Abstract:Algae and bacteria are both the oldest forms of life on our planet, and billions of years of natural evolution have driven the algae and their microbiomes to evolve into interactive phycobionts. Through complex, flexible, intelligent, and multi-interface interactions between algae and bacteria, the functions of both sides of the phycobionts are exquisitely regulated. The creation, innovation, and development of the phycobiont theory shows vital scientific value for revealing the mystery of the origin and evolution of the life on Earth, and this theory is also being transformed into diverse practical applications in significant fields for sustainable development. After centuries of incubation, knowledge accumulation and development, currently, it is the right time to promote Phycosphere Microbiology to develop into an emerging interdiscipline. In this review, we comprehensively discussed the core concepts of Phycosphere Microbiology, sorted out its vital relationships with environment protection, human health maintenance, resource utilization, and green-oriented transition of energy, then reviewed its development history, and summarized the main research achievements during three development periods. Finally, we also proposed and discussed the future development trends and potential research directions for this emerging interdiscipline.

Abstract:Activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme catalytic peptide (APOBEC) constitute a conserved family of cytidine deaminase enzymes. The family members have different functions in the body, and they play an important role in the immune defense of the host. AID plays a role mainly in the adaptive immune systems of vertebrates, mediating class switch recombination, antibody affinity maturation, and antibody diversity generation. APOBEC1 capable of catalyzing cytosine deamination, mediating RNA editing for cellular regulation, and resisting retroviral infection is involved in tumorigenesis and cancer development. APOBEC2, most abundant in cardiac and skeletal muscle, is associated with muscle fiber type switch, loss of weight, muscle development, and myopathy. Moreover, it may have potential indirect effects in controlling gene expression. APOBEC3s play key roles in both innate and adaptive immune responses. They are involved in the inhibition of retrotransposon functioning and viral infection, DNA degradation, RNA editing, and cell cycle regulation. The APOBEC4 gene is conserved in various animal species, with the active center sequence different from those of other APOBEC proteins. It is widely recognized that APOBEC4 is a uridine-editing enzyme, which has antiviral activity. The research is limited regarding the animal-derived APOBEC family members. This review describes the structural characteristics and biological functions of APOBEC family members, providing reference for research on the roles of animal-derived APOBEC family members in the immune responses and disease control. In addition, this review provides new ideas for the development of antivirals by enhancing the activities of APOBEC family members.

Abstract:Bacillus subtilis is a generally recognized as safe (GRAS) probiotic and an excellent industrial chassis strain. It possesses advantages such as strong heterologous protein secretion capability, robust growth with low-quality carbon sources, and negligible codon bias. Since 2016, clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has been successfully applied to B. subtilis, enabling precise genetic modifications, including point mutations, gene knockout, foreign gene insertion, gene expression regulation, and base editing. These advancements have significantly promoted the development of B. subtilis as an efficient microbial cell factory and have shown broad application potential in agriculture, pharmaceuticals, food production, and synthetic biology. This paper systematically review the development of the CRISPR system in B. subtilis and summarize its application in the efficient production of various products. The aim is to provide insights into the targeted optimization of metabolic pathways in B. subtilis via the CRISPR system to achieve efficient and stable industrial production of target products, as well as to offer references for the further development and application of novel gene editing systems.

Abstract:Organic contamination of crops poses a threat to the safety of food products and human health, and it is urgently needed to be solved. Endophytic bacteria are indispensable in microecosystems. In recent years, researchers have screened and isolated endophytic bacteria with the function of degrading pollutants from the environment. These bacteria have been used to regulate the metabolic processes of organic pollutants in crops, which achieve the efficient reducing of toxic organic pollutants in crops. This paper reviews the research progress in the reduction of toxic organic pollutant accumulation in crops by functional endophytic bacteria, focusing on the degradation genes, products, and pathways of toxic organic pollutants in crops after the colonization of functional endophytic bacteria. Furthermore, it discusses the factors affecting the degradation efficiency of functional endophytic bacteria and emphasizes the importance of these bacteria in reducing organic pollutants in crops. This review provides ideas and a basis for the further utilization of endophytic bacteria to control the risk of organic contamination in crops.

Abstract:Tuberculosis is an ancient zoonotic disease that poses a serious threat to the health of humans and animals worldwide. Mycobacterium tuberculosis (Mtb) is the primary causative agent of tuberculosis. As a bridge between innate and adaptive immunity, dendritic cells (DCs) play a pivotal role in controlling Mtb infection by utilizing their potent antigen-presenting capacity to activate the adaptive immune response of the host and thus resist further infection. In recent years, more and more studies have shown that Mtb can evade host immune defenses by regulating DC differentiation and maturation, interfering with phagocytosis and autophagy, and inhibiting the expression of antigen presentation-related molecules, thus causing persistent infection. This review summarizes the current research on the molecular mechanisms of Mtb in regulating DC antigen presentation, aiming to provide insights for further study of Mtb-DC interaction mechanism and development of prevention and control strategies for tuberculosis.

Abstract:Cryptococcus neoformans is a common opportunistic pathogen, exhibiting pronounced neurotropism that often results in cryptococcal meningitis. Its invasive ability is closely associated with multiple factors, including capsular polysaccharides, melanin, hydrolases, and adaptability to the host environment. Conventional diagnostic methods such as fungal culture and India ink staining, though still in use, have notable limitations, whereas emerging techniques like molecular diagnostics, imaging technologies, and biochips have significantly enhanced the diagnostic accuracy and sensitivity. In clinical treatment, amphotericin B and fluconazole are widely used as first-line antifungals, while the resistance to azoles is a growing problem and results in an elevated rate of clinical treatment failure. This is mainly attributed to target alterations, upregulation of efflux pump expression and genomic ploidy changes. Recent studies on virulence factors and resistance mechanisms have driven the development of novel antifungal therapies, including drug repurposing, novel drug development, and innovative drug delivery strategies. This article reviews the latest research in the virulence factors, diagnostic techniques, antifungal resistance mechanisms, and therapeutic development of C. neoformans, providing insights into the clinical management of cryptococcosis.

