Ferric uptake regulator (Fur) is a key regulatory factor of iron metabolism and virulence inPseudomonas aeruginosa. Many research groups have failed to construct thefur-deleted mutant ofP.aeruginosa, sofur has always been considered to be an essential gene inP.aeruginosa, and the knowledge of its biological function is limited. [Objective] This study aims to construct afur-deleted mutant ofP.aeruginosa and analyze its phenotypes. [Methods] WithP.aeruginosa PAO1 as the parental strain, thefur-deleted mutant was constructed by homologous recombination. After that, we studied the effects offur on the growth, siderophore biosynthesis, resistance to oxygen stress, flagella formation, biofilm formation, and virulence ofP.aeruginosa. In addition, we explored the cause of the growth defect phenotype of thefur-deleted mutant by genetic analysis. [Results] Thefur-deleted mutant ofP.aeruginosa was successfully constructed. The deletion offur greatly limited the growth ofP.aeruginosa and reduced the growth adaptability ofP.aeruginosa to the iron-limited environment, while it did not affect the growth adaptability ofP.aeruginosa to the iron-rich environment. This growth defect phenotype of Δfur was caused by the slow cell growth and proliferation, rather than by cell death. Interestingly, heterologousfur could completely complement the growth defect phenotype of Δfur, suggesting that the Fur ofP.aeruginosa was not functionally unique. Although there was a functional relationship between Fur and the toxin-antitoxin system PacTA, the growth defect phenotype ofP.aeruginosa Δfur was not associated with PacT toxin. In addition to affecting the growth phenotype ofP.aeruginosa, the deletion offur also madeP.aeruginosa lose the inhibitory effect on siderophore biosynthesis and the ability to form flagella and have increased sensitivity to H₂O₂ and reduced virulence toGalleria mellonella larvae. Moreover, the deletion offur increased the intracellular cyclic diguanylate (c-di-GMP) level ofP.aeruginosa to induce the expression ofpelF andpslA, thereby promoting the biofilm formation ofP.aeruginosa. [Conclusion] fur is a non-essential gene that can be deleted and plays a crucial role in the normal growth, siderophore biosynthesis, resistance to oxygen stress, flagellum formation, biofilm formation, and virulence ofP.aeruginosa, which lays a foundation for the development of vaccines and agents againstP.aeruginosa.

[Objective] Since the accumulation of polyethylene terephthalate (PET) waste causes a major threat to the health of the natural environment, the degradation of PET has become a global hot issue. Enzymatic degradation of PET has garnered considerable attention because of its eco-friendly properties. However, due to the low catalytic activity, natural PET-degrading enzymes remain to be modified according to specific needs. Directed evolution enables the rapidly enhancement of the catalytic activities of PET-degrading enzymes, in which screening methods are the key for obtaining high-performance mutants. This study develops a novel, efficient, and sensitive screening method and applies it to direct modification ofThermobifida fusca cutinase Tfu-0883 to obtain the mutants with improved PET-degrading activity. [Methods] A mutant library constructed by error-prone PCR was coated on phospholipid plates. The mutant with improved PET-degrading activity was screened out based on the size of the hydrolytic circle. The enzymatic properties of the mutant were determined, and the rational modification sites were identified. Finally, a forward mutant was obtained. [Results] The single colony with the largest hydrolysis circle, mutant H10 (N2D/D94H/A149E), was selected from the phospholipid plate, with the PET-degrading activity 1.5 times that of the wild type. The mutant H10 exhibited the best performance at 60 ℃ and pH 8.0. The residues at positions 2 and 149 in the mutant H10 were distantly located from the substrate-binding groove, and any mutation in the residues would result in decreased enzyme stability. The residue at position 94 was situated near the substrate-binding groove, where it underwent a change from negatively charged Asp to positively charged His. This alteration facilitated adsorption onto the negatively charged PET surface and played a crucial role in enhancing the degradation ability of mutant H10. With the wild type as a template, the 94th amino acid residue was mutated to His, Lys, and Arg, which possess positive charges but exhibit reduced steric hindrance. The mutants D94H, D94K, and D94R all exhibited enhanced PET-degrading ability. Notably, among these mutants, D94K demonstrated a 3.6-fold higher rate of PET degradation than the wild type. [Conclusion] We developed a method for screening PET-degrading enzymes based on the phospholipase cycle and obtained the mutants with enhanced PET-degrading activity. The 94th residue of the cutinase Tfu-0883 is demonstrated as the first to possess the potential for enhancing the PET-degrading activity.

