[Objective] To apply multifunctional plant growth-promoting rhizobacteria to enhance peanut growth and mitigate the inhibitory effects caused by continuous cropping. [Methods] Plant growth-promoting rhizobacteria were screened from the rhizosphere soil of peanut plants in a system with continuous cropping for ten years, and their growth-promoting and antagonistic abilities were determined. The strains were identified by 16S rRNA gene sequencing. Three plant growth-promoting rhizobacterial strains with complementary functions and no growth inhibition between each other were selected to prepare a compound microbial inoculant, the plant growth-promoting effect of which was examined by seed germination and pot experiments. High-throughput sequencing was carried out for the V3–V4 region of bacterial 16S rRNA gene. [Results] A total of 37 plant growth-promoting rhizobacterial strains capable of promoting plant growth and inhibiting pathogen growth were screened from the rhizosphere of peanut plants in a continuous cropping system. Three strains were selected to prepare the compound inoculant. Compared with the blank control, the compound inoculant increased the germination rate of peanut by 13.22%. Compared with the treatments with the three strains alone, the compound inoculant increased the germination rate by 6.99%, 7.51%, and 8.87%, respectively. The application of the compound inoculant had significant promoting effects on the root morphology, number of nodules, chlorophyll relative content (SPAD), photosynthetic parameters, and antioxidant enzyme activity of peanut plants. Specifically, it increased the total root length, number of root tips, taproot diameter, root volume, and root activity by 43.50%, 49.31%, 15.11%, 16.92%, and 112.16%, respectively. The application of the compound inoculant significantly increased the leaf SPAD value and promoted the photosynthesis of peanut plants at seedling stage and flowering stage. Furthermore, it increased the number of root nodules by 34 nodules per plant. However, the application of the compound inoculant had no significant effect on the bacterial diversity in peanut rhizosphere. The dominant phyla wereProteobacteria,Actinobacteriota, andBacteroidota, accounting for more than 70%.Novosphingobium andSphingomonas were the dominant genera. [Conclusion] The compound inoculant of plant growth-promoting rhizobacteria improved the seed germination, root growth, leaf SPAD value, and photosynthesis of peanut plants, providing technical support for alleviating continuous cropping obstacles and promoting the healthy growth of peanut plants.

[Objective] To investigate the role of the small RNA (sRNA) RybB and the chaperone protein Hfq in regulating the expression of porin OmpD inSalmonella. [Methods] In this study,Salmonella Typhimurium (STM) was used as the research object. The pCE40 plasmid carrying the reporter genelacZ encoding β-galactosidase was transferred into the single mutant lackingompD to obtain thelacZ reporter strain. On this basis, we employed P22 phage-mediated transduction to construct the double mutants lacking full-lengthrybB, full-lengthhfq, partial sequence ofhfq, or truncatedhfq sequence and the triple mutantlacking full-lengthrybB and full-lengthhfq. The regulatory effects of RybB and Hfq on the expression of OmpD were probed by β-galactosidase activity assay and RT-qPCR. [Results] We successfully constructed the double and the triple mutant. Compared with that in the wild type (WT), the OmpD activity was down-regulated by 2.16% in thelacZ reporter strain with truncated sequence (87 residues) ofhfq, and the β-galactosidase activity of OmpD increased in the rest strains. Compared with WT, except for STM LT2∆ompD::lacZ∆hfq6, all the mutants showed up-regulated transcript level ofompD (P < 0.05), with the most significant up-regulation of 1.83-folds in the triple mutant. [Conclusion] The transcription and translation ofompD are mainly regulated by the negative feedback ofhfq and RybB. The distal end of Hfq plays a key role in the transcriptional repression ofompD. By construction of several mutants, this article illustrated the interactions of RybB and Hfq with OmpD and explored the key regions of Hfq in regulatingompD, which enriched the theory of sRNA regulation.