Abstract:Collagen is the most abundant protein in mammals, accounting for about one-third of human protein. As an important component of the connective tissue and extracellular matrix, collagen is essential for maintaining physiological functions and repairing injuries and has important applications in the fields of medicine, food, and beauty. The main methods for producing collagen are natural extraction, chemical synthesis, and biosynthesis. Natural extraction from animal connective tissue has ethical issues, unstable quality, and infectious disease risks. Chemical synthesis is costly and it is not easy to synthesize complex collagen structures. Biosynthesis enables the production of recombinant collagen for different purposes by genetic engineering in a more controllable, safer, and more precise manner. However, due to the complex structure of collagen, its biosynthesis depends on specific molecular chaperones and modifying enzymes, and thus the production of recombinant collagen is challenging. In addition, different types of collagen need to form particular tissue structures, such as fibril, reticular, or transmembrane structures, which further increases the difficulty of production. This article clarifies the multifunctionality of recombinant human collagen, reviews the latest progress and challenges in its biosynthesis, and looks forward to future development directions. This review aims to help researchers, engineers, and industry practitioners understand the research trends of recombinant collagen and promote its further development and commercialization in different application fields.

Abstract:The bacterial stringent response refers to the adaptive reaction that bacteria exhibit when faced with adverse environmental conditions, altering their metabolism and reducing the growth rate to enhance survival and adaptability. The rapid accumulation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp in this article, mediates the stringent response, playing a crucial role in microbial adaptation to environmental changes. The levels of (p)ppGpp within bacteria are regulated by RelA/SpoT homologue (RSH) proteins, which include small alarmone synthetases (SASs), small alarmone hydrolases (SAHs), and bifunctional proteins such as Rel. Furthermore, recent studies have identified a new bacterial alarmone, adenosine tetraphosphate (ppApp) and adenosine pentaphosphate (pppApp), collectively referred to as (p)ppApp, which is involved in the regulation of various biological processes in bacteria. The enzymes involved in (p)ppGpp metabolism vary among different bacterial species. This study systematically classifies and reviews the structural and biochemical characteristics of the known RSH proteins and summarizes their biochemical functions, aiming to promote further exploration and development in this field.

Abstract:Objective To provide environmental sustainable, safe, and efficacious management approaches for root rot impacting a range of crops in the unique agro-ecosystems of Gansu and Qinghai Provinces.Methods The plate confrontation method and the organophosphorus agar plate were used for preliminary screening of 305 strains of tested bacteria, and the strains obtained from preliminary screening were re-screened with the fermentation broth method. Subsequently, the nitrogen-fixing, phosphate-solubilizing, and potassium-solubilizing abilities of the strains were determined by the Kjeldahl method, ultraviolet spectrophotometry, and flame photometry, respectively. The siderophore-producing activity, the IAA content in the fermentation broth, as well as the acid, alkali, and salt tolerance of the strains, were determined by spectrophotometric methods. Finally, targeting the pathogens causing root rot in various crops in Gansu and Qinghai Provinces, bacterial consortia were constructed with different functional strains for disease prevention and plant growth promotion. The plant growth-promoting and antifungal effects of different consortia were evaluated, and the best consortium was selected. Furthermore, the 16S rRNA gene and gyrB of the strains in the best consortia were sequenced for identification. The root rot-preventing and plant growth-promoting effects of the best consortium were evaluated by the pot culture method.Results A total of 86 antagonistic strains and 134 phosphate-solubilizing strains were preliminarily screened out, and 20 antagonistic strains were selected after re-screening, among which strains K87 and LB17 demonstrated excellent broad-spectrum antifungal effects. Specifically, K87 showed inhibition rates of 87.53%, 74.90%, 75.15%, 79.69%, and 88.43% against Fusarium avenaceum, F. equiseti, F. oxysporum, F. solani, and Microdochium bolleyi, respectively. LB17 exhibited inhibition rates of 61.89%, 87.52%, and 87.23% against F. oxysporum, F. solani, and Bipolaris sorokiniana, respectively. Among the 8 strains with superior plant growth-promoting abilities, LB17 had the strongest siderophore-producing activity, with an iron carrier activity unit (su) value of 0.32, and K113 exhibited a good nitrogen-fixing capability, fixing nitrogen at a rate of 0.08 g/L. K87 secreted the highest amount of IAA, which reached 9.87 mg/L. MP6 had the greatest ability to solubilize inorganic phosphorus, with a solubilization rate of 1 470.69 μg/mL, while K85 showed the best performance in solubilizing organic phosphorus, with a solubilization rate of 1 321.23 μg/mL. MP41 excelled in potassium solubilization, with a solubilization rate of 140.33 mg/L. Ultimately, 14 bacterial consortia were constructed, in which T2 exhibited the best synthetic performance, with a nitrogen-fixing rate of 0.212 g/L, a potassium solubilization rate of 86.28 mg/L, and an IAA secretion rate of 16.91 mg/L. Moreover, its inhibition rates against the 6 pathogenic fungi all reached over 60.00%, and even 87.69% against F. equiseti. Strains LB17, K87, and MP6 in this consortium were all identified as Bacillus velezensis. T2 demonstrated significant biocontrol efficacy against root rot in naked barley, with the control effects exceeding 70.00%, and exhibited remarkable plant growth-promoting properties.Conclusion This study developed an efficient bacterial consortium for the management of crop root rot and the promotion of crop growth in the unique agro-ecosystems in Gansu and Qinghai Provinces.

