[Objective] To construct the γ-aminobutyrate-producing recombinant strains ofEscherichia coli and investigate their fermentation characteristics.[Methods] We constructed two recombinant plasmids pTrc99a-gadB and pTrc99a-gadB-SNO1-SNZ1 and then respectively transformed them into the gene-knockout strainE.coli K12/ΔgabTΔgabPΔpuuE. We investigated and optimized the fermentation process of the recombinant strains for producing γ-aminobutyrate.[Results] The target proteins were highly expressed in the recombinant strains harboring the constructed plasmids. The highest concentration of γ-aminobutyrate was 4.6 g/L in the fermentation broth ofE.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB cultured in the medium containing 10 g/L l-monosodium glutamate and was 21.9 times higher than that in the fermentation broth of the wild type strain. At the l-monosodium glutamate concentration of 20 g/L, the conversion rate of substrate was the highest and the concentration of γ-aminobutyrate reached 8.4 g/L. The concentration of γ-aminobutyrate was slightly lower in the fermentation broth of the recombinant strainE.coli K12ΔgabTΔgabPΔpuuE/pTrc99a-gadB-SNO1-SNZ1, probably due to the excessive consumption of energy. The highest concentration of γ-aminobutyrate was 9.4 g/L whenE.coli K12ΔgabTΔgabPΔpuuE/ pTrc99a-gadB was cultured in 1 L fermentation medium containing 20 g/L l-monosodium glutamate.[Conclusion] We obviously increased the yield of γ-aminobutyrate produced by the recombinant strain. This finding lays a foundation for the industrial production of γ-aminobutyrate.

[Objective] The cell wall structure characteristics make it a challenge to obtain the whole proteins ofPaenibacillus sp. as a Gram-positive bacterium. We employed three representative lysis methods: lysozyme combined with ultrasonic treatment (method 1), lysozyme combined with SDS heat treatment (method 2), and liquid nitrogen combined with ultrasonic treatment (method 3), to obtain the samples for mass spectrometry-based proteomics research onPaenibacillus sp.[Methods] We compared the protein extraction efficiency of the three methods based on protein yield and SDS-PAGE. Furthermore, we analyzed the obtained protein samples by mass spectrometry.[Results] The protein extraction efficiency of the three methods was similar. The number of proteins identified in the sample prepared with method 3 was the highest (2 638 proteins), followed by that with method 1 (2 452 proteins), and that with method 2 was the lowest (2 003 proteins). The Venn diagram was established to compare the protein identification flux of the samples prepared with different methods. Considering the protein extraction efficiency and the shortcomings of method 3, we finally chose method 1 as the most suitable method to extract the proteins for mass spectrometry-based proteomic analysis. Finally, the basic properties of proteins identified by mass spectrometry, including molecular weight, isoelectric point, and hydrophobicity, were analyzed. The proportion of each component of the proteins obtained by the three methods was the same as that of the predicted proteins of the model strainPaenibacillus polymyxa.[Conclusion] For the proteomics research on Gram-positivePaenibacillus sp. by mass spectrometry, lysozyme combined with ultrasonic treatment is the most suitable method for sample preparation.

Outer membrane vesicles (OMVs) are spherical structures secreted by Gram-negative bacteria, with diameters of 10–250 nm. OMVs have gradually been recognized as a novel secretion system capable of transporting various substances such as lipids, proteins, nucleic acids, cytotoxins, and signaling molecules. OMVs possess a range of biological functions, including inter-bacterial communication, transmission of pathogenic factors, resistance to adverse external environments, and modulation of immune responses. Due to the distinct biological characteristics of OMVs, the antibiotic resistance transmission mediated by bacterial OMVs, as well as the potential applications of OMVs, has garnered increasing attention in recent years. This paper reviews the origins and substance delivery functions of bacterial OMVs and the protective effects of OMVs on the bacteria exposed to antibiotics and in adverse environments. Furthermore, this paper summarizes the potential applications of bacterial OMVs in the treatment of diseases, aiming to enrich the knowledge about bacterial OMVs.

