Ion transporters play an important role in maintaining intracellular pH homeostasis and ionic equilibrium. Sodium ion transporters and potassium ion transporters exist widely in halophilic and halotolerant microorganisms, and their function of retaining potassium and excreting sodium is one of the two major strategies for microbial tolerance to salt stress. In recent years, new sodium and potassium ion transporters, such as RDD, UPF0118, DUF, and KimA, have been discovered in halophilic and halotolerant microorganisms. The transporters of other metal ions, such as Fe³⁺ and Mg²⁺, have been proved to play a role in microbial osmoregulation by participating in the synthesis of intracellular compatible solutes. This paper reviews the ion transporters associated with salt stress tolerance in halophilic and halotolerant microorganisms, analyzes their molecular structures and working mechanisms, and prospects for their applications in agriculture. Discovering new ion transporters, revealing the structures and mechanisms of ion transporters associated with salt stress tolerance, and analyzing the synergistic effect of coexisting transporter systems and their regulation mechanisms will deepen the understanding of the regulatory mechanisms of salt stress tolerance of halophilic and halotolerant microorganisms and provide new ideas for the improvement of crops in saline-alkali land.

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.

Antibiotics are secondary metabolites produced by microorganisms during the stationary phase. They are widely used in the clinical treatment of bacterial infections because of their ability to kill bacteria or inhibit bacterial growth. In the long-term evolutionary process, bacteria have adopted several strategies to cope with the threats of antibiotics in the environment. In addition to the well-known antibiotic resistance, bacteria can develop tolerance and persistence to antibiotics, which seriously affects the clinical efficacy of antibiotics. Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (herein collectively referred to as ppGpp) are the alarmone signal molecules produced by bacteria in response to unfavorable environmental conditions such as nutritional starvation. ppGpp can regulate transcription globally and enable bacteria to survive in unfavorable conditions. An increasing number of studies have shown that ppGpp is closely related to antibiotic stress response. On this basis, this review summarizes the synthesis, hydrolysis, and mechanism of action of ppGpp in bacteria, with emphasis on the role of ppGpp in antibiotic stress response. This review aims to provide new ideas for the development of novel antibiotics.

The non-biodegradable nature of heavy metals (HMs) results in their long-term presence in the environment, leading to severe environmental pollution and posing a threat to human health and ecosystems. Compared with physical and chemical remediation techniques, microbial remediation is praised for the low cost, environmental friendliness, and high efficiency. When facing heavy metal stress or nutrient imbalance, microorganisms are stimulated to produce and secrete extracellular polysaccharides (EPSs). Therefore, the production of EPSs is regarded as one of the important strategies employed by microorganisms to combat HM stress. EPSs not only protect microorganisms in extreme conditions such as low temperature, high temperature, high salinity, or exposure to toxic compounds but also facilitate the communication and transfer of information and substances both inside and outside the cells. EPSs serve as a protective barrier to restrict the entry of HM ions into the cells and as a medium for communication. EPSs contain multiple negatively charged functional groups capable of complexing with HM ions, undergoing ion exchange, and participating in redox reactions, thereby reducing the bioavailability and toxicity of HMs. Microbial EPSs play a significant role in the remediation of HM-contaminated environments. However, there is currently a lack of a systematic review on the synthesis process of microbial EPSs, the mechanisms of the interaction of EPSs with HMs, and the application status of EPSs in the environments with HM stress. This article provides an overview of microbial EPSs and their classification, elaborates on the intracellular and extracellular biosynthesis mechanisms of bacterial EPSs, explores the interactions between microbial EPSs and HMs, and discusses research advances in the use of microbial EPSs for the remediation of HM pollution in water and soil environments. Finally, it looks ahead to the synthesis of EPSs and the role of EPSs in HM remediation, offering support for the further application of microbial EPSs in the remediation of environmental HM pollution.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a severe infectious disease affecting both domestic pigs and wild boar. ASFV has a large genome, and the non-structural protein pD1133L is predicted to be one of the six helicases the genome encodes. We used the IP-MASS technology to screen the host proteins interacting with pD1133L and found that vimentin (VIM) is one of the host proteins that interacted with pD1133L. However, it remains unclear how the VIM affects ASFV replication. [Objective] To investigate the mutual regulation between ASFV and VIM and disclose the mechanism by which VIM enhances ASFV replication. [Methods] We employed the Co-IP assay to examine the interaction between pD1133L and VIM. Furthermore, we examined the effects of VIM on ASFV replication by designing and synthetizing VIM siRNAs and overexpressing VIM. Western blotting and quantitative real-time PCR (qPCR) were employed to determine the impact of ASFV on the protein and mRNA levels of VIM. Western blotting and indirect immunofluorescence assay (IFA) were used to explore the changes in the phosphorylation level and subcellular localization of VIM in macrophages infected with ASFV. The CCK-8 kit was used to determine the optimal concentration of KN-93, a VIM phosphorylation inhibitor, for treatment. The effects of KN-93 on the phosphorylation and subcellular localization of VIM and the replication of ASFV were examined by Western blotting and IFA. [Results] The overexpression of VIM promoted the replication of ASFV, while the knockdown of VIM inhibited ASFV replication. In addition, ASFV infection down-regulated both the protein and mRNA levels of VIM in a time-dependent manner. After ASFV infection, VIM was modified by phosphorylation and changed in subcellular localization, thereby promoting ASFV replication. [Conclusion] This study confirms the interaction between ASFV and the host protein VIM. After ASFV infection, pD1133L leads to the rearrangement of the subcellular localization of VIM towards paranuclear aggregation, which promotes ASFV replication.