Abstract:Objective The rhizosphere microorganism-plant combined approach has high application potential for the remediation of heavy metal-contaminated soil. This study observed the effects of adding exogenous plant growth-promoting bacteria (PGPB) on the growth and molybdenum (Mo) accumulation of alfalfa (Medicago sativa), aiming to provide theoretical references for plant-microbial remediation of Mo-contaminated soil.Methods The endophytic bacteria were isolated from dominant plants of Mo tailing and they were identified based on morphological characteristics and molecular evidence. The plant growth-promoting (PGP) properties of molybdate-reducing strains were determined. By adding exogenous PGPB into the soil, we investigated the effects of adding exogenous PGPB on the biomass, physiological activity, and Mo accumulation of alfalfa.Results Two molybdate-reducing strains M9 and M13 were obtained and identified as Serratia plymuthica based on morphological characteristics, 16S rRNA gene sequence, and gyrB sequence. M9 and M13 had the abilities to fix nitrogen, solubilize phosphorus, solubilize potassium, and secrete indole-3-acetic acid (IAA), siderophores, and 1-amino cyclopropane-1-carboxylic acid (ACC) deaminase. Under Mo stress, the inoculation of M9, M13, and M9+M13 significantly promoted the growth of alfalfa, increasing the plant height, root length, and fresh weight of alfalfa compared with the non-inoculation control group. At the same time, the inoculation increased the chlorophyll content and peroxidase (POD) activity while decreasing the malondialdehyde (MDA) content in alfalfa. M9 and M13 significantly affected the Mo accumulation of alfalfa. The Mo content in the above-ground and under-ground parts of alfalfa inoculated with M9, M13, and M9+M13 significantly decreased compared with that in the non-inoculation control group. The decreased enrichment factor of Mo in alfalfa indicated that inoculation with molybdate-reducing strains reduced the uptake and transport of Mo in alfalfa.Conclusion The molybdate-reducing strains M9 and M13 can promote the growth and reduce the Mo content of alfalfa in Mo-contaminated soil. This finding can provide theoretical reference for revealing the mechanism of microbial-enhanced Mo remediation by plants as well as the joint remediation of Mo-contaminated soil by plants and microorganisms.

Abstract:Objective Phosphate-solubilizing bacteria (PSB) can increase available phosphorus by promoting the transformation of different phosphorus forms in soil. However, the phosphate-solubilizing ability of PSB is influenced by soil texture. Therefore, we studied the changes of phosphorus fractions in red soil with different textures and treated with PSB, with the aims of improving the efficient utilization of soil phosphorus.Methods We collected the rhizosphere soil samples of four main Camellia oleifera varieties (‘Huashuo’ ‘Huaxin’ ‘Huajin’ and ‘Changlin No. 40’) in Liuyang City, Hunan Province. PSB strains were isolated, purified, screened, and identified by plate coating, transparent circle method, molybdenum-antimony anti-spectrophotometric method, and 16S rRNA gene sequencing, and a batch of highly efficient PSB strains were obtained. The phosphate-solubilizing abilities of the obtained PSB strains were investigated with different phosphorus sources (iron phosphate, aluminum phosphate, calcium phosphate, and calcium phytate). PSB were inoculated into the red soil samples added with 0, 20% and 40% perlite (0%PR, 20%PR and 40PR%) to clarify the changes in phosphorus fractions in the red soil samples with different textures.Results A total of 57 strains of PSB were isolated from the rhizosphere of C. oleifera, of which strains CL37, HS5, and CL36 exhibited stronger phosphate-solubilizing abilities. The three strains were identified by 16S rRNA gene sequencing and named Pantoea sp. CL37, Burkholderia sp. HS5, and Burkholderia sp. CL36, respectively. The three strains showed significant differences in their ability to solubilize different phosphorus sources, with the highest solubilizing ability for calcium phytate and calcium phosphate. Compared with CK, the inoculation of PSB increased the available phosphorus (AP) in soil by 8.90%-54.60% and 1.90%-56.00% in 20%PR and 40%PR, respectively. The inoculation with PSB increased Fe-P, Resin-P, NaHCO₃-Pi, and NaOH-Pi, which showed a tendency of first increasing and then decreasing along with the increase in the addition of perlite in red soil. Meanwhile, PSB decreased the content of HCl-Pi and Residual-P, and the decrease in HCl-Pi was more pronounced in 20%PR than in 0%PR and 40%PR. Of all treatments, the inoculation with HS5 led to the highest increases in Fe-P, Resin-P, NaHCO₃-Pi, and NaOH-Pi in the soil samples analyzed. Correlation analysis and random forest analysis suggested that AP was mainly affected by Resin-P, Al-P, NaOH-Pi, acid phosphatase, urease, pH, and NaOH-Po.Conclusion The phosphate-solubilizing pathways of PSB vary in red soil with different textures. Strain HS5 has a strong phosphorus-transforming ability and is more conducive to phosphorus transformation in the red soil with 20%PR.

Abstract:As autotoxic substances secreted by plant roots, phenolic acids such as p-hydroxybenzoic acid (PHBA), are the main factors causing continuous cropping obstacles.Objective To study the effects of Microbacterium aurantiacum on the growth of tomato plants and the microbial community structure in rhizosphere soil under PHBA stress.Methods We irrigated tomato rhizosphere with the suspension of M. aurantiacum GX14001 and then measured the growth traits of tomato plants and the soil microbial community changes in the rhizosphere soil.Results Under the PHBA treatment, GX14001 significantly promoted the growth of tomato plants, increasing the leaf area, stem diameter, and plant height by 244.0%, 156.5%, and 128.0%, respectively. GX14001 increased the richness but did not cause changes in the diversity of bacteria in the rhizosphere soil. Meanwhile, it decreased the richness and diversity of fungi in the rhizosphere soil. At the phylum level, compared with the control group, the GX14001 group showed increased relative abundance of Actinobacteriota, Chloroflexi, and Proteobacteria, with Ascomycetes as the dominant fungal phylum.Conclusion M. aurantiacum GX14001 promotes the growth of tomato plants by changing the microbial community structure in the rhizosphere soil. It increases the relative abundance of beneficial microorganisms in the soil to create a favorable environment for tomato growth.