[Objective] To obtain the proteins of acetyl-CoA synthase (ACS_MU) and PHA synthase (PhaC_MU) fromMassilia sp. UMI-21 by structuring anin vitro recombinant expression system, and to elucidate their roles in the biosynthesis of polyhydroxybutyrate (PHB) using the one-phase reaction system (OPRS). [Methods] Seamless cloning was employed to ligate the acetyl-CoA synthase geneacs_MU and the PHA synthase genephaC_MU amplified fromMassilia sp. UMI-21 to the pQE-80L plasmid to construct the recombinant plasmids. The recombinant plasmids were transformed intoEscherichia coli BL21(DE3), and the recombinant strains were obtained. ACS_MU and PhaC_MU were purified using a 6×His tag, and their activities were determined by the 5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB) method. With 3HB as a substrate, the one-phase reaction system (OPRS) was employed to validate the functions of ACS_MU and PhaC_MU in the biosynthesis of PHB. [Results] The recombinant strains BL21-pQE-80L-acs_MU and BL21-pQE-80L-phaC_MU were successfully engineered, with the ACS_MU and PhaC_MU yields of 24.8 mg/L and 25.6 mg/L, respectively. The specific activity of ACS_MU was (0.148±0.011) U/mg, and that of PhaC_MU for (R)-3HBCoA was (0.102±0.011) U/mg. Nuclear magnetic resonance hydrogen spectroscopy (¹H-NMR) results showed that products from the all three PHB synthesis pathways, ACS_Pt-PCT_CP-PhaC_Re, ACS_MU-PCT_CP-PhaC_Re, and ACS_MU-PCT_CP-PhaC_MU, in OPRS were PHB. The yields of PHBvia the three pathways were 0.62, 0.76, and 0.64 g/L, respectively. [Conclusion] The genesacs_MU andphaC_MU can be overexpressed in theE.coli expression system to yield active soluble proteins. Compared with the ACS_Pt-PCT_CP-PhaC_Re pathway, substitution of ACS_Pt with ACS_MU increased the PHB yield by 22.58%. The yield of PHB was contingent upon the stability of acetyl-CoA synthase (ACS), which provided acetyl-CoA for reaction under identical PhaC. Replacing PhaC_Re with PhaC_MU decreased the PHB yield by 15.79% compared with ACS_MU-PCT_CP-PhaC_Re. The polymerase PhaC plays a crucial role in PHB synthesis under identical precursor concentrations.

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.

[Objective] To generateListeriamonocytogenes strains withlmo0880 deleted and complemented strains, so as to investigate the roles of Lmo0880 in bacterial infection in a host. [Methods] Thelmo0880-deleted strain was generated by homologous recombination, and the complementary strain was constructed by introducing an integrative plasmid carryinglmo0880 into thelmo0880-deleted strain. The growth, adhesion, invasion, and intracellular proliferation were compared between thelmo0880-deleted strain, complementary strain, and the wild type. [Results] The deletion oflmo0880 did not significantly impact bacterial growth or adhesion. However, it led to notable decreases in cell invasion, proliferation, and colonization in the liver and spleen, ultimately diminishing the pathogenicity in mice. [Conclusion] The LPXTG-anchored protein Lmo0880 plays a crucial role in bacterial invasion, proliferation, and colonization in a host. These findings provide a solid foundation for deeply understanding the pathogen-host interaction duringL.monocytogenes infection.

[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.

[Objective] Episomal expression vectors typically have higher copy number to achieve strong gene expression than chromosomal expression vectors. Moreover, they are more convenient and flexible for DNA manipulation. However, the episomal plasmids suitable for the application inRhodosporidium toruloides remain to be determined, and the expression of heterologous genes or CRISPR/Cas9-based genome editing needs to be achieved by integration, which is a key reason for the slow progress in its genetic modification. Thus, this work aims to construct an episomal plasmid ofR.toruloides, which facilitates the expression of heterologous genes and promotes the gene editing in a time-saving manner. [Methods] First, the possible autonomously replicating sequences (ARSs) in the phenylalanine ammonia-lyase gene (PAL) ofR.toruloides were mined. Specifically,PAL and its upstream and downstream sequences were amplified in segments and constructed into a plasmid containing the β-isopropyl malate dehydrogenase gene (LEU2). The recombinant plasmids were then introduced intoLEU2-deficientR.toruloides by the electroporation method. An ARS was then identified according to transformation efficiency. Then, theBTS1 gene encoding geranylgeranyl pyrophosphate synthase was selected as the knockout target, and its gRNA was constructed into the episomal plasmid based on the identified ARS. The color change of the transformant was observed to verify whether the episomal plasmid was successfully applied to the CRISPR/Cas9 system ofR.toruloides. [Results] In this work, an ARS was identified, based on which an episomal plasmid was constructed and applied to CRISPR/Cas9 editing inR.toruloides. Finally, the episomal plasmid-based gene knockout ofR.toruloides was successfully achieved. [Conclusion] This work enriched the existing tool library and provided a research basis and technical support for the application ofR.toruloides in synthetic biology.