[Objective] ALTA4H-deleted porcine kidney cell line (PK-15) was constructed by CRISPR/Cas9 to study the effect ofLTA4H on the replication of foot-and-mouth disease virus (FMDV), with a view to providing a theoretical basis for revealing the functions ofLTA4H and the mechanism of regulating virus replication. [Methods] Two small guide RNAs (sgRNAs) targeting porcineLTA4H were designed and integrated into the vector pX459-puro-MCS, respectively. The recombinant plasmid was transfected into PK-15 cells, which were then cultured in the medium supplemented with puromycin, and the monoclonal cells were selected by limiting dilution method. Western blotting and sequencing were performed to detectLTA4H knockout, andLTA4H-deleted cells were obtained. Furthermore, Western blotting, RT-qPCR, and virus titer assay were employed to examine FMDV replication and protein expression afterLTA4H knockout. [Results] Compared with the wild type cells, theLTA4H-deleted PK-15 cells exhibited inhibited FMDV replication. [Conclusion] This study successfully constructed a PK-15 cell line with LTA4H gene knockout, demonstrating that LTA4H can promote the replication of FMDV, and the results provided a theoretical basis for the subsequent research on the function ofLTA4H.

[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.

[Objective] Glucanases serve as one of the main components in feed additives. This study identified and characterized a novel GH9 glucanase gene derived from rumen microbiota in herbivores, aiming to provide a reference for the research and development of feed enzymes. [Methods] We obtained theIDSGLUC9-25 gene from the rumen fluid cDNA of Hu sheep and heterologously expressed it inEscherichia coli. The recombinant protein was induced for expression by isopropyl β-d-thiogalactopyranoside, purified, and then subjected to functional characterization. [Results] IDSGLUC9-25 encoded a protein consisting of 527 amino acid residues, which included a CelD_N domain and a GH9 family catalytic domain. The recombinant rIDSGLUC9-25 protein exhibited a molecular weight of approximately 62.7 kDa and the highest enzymatic activity at 40 ℃ and pH 6.0. The enzyme displayed robust catalytic activity within the temperature range of 30–50 ℃. After preincubation at pH 4.0–8.0 for 1 h, rIDSGLUC9-25 retained the relative activity over 90%. The substrate spectrum analysis revealed that rIDSGLUC9-25 exhibited specific activities against barley β-glucan, moss lichenan, konjac gum, and xyloglucan, with the activities of (443.55±24.48), (65.56±5.98), (122.37±2.85), and (159.16±7.73) U/mg, respectively. The hydrolysis assay showed that rIDSGLUC9-25 primarily catalyzed the hydrolysis of β-glucan into cellotriose (representing 64.19%±1.19% of total reducing sugars) and cellotetraose (representing 26.24%±0.12% of total reducing sugars). Additionally, the enzyme predominantly generated cellotriose from the hydrolysis of lichenan (representing 78.46%±0.89% of total reducing sugars). [Conclusion] This study characterizes IDSGLUC9-25, an endo-β-1,4-glucanase (EC 3.2.1.4) derived fromTreponema sp. The enzyme exhibited robust activity in the conversion of polysaccharides into cellotriose and cellotetraose, establishing a foundation for the development of feed enzymes and functional oligosaccharides preparation.

[Objective] To construct a chromosome-plasmid balanced lethal system based on the glutamate racemase (MurI) gene for the expression of exogenous antigens in the attenuated vaccine strain ofPseudomonas plecoglossicida (Pp ΔtssD-1), so as to provide new ideas and methods for the development of multi-component live vaccines. [Methods] We constructed amurI-deleted strain fromPp ΔtssD-1 by homologous recombination. First, we replaced the kanamycin resistance gene of the pBBR1MCS-2 plasmid withmurI to construct a balanced lethal plasmid. Subsequently, we inserted the green fluorescent protein gene into the multicloning site of the plasmid to examine the expression stability of the exogenous antigen. Finally, we characterized the recombinant strain in terms of the growth curve, plasmid stability, and expression of the exogenous antigen. [Results] ThemurI-deleted strain was unable to grow in the lysogeny broth medium without D-glutamate. The non-resistant complemented strain regained growth capability in the lysogeny broth medium without D-glutamate. However, its growth was slower than that of the starting strain. Exogenously introduced antigens were identified as stable in the absence of antibiotic selection, and distinct green fluorescence signals were observed under a fluorescence microscope. Additionally, the balanced lethal plasmid exhibited high genetic stability within the recombinant strain. [Conclusion] A novel chromosome-plasmid balanced lethal system targetingmurI was developed in this study. It enabled the expression of exogenous antigens inPp ΔtssD-1 without the need for antibiotic selection. This system provides a new method for the development of multi-component live vaccines, with no need of antibiotic resistance markers and high plasmid stability.

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.