Abstract:Objective Vibrio parahaemolyticus is a major foodborne pathogen that causes acute gastroenteritis in humans. Here, we aim to decipher the mechanisms by which the histidine kinase EnvZ regulates the swarming motility and biofilm formation of V. parahaemolyticus.Methods The plasmids pBAD24 and pMal carrying inducible promoters were used to construct the plasmids carrying target genes for overexpression. The recombinant plasmids were introduced into the wild-type strain (WT) and envZ-deleted strain (ΔenvZ) of V. parahaemolyticus. Swarming plates were prepared to measure swarming motility, while biofilm formation was detected by crystal violet staining. The RT-qPCR and bioluminescence reporter assays were employed to explore the mechanisms by which EnvZ regulated the expression of downstream genes.Results The swarming motility of ΔenvZ was significantly lower than that of WT, while the introduction of the pBAD24-envZ plasmid into ΔenvZ restored its swarming ability. The transcription levels of the lateral flagellar genes were positively correlated with the swarming motility. The promoter activities of P_scrABC-lux in ΔenvZ and ΔenvZΔompR were lower than those in WT and ΔompR. The swarming ability of ΔenvZ was significantly increased when the Scr system was overexpressed via the pMal-scrABC plasmid, while the overexpression of EnvZ in the strain without scrABC did not change the swarming motility. In addition, ΔenvZ exhibited a significant decrease in biofilm formation compared with WT. The pMal-envZ plasmid restored the biofilm formation of ΔenvZ to the level of WT, whereas the pMal-scrABC plasmid did not have this effect. The promoter activity of the extracellular polysaccharide operon (epsA-J) and the transcription levels of the extracellular polysaccharide genes in ΔenvZ were both significantly lower than those in WT.Conclusion The histidine kinase EnvZ enhances the swarming motility of V. parahaemolyticus by regulating the expression of the Scr system and promotes the biofilm formation by regulating the expression of extracellular polysaccharides.

Abstract:Objective To investigate the inhibitory effect and mechanism of thymol on the biofilm formation of Proteus mirabilis (PM).Methods The minimal inhibitory concentration (MIC) and minimal biofilm inhibitory concentration (MBIC) of thymol were determined by the broth micro-dilution method. The time-kill curve of thymol was established by the colony counting method. Crystal violet staining was performed to determine the inhibitory effect of thymol on the biofilm formation of PM. The effects of thymol on the swarming and swimming of PM and the extracellular polymeric substances (extracellular polysaccharides, extracellular proteins, and eDNA) of the biofilm were determined. RT-qPCR was employed to investigate the effects of thymol on the expression levels of PM biofilm-related genes: rsbA (encoding histidine-containing phosphotransmitter of the bacterial two-component system), flhD (encoding the flagellar regulon), and mrpA (encoding mannose-resistant Proteus-like fimbriae).Results The MIC of thymol against PM was 0.156 mg/mL. Thymol significantly inhibited the biofilm formation of PM, with the MBIC of 0.313 mg/mL. Thymol inhibited the motility of PM, reduced the formation of extracellular polysaccharides, proteins, and eDNA in PM biofilm, and decreased the expression levels of rsbA, flhD, and mrpA.Conclusion Thymol inhibits the biofilm formation of PM by suppressing the motility and formation of extracellular polymers and down-regulating the expression of biofilm-related genes.

Abstract:Objective Staphylococcus epidermidis is a coagulase-negative, Gram-positive coccobacillus that is widely found in the skin, breast milk, and blood. Bacteria in breast milk play a crucial role in the establishment of the gut microbiota in the intestinal tract and in the enhancement of immunity of infants. We then performed comparative genomic analyses to understand the genetic diversity and functional genes of breast milk-derived S. epidermidis.Methods We used the Illumina NovaSeq platform to sequence the genomes of 110 strains of S. epidermidis preliminarily isolated from healthy breast milk by our research team. We then performed comparative genomic analyses for the 110 strains and 263 skin, blood, and breast milk-derived S. epidermidis strains publicly available from the NCBI.Results The genome size of the 373 strains of S. epidermidis was (2.50±0.33) Mb, with the G+C content was (32.0±0.1)%, and the number of coding sequences (CDs) being 2 331±368. Differences existed in genome size and number of CDs among breast, blood, and skin isolates (P<0.05), with blood isolates having the largest genome size and the highest number of CDs. The phylogenetic tree showed that the S. epidermidis isolates of the same source had obvious aggregation, and the breast milk isolates and blood isolates were more closely related. There were differences in the number of virulence factors and drug resistance genes among the strains of three sources (P<0.05). The blood-derived strains exhibited the highest diversity of virulence and resistance genes, whereas the breast milk-derived strains displayed the lowest diversity of such genes.Conclusion S. epidermidis has undergone adaptive evolution to different habitats. Compared with skin- and blood-derived strains, breast milk-derived strains carry few genes related to biofilm synthesis, drug resistance, and virulence. This study gives new insights into the adaptive evolution of S. epidermidis and provides a theoretical basis for subsequent research on the genetic background of breast milk isolates.

Abstract:Insects have a mutually dependent symbiotic relationship with their gut microbiota, which plays an important role in the insect metabolism, immunity, development, and pesticide resistance. Gut microbiota is influenced by factors such as diet, sex, and rearing environment. Currently, little is known about the gut microbiota differences between males and females of the phytophagous insect Dolycoris baccarum.Objective To study the effects of sex on the composition and abundance of gut microbiota in D. baccarum, explore sex-related microorganisms, and provide a basis for utilizing different sex-associated strains in the biocontrol of D. baccarum.Methods The PacBio platform was used for third-generation 16S rRNA gene amplicon sequencing and the bioinformatics analysis was performed to reveal the diversity and composition of gut microbiota in male and female D. baccarum. The bacterial isolation and culture method was employed to obtain gut microbiota strains from D. baccarum, which were then identified by morphological analysis and 16S rRNA gene sequencing.Results The gut microbiota of D. baccarum was diverse, including a total of 165 genera belonging to 109 families, 60 orders, 29 classes of 14 phyla. The gut microbiota was analyzed at six taxonomic levels (phylum, class, order, family, genus, and species). Only the phylum Deinococcota and the class Deinococci were specific to males, while the phyla Chloroflexi, Desulfobacterota, and Cyanobacteria, the class Cyanobacteriia, the genus Lacticaseibacillus, and the species Glutamicibacter creatinolyticus, Acinetobacter, and Lacticaseibacillus paracasei were specific to females. The relative abundance of Serratia marcescens was significantly higher in males than in females. In the guts of females and males, the relative abundance of Gammaproteobacteria, Enterobacterales, Yersiniaceae, Serratia, and S. marcescens was 51.223% and 95.512%, 49.784% and 95.492%, 2.385% and 10.377%, 2.383% and 10.372%, and 2.310% and 10.375%, respectively. All 28 strains isolated in vitro and identified based on morphological characteristics and 16S rRNA sequences belonged to Serratia.Conclusion There are significant differences in the gut microbiota composition between male and female D. baccarum. The diversity of gut microbiota in male adults of D. baccarum is significantly higher than that in female adults. All strains isolated belong to Serratia. This study provides theoretical support for investigating the potential functions of gut microbiota in male and female D. baccarum as well as for the biocontrol of D. baccarum.