Methanotrophs can utilize methane as the only carbon source and energy, and they can survive and participate in material circulation and energy flow in ecosystems. [Objective] To unveil the structure and functions of methanotrophs community in the sediments from the Aha Lake Reservoir (referred to as the Reservoir), a typical karst lake reservoir in Guiyang City, Guizhou Province. [Methods] We used metagenomics to analyze the sediments collected from the edge and the center of the Reservoir. [Results] The dominant aerobic methanotrophs wereMethylobacter (0.37%) andMethylomonas (0.12%), and the dominant anaerobic methanotrophs wereCandidatus_Methylomirabilis (0.12%), being NC10 denitrifying anaerobic methanotrophs. The genepmoA encoding particulate methane monooxygenase of aerobic methanotrophs had the relative abundance of 6.16×10⁷ copies/g and the 16S rRNA gene had the relative abundance of 2.84×10⁷ copies/g in denitrifying anaerobic methanotrophs. The diversity of four metabolic functional genes followed a trend of nitrogen metabolism > carbon metabolism > sulfur metabolism > methane metabolism. Kyoto encyclopedia of genes and genomes (KEGG) annotation revealed six functions and 18 complete pathways involving carbon (including methane), nitrogen, and sulfur metabolism. The results of principal coordinate analysis (PCoA) showed huge discrepancies in the distribution and functions of methanotrophs between sediments from the edge and the center of the Reservoir. Moreover, redox potential, conductivity, and sulfate were primary environmental factors affecting methanotroph distribution. [Conclusion] Type I aerobic methanotrophs dominated the Reservoir with abundant metabolic pathways. Types I and II methanotrophs exhibited huge discrepancies in terms of their adaptability to O₂. All these fundings are expected to provide theoretical support for lake water environment conservation and microbial utilizationation.

Metals like iron, copper, zinc, and manganese are trace elements essential for the survival and growth of diverse organisms. They influence the protease activity, immune response, physiological processes, and anti-infection mechanism in organisms. During bacterial infection, the host can limit or increase the availability of metal ions in the internal environment to inhibit bacterial proliferation. Meanwhile, bacteria have evolved various transport systems to adapt to the changes in metal ion levels in the host. The metal ion efflux systems exhibit distinctive efflux patterns due to variations in the structural and biochemical properties. We reviewed the available articles and our own research findings about the bacterial efflux systems of iron, copper, zinc, and manganese ions, aiming to provide an overview of the progress in the research on the regulatory mechanisms governing bacterial metal homeostasis. This review of metal ion efflux systems across different bacteria highlights the adaptation that enables bacterial survival in diverse host environments.

Rhizobia would encounter oxidative stress of reactive oxygen species (ROS) in the process of infecting leguminous plants. Methionine-containing proteins are easy to be oxidized to methionine sulfoxide, leading to changes in protein structure and function. Methionine sulfoxide reductases (Msrs) can reduce methionine sulfoxide to methionine, restoring protein structure and function. We have identified four Msrs in the genome ofMesorhizobium huakuii 7653R that are involved in oxidative stress response, while the mechanism remains unclear. [Objective] To identify the substrates of four Msrs and elucidate the roles of the four Msrs inM.huakuii 7653R. [Methods] According to the methionine content, we determined the distribution of all the proteins inM.huakuii 7653R. Then, we used the online protein interaction prediction tools to predict the substrates of the four Msrs, and performed gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment for the predicted substrates. Finally, we verified the interaction between them by using the bacterial two-hybrid system. [Results] The methionine content followed the normal distribution, with most proteins in the middle and few proteins on both sides. Six antioxidant enzymes and six transcription factors were selected as the candidate substrates of the four Msrs. Finally, the bacterial two hybrid results showed that two antioxidant enzymes and five transcription factors can interact with the four Msrs to different degrees. [Conclusion] We provided the proof in illustrating the role of Msrs in the oxidative stress response toM.huakuii 7653R and provided a new idea for the research on the mechanism of rhizobia in response to ROS.

[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.

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.

Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis (TB), threatens the health of millions of people worldwide. The pattern recognition receptors (PRRs) including DNA and RNA sensors on immune cells recognize the invaded Mtb to activate the innate immune system and induce the production of interferon-beta (IFN-β). IFN-β is a major effector cytokine in innate antiviral response, while its role in the host response to Mtb infection remains controversial. IFN-β induced by Mtb can promote bacterial growth and improve the bacterial survival in the host. However, IFN-β treatment before Mtb infection can protect the host from bacterial infection. Focusing on the PRR signaling pathways that can recognize Mtb and mediate the IFN-β production, this review expounds the role of IFN-β in mediating the regulation of immune function by Mtb, especially the mutual inhibitory effect between IFN-β and IL-1β, aiming to reveal the pathogenic mechanism of Mtb and facilitate future research and development of anti-TB drugs.

[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.