2023, 63(8):2931-2932.
Abstract:
ZHAO Min , OUYANG Zirou , ZHAO Jianhong
2023, 63(8):2935-2947. DOI: 10.13343/j.cnki.wsxb.20220861
Abstract:Clostridioides difficile, a spore-forming Gram-positive obligate anaerobic bacterium, is the main pathogen causing antibiotic-associated diarrhea. Two toxins produced by C. difficile, toxin A and toxin B, play a key role in its pathogenesis. The roles of toxins depend on four functional domains: glucosyltransferase domain, cysteine protease domain, transmembrane domain, and receptor-binding domain. The receptor-binding domain recognizes and binds to the receptors on cell surface, mediating toxin endocytosis to form endosomes. After autocatalytic cleavage, the toxic fragment, glucosyltransferase domain, is released into the cytoplasm. Glucosyltransferase can inactivate GTPase in host intestinal epithelial cells, leading to cytoskeletal depolymerization and necrosis, which causes clinical symptoms such as diarrhea and pseudomembranous colitis. The production of toxins is regulated by a variety of factors within and outside the pathogenicity locus. Located in the pathogenicity locus, tcdR promotes and tcdC inhibits the expression of the toxin genes, respectively. The genes outside the locus, such as spo0A and codY, indirectly affect the production of toxins by inhibiting the expression of tcdR. This review focuses on the pathogenic process of toxins and the molecular mechanism of the regulation of toxin gene expression, hoping to provide new ideas for the development of toxin-targeted therapies.
CHEN Ying , XU Ping , DAI Erhei , ZHANG Yao
2023, 63(8):2948-2966. DOI: 10.13343/j.cnki.wsxb.20220896
Abstract:Tuberculosis (TB), the second (after COVID-19) deadliest infectious killer, is caused by Mycobacterium tuberculosis (MTB). COVID-19 pandemic has shown devastating effect on the diagnosis and treatment of TB, posing a huge challenge to the ending of TB. Thus, early diagnosis and treatment is still the key to prevention and control of TB spread. The accurate diagnosis of TB depends on the specificity of MTB antigen and the specificity and sensitivity of detection techniques. Therefore, it is urgent to develop highly specific antigens and detection techniques. The advanced proteogenomics and mass spectrometry make it easy to detect known or new MTB-specific antigens from clinical body fluids and tissue samples and monitor the dynamic expression of antigen during treatment. Among 4 008 annotated genes of MTB in NCBI (NC_000 962.3), more than 140 known genes have been listed as potential antigens for TB diagnosis, while only a few annotated antigens have been used in the screening and auxiliary diagnosis of TB, which are still far from the WHO diagnostic standards. In this paper, we reviewed the reported antigens of MTB and the potential of screening specific neoantigens based on proteogenomic technologies for better understanding them and developing new efficient antigens.
WANG Xiujuan , GU Yufeng , WANG Wenhui , LI Jie , HAO Haihong
2023, 63(8):2967-2979. DOI: 10.13343/j.cnki.wsxb.20220899
Abstract:Disinfectants are organic molecules that kill pathogenic microorganisms on object surface, equipment, and skin, in the air and water, and carried by other vectors. They can kill pathogenic microorganisms in vitro and cut off their transmission routes to achieve contamination control, playing a role in ensuring life safety. However, the irrational use of disinfectants leads to resistance of bacteria to disinfectants. Horizontal transfer of disinfectant resistance genes between different species exacerbates the risk of transmission, aggravating resistance to disinfectants. Moreover, bacterial resistance to disinfectants may lead to co-resistance to antibiotics, posing a huge threat to public safety. However, the knowledge about resistance to disinfectants and co-resistance to disinfectants and antibiotics is limited. We review the research reports on the resistance of bacteria to disinfectants, introduce the mechanisms of disinfectants and the mechanisms of bacterial resistance to disinfectants, and expound the spread of disinfectant resistance genes and the co-resistance of bacteria to disinfectants and antibiotics. This review lays a foundation for reducing the emergence of disinfectant resistance and formulating reasonable specifications for the use of disinfectants.
ZUO Wei , WANG Xinyu , HU Jiangang , WANG Shaohui
2023, 63(8):2980-2993. DOI: 10.13343/j.cnki.wsxb.20220900
Abstract:Ubiquitination is a common post-translational modification of proteins in eukaryotic cells, which is involved in a variety of physiological and pathological processes such as DNA repair, cell cycle, immunological response, and signal transduction. Meanwhile, host cells use deubiquitinases (DUBs) to reverse ubiquitin signals, ensuring the dynamic balance of the ubiquitination system and physiological functions. In the case of bacterial infection, host cells mount a defense response by ubiquitination. However, pathogenic bacteria can encode and secrete effectors to regulate the ubiquitination of the host, thereby interfering with the host cellular immune response and bolstering their survival and virulence. This review outlines the research on the effectors of pathogenic bacteria that regulate ubiquitination pathways in host cells, which is expected to enhance the understanding of bacteria’s regulation of host ubiquitination for the infection.
CHEN Yanghui , LI Yuan , WANG Bei
2023, 63(8):2994-3008. DOI: 10.13343/j.cnki.wsxb.20220905
Abstract:Intracellular pathogenic bacteria can invade the host cells and survive and reproduce in the cells. They infect host cells by adhesion, invasion, and survival, finally causing damage to the host cells. Studies have demonstrated that most intracellular pathogenic bacteria are passively ingested by phagocytes. With the development of molecular biology and immunology, increasing intracellular pathogenic bacteria have been proved to actively invade the host cells and evolve a variety of ways to regulate the host cell signaling pathways for escaping cellular immunity. We provided a brief overview of the common molecular mechanisms of intracellular pathogenic bacteria invading the host cells. Further, we introduced the invasion strategies adopted by common intracellular pathogenic bacteria and summarized the main research advances in recent years.