Abstract:Aeromonas hydrophila is a pathogen that can infect both fish and mammals, including humans. Objective To construct the tolA-deleted strain of A. hydrophila ATCC 7966 and use this strain to explore the biological functions of tolA. Methods We constructed the tolA-deleted strain AhΔ tolA by homologous recombination and characterized the physiological phenotype of AhΔ tolA. Transcriptome sequencing was performed to compare the gene expression between the wild type (WT) and AhΔ tolA. Results The cell morphology of AhΔ tolA was changed. The deletion of tolA significantly enhanced the sensitivity to sodium deoxycholate and oxidative stress, while significantly reducing the biofilm formation and the expression levels of several virulence genes. The yield of outer membrane vesicles was significantly increased in AhΔ tolA. Transcriptomic analysis data showed that a total of 300 differentially expressed genes (DEGs) were screened between WT and AhΔ tolA, including 171 genes with up-regulated expression and 129 genes with down-regulated expression. GO enrichment analysis showed that the DEGs were mainly enriched in the oxidation-reduction process, metabolic process, outer membrane, and oxidoreductase activity. KEGG pathway enrichment analysis showed that the DEGs were mainly enriched in the biosynthesis of secondary metabolites, microbial metabolism in diverse environments, biosynthesis of cofactors, and biosynthesis of amino acids. Conclusion This study gives an insight into the roles of tolA in A. hydrophila and provides information about the metabolic pathways involving tolA. These results provide a theoretical reference for the prevention and control of A. hydrophila.

Abstract:N-Heterocyclic carbine-silver (Ag-NHC) complexes possessing excellent stability, water solubility, and bactericidal activity are antimicrobial candidates with great potential. Objective To study the inhibitory activity and mechanism of a novel Ag-NHC complex 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene silver (I) acetate (SBC3), synthesized by Matthias Tacke’s team against Escherichia coli. Methods Visible spectrophotometry was employed to examine the antimicrobial activity of SBC3. Transmission microscopy was employed to observe the morphological changes of DHB4 cells post SBC3 treatment. Flow cytometry was performed to detect the effect of SBC3 on the intracellular reactive oxygen species (ROS) content. The 5,5′- dithiobis-(2-nitrobenzoic acid) (DTNB) assay was used to determine the intracellular thioredoxin (Trx) and thioredoxin reductase (TrxR) activities and the content of glutathione (GSH) post SBC3 treatment. Dithiothreitol (DTT), the ROS scavenger, was added to rescue DHB4 from ROS. SBC3-resistant strains (SRSs) were obtained by successive passaging in the laboratory. The obtained strains showed the minimal inhibitory concentrations (MICs) against SBC3 being 24, 32, and 56 μg/mL, respectively, which were 3, 4, and 7 folds of the MIC of WT. The three strains were named SRS3, SRS4, and SRS7 and then used to retest the above indicators. Western blotting was performed to determine the expression levels of Trx1 and S-glutathionylated proteins ( S-PSSG) post SBC3 treatment. Results The MIC values of SBC3 against tested pathogens were 8.0–30.0 μg/mL. The DHB4 cells treated with SBC3 underwent swelling, which was accompanied by contents leakage. SBC3 significantly inhibited the Trx and TrxR activities, reduced the GSH content, and elevated the ROS level in DHB4. SBC3 treatment decreased the Trx and TrxR activities, reduced the GSH content, and down-regulated the expression of S-PSSG in SRS3, SRS4, and SRS7. However, all the above indicators were increased to different extent compared with those in DHB4. Conclusion SBC3 can target the thiol-dependent redox system (TDRS) of E. coli to exert antibacterial effects. This study provides a new idea for the design of SBC3 as a novel antimicrobial agent.

Abstract:Salmonella Enteritidis is a major foodborne pathogen that can cause gastrointestinal infections in both humans and animals. As one of the key genes encoding the iron-sulfur cluster assembly, iscA plays a role in the transport of iron ions and energy metabolism. IscA is a conserved A-type iron-binding protein. Objective To study the role of iscA in the infection process of Salmonella by constructing an iscA-deleted mutant (Δ iscA) of Salmonella Enteritidis Z11. Methods The unmarked in-frame gene deletion method was employed to construct Δ iscA from the laboratory-preserved Salmonella Enteritidis Z11 strain. The wild type (WT) and Δ iscA were compared in terms of motility and biofilm formation. Additionally, the impact of IscA on the virulence of Salmonella Enteritidis was explored in both RAW264.7 cells and a mouse model. Results The deletion mutant Δ iscA was successfully constructed. No significant difference in the growth or biofilm formation was observed between Δ iscA and WT, indicating that the deletion of iscA did not affect the normal growth or biofilm formation of Salmonella Enteritidis. However, Δ iscA exhibited a significantly smaller zone of motility than WT at the time point of 6 h, suggesting that the loss of iscA reduced the motility of Salmonella Enteritidis Z11. In RAW264.7 cells, the adhesion and invasion of Δ iscA significantly decreased to 37% and 20%, respectively, of those of WT. Furthermore, the proliferation rate of Δ iscA in the cells was significantly lower than that of WT. Mouse infection experiments revealed that Δ iscA demonstrated reduced colonization in the jejunum and cecum compared with WT. Conclusion iscA is closely associated with the virulence of Salmonella Enteritidis. Its deletion affects the motility, adhesion, invasion, and proliferation, ultimately reducing the colonization in the host intestine and influencing the infection process of Salmonella Enteritidis.

Abstract:Objective Waste printed circuit boards (PCBs) contain about 40% non-ferrous metals, which is more than 40 times that of natural ores, and thus they are known as a veritable “urban mine”. To achieve efficient and green recycling of PCBs, this study developed a leaching system that can efficiently leach non-ferrous metals from PCBs.Methods Acidithiobacillus ferrooxidans MA-Y1 was used to construct a bioleaching system for PCBs. Additionally, an electric current was introduced to strengthen the Cu and Ni leaching from PCBs.Results The optimal leaching parameters were addition of PCBs at 80.0 g/L, particle size of 4.0 cm, liquid velocity of 2.0 L/min, and electric current of 70.0 mA. Under these conditions, the leaching ratios of Cu and Ni from PCBs were 84.0% and 75.3%, respectively, which represented increases of 15.8% and 17.1% compared with the case without electric current.Conclusion An electric current improves the ability of A. ferrooxidans MA-Y1 to recover Cu and Ni from PCBs.

