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 drug resistance mechanism and gene structure characteristics of a carbapenemase-producing novel incompatibility group plasmid pNY2385-KPC from Citrobacter freundii. Methods: A multi-drug resistant strain was obtained from urine samples of patients with fever in the emergency ward of Li Huili Hospital, Ningbo Medical Center. Bacterial species was preliminary identified and finally confirmed by 16S rRNA gene amplification and the average nucleotide identity alignment, respectively. The minimum inhibitory concentrations of the antimicrobial agents were determined by VITEK 2 Compact System. The complete genome sequence was obtained by "third-generation" sequencing methods, and then detailed annotation of gene function and comparative genomic analysis of plasmid structure were carried out by BLASTP/BLASTN, RefSeq, ConservedDomains, ResFinder, Isfinder, etc. Results: The pNY2385-KPC carried by citrobacter freundii NY2385 belonged a novel incompatibility group, and contained bla_KPC-2 and conjugative transfer (type Ⅳ secretory system, T4SS) genes, which could induce conjugative transfer. A total of 15 plasmids of the same type as pNY2385-KPC were retrieved by NCBI, which were from Citrobacter freundii, and the rest were from Serratia marcescens, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Raoultella planticola and other bacteria, and were broad-host-range plasmids. The sequence comparative analysis of all 6 of the novel plasmid from Citrobacter freundii showed that the structure of the novel plasmid had certain conserved property, with Tn6296 variant structure carrying bla_KPC-2, and plasmid pCF1807-3 had both repA_pNY2385-KPC and repA_IncX8. Conclusion: The pNY2385-KPC type plasmids in Citrobacter freundii carried bla_KPC-2 resistance gene, which were divided into two subtypes: repA_pNY2385-KPC single replicator and repA_pNY2385-KPC/repA_IncX8 complex replicator, belonging to broad-host-range plasmids. And as a mobile genetic element, the plasmids promote the spread of bla_KPC-2.
Humans ; Citrobacter freundii/genetics* ; RNA, Ribosomal, 16S/genetics* ; Emergency Service, Hospital ; Escherichia coli ; Genomics

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

Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.
Male ; Rabbits ; Animals ; Mice ; Ubiquitins ; Escherichia coli/genetics* ; Isopropyl Thiogalactoside ; Antibodies ; Enzyme-Linked Immunosorbent Assay

Objective To prepare rabbit polyclonal antibody specifically against human lactate dehydrogenase C4 (LDHC4). Methods Site-directed mutation was performed by PCR to generate the mutated LDHC gene, and the mutated gene was ligated into the pET-28a vector to form the pET-28a-LDHC recombinant expression vector. The recombinant vector was introduced into E. coli BL21 (DE3), and LDHC4 protein was obtained by induced expression. The recombinant protein was used as an antigen to immunize New Zealand rabbits, and the antiserum was obtained after three boosted immunizations. The titer of the antiserum against LDHC4 were detected by ELISA. Western blot was used to detect the specificity of the antiserum, and immunohistochemistry was used to detect the expression of LDHC4 in human triple-negative breast cancer tissue. Results A specific rabbit anti-human LDHC4 polyclonal antibody was obtained with an antibody titer of 1:51 200. The antibody can be used for Western blot and immunohistochemistry. Conclusion The specific rabbit anti-human LDHC4 polyclonal antibody is successfully prepared.
Humans ; Rabbits ; Animals ; Escherichia coli/genetics* ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; L-Lactate Dehydrogenase/metabolism* ; Blotting, Western ; Antibody Specificity

OBJECTIVES: The prevalence of carbapenem-resistant Enterobacterales (CRE) presents a significant challenge in clinical anti-infective treatment. This study aims to investigate drug resistance and the molecular epidemiological characteristics of CRE in our area. Additionally, we seek to evaluate practicality of utilizing carbapenemase inhibitor enhancement test in clinical laboratory. METHODS: Non-repeated CREs isolated from clinical specimens at Xiangya Hospital, Central South University, were collected. Minimum inhibitory concentration (MIC) combined with Kirby-Bauer (KB) assay was used to detect the drug susceptibility of the strains, and 13 carbapenemase-producing genes were detected by PCR. The phenotype of 126 strains of carbapenemase-producing Enterobacterales identified by PCR was detected by the carbapenemase inhibitor enhancement test to understand the agreement between the method and the gold standard PCR results. RESULTS: Among 704 CRE strains examined, we observed significant drug resistance in 501 strains dentified as carbapenemase-producing Enterobacterales (CPE). Klebsiella pneumoniae was the predominant CPE strain, followed by Enterobacter cloacae and Escherichia coli. A total of 9 carbapenemase types were detected, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integron- encoded metallo-β-lactamases (VIM), imipenemase (IMP), oxacillinase-48 (OXA-48), and rare imipenem-hydrolyzing β-lactamase (IMI), adelaide imipenemase (AIM), Bicêtre carbapenemase (BIC), and guiana extended-spectrum β-lactamase (GES). The detection rate of KPC serine carbapenemase was 61.7% (309/501). The carbapenemase inhibitor enhancement test exhibited a 100% consistency rate for the strains producing Class A serine carbapenemase and/or Class B metallo-β-lactamases. CONCLUSIONS CRE strains in Changsha, Hunan, China, are wide distribution and exhibit carbapenemase production. The main mechanism of carbapenem resistance in these bacterias is predominatly attributed to the production of KPC serine carbapenemase. The presence of GES and IMI genes carried by Enterobacterales has been detected for the first time in this region. The carbapenemase inhibitor enhancement test has been proven to be an accurate method for detecting CRE producing Class A serine carbapenemase and/or Class B metallo-β-lactamases. This method offers simpicity of operation and ease of results interpretation, making it weel-suited meeting the clinical microbiology laboratory's reguirements for the detection of serine carbapenemase and metallo-β-lactamases.
Humans ; Carbapenems/pharmacology* ; Molecular Epidemiology ; Bacterial Proteins/analysis* ; beta-Lactamases/analysis* ; Klebsiella pneumoniae/genetics* ; Escherichia coli ; Microbial Sensitivity Tests ; Serine ; Anti-Bacterial Agents/pharmacology*

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