ZHAO Yanni , YU Rui , LIU Huan , WANG Yongbo
2023, 63(8):3009-3025. DOI: 10.13343/j.cnki.wsxb.20220926
Abstract:Functional metabolomics studies the functions of differential metabolites and related proteins, enzymes, and genes by metabolomics tools, in vitro and in vivo experiments, and molecular techniques based on the discovery of key metabolites, so as to reveal the molecular regulation mechanism in organisms. Being capable of accurately identifying the key regulatory metabolites and the related genes or enzymes, functional metabolomics has received extensive attention in the prevention and control of microorganism-induced diseases and the industrial production. This article introduces the research process, methods, and platforms of functional metabolomics and the application of this technology in microbial research, with focus on the metabolic characteristics, regulatory targets, and related prevention and control strategies of eukaryotes, prokaryotes, and viruses. Finally, we put forward the problems and challenges in the research of functional metabolomics, aiming to provide new ideas for the follow-up study and the development of functional metabolomics.
MA Dongxue , ZHAO Xuning , ZHAO Yangguo
2023, 63(8):3026-3040. DOI: 10.13343/j.cnki.wsxb.20220854
Abstract:[Objective] The biodegradation of sulfur-containing amino acids is an important factor in the rise of toxic sulfides in the mariculture environment, and the analysis of the mechanisms and influencing factors of microbial degradation of sulfur-containing amino acids is a key link to control the sulfide concentration in this system. [Methods] This study used the dilution coating-stacked dish clamp method to isolate a sulfide-producing anaerobic strain from the sediment of the mariculture environment in this laboratory, and the mechanism and pathway of sulfide production with cysteine as substrate were investigated by metabolomics. [Results] The strain was identified as Citrobacter freundii, which reduced sulfate under anaerobic conditions and degraded cysteine to produce sulfide. The addition of l-cysteine improved its ability to reduce sulfate. The maximum accumulation of sulfide in this strain was 302.4 mg/L at 35 ℃, salinity 10, and pH 8.0 with 1 g/L l-cysteine as substrate. The study of cysteine desulfhydrase, the important contributor to sulfide production in the strain, revealed that the optimum temperature of this enzyme was 35 ℃, and it had high activity at pH 6.0-8.0, which could rapidly degrade cysteine to produce sulfide. Combined with the metabolomics study, it was found that the strain contained 3-mercaptopyruvate sulfotransferase, cystathionine gamma-lyase, and cysteine desulfhydrase catalyzing the degradation of cysteine was the main pathway to produce hydrogen sulfide, and sulfite reductase reducing sulfate and sulfite was its secondary pathway to produce hydrogen sulfide.[Conclusion] By revealing the mechanism of sulfide production from cysteine degradation by Citrobacter, this paper provides a theoretical basis for the prevention and control of sulfide in mariculture.
ZHOU Qingqing , YANG Qinglin , LI Yue , LI Yanhong , YANG Dacheng , WU Zhengli
2023, 63(8):3041-3053. DOI: 10.13343/j.cnki.wsxb.20220859
Abstract:[Objective] The antibiotic resistance poses great challenges to the prevention and treatment of bacterial infections in animals, and biofilm formation is one of the main factors inducing the resistance. We determined the effects of chloropropanoyl clinafloxacin on the growth and biofilm formation of seven strains of bacteria, with a view to finding out whether chloropropanoyl clinafloxacin has antibacterial activity.[Methods] The inhibitory activity of chloropropanoyl clinafloxacin was determined by routine drug sensitivity test. The perforation method and microbroth twofold dilution method was employed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The biofilm-forming abilities and growth rates of the seven strains exposed to sub-inhibitory concentrations of the drug were examined by crystalline violet staining. [Results] The fluoroquinolone derivative chloropropanoyl clinafloxacin showed the MIC≤10 mg/L and MBC≤48 mg/L against four Gram-negative strains and was sensitive to three Gram-positive strains (MIC≤10 mg/L and MBC≤10 mg/L). The biofilm formation and growth of the seven strains were significantly inhibited by chloropropanoyl clinafloxacin at the sub-inhibitory concentrations, which indicated that chloropropanoyl clinafloxacin had good antibacterial activity at sub-inhibitory concentrations. [Conclusion] Chloropropanoyl clinafloxacin can be used as an antibacterial agent and provides a basis for the development of new agents or drugs targeting biofilm for the treatment of bacterial infections.
WANG Zhen , ZHOU Yazhou , WANG Likun , HUANG Ziran , CHANG Yuxiao , HOU Fengyi , ZHANG Huan , LING Hui , SUN Yifan , TAN Yafang , YANG Ruifu , BI Yujing , PAN Zhiyuan
2023, 63(8):3054-3067. DOI: 10.13343/j.cnki.wsxb.20220862
Abstract:[Objective] To investigate the alterations in gut microbiota and their associations with the biomarkers of myocardial hypertrophy induced by continuous normobaric hypoxia in rats. [Methods] Sixteen specific-pathogen-free female Sprague-Dawley rats were randomly assigned into two groups: the normoxia group and the hypoxia group. After adaptation for 1 week, the rats in the hypoxia group were immediately placed into the hypoxic chamber with the oxygen concentration at 10% for 30 d, and the rats without hypoxia treatment were set as the control. We monitored the body weight of each rat daily, determined the haematological and biochemical parameters as well as the organ coefficients at the end of the experiments. Further, we collected stools from each rat before (day 0) and after hypoxia challenge (day 30) to test the gut microbiota by using 16S rRNA gene sequencing. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the mRNA levels of gene ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), Myh6 (myosin heavy chain 6), and Myh7 (myosin heavy chain 7), the biomarkers of myocardial hypertrophy in the right ventricular tissue. Spearman correlation analysis was performed to explore the links between intestinal microbiomes and parameters. [Results] Hypoxia reduced the body weight, increased the erythrocyte count, hemoglobin level, and hematocrit, and failed to affect the counts of leukocytes and platelets. Hypoxia increased the right ventricular index, enhanced the activities of creatine kinase (CK) and lactate dehydrogenase (LDH) in the serum, and up-regulated the mRNA levels of BNP and Myh7 in right ventricular tissue, which suggested pathologically myocardial hypertrophy after hypoxia challenge for 30 d. Hypoxia significantly altered the alpha diversity and beta diversity of gut microbiota in rats. The results of linear discriminant analysis effect size (LEfSe) showed that the rats exposed to hypoxia had lower relative abundance of Prevotellaceae and Lachnospiraceae and higher relative abundance of Lactobacilliaceae and Lactobacillus than the control rats. LDH was positively correlated with Monoglobus and Papilibacter and negatively correlated with Defluvialeaceae_UCG-011. CK was positively correlated with strain RF39. The expression of BNP was positively correlated with Ruminocochaceae_UCG_009, and that of Myh6 was positively correlated with Prevotellaceae_NK3B31_group and Helicobacter.[Conclusion] The species richness, evenness, and composition of gut microbiota in rats are greatly affected after exposure to continuous normobaric hypoxia. The changes of gut microbiota have correlations with the biomarkers of myocardial hypertrophy induced by hypoxia, indicating that gut microbiota plays a crucial role in hypoxia-induced myocardial hypertrophy.