Abstract:Objective A Talaromyces purpureogenus strain with solubilizing effects on tricalcium phosphate and calcium phytate was screened from the rhizosphere soil of maize and named XZY3PSF. To promote the application of this strain in serving agricultural production, we optimized the culture conditions for phosphorus solubilization of this strain and studied the effects of this strain on the growth and quality of red cluster pepper.Methods We evaluated the solubilizing effects of strain XZY3PSF on Ca₃(PO₄)₂, FePO₄, AlPO₄, phosphate rock powder, soybean lecithin, and egg yolk by measuring the available phosphorus content in the solution after liquid shaking culture. The effects of carbon source, nitrogen source, pH, and liquid loading volume on the phosphorus solubilization of this strain were studied. Pot experiments were carried out in a greenhouse to evaluate the effects of this strain on the growth and fruit quality of red cluster pepper.Results The carbon sources used by the strain were in an order of fructose>glucose>maltose>sucrose, and the nitrogen sources followed an order of ammonium chloride>ammonium nitrate>ammonium sulfate>potassium nitrate>urea. The phosphorus-solubilizing ability of the strain was higher under the condition of initial pH 6.0-7.0, and it was not affected by the liquid loading volume. The strain achieved higher phosphorus solubilization with tricalcium phosphate and phosphate rock powder as the phosphorus sources. With tricalcium phosphate as the phosphorus source, the strain showed the highest phosphorus solubilization on days 5, which was higher than that with other phosphorus sources (P<0.01). With phosphate rock powder as the phosphorus source, the strain showed the highest phosphorus solubilization on days 21 (P<0.01). Compared with the application of compound fertilizer, the inoculation of this strain increased the plant height, yield, vitamin C content, and total capsaicinoid content of red cluster pepper (P<0.05). The results of Pearson correlation analysis showed that the phosphorus solubilization of strain XZY3PSF in the culture medium with different nitrogen or carbon sources was negatively correlated with pH and the dry weight of mycelia. The capsaicinoid content was positively correlated with vitamin C and sugar content of pepper fruits.Conclusion Strain XZY3PSF has a strong ability to solubilize phosphorus, especially aluminum phosphate and iron phosphate, while improving the main nutritional indicators of crops. It is hypothesized that the strain will have a strong ability to solubilize phosphorus in the laterite soil in southern China. These findings lay a foundation for subsequent application of this strain in the fieled.

Abstract:Objective To analyze the metabolic substrates required for the sclerotial formation of Rhizoctonia solani and understand the influence of nutritional elements and environmental factors on this process.Methods Biolog phenotypic microarray was used to study the effects of 663 nutritional substances, 96 osmotic environments, and 96 pH environments on the sclerotial formation of R. solani.Results Among the tested nutritional substances and environmental conditions, 19/95 carbon sources, 21/95 nitrogen sources, 16/94 phosphorus and sulfur sources, 69/94 nutritional supplements, 61/282 peptide nitrogen sources, 28/96 osmotic environments, and 40/96 pH environments induced the sclerotial formation of R. solani. Notably, N-acetyl-d-glucosamine, uridine 3′-monophosphate, phosphoryl choline, and five dipeptides (Arg-Trp, Met-Arg, Pro-Phe, Val-Tyr, and Val-Met), as well as three environmental conditions of 10 mmol/L and 20 mmol/L ammonium sulfate at pH 8.0, and pH 4.5+l-proline, significantly induced the sclerotial formation of R. solani. R. solani formed sclerotia in the environments with a broad range of pH 4.0-10.0. The KEGG analysis indicated that the substances inducing sclerotial formation were primarily involved in metabolic pathways, ABC transporters, secondary metabolite biosynthesis, and d-amino acid metabolism.Conclusion Nutrient limitation and environmental stress are key factors inducing the sclerotial formation of R. solani. Under nutrient-restricted conditions, the suitable substances for inducing sclerotial formation include five carbon sources (d-sorbitol, d-xylose, N-acetyl-d-galactosamine, d-arabinose, and d-melezitose), three nitrogen sources (N-acetyl-d-glucosamine, adenosine, and thymidine), two phosphorus sources (uridine 3′-monophosphate and phosphoryl choline), one nutritional supplement (Tween-80), and five peptide nitrogen sources (Arg-Trp, Met-Arg, Pro-Phe, Val-Tyr, and Val-Met). The suitable osmotic environments were 10 mmol/L and 20 mmol/L ammonium sulfate at pH 8.0, and the suitable pH environments were pH 4.0-4.5 and pH 9.5-10.0. These findings provide a foundation for understanding the sclerotial formation mechanism of R. solani.

Abstract:Objective This study characterized a novel esterase EstE from Streptomyces griseus by heterologous expression in Escherichia coli and systematically evaluates its thermostability, alkaline stability, and the effects of various additives (metal ions, detergents, and organic solvents) on its enzymatic activity to explore its potential for industrial applications.Methods We synthesized the gene estE' encoding the same amino acid sequence as the native gene by optimizing the original sequence of estE from S. griseus. We then constructed the recombinant plasmid carrying the optimized gene by ligating the gene into the pET-28b(+) vector. The esterase EstE was then expressed under the induction of IPTG and purified via Co²⁺ affinity chromatography. Furthermore, the enzymatic properties of the purified EstE were determined by the p-nitrophenol method, and bioinformatics analysis was performed for this enzyme.Results EstE consisted of 289 amino acid residues, with a molecular weight of 31.6 kDa. It belonged to the GDS(L) family, with Ser¹⁶, Asp¹⁹⁴, and His²²⁴ forming its catalytic triad. The enzyme showed the optimal activity at 40 ℃ and pH 8.5, with the highest catalytic efficiency (specific activity of 61.03 U/mg) observed in the case of p-nitrophenyl acetate as a substrate. EstE demonstrated robust thermostability, with the relative activity of 50% after 156.11 h of incubation at 40 ℃ and 2.67 h of incubation at 100 ℃. Moreover, it showed excellent alkaline stability, with the relative activity exceeding 80% after incubation at pH 8.5 for 100 h. In addition, this enzyme exhibited excellent tolerance to organic solvents, maintaining stable activity in the presence of 30% DMSO.Conclusion A novel esterase EstE from S. griseus is successfully obtained through heterologous expression, demonstrating excellent catalytic properties, thermostability, alkaline stability, and organic solvent tolerance, positioning it as a promising candidate for industrial applications.

