transmitting the ARGs. In the future, we will continue to strengthen the monitoring of ARGs and MDR, and increase the number of strains to further verify the accuracy of the MDR markers.
Humans ; Escherichia coli/genetics* ; Escherichia coli Infections/epidemiology* ; Multilocus Sequence Typing ; Genotype ; Beijing ; Drug Resistance, Multiple, Bacterial/genetics* ; Anti-Bacterial Agents/pharmacology* ; Diarrhea ; Microbial Sensitivity Tests