TANG Miaomiao , WANG Yingeng , WANG Chunyuan , ZHANG Zheng , LIAO Meijie , LI Bin , RONG Xiaojun , GE Jianlong , YU Yongxiang
2023, 63(8):3068-3082. DOI: 10.13343/j.cnki.wsxb.20220864
Abstract:[Objective] To investigate the characteristics of culturable microorganisms and the genetic diversity of dominant pathogens during shrimp postlarva bacterial vitrified syndrome (BVS) of Litopenaeus vannamei in the factory breeding system. [Methods] The composition and structural characteristics of culturable bacterial communities in shrimp (parent shrimp, fertilized egg, nauplius, zoea, mysis, and postlarva), water, and bait samples at different breeding stages were studied by in vitro bacterial culture method combined with gene sequencing technology, and the genetic diversity of the pathogenic bacteria was studied by multilocus sequence analysis (MLSA). [Results] The 526 isolates with typical morphological differences and community dominance belonged to 113 species, 38 genera, 24 families, 16 orders, 5 classes, and 4 phyla. At the class level, Gammaproteobacteria had the highest abundance, with 453 isolates (86.1% of the total isolates). At the genus level, Vibrio had the highest abundance, with 369 isolates (70.2%). At the species level, V. alginolyticus was the most dominant species, with 112 isolates (21.3%). V. alginolyticus was distributed in the whole breeding system and had the highest abundance in the baits. The multiple correlation analysis showed that the influence of baits on the structure of culturable bacterial community increased gradually with the development of larvae. The MLSA confirmed 100 out of the 112 potential isolates as V. alginolyticus. MLSA was further employed to construct a phylogenetic tree for revealing the genetic diversity of the isolates. The 100 V. alginolyticus isolates were classified into 9 clusters, and the isolates from the same type of samples showed dispersed distribution in different clusters. [Conclusion] During the occurrence of BVS, abundant culturable microorganisms existed in the factory breeding system of L. vannamei. The baits exerted effect on the structure of culturable bacterial community in the larvae. V. alginolyticus was the dominant species of Vibrio in the factory breeding system of L. vannamei, with wide distribution and high genetic diversity in the breeding system. The results of this study provide data support for analyzing the succession rules of culturable microorganisms in shrimp breeding system and lays a foundation for pathogen prevention and control and healthy breeding of shrimp.
WANG Mengying , LIU Mafeng , CHENG Anchun
2023, 63(8):3083-3095. DOI: 10.13343/j.cnki.wsxb.20220866
Abstract:Heme is an essential nutrient for the growth and proliferation of most bacteria since it is involved in a variety of physiological processes. Bacteria can obtain heme through biosynthesis and acquisition from the host. However, excessive heme is toxic, and bacteria can alleviate heme toxicity by efflux, sequestration, and degradation. Riemerella anatipestifer (RA), a Gram-negative bacterium that infects birds, can transport heme from hemoglobin. However, whether RA can synthesize heme remains unknown. The genome analysis revealed that the gene RA0C_RS08070 of RA ATCC 11845 strain encodes the ferrochelatase HemH, which is a key enzyme that participates in the insertion of iron into porphyrin center to form heme. The loss of hemH leads to the accumulation of iron and porphyrin, causing toxicity to bacteria. [Objective] To identify the role of HemH in the synthesis of heme and identify the genes involved in the detoxification of iron and porphyrin in RA. [Methods] In this study, ΔhemH, the hemH-deleted mutant of RA ATCC 11845, was constructed, and the growth curves of the parental strain and ΔhemH in the GCB liquid medium and the GCB liquid medium supplemented with hemoglobin (Hb) were established. Further, the transcriptomes of the parental strain and ΔhemH were sequenced and analyzed. [Results] RA ATCC 11845ΔhemH did not grow in the GCB medium, while it grew well in the GCB medium supplemented with Hb. Transcriptome analysis revealed 354 differentially expressed genes (DEGs) between ΔhemH and the parental strain. Gene ontology (GO) functional annotation showed that the DEGs were mainly involved in catalytic activity, biological regulation, and metabolic processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly enriched in amino acid metabolism, oxidative phosphorylation, and tricarboxylic acid cycle (TCA cycle). [Conclusion] HemH is involved in heme synthesis, and the mutant with the deletion of hemH changed the expression of the genes to adapt to the disorder of metabolism. This study lays a foundation for further studying the role of HemH in RA.
