Qian Yu, Hailin Li, Ling Du, Lifei Shen, Jiaxue Zhang, Lingyue Yuan, Huang Yao, Hong Xiao, Qunhua Bai, Yan Jia, Jingfu Qiu, Yingli Li
The ferric uptake regulator (Fur) is a global regulator that influences the expression of virulence genes in Klebsiella pneumoniae. Bioinformatics analysis suggests Fur may involve in iron acquisition via the identified regulatory box upstream of the yersiniabactin receptor gene fyuA. To observe the impact of the gene fyuA on the virulence of K. pneumoniae, the gene fyuA knockout strain and complementation strain were constructed and then conducted a series of phenotypic experiments including chrome azurol S (CAS) detection, crystal violet staining, and wax moth virulence experiment. To examine the regulatory relationship between Fur and the gene fyuA, green fluorescent protein (GFP) reporter gene fusion assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), gel migration assay (EMSA), and DNase I footprinting assay were used to clarify the regulatory mechanism of Fur on fyuA. CAS detection revealed that the gene fyuA could affect the generation of iron carriers in K. pneumoniae. Crystal violet staining experiment showed that fyuA could positively influence biofilm formation. Wax moth virulence experiment indicated that the deletion of the fyuA could weaken bacterial virulence. GFP reporter gene fusion experiment and RT-qPCR analysis revealed that Fur negatively regulated the expression of fyuA in iron-sufficient environment. EMSA experiment demonstrated that Fur could directly bind to the promoter region of fyuA, and DNase I footprinting assay further identified the specific binding site sequences. The study showed that Fur negatively regulated the transcriptional expression of fyuA by binding to upstream of the gene promoter region, and then affected the virulence of K. pneumoniae.
{"title":"Transcriptional regulation of the yersiniabactin receptor fyuA gene by the ferric uptake regulator in Klebsiella pneumoniae NTUH-K2044","authors":"Qian Yu, Hailin Li, Ling Du, Lifei Shen, Jiaxue Zhang, Lingyue Yuan, Huang Yao, Hong Xiao, Qunhua Bai, Yan Jia, Jingfu Qiu, Yingli Li","doi":"10.1002/jobm.202400001","DOIUrl":"10.1002/jobm.202400001","url":null,"abstract":"<p>The ferric uptake regulator (Fur) is a global regulator that influences the expression of virulence genes in <i>Klebsiella pneumoniae</i>. Bioinformatics analysis suggests Fur may involve in iron acquisition via the identified regulatory box upstream of the yersiniabactin receptor gene <i>fyuA</i>. To observe the impact of the gene <i>fyuA</i> on the virulence of <i>K. pneumoniae</i>, the gene <i>fyuA</i> knockout strain and complementation strain were constructed and then conducted a series of phenotypic experiments including chrome azurol S (CAS) detection, crystal violet staining, and wax moth virulence experiment. To examine the regulatory relationship between Fur and the gene <i>fyuA</i>, green fluorescent protein (GFP) reporter gene fusion assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), gel migration assay (EMSA), and DNase I footprinting assay were used to clarify the regulatory mechanism of Fur on <i>fyuA</i>. CAS detection revealed that the gene <i>fyuA</i> could affect the generation of iron carriers in <i>K. pneumoniae</i>. Crystal violet staining experiment showed that <i>fyuA</i> could positively influence biofilm formation. Wax moth virulence experiment indicated that the deletion of the <i>fyuA</i> could weaken bacterial virulence. GFP reporter gene fusion experiment and RT-qPCR analysis revealed that Fur negatively regulated the expression of <i>fyuA</i> in iron-sufficient environment. EMSA experiment demonstrated that Fur could directly bind to the promoter region of <i>fyuA</i>, and DNase I footprinting assay further identified the specific binding site sequences. The study showed that Fur negatively regulated the transcriptional expression of <i>fyuA</i> by binding to upstream of the gene promoter region, and then affected the virulence of <i>K. pneumoniae</i>.</p>","PeriodicalId":15101,"journal":{"name":"Journal of Basic Microbiology","volume":"64 8","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140834622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Brevibacillus thermoruber strain Nabari cells grow as widely spreading dendritic colonies on reasoner's 2A-agar (1.5%) plates at around 55°C but as small motile colonies at 37°C. Motile colonies can be divided into colonies that move in straight or curved lines over long distances (wandering colonies), and colonies that rotate at a fixed location (rotating colonies). The addition of surfactant to the agar medium greatly increased the frequency of wandering colonies and facilitated the study of such colonies. The morphology of the wandering colonies varied: circular at the tip and pointed at the back, lemon-shaped with pointed ends, crescent-shaped, bullet-shaped, fish-like, and so on. A single colony may split into multiple colonies as it moves, or multiple colonies may merge into a single colony. The most surprising aspect of the movement of wandering colonies was that when a moving colony collides with another colony, it sometimes does not make a U-turn, but instead retreats straight back, as if bouncing back. The migration mechanisms of wandering colonies are discussed based on optical microscopic observations of swimming patterns of single cells in water and scanning electron microscopy of the arrangement of bacterial cells in wandering colonies.