[Objective] This study explored the proliferation dynamics and persistent spread characteristics ofCnaphalocrocis medinalis granulovirus (CnmeGV) inCnaphalocrocis medinalis, aiming to enrich the epidemiological knowledge about baculovirus and provide theoretical support for the efficient application of CnmeGV formulations. [Methods] Transcriptome sequencing was employed to determine the transcriptional levels of viral genes in the larvae infected with CnmeGV for 96 h and in the emerged adults. A quantitative detection marker based on the specific geneCmorf123 of CnmeGV was established to measure the proliferation dynamics and persistent spread characteristics of CnmeGV inC.medinalis. [Results] All the genes of CnmeGV were transcribed in the infected larvae, with the acetyltransferase gene showing the highest transcription level. However, no transcription of viral gene was detected in the transcriptome of adults. The measurement with the quantitative detection marker of CnmeGV showed that the replication level of viral gene remained stable within 48 h post infection. The copies of viral genes significantly increased 2 and 4 days post infection, and each nanogram of DNA contained 83 copies of viral genes 4 days post infection. After infection, no viral transcription was detected in the adults or eggs, while a few transcripts were detected in the pupae. Viral DNA was detected in more than 86.7% of pupa and pupal slough samples and while 13.3% of adults. Viral DNA was detected in both the eggs and the second-generation larvae after infection, while no viral DNA was detected on the egg after the surface treatment. [Conclusion] The proliferation level of CnmeGV inC.medinalis gradually increased and then remained stable within 4 days post infection. CnmeGV particles can be spread for two generations by the egg surface. The eclosion ofC.medinalis is the main pathway for clearing CnmeGV.

[Objective] Antibiotics as emerging pollutants have aroused wide concern. In view of the shortage of effective tetracycline-degrading strains, this study aims to screen and identify the strains for tetracycline degradation, analyze degradation properties and type, pinpoint the localization of active substances for bio-degradation, and evaluate the physiological toxicity of degradation products. [Methods] Tetracycline was used as the sole carbon source to screen out the target strain from tetracycline-contaminated pig sludge. The strain was identified based on colony morphology, physiological and biochemical characteristics, scanning electron microscopy images, and the 16S rRNA gene sequence. Different carbon sources, pH, and removal kinetics were employed to characterize the degradation process of the strain. Different components of the strain were extracted to determine the degradation type of tetracycline by the strain. Furthermore, the intracellular and extracellular fluids of the strain were used to degrade tetracycline, so as to determine the location of the active substance for degradation. Finally, the toxicity of the degradation products was assessed. [Results] The strain MEH2305 was screened out and identified asEnterobacter hormaechei, which showed the best degradation performance at pH 7.0 and with tryptone as the carbon source. Strain MEH2305 showed a total tetracycline removal rate of 68% on the 7th day of culturevia abiotic degradation and bio-degradation, and the removal rates of oxytetracycline and doxycycline hydrochloride were 53% and 56%, respectively. The tetracycline removal efficiency by the intracellular and extracellular fluids of MEH2305 was 40.77% and 31.18%, respectively. Compared with tetracycline control without MEH2305, the tetracycline degradation products of MEH2305 had reduced physiological toxicity on Gram-negativeEscherichia coli K88 and Gram-positiveBacillus subtilis 168. [Conclusion] The strain MEH2305 can be used as an effective and safe tetracycline-degrading strain for the treatment of antibiotics in the environment.

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.