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.

[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] 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.

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.

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] 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] 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.

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] Colony extraction and counting is essential in agriculture, food, and health industries. Currently, most of the available algorithms for automatic counting of colonies use colony culture dishes and has poor applicability to colony count plates. In addition, the current technologies have good performance in conventional segmentation of adherent objects, while their accuracy remains to be improved for the segmentation and counting of adherent colonies due to the unique morphological characteristics of colonies. [Methods] To solve such problems, we proposed a colony segmentation and counting algorithm based on target color base and gradient direction matching. Firstly, the color feature of the colony in the image was used as a base to convert the image into a base space to enhance the difference between the colony and the background. Secondly, the gradient magnitude feature of the colony image was used to filter the gradient direction, and then the matching was performed through the gradient direction, thereby segmenting the adherent colonies. Finally, non-maximum suppression was employed to screen and count the colonies. [Results] Through experiments, the counting accuracy of the algorithm in this study reaches 98.00%, demonstrating its capability to meet practical requirements. [Conclusion] In the context of targeted segmentation and counting of colonies, the algorithm studied in this paper not only exhibits high counting accuracy but also demonstrates good robustness. This algorithm had not only high counting accuracy but also good robustness, producing excellent results in the colony segmentation and counting of colony count plates from different manufacturers. However, it showed decreased counting accuracy in the detection and segmentation of large-area targets. Therefore, this algorithm is suitable for the detection and segmentation of small targets such as colonies.

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.