{"title":"Unique behavioral patterns of wandering colonies of Brevibacillus thermoruber on agar plates","authors":"Kazuo Sakka, Masaki Kihira, Wataru Kuhara, Akihiro Mochida, Satoru Ogawa, Tetsuya Kimura, Makiko Sakka","doi":"10.1002/jobm.202400091","DOIUrl":"10.1002/jobm.202400091","url":null,"abstract":"<p><i>Brevibacillus thermoruber</i> strain Nabari cells grow as widely spreading dendritic colonies on reasoner's 2A-agar (1.5%) plates at around 55°C but as small motile colonies at 37°C. Motile colonies can be divided into colonies that move in straight or curved lines over long distances (wandering colonies), and colonies that rotate at a fixed location (rotating colonies). The addition of surfactant to the agar medium greatly increased the frequency of wandering colonies and facilitated the study of such colonies. The morphology of the wandering colonies varied: circular at the tip and pointed at the back, lemon-shaped with pointed ends, crescent-shaped, bullet-shaped, fish-like, and so on. A single colony may split into multiple colonies as it moves, or multiple colonies may merge into a single colony. The most surprising aspect of the movement of wandering colonies was that when a moving colony collides with another colony, it sometimes does not make a U-turn, but instead retreats straight back, as if bouncing back. The migration mechanisms of wandering colonies are discussed based on optical microscopic observations of swimming patterns of single cells in water and scanning electron microscopy of the arrangement of bacterial cells in wandering colonies.</p>","PeriodicalId":15101,"journal":{"name":"Journal of Basic Microbiology","volume":"64 6","pages":""},"PeriodicalIF":3.1,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jobm.202400091","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140668054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
NAD+-dependent (2 R,3 R)‑2,3‑butanediol dehydrogenase (BDH) from Neisseria gonorrhoeae (NgBDH) is a representative member of the medium-chain dehydrogenase/reductase (MDR) superfamily. To date, little information is available on the substrate binding sites and catalytic residues of BDHs from this superfamily. In this work, according to molecular docking studies, we found that conserved residues Phe120 and Val161 form strong hydrophobic interactions with both (2 R,3 R)‑2,3‑butanediol (RR-BD) and meso-2,3‑butanediol (meso-BD) and that mutations of these residues to alanine or threonine impair substrate binding. To further evaluate the roles of these two residues, Phe120 and Val161 were mutated to alanine or threonine. Kinetic analysis revealed that, relative to those of wild type, the apparent KM values of the Phe120Ala mutant for RR-BD and meso-BD increased 36- and 369-fold, respectively; the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 586- and 3528-fold, respectively; and the apparent KM values of the Val161Ala mutant for RR-BD and meso-BD increased 4- and 37-fold, respectively, the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 3- and 28-fold, respectively. Additionally, the Val161Thr mutant slightly decreased catalytic efficiencies (twofold with RR-BD; 7.3-fold with meso-BD) due to an increase in KM (sixfold for RR-BD; 24-fold for meso-BD) and a slight increase (2.8-fold with RR-BD; 3.3-fold with meso-BD) in kcat. These findings validate the critical roles of Phe120 and Val161 of NgBDH in substrate binding and catalysis. Overall, the current study provides a better understanding of the substrate binding and catalysis of BDHs within the MDR superfamily.