The type VI secretion system (T6SS) as a dynamic multi-protein complex has a clear division of labor among its components, transporting effector proteins to compete for bacterial growth. Studies have shown that T6SS mediates the competitiveness ofAcinetobacter baumannii in the microbial community and affects the drug resistance evolution and invasion in the host. Particularly, the valine-glycine repeat protein G (VgrG), the proline-alanine-alanine-arginine (PAAR), the hemolysin-coregulated protein (Hcp), and the effector-immunity (E-I) pair play a key role. Although T6SS has been extensively studied, there are few articles about its clinical application prospects, as this poses challenges to the identification, characterization, transport mechanism revealing, and other basic research on their functional proteins. We reviewed the research progress in the distribution, functional protein characteristics, and transport mechanism of T6SS inA.baumannii and provided evidence for its application based on the application cases of T6SS. This review aims to promote the research on the genes and functions of T6SS inA.baumannii and provide new targets and ideas for developing new anti-infective vaccines, screening suitable inhibitors, and producing engineered drug delivery tools.

Guanosine tetraphosphate (ppGpp)/guanosine pentaphosphate (pppGpp) is a signaling molecule in the bacterial stringent response, whose synthesis and hydrolysis are controlled by the synthesis and hydrolysis activities of proteins in the RelA/SpoT homologue (RSH) family. The (p)ppGpp-mediated stringent response enhanced bacterial adaptation to nutrient deprivation and antibiotic resistance. In recent years, (p)ppGpp has been found to associate with bacterial growth, cell division, and antibiotic synthesis, which was an important global regulator in the bacteria. (p)ppGpp has many target sites in the cell, which allow it to regulate DNA replication, transcription, cell cycle, ribosome biosynthesis, and the expression of antibiotic synthesis gene clusters. However, how (p)ppGpp controls transcription and other metabolic processes depends on the bacterial species, and (p)ppGpp regulates the same processes in different bacteria species through different mechanisms. Therefore, this manuscript reviewed the types of (p)ppGpp synthetic and hydrolytic enzymes, the mechanisms of (p)ppGpp regulation on microbial metabolism and the cell cycle, as well as the regulation mechanisms of antibiotic synthesis and tolerance, which lays the foundation for bacterial resistance and cell physiology researches.

Terpenoids, a group of natural products possessing diverse chemical structures and biological activities, have been widely used in the production of food additives, medicines, and cosmetics. Terpenoids are mainly produced by plants. With the rapid development of synthetic biology, heterologous production of terpenoids by engineered microbial strains is more economical and environmentally friendly compared with the conventional methods of extraction from plants and chemical synthesis. The catalytic activities of terpene synthases and the structural specificity of their products are pivotal for the heterologous biosynthesis of terpenoids. Directed evolution and rational design can be adopted to optimize the catalytic properties and product profile of target terpene synthases. However, this requires a feasible high-throughput method to screen mutant libraries. In recent years, various high-throughput screening methods have been developed to boost the sensitivity and efficiency in screening terpene synthases. We review recently established high-throughput screening methods for terpene synthases and briefly outline the principles and advantages and disadvantages of each method. Eventually, we envision the future directions of the application of high-throughput screening in the engineering of terpene synthases.

[Objective] Pseudomonas aeruginosa is a major Gram-negative pathogen that can exacerbate lung infections in the patients with cystic fibrosis, which can ultimately lead to death. The emerging multi-drug resistant and pan-drug resistant strains pose challenges to the prevention and control ofP.aeruginosa infection.[Methods] A phage capable of lysing multi-drug resistantP.aeruginosa was isolated from farm sewage. The morphological characteristics, biological characteristics, host range, genomic features, andin vitro inhibitory ability of the isolate were studied. The biofilm inhibition test was conducted with both the phage and antibiotics.[Results] The phage PH826 was identified to be a member of the genus Nankokuvirus and it can keep stability within a wide temperature range from 4 ℃ to 60 ℃ and pH range (pH 3.0–11.0). The host range test showed that PH826 could lyse 13P.aeruginosa strains from human and animals. The antibacterial testin vitro showed that PH826 was strongly lytic againstP.aeruginosa at multiplicity of infection (MOI) of 10, 1, and 0.1. The genome of PH826 was 87 956 bp in length, with the G+C content of 54.70%, encoding 165 open reading frames (ORFs) including an endolysin gene. The combination of PH826 at 1×MIC (minimum inhibitory concentration, MIC), 2×MIC, or 4×MIC and ciprofloxacin reduced biofilm formation by more than 80% at the time points of 24 h and 48 h. The combination of PH826 at 2×MIC or 4×MIC and meropenem reduced biofilm formation by more than 80% at the time points of 24 h and 48 h.[Conclusion] PH826 may be a potential disinfectant and therapeutic agent that can be used alone or in combination with antibiotics for the prevention and treatment of pathogenicP.aeruginosa.