TAO Yu , YE Ting , FEI Qingru , FU Xiaojie , ZHOU Yu
2023, 63(8):3096-3109. DOI: 10.13343/j.cnki.wsxb.20220867
Abstract:[Objective] To reveal the role of the phosphopyridoxamine oxidase gene pnpox in vitamin B6 (VB6) synthesis and aflatoxin B1 (AFB1) degradation in Stenotrophomonas acidaminiphila CW117. [Methods] The phosphopyridoxamine oxidase gene was mutated in strain Stenotrophomonas acidaminiphila CW117 by the recombinant plasmid pK19mobΩ2HMB/P-pnpox, and thus the mutant pnpox::pK19mobΩ2HMB was constructed. The vitamin B6 (i.e., pyridoxine and pyridoxal) synthesis and AFB1 degradation activities of the mutant and the wild-type strain CW117 were detected by high performance liquid chromatography (HPLC). The detemination results were compared to reveal the role of pnpox in VB6 synthesis and AFB1 degradation. [Results] The mutant pnpox::pK19mobΩ2HMB was successfully constructed. It had significantly lower yield of pyridoxal than the wild-type strain CW117, while no significant difference was observed in the yield of pyridoxine between the mutant and CW117. Meanwhile, the mutant showed no significant difference in AFB1 degradation from CW117.[Conclusion] Phosphopyridoxamine oxidase plays an important role in the pyridoxal synthesis of CW117. The mutation of pnpox reduced the VB6 production and thus seriously delayed the growth of the strain, while the gene showed no significant contribution to AFB1 degradation in strain CW117.
WANG Meng , WANG Xue , ZHAO Jia'nan , ZENG Jun , ZUO Junhao , GUO Zhiliang , JI Fang , SHAO Jianli , ZHANG Limin , WANG Chengmin , QIN Jianhua
2023, 63(8):3110-3128. DOI: 10.13343/j.cnki.wsxb.20220878
Abstract:[Objective] As a major pathogen of human infection, multidrug-resistant Klebsiella poses a great challenge in clinical treatment. To provide more scientific data for the application of phages, we analyzed and compared the biological and genomic characteristics of several strains of Klebsiella phages. [Methods] Klebsiella phages were isolated from human and animal fresh feces and sewage by the double plate method. The phage morphology was observed by phosphotungstic acid staining and transmission electron microscopy. The host spectrum, thermal stability, and pH stability of the phages were measured, and the one-step growth curve and in vitro bacteriostatic curve were established. The comparative genomics analysis was carried out for the whole genomes of the phage isolates. We further evaluated the protective effect of the phage isolates by the survival rate of Galleria mellonella larvae infected with the multidrug-resistant mutant Klebsiella variicola BS375-3 in vivo. [Results] The five phage isolates belonged to Schitoviridae (pKP-BM327-1.2), Autographiviridae (pKP-M186-2.1, pKP-M186-2.2, and pKV-BS375-3.1), and Drexlerviridae (pKP-BS317-1.1). The phage isolates pKV-BS375-3.1 and pKP-BM327-1.2 could lyse eight and three bacterial hosts, respectively, while pKP-M186-2.1, pKP-M186-2.2, and pKP-BS317-1.1 could only lyse one bacterial host. The five phage isolates entered the exponential growth stage 10-20 min after inoculation and maintained stable activity at -20 ℃-37 ℃ and pH 6-10. The survival rate of G. mellonella larvae infected with the K. variicola BS375-3 treated with pKV-BS375-3.1 (MOI=100) reached 80% (8/10) after 96 h. The genomes of the five phage isolates had the length of 42-77 kb and did not carry resistance genes or virulence genes. The traceability analysis based on endolysin showed that the protein demonstrated diversity in Klebsiella phages and was conserved within the genus. [Conclusion] All the five Klebsiella phage isolates had good antibacterial activity in vitro and stable biological characteristics. The endolysins of the phage isolates were conserved within the genus. The phage pKV-BS375-3.1 with a wide host range and short latency has a potential application prospect in the clinical treatment of Klebsiella pneumoniae and K. variicola infections.
MENG Jiayi , ZHANG Guojing , ZHOU Feng'en , WANG Nan , ZHOU Yifa , YUAN Ye
2023, 63(8):3129-3143. DOI: 10.13343/j.cnki.wsxb.20220880
Abstract:[Objective] We cloned, expressed, and characterized the glucomannan-degrading enzymes from Paenibacillus macerans, aiming to enrich the glucomannan-degrading enzyme resources and decipher the mechanism of glucomannan degradation by P. macerans. [Methods] We retrieved the glucomannan-degrading enzyme genes and constructed recombinant strains to express the recombinant enzymes. The roles of the purified proteins in the degradation of glucomannan were then studied. [Results] We cloned and expressed five glucomannan-degrading enzymes, among which PmMan1 and PmMan2 were endo-β-mannanases, and PmGlc1, PmGlc2, and PmGlc3 were exo-β-glucosidases. PmGlc1 only hydrolyzed pNPβGlc, and PmGlc2 hydrolyzed β-1,6-glucosidic bonds in disaccharides and ginsenosides. PmGlc3 hydrolyzed a wide range of β-glucosidic bonds. PmMan1, PmMan2, PmGlc2, and PmGlc3 degraded glucomannan oligosaccharides, and PmMan1 and PmMan2 degraded glucomannan. In the degradation of glucomannan, PmGlc2 and PmGlc3 had synergistic effects with PmMan2, and the synergistic effect was more significant between PmGlc3 and PmMan2. [Conclusion] We obtained four glucomannan-degrading enzymes from P. macerans and clarified the role of the enzymes in glucomannan degradation. The findings enrich the enzyme resources and theoretical research achievements and provide an effective tool for enzymatic preparation of active glucomannan oligosaccharides.