Abstract:Objective Streptomyces rapamycinicus has large biosynthetic potential with 55 natural product biosynthetic gene clusters (BGCs), most of which have not yet been identified. This study aims to obtain a series of novel compounds by Bacterial Artificial Chromosome (BAC) library-based cloning of novel BGCs from S. rapamycinicus on a large scale and then heterologously expressing them in model Streptomyces strains.Methods The bioinformatics analysis of the novelty of BGCs screened out 11 unknown BGCs encoding non-ribosomal peptides, polyketides or terpenoids, from S. rapamycinicus SIPI RP202. Then, we cloned these BGCs by constructing a BAC library and screening via PCR, and then introduced them into three heterologous expression hosts by conjugative transfer. Finally, LC-MS was employed to detect whether these BGCs were successfully expressed after fermentation in three media and fermentation broth extraction with two approaches. Novel compounds were separated, purified, and structurally elucidated.Results An unknown terpenoid BGC was successfully expressed in Streptomyces albus Del14. Three novel aromatic meroterpenoids, rapamylic acids A-C, were identified. Then, the potential biosynthetic pathways of rapamylic acids A-C were proposed based on their structural features and BGC.Conclusion We successfully unlocked a silent BGC from S. rapamycinicus by large-scale BGC cloning and heterologous expression, providing an alternative strategy for the activation of silent BGCs from other Streptomyces strains. Meanwhile, the discovery of this kind of novel meroterpenoids expands the structural diversity of bacterial terpenoids.

Abstract:Paranosema locustae, an environmentally friendly biocontrol agent, holds significant potential for managing locusts. However, its application is affected by various environmental factors. Understanding the resistance differences of P. locustae under different conditions is essential for its application in biocontrol.Objective To study the effects of different environmental conditions on the energy metabolism and stress responses of P. locustae, providing a theoretical foundation for its environmental adaptability.Methods Spores of P. locustae were exposed to three controlled experimental environments: a dry environment at 40 °C (40 °C GR), a wet environment at 20 °C (20 °C SR), and ultraviolet irradiation under dry conditions (≥100 μW/cm²) (ZW) for varying time periods. We employed a microplate reader, laser confocal microscopy, and differential interference contrast microscopy to assess the survival curves of infected locusts and the spore germination rate, ATP level, protein content, reactive oxygen species (ROS) level, and trehalose level of P. locustae.Results In the 20 °C SR group, P. locustae showed a decrease in the germination rate and notable rises in ATP and active oxygen (ROS) levels, and the median survival time of infected locusts increased. The 40 °C GR group showed no significant changes in the ATP level, ROS level, spore germination rate, or the survival curve of infected locusts compared with the control group. The ZW group showed increases in the ATP and ROS levels, but no significant change in the germination rate or the survival curve of infected locusts.Conclusions Dry spores of P. locustae exhibit greater resistance to environmental stress, while prolonged exposure to liquid conditions leads to a decrease or even loss of spore viability. These findings provide insights for the preservation and application of P. locustae formulations, establishing a theoretical basis for revealing its environmental adaptability.

Abstract:Objective To explore the technical feasibility of using transgenic microalgae to control Aedes that transmit diseases such as dengue fever.Methods Taking the neurotransmitter transporter gamma-aminobutyric acid receptor gene (gat) of Aedes as a target, we constructed a shRNA-expressing vector and then transferred the vector into Chlamydomonas reinhardtiii CC124 and Chlorella vulgaris HOC5 through electroporation. The recombinant microalgae were used to feed the larvae and adults of Aedes albopictus.Results The transgenic algae delayed the larval development and had obvious lethal effects on the larvae and adults of A. albopictus, causing the mortality rates of 75.56% and 58.67%, respectively. The expression level of gat in the larvae was significantly down-regulated.Conclusion Deploying gat-shRNA transgenic microalgae in enclosed water environments to suppress mosquito populations in the vicinity is technically feasible. This strategy provides a new perspective on using biological methods to control mosquitoes and block the transmission of severe infectious diseases such as dengue fever and Zika virus disease.

Abstract:Objective To explore the physiochemical response mechanism of a model algal strain Chlamydomonas reinhardtii that hyper-accumulates oils to exposure of naphthenic acids (NAs).Methods The impacts of a typical NA, cyclohexanecarboxylic acid (CHCA), on the physiochemical parameters, including growth, photosynthetic activity, pH value of the culture, uptake of nitrogen and phosphorus, and biochemical constituents (lipids, carbohydrates, proteins, and pigments), of C. reinhardtii with high initial cell density under high light-nitrogen repletion (HL+N) and high light-nitrogen deprivation (HL-N) conditions were studied.Results The exposure to CHCA prominently promoted the uptake of phosphorus by C. reinhardtii under HL+N, while significantly inceasing the relative abundance of saturated C16:0 and decreasing the relative abundance of polyunsaturated C18:3n3. In contrast, CHCA treatment significantly inhibited the photosynthetic activity and phosphorus uptake but did not affect the fatty acid profile of C. reinhardtii under HL-N stress. In addition, the growth, pH value of the culture, and content of lipids, carbohydrates, proteins, and pigments, in C. reinhardtii under both HL+N and HL-N conditions all remained relatively constant when subjected to CHCA exposure.Conclusion The tolerance of microalgae to environmental stress can be visualized by growth curves, photosythetic activity, uptake of nitrogen and phosphorus, and key biochemical constituents. HL+N improves the tolerance of C. reinhardtii to NAs by promoting the uptake of phosphorus and altering the fatty acid profile, while the case was contrary under HL-N. These findings are beneficial for establishing strategies on effective cultivation of microalgae that highly tolerate NAs.