[Objective] To study the transcriptional regulation of type Ⅵ secretion system 1 (T6SS1) genes by QsvR inVibrio parahaemolyticus.[Methods] Total RNA was extracted from the wild type (WT) andqsvR mutant (ΔqsvR). Quantitative real-time PCR (qPCR) was employed to investigate the transcriptional regulation of target genes by QsvR. Primer extension was carried out to detect the transcription initiation site and core promoter for each target gene and calculate the transcriptional variations between WT and ΔqsvR. The regulatory DNA region of each target gene was cloned into the restriction endonuclease sites of pHRP309 harboring a promoterless genelacZ, and then each recombinant plasmid was transferred into WT and ΔqsvR, respectively. A β-Galactosidase Enzyme Assay System (Promega) was used to measure the β-galactosidase activity in cell lysates. The recombinant pHRP309 vector containing the regulatory DNA region of one of the target gene was transferred intoEscherichia coli 100λpir harboring an empty pBAD33 or pBAD33-qsvR to test whether QsvR can regulate the target genes in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and His-QsvR was over-expressed and then purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay (EMSA) was employed to determine the DNA-binding activity of His-QsvR to each target DNA fragmentin vitro.[Results] The mRNA levels of T6SS1-associated genes, VP1388 (the first gene of VP1388–1390 operon) andhcp1 (the first gene of VP1393–1406 operon), were significantly up-regulated in ΔqsvR relative to those in WT, indicating that QsvR activated the transcription of VP1388 andhcp1. Only one transcription initiation site was detected for VP1388 orhcp1, locating at 64 bp upstream of VP1388 and 62 bp upstream ofhcp1, respectively, and their transcriptional activities were all repressed by QsvR. QsvR repressed the promoter activities of VP1388 andhcp1 in bothV.parahaemolyticus andE.coli 100λpir. His-QsvR was able to bind to the regulatory DNA regions of VP1388 andhcp1.[Conclusion] QsvR directly repressed the transcription of T6SS1-associated operons, VP1388–1390 and VP1393–1406, inV.parahaemolyticus.

The term "microorganism" refers to tiny organisms such as archaea, bacteria, protists, fungi, and viruses, and the term "microbiome" refers to a collection of microorganisms. Although they share the body space of the host, their roles as determinants in host health and diseases are ignored. As a collection of information, the microbiome includes the genomic data, structural elements, metabolites, and environmental conditions of microorganisms. Studies have demonstrated that the microbiome plays an essential role in maintaining host homeostasis and regulating host phenotypes. With the advent of new technologies, including next-generation sequencing (NGS) and sequencing-based microbiome profiling, researchers have probed into the relationship between the microbiome and host phenotypes. By an overview of microbiome, this paper elaborated on the microbiome-host genetics interactions based on genome-wide association analysis and made an outlook on the future of this field.

With the evolution of bacteria and the overuse of certain antibiotics, drug-resistant bacterial infections have emerged as a major public health challenge in the 21st century.Klebsiella pneumoniae, in particular, has aroused wide concern due to its drug-resistant nature. Bacteriophages have demonstrated the potential and advantages in treating bacterial infections caused by drug-resistant strains, while there are no clinical guidelines for phage therapy. Notably, even though a few successful cases of bacteriophage therapy have been documented in the treatment ofK.pneumoniae infections, bacteriophages are used in combination with antibiotics in most cases and the role bacteriophages play remains unclear. This article provides an overview of bacteriophage therapies forK.pneumoniae infections, including their characteristics and the factors influencing their efficacy. We compiled and analyzed data from available studies, with the intention of offering valuable insights for the application of bacteriophage therapy in combatingK.pneumoniae and other drug-resistant bacteria.