TIAN Xuping , LI Lingyan , LI Jie , GAO Jian , DENG Kai , DONG Xiuzhu
2023, 63(8):3144-3156. DOI: 10.13343/j.cnki.wsxb.20220881
Abstract:[Objective] To explore the feasibility of using Clostridium and Methanomassiliicoccus from human intestine to synergistically convert betaine and choline to methane. [Methods] Illumina sequencing of the 16S rRNA gene was performed to survey the diversity of archaea in the feces from healthy people of 20-40 years old and over 40 years old. The Methanomassiliicoccus-specific mtaB gene and Methanomassiliicoccus-specific 16S rRNA gene were quantitated by quantitative PCR (qPCR) to quantify the trimethylamine-utilizing methanogens in human intestine. Metagenome-assembled genomes (MAGs) were reconstructed from metagenome data for the identification of the intestinal bacteria carrying the betaine reductase gene grdH and choline trimethylamine-lyase gene cutC. The bacteria that reduced betaine and choline were isolated from feces and used to construct the coculture with Methanomassiliicoccus. The potential of the coculture for producing methane from betaine and choline was then determined. [Results] The main methanogenic archaea in the intestine of the 20-40 years old included Methanobrevibacter (49.18%) and Methanobacterium (33.34%) affiliating to Methanobacteriaceae (82.16%), Methanosarcina (5.70%) of Methanosarcinaceae (5.67%), and Methanomassiliicoccus (3.14%) of Methanosassilicoccaceae (3.13%). The methanogen diversity was lower in the feces from the people over 40 years old, from whom Methanosassilicoccaceae was not detected. Quantitative PCR determined that the total abundance of archaea and bacteria in the people of 20-40 years old was 3.11 and 2.90 folds, respectively, higher than in those over 40 years old. Specifically, the abundance of Methanomassiliicoccus and Methanosarcina was 6.53 and 5.52 folds higher, respectively. A total of 229 bacterial MAGs were obtained from the fecal specimens, in which 42 MAGs carried genes grdH and cutC and were affiliated to Lachnospiraceae, Enterobacteriaceae, and Clostridiaceae. Clostridium malenominatum B8 was isolated from the fecal specimens. The co-culture of this strain with Methanomassiliicoccus luminyensis B10 in the medium with 20 mmol/L betaine or choline degraded 47.03% betaine and 25.83% choline to produce methane, during which trimethylamine was detected as the intermediate. [Conclusion] The human intestinal Clostridium B8 and M. luminyensis B10 synergistically convert betaine and choline to methane. Therefore, we hypothesize that they play a role in reducing the trimethylamine in human intestine.
LIU Ran , JIANG Yunbin , DENG Huan , ZHANG Yinping , HAN Cheng , ZHONG Wenhui
2023, 63(8):3157-3172. DOI: 10.13343/j.cnki.wsxb.20220889
Abstract:Inorganic nitrogen assimilation performed by microorganisms can immobilize chemical fertilizer nitrogen that is not directly absorbed by crops after application to the soil, which can reduce the losses of chemical nitrogen fertilizer and the risk of environmental nitrogen pollution. Soil inorganic nitrogen assimilation is performed by functional microbial populations rather than a large number of redundant microorganisms. [Objective] The enrichment, isolation, identification, and whole genome sequencing of dominant inorganic nitrogen-assimilating bacteria in acidic dryland red soil and clarification of the nitrogen assimilation capacity of the strains in soil can provide strain resources and a theoretical basis for the application of chemical nitrogen fertilizer in acidic soil and the research on the nitrogen transformation process. [Methods] We added KNO3 or (NH4)2SO4 as the inorganic nitrogen source and glucose as the carbon source into the acidic dryland red soil. Then, we performed strain enrichment under aerobic conditions and screened the dominant bacterial strains assimilating inorganic nitrogen by the gradient dilution isolation method. We verified the inorganic nitrogen assimilation ability of the strains by soil recolonization experiments, and employed whole genome sequencing to analyze the nitrogen metabolic pathways of different strains. [Results] The relative abundance of 16S rRNA genes of dominant inorganic nitrogen-assimilating microorganisms in acidic dryland red soils increased from 0.20%-0.94% to 20.2%-30.2% after one week of enrichment. We isolated three dominant inorganic nitrogen-assimilating strains, which were identified as Burkholderia sp. M6-3, Bacillus funiculus M2-4, and Arthrobacter sp. M7-15. The inorganic nitrogen assimilation rates of strains M6-3, M2-4, and M7-15 in sterilized soil were (1.28±0.61), (0.17±0.07), and (0.16±0.02) mg/(kg·d), respectively. M6-3 possessed a more complete metabolic pathway and more functional genes related to nitrogen assimilation than the other two strains. In terms of nitrogen metabolic pathways and functional activity, Burkholderia sp. M6-3 was dominant in the assimilation of inorganic nitrogen in acidic dryland red soils. [Conclusion] This study confirmed that low-abundance microbial taxa play a dominant role in the inorganic nitrogen assimilation of acidic dryland red soil, and revealed the metabolic process of inorganic nitrogen assimilation at the genomic level of the strains. The above results provide strain resources and a theoretical basis for the study of chemical nitrogen fertilizer application and transformation process in the acidic dryland red soil.
ZHANG Lei , ZHAO Zhixin , ZHAO Feiyan , SUN Zhihong
2023, 63(8):3173-3186. DOI: 10.13343/j.cnki.wsxb.20220893
Abstract:The microorganisms in colostrum have a variety of beneficial effects on the growth and development of infants. [Objective] We analyzed several factors affecting the flora composition of colostrum, aiming to provide a new idea for the follow-up study of human colostrum flora. [Methods] We used PacBio SMRT sequencing platform to complete the bacterial 16S rRNA gene sequencing of 37 colostrum samples collected from Enshi, Hubei Province. We then used the sequence data and the published data of 62 human colostrum samples from other regions in the public database to explore the composition and structure of bacterial flora in the human colostrum samples from different regions. [Results] There were 345 genera and 937 species in 99 colostrum samples. Lactococcus lactis, Ralstonia insidiosa, and Gemella haemolysans were the dominant species in human colostrum. The principal coordinate analysis (PCoA) based on jaccard distance showed a significant separation trend of flora in human colostrum samples between different regions. The R2 of the factors affecting the composition of colostrum flora followed the trend of region>infant feeding modes>maternal health during pregnancy>mode of delivery>parity. [Conclusion] Region was the main factor affecting the flora composition of human colostrum.