{"title":"The critical role of residues Phe120 and Val161 of (2 R,3 R)‑2,3‑butanediol dehydrogenase from Neisseria gonorrhoeae as probed by molecular docking and site-directed mutagenesis","authors":"Xue Dong, Tingting Zhang, Chuanyue Gui, Shuping Fei, Haonan Xu, Jianrong Chang, Chaoqun Lian, Wanggang Tang","doi":"10.1002/jobm.202300751","DOIUrl":"10.1002/jobm.202300751","url":null,"abstract":"<p>NAD<sup>+</sup>-dependent (2 <i>R</i>,3 <i>R</i>)‑2,3‑butanediol dehydrogenase (BDH) from <i>Neisseria gonorrhoeae</i> (NgBDH) is a representative member of the medium-chain dehydrogenase/reductase (MDR) superfamily. To date, little information is available on the substrate binding sites and catalytic residues of BDHs from this superfamily. In this work, according to molecular docking studies, we found that conserved residues Phe120 and Val161 form strong hydrophobic interactions with both (2 <i>R</i>,3 <i>R</i>)‑2,3‑butanediol (RR-BD) and <i>meso</i>-2,3‑butanediol (meso-BD) and that mutations of these residues to alanine or threonine impair substrate binding. To further evaluate the roles of these two residues, Phe120 and Val161 were mutated to alanine or threonine. Kinetic analysis revealed that, relative to those of wild type, the apparent <i>K</i><sub>M</sub> values of the Phe120Ala mutant for RR-BD and meso-BD increased 36- and 369-fold, respectively; the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 586- and 3528-fold, respectively; and the apparent <i>K</i><sub>M</sub> values of the Val161Ala mutant for RR-BD and meso-BD increased 4- and 37-fold, respectively, the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 3- and 28-fold, respectively. Additionally, the Val161Thr mutant slightly decreased catalytic efficiencies (twofold with RR-BD; 7.3-fold with meso-BD) due to an increase in <i>K</i><sub>M</sub> (sixfold for RR-BD; 24-fold for meso-BD) and a slight increase (2.8-fold with RR-BD; 3.3-fold with meso-BD) in <i>k</i><sub>cat</sub>. These findings validate the critical roles of Phe120 and Val161 of NgBDH in substrate binding and catalysis. Overall, the current study provides a better understanding of the substrate binding and catalysis of BDHs within the MDR superfamily.</p>","PeriodicalId":15101,"journal":{"name":"Journal of Basic Microbiology","volume":"64 6","pages":""},"PeriodicalIF":3.1,"publicationDate":"2024-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140636202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Asmaa E. Ahmed, Hassan Abol-enein, Adel A. El-Morsi, Ahmed S. El-Hefnawy, Ashraf A. Elsayed, Sherry Khater, Abdelwahab Hashem, Abdel-Rahman N. Zekri, Samia A. Haroun, Ahmed A. Shokeir, Amira Awadalla
Hepatitis C virus (HCV) is the most common infection worldwide. The correlation between HCV and renal cell carcinoma (RCC) is still mysterious. Therefore, the relationship between HCV and RCC was investigated. The study included 100 patients with RCC; 32 with HCV infection, and 68 without HCV infection. Expressions of viral proteins (NS3 and NS5A) were tested using an immune electron-microscope (IEM) and immunohistochemistry (IHC). IHC and quantitative real time-PCR investigated the presentation of human proteins TP53 and p21 genes. Transmission electron (TEM) detected viral-like particles in infected RCC tissues. The gene and protein expression of P53 was higher in HCV positive versus HCV negative patients and p21 was lower in HCV positive versus HCV negative in both tumor and normal tissue samples. Viral like particles were observed by TEM in the infected tumor and normal portion of the RCC tissues and the plasma samples. The IEM showed the depositions of NS3 and NS5A in infected renal tissues, while in noninfected samples, were not observed. The study hypothesizes that a correlation between HCV and RCC could exist through successfully detecting HCV-like particles, HCV proteins, and (p53 and p21) in RCC-infected patients.
{"title":"Association between hepatitis C virus genotype 4 and renal cell carcinoma: Molecular and virological studies","authors":"Asmaa E. Ahmed, Hassan Abol-enein, Adel A. El-Morsi, Ahmed S. El-Hefnawy, Ashraf A. Elsayed, Sherry Khater, Abdelwahab Hashem, Abdel-Rahman N. Zekri, Samia A. Haroun, Ahmed A. Shokeir, Amira Awadalla","doi":"10.1002/jobm.202300279","DOIUrl":"10.1002/jobm.202300279","url":null,"abstract":"<p>Hepatitis C virus (HCV) is the most common infection worldwide. The correlation between HCV and renal cell carcinoma (RCC) is still mysterious. Therefore, the relationship between HCV and RCC was investigated. The study included 100 patients with RCC; 32 with HCV infection, and 68 without HCV infection. Expressions of viral proteins (NS3 and NS5A) were tested using an immune electron-microscope (IEM) and immunohistochemistry (IHC). IHC and quantitative real time-PCR investigated the presentation of human proteins TP53 and p21 genes. Transmission electron (TEM) detected viral-like particles in infected RCC tissues. The gene and protein expression of P53 was higher in HCV positive versus HCV negative patients and p21 was lower in HCV positive versus HCV negative in both tumor and normal tissue samples. Viral like particles were observed by TEM in the infected tumor and normal portion of the RCC tissues and the plasma samples. The IEM showed the depositions of NS3 and NS5A in infected renal tissues, while in noninfected samples, were not observed. The study hypothesizes that a correlation between HCV and RCC could exist through successfully detecting HCV-like particles, HCV proteins, and (p53 and p21) in RCC-infected patients.</p>","PeriodicalId":15101,"journal":{"name":"Journal of Basic Microbiology","volume":"64 6","pages":""},"PeriodicalIF":3.1,"publicationDate":"2024-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140570260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}