Abstract:Objective To express the recombinant nonstructural protein NSP1 of the porcine reproductive and respiratory syndrome virus (PRRSV) strain NADC 30, evaluate its immune effect in mice, and explore the potential value of the nonstructural proteins of PRRSV as vaccine antigens.Methods The prokaryotic expression system was used to express NSP1 of NADC30. After purification, the expression and antibody reactivity of NSP1 in vitro were identified by Western blotting. After mice were immunized with NSP1, the levels of cellular and humoral immunity induced by NSP1 were measured. The level of neutralizing antibody induced by NSP1 was evaluated by the virus neutralization assay.Results The target gene of NSP1 was connected to the Escherichia coli pET-28a vector for prokaryotic expression. Western blotting identified that the recombinant protein NSP1 was correctly expressed and had antibody reactivity. After mice were immunized with the confirmed recombinant protein NSP1, the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the spleen lymphocytes of mice were elevated, and cellular immunity was stimulated. At the same time, the recombinant protein NSP1 significantly improved the proliferation of spleen lymphocytes in mice. ELISA results suggested that the level of specific antibodies in the serum rose after immunization. Further analysis of the specific antibody subtypes (IgG2a and IgG1) produced showed that the type of immunity stimulated by recombinant NSP1 was biased to Th2 humoral immunity. In addition, the virus neutralization assay showed that the recombinant protein NSP1 had a good virus neutralization ability, with the neutralization titer of 1:37 on day 28 and 1:31 on day 42, which were significantly higher than those of the PBS control group and had no difference from those of the commercial vaccine group.Conclusion The recombinant nonstructural protein NSP1 of PRRSV can stimulate cellular and humoral immunity in mice and has a good virus neutralization ability, which provides a new idea for the development of next-generation PRRSV vaccines.

Abstract:Objective To study the composition, functional characteristics, and vertical distribution features of microbial communities of three different habitats in the Yellow River Delta wetland ecosystem, and provide theoretical support and potential microbial resources for targeted restoration and sustainable management of wetland ecology.Methods By using 16S rRNA gene amplicon sequencing and metabolomics analyses, we compared the composition and structures of soil bacterial communities in three habitats (vegetation-covered area, bare land, and biohabitat), and analyzed the characteristics of bacterial communities at varying soil depths as well as the potential interactions between habitat-specific bacteria and metabolites.Results The dominant phyla in the three habitats were Proteobacteria and Bacteroidota. The dominant phyla specific to the shallow and deep soil layers were Gemmatimonadota and Firmicutes, respectively. The unidentified MBNT15 in the vegetation-covered area, Halomonas in the bare land, and unidentified Rhodobacteraceae and Woeseia in the biohabitat showed significantly different abundance between different depths, and Bacillus was enriched in the deeper soil layer of all the three habitats. Metabolomic analysis revealed that the vegetation-covered area showed higher levels of sphinganine, 3-indoleacrylic acid, 2,4-dihydroxybenzoic acid, and perfluorooctanoic acid. Deoxycholic acid had the highest level in the bare land, while sulfamethoxazole was the highest in the biohabitat, which had lower level of l-tryptophan. Correlation analysis revealed that in the vegetation-covered area, Micrococcus luteus and Pseudomonas geniculata showed significantly positive correlations with sphinganine and perfluorooctanoic acid. Saccharospirillum salsuginis had significantly positive correlations with 3-indoleacrylic acid and 2,4-dihydroxybenzoic acid. In the bare land, Bacillus horikoshii showed a significantly positive correlation with deoxycholic acid. In the biohabitat, Halomonas ventosae had a significantly positive correlation with l-tryptophan, while Halomonas korlensis showed a significantly positive correlation with sulfamethoxazole.Conclusion Our study demonstrated that varying soil depths significantly impact the structure of microbial communities, and the structural and functional characteristics of soil microbial communities exhibit habitat specificity. The enriched bacteria such as M. luteus in the vegetation-covered area may promote plant growth and enhance stress resistance by regulating metabolites. The enriched Bacillus in the bare land plays a role in decomposing bird feces. The unique bacteria such as H. ventosae in the biohabitat demonstrate the potential for maintaining the ecological health of crab habitats through metabolite regulation. These findings offer new insights into the microbial regulation and management of wetland ecosystems.

Abstract:Leaf photosynthesis forms the foundation of plant energy and material cycles. Mycorrhizal fungi, as a crucial component associated with roots, play a significant role in regulating the nutrient absorption, water utilization, and stress resistance of plants and are key ecological factors affecting the function and stability of forest ecosystems. However, at present, knowledge is limited regarding the effects of different mycorrhizal types on the photosynthetic capacity of woody plants and their responses to environmental changes.Objective To explore the differences in photosynthetic capacity among woody plants with different mycorrhizal types and their responses to changes in leaf characteristics and environmental factors.Methods According to the China Plant Trait Database and available articles, we identified three mycorrhizal types of woody plants, which included arbuscular mycorrhiza (AM), ectomycorrhiza (ECM), and AM+ECM. On this foundation, a database of woody plant traits for different mycorrhizal types in China was established, with the data of each sample encompassing photosynthetic capacity, photosynthetic physiology, leaf structure, nutrient characteristics, and environmental factors.Results The woody plants with ECM had higher photosynthetic capacity than those with AM. Mycorrhizal types significantly influenced the relationship between leaf traits and photosynthetic capacity. The photosynthetic capacity of woody plants was primarily affected by the stomatal conductance and transpiration rate of leaves. In addition, the photosynthetic capacity of plants with AM were influenced by leaf area, specific leaf area (SLA), and nitrogen and phosphorous content. The photosynthetic capacity of plants with AM+ECM were affected by SLA, specific leaf weight, and carbon and phosphorous content, while that of plants with ECM was influenced by the vapor pressure deficit. The maximum net photosynthetic rate and maximum electron transport rate of plants with ECM were more susceptible to temperature and precipitation than plants with AM and AM+ECM.Conclusion Mycorrhizal types significantly affect the maximum net photosynthetic rate of woody plants, and leaf characteristics primarily influence the maximum net photosynthetic rate by regulating the maximum electron transport rate. Moreover, the effects of environmental factors on the morphological and physiological traits of woody plant leaves depend on mycorrhizal types.