LIU Siyu , MA Yushu , WEI Dongzhi
2023, 63(8):3187-3202. DOI: 10.13343/j.cnki.wsxb.20220894
Abstract:The genome of Gluconobacter oxydans encodes a large number of sensor kinases and response regulators, potential members of two-component signal transduction systems (TCSs), by which bacteria recognize and respond to a variety of environmental stimuli. However, little is known about the structures and functions of these proteins in G. oxydans. [Objective] To study the autophosphorylation activity of a potential TCS protein encoded by GOX0645 in G. oxydans 621H, and to reveal the role of this protein in bacterial chemotaxis.[Methods] We performed sequence analysis on GOX0645 from G. oxydans 621H that might encode a TCS member, detected the autophosphorylation activity of the encoded protein by in vitro luminescent kinase assay, and then identified the amino acid residues critical to the autophosphorylation by site-directed mutagenesis. We employed differential centrifugation to study the subcellular localization of the protein, and BiFC assay and Octet® system to explore the interaction between the protein and a flagellar motor protein of G. oxydans. [Results] The protein encoded by GOX0645 contained the conserved domains of both histidine kinases and response regulators, with a conserved Asp and three His residues associated with autophosphorylation activity. Furthermore, the protein, as a hybrid protein, showed the activity of autophosphorylation which was affected by the concentration of cAMP molecule. The protein was localized in the cytoplasm and had a moderate affinity with the flagellar motor protein, which implied its regulatory role in bacterial chemotaxis. [Conclusion] This study analyzed a potential TCS that might be involved in the regulation of chemotaxis in G. oxydans 621H. This finding contributes to gaining insight into the molecular mechanism of two-component systems in G.oxydans.
LI Jin , WANG Yuxin , WANG Xuhang , LI Jing , FU Kailai , DAI Jianjun , TANG Fang , XI Jing
2023, 63(8):3203-3218. DOI: 10.13343/j.cnki.wsxb.20220897
Abstract:[Objective] To improve the storage stability of the phage at room temperature, solve the problem of inactivation of phage cocktail in vivo, and provide references for the effective treatment of gastrointestinal diseases by phages. [Methods] This study prepared phage cocktail microsphere powder by spray drying technology. The optimal preparation conditions were determined by single factor experiment and orthogonal experiment. The characteristics were studied, and the stability of the phage was compared with that of free phage at room temperature and in vivo was compared. Gastrointestinal diseases caused by Escherichia coli O157:H7 were treated by oral administration. [Results] In this study, a combination of trehalose and leucine was used to prepare a thermally stable phage cocktail microsphere powder. The results showed that the optimal mass ratio of trehalose and leucine was 9:1, the optimal speed of wriggling pump was 7.5 mL/min, the optimal trehalose concentration was 2%, the optimal inlet temperature was 130 ℃, and the optimal volume ratio of the phage cocktail suspension to the protectant solution was 1:50. Under the optimal conditions above, the titer loss was the lowest, only decreased by (0.623±0.235) log10 PFU/g. After 6 months of storage at room temperature, the titer loss of the phage cocktail microsphere powder was only (0.862±0.082) log10 PFU/g, which had longer storage stability than the free phage, and the stability in vivo and therapeutic effect were much better than the free phage. [Conclusion] Studies have shown that the spray drying method and suitable protective agent formula can be used to prepare phage cocktail dry powder with biological activity and thermal stability, which can extend retention time, facilitating the transportation under normal temperature conditions. This provides references for the transformation of phage preparations from the laboratory to the industrial large-scale production. Moreover, the phage microsphere powder shows an excellent ability to remove E. coli in the intestinal tract with a high speed, indicating its potential as an orally therapeutic agent in vivo.
ZHAI Ligong , LI Ganghui , HUANG Ju , CAI Qiuhui , WEI Zhaohui , HE Xiaodong , CAO Rui , WANG Junying
2023, 63(8):3219-3234. DOI: 10.13343/j.cnki.wsxb.20220901
Abstract:[Objective] This study aims to decipher the mechanism of Salmonella enterica subsp. enterica Derby (S. Derby) adapting to hypertonic stress at the transcriptional level. We mined the differentially expressed genes (DEGs) to explore the metabolic pathways associated with the response of S. Derby to the stress. [Methods] After the hyperosmotic tolerance of S. Derby was induced, we extracted the total RNA, removed the rRNA, and constructed a cDNA library. The relevant DEGs were identified by transcriptome sequencing and bioinformatics tools and verified by real-time fluorescence quantitative PCR. [Results] After hyperosmotic stimulation, 3 950 DEGs were identified by transcriptome sequencing, which included 21 significantly up-regulated genes and 38 significantly down-regulated genes. The genes involved in the efflux of Na+ from the cell membrane and the metabolism of amino acids were up-regulated, which can provide energy and help S. Derby survive in a hyperosmotic environment. The genes associated with the sugar transport system (PTS), glycolysis, and anti-oxidation of S. Derby were significantly down-regulated in the stress group. Under hyperosmotic stress, the bacteria cannot take up carbohydrates from the external environment and thus the synthesis of lipopolysaccharide in the outer membrane of the cell is inhibited, which reduces the invasiveness of S. Derby, thereby increasing the toxicity of S. Derby. [Conclusion] Under saturated NaCl stress, the osmotic tolerance of S. Derby is significantly improved, during which the Na+/H+ antiporter and the glutamate metabolic pathway play a key role. The findings provide a theoretical basis for the further understanding and control of S. Derby contamination in food.
XIANG Xin , YIN Hengxia , ZHU Zhaoyu , QIU Qinghui , LIU Yurui , FAN Jiakai , DENG Jiawen , ZHANG Dejun , ZHANG Benyin
2023, 63(8):3235-3251. DOI: 10.13343/j.cnki.wsxb.20220908
Abstract:Soil microorganisms play a key role in the sustainability of ecosystems. There are diverse extreme habitats on the Qinghai-Tibetan Plateau (QTP), where the differences of soil bacterial composition and the driving factors remain to be studied. [Objective] To explore the differences and influencing factors of soil bacterial diversity in different extreme habitats on the QTP. [Methods] High-throughput sequencing of 16S rRNA genes was performed for the soil samples from seven typical extreme habitats on the QTP. The bioinformatics tools were employed to analyze the differences in bacterial composition and functions among different habitats. The potential soil factors influencing bacterial composition were further analyzed. [Results] A total of 16 323 712 high-quality reads and 26 504 operational taxonomic units (OTUs) were obtained for the 36 soil samples collected from seven different habitats. At the phylum level, the relative abundance of Actinomycetota and Proteobacteria were the highest in all the habitats. Bacillus, Ambiguous_taxa, Solirubacter, and Pseudoarthrobacter were the dominant genera. In addition, there was no significant difference in bacterial alpha diversity among different habitats, while the bacterial beta diversity showed significant differences, which was further confirmed by Linear discriminant analysis Effect Size (LEfSe). Redundancy analysis (RDA) identified Mg2+, Na+, and K+ as the main factors affecting the bacterial community structure, and the effects of other soil physicochemical factors on the distribution of dominant flora varied in different habitats. Finally, FAPROTAX tool was used to predict the bacterial function, which suggested that the roles of bacteria in the biogeochemical cycling processes of nitrogen and sulfur varied in different habitats. [Conclusion] The bacterial community structure varies greatly in different extreme habitats of the QTP, which is driven by different soil physicochemical factors. The presence of rich unannotated genera in each habitat indicates that the QTP has rich potential new bacterial resources.
GE Huihua , LIU Ting , YANG Chun , ZHANG Guangya
2023, 63(8):3252-3263. DOI: 10.13343/j.cnki.wsxb.20220910
Abstract:[Objective] β-1,4-xylanase is one of the key enzymes in the biodegradation of xylan. Psychrophilic and acidophilic xylanases play an important role in preparing functional xylooligosaccharides, whereas little is known about these enzymes. [Methods] We discovered a novel xylanase gene by functional annotation of the genome of the deep-sea bacterium Flammeovirga pacifica strain WPAGA1. The sequence alignment suggested 60% identity of this sequence with the verified β-1,4-xylanase from Clostridium saccharobutylicum (ID: P17137). Then, we constructed the recombinant plasmid and transformed it into the host cells for expression. After purifying the enzyme by nickel column, we examined the enzyme properties. [Results] The full-length β-1,4-xylanase (Xyl4513) had two conserved domains: a catalytic module belonging to the glycoside hydrolase family 11 (Xy14513-T) and a carbohydrate-binding module (CBM) belonging to family 60 (CBM4513). This was a rare phenomenon that the GH11 xylanase contained CBM. The purified Xyl4513 showed the highest activity at 30 °C and pH 3.0, being a psychrophilic and acidophilic β-1,4-xylanase. The truncated β-1,4-xylanase (Xy14513-T) demonstrated the highest activity at 20 °C and pH 4.0 and the catalytic efficiency (kcat/Km) 20% lower than that of Xyl4513, indicating the positive effects of CBM on the stability and catalytic performance of β-1,4-xylanase. In addition, Ca2+, Mg2+, and Ni2+ improved the catalytic activities, and Ca2+ showed the best performance. Only in the presence of Ca2+, CBM4513 had the specific binding ability to β-1,4-xylan, demonstrating a Ca2+-dependent CBM, and the maximum binding amount was 9.13 μmol/g. [Conclusion] We obtained a novel psychrophilic and acidophilic β-1,4-xylanase with a Ca2+-dependent CBM, which enriched the related gene and protein resources. The findings of this study will provide valuable information for exploring the stability, catalytic mechanism, and engineering of xylanases and CBMs.
HUANG Chen , JIANG Lin , LIANG Yinping , HAN Lingjuan , YANG Kaiyuan , QIN Yunmeng , GAO Peng , ZHAO Xiang
2023, 63(8):3264-3278. DOI: 10.13343/j.cnki.wsxb.20220911
Abstract:Lespedeza davurica is a grass for ecological restoration in the Loess Plateau. The saline-alkaline soil in the Loess Plateau greatly affects the growth and restricted the economic and ecological values of L. davurica. Inoculation of growth-promoting rhizobacteria is an important way to improve plant tolerance to saline-alkaline stress. [Objective] This study aims to reveal the effect and mechanism of Acinetobacter calcoaceticus in promoting the seedling growth of L. davurica under saline-alkaline stress, preliminarily disclose the potential growth-promoting mechanism of legumes, and provide a theoretical basis for the field application of this bacterium. [Methods] The potassium-containing medium was used to measure the potassium-solubilizing ability of two A. calcoaceticus strains isolated from L. davurica rhizosphere. The liquid fermentation method was employed to determine the production of siderophore and indole-3-acetic acid (IAA) by the two strains. Furthermore, we carried out petri dish and pot experiments to determine the growth-promoting effects of A. calcoaceticus on L. davurica seedlings under saline-alkaline stress. [Results] Strains DP25 and DP27 had the abilities to solubilize potassium and showed the siderophore production of 53.13% and 86.67% and the IAA production of 1.01 mg/L and 17.31 mg/L, respectively. Compared with the control, the inoculation of DP25 significantly improved the stem length and root length of L. davurica seedlings in petri dishes. The pot experiments showed that strains DP25 and DP27 had positive effects on the photosynthetic pigment synthesis and photosynthesis of the plants under saline-alkaline stress. Compared with the control, the inoculation with strains DP25 and DP27 increased the plant height, stem diameter, root surface area, total root length, and bifurcation number of L. davurica plants in pots. In addition, strain DP27 increased the aboveground biomass, underground biomass, root volume, total root tips, and root activity. [Conclusion] A. calcoaceticus demonstrates growth-promoting effect on the seedlings and adults of L. davurica. It secretes IAA to promote the growth of seedlings and improves the photosynthetic capacity and root morphogenesis to facilitate the growth of the adults under saline-alkaline stress. A. calcoaceticus can be used as a growth-promoting bacterium for planting L. daurica in the saline-alkaline area of Loess Plateau.
SUN Minghui , ZHANG Jiaxin , ZHANG Yiqiong , ZHAO Haodong , JING Xin , GAO Xuze , GUO Sijia , FENG Peilin , LIU Xiaoyu , CHEN Dafu , FU Zhongmin , GUO Rui
2023, 63(8):3279-3291. DOI: 10.13343/j.cnki.wsxb.20220914
Abstract:[Objective] Nosema ceranae exclusively infects adult honey bees and causes nosemosis, resulting in great losses for the beekeeping industry. Little is known about the N. ceranae N6-adenine-specific methyltransferase gene NcN6AMT. This study cloned the coding sequence (CDS) region of NcN6AMT, investigated the physicochemical properties and molecular characteristics of NcN6AMT, and then determined the relative expression level of NcN6AMT during the infection process of N. ceranae in Apis mellifera ligustica and Apis cerana cerana workers. This study aim to enrich the information about NcN6AMT and lay a foundation for exploring the function and epigenetic regulation mechanism of NcN6AMT during the infection process of N. ceranae. [Methods] Protparam and ProtScale were used to analyze the isoelectric point and hydrophilia of NcN6AMT. The signal peptide, phosphorylation site, transmembrane domain, secondary structure, and tertiary structure of NcN6AMT were predicted by SignalP 5.0, NetPhos 3.1, TMHMM-2.0, SOPMA, and SWISS-MODEL, respectively. WoLF PSORT II was employed to predict the subcellular localization of NcN6AMT. TBtools was employed to predict the domains in the amino acid sequences of N6AMTs in Homo sapiens, Mus musculus, Nilaparvata lugens, Encephalitozoon cuniculi, Encephalitozoon intestinalis ATCC 50506, Encephalitozoon Romaleae SJ-2008, Spraguea lophii 42_110, Nosema bombycis CQ1, Nitzschia inconspicua, and N. ceranae. MEME and MEGA 11.0 were employed to predict the conserved motifs and build the phylogenetic tree of N6AMTs. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed to determine the relative expression level of NcN6AMT during the N. ceranae infection of A. m. ligustica and A. c. cerana workers. [Results] The target fragment with a size of about 500 bp was amplified via PCR, and the cloning and sequencing results showed that it was consistent with the predicted sequence in GenBank. The deduced NcN6AMT protein was composed of 166 residues and had the molecular weight of 18.7 kDa, the formula of C845H1374N214O249S6, the theoretical isoelectric point of 5.88, the lipid solubility coefficient of 119.76, the instability coefficient of 37.47, the average hydrophilic coefficient of 0.025, and 15 phosphorylation sites, with no typical transmembrane domain or signal peptide. NcN6AMT was located in cytoplasm, mitochondria, nucleus, and vacuole membrane. It had one structural domain MTS, which also existed in the N6AMTs of other eight species such as N. bombycis CQ1 and E. cuniculi. Five same conserved motifs were predicted in the N6AMTs of N. ceranae, E. cuniculi,E. intestinalis ATCC 50506, and E. romaleae SJ-2008. The sequence identity of N6AMTs in N. ceranae, N. bombycis CQ1, E. cuniculi, E. intestinalis ATCC 50506, and E. romaleae SJ-2008 was 70.92%. The N6AMTs in N. ceranae and N. bombycis CQ1 were grouped into one clade. The expression of NcN6AMT was first up-regulated and then down-regulated within 1-4 days post inoculation (dpi) of N. ceranae in A. m. ligustica and A. c. cerana workers. [Conclusion] The CDS region of NcN6AMT was successfully cloned, and the physiochemical properties and molecular characteristics of NcN6AMT protein were clarified. The N6AMT proteins in N. ceranae and N. bombycis were highly conserved. During the first proliferation cycle (1-4 dpi) of N. ceranae in A. m. ligustica and A. c. cerana workers, the expression of NcN6AMT exhibited a bell-shaped pattern.
HAO Ruiying , LI Liang , YANG Xiurong , MA Haoxuan , SHI Shuo , FENG Yu
2023, 63(8):3292-3309. DOI: 10.13343/j.cnki.wsxb.20220921
Abstract:[Objective] To clarify the mechanism of Piriformospora indica in inducing wheats against root rot. [Methods] Seeds were soaked in the suspension of P. indica, and the medium without P. indica was used as control. After Fusarium graminearum infecting wheats, this study analyzed the physiological and biochemical indexes and transcriptome changes. [Results] The pathogen F. graminearum induced the production of hydrogen peroxide in wheats, reduced the intracellular water content, and destroyed the stability of the membrane. The colonization of P. indica increased the activity of intracellular antioxidant enzymes and reduced the accumulation of oxygen free radicals in cells. Intracellular water content was further maintained, and the stability of membranes was enhanced. The colonization of P. indica also reprogrammed the mRNA transcriptome changes caused by F. graminearum. The expression of resistance-related genes was enhanced. Based on the above results, P. indica effectively improved the resistance of wheats to F. graminearum. [Conclusion] The implementation of the work will provide theoretical basis and experimental basis for in-depth understanding of plant-microbe interaction and development of new efficient and environmentally friendly root rot agents.
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