Aggeliki Skagia, Eleni Vezyri, Konstantinos Grados, Anastasia Venieraki, Michael Karpusas, Panagiotis Katinakis, Maria Dimou
{"title":"Structure-Function Analysis of the Periplasmic Escherichia coli Cyclophilin PpiA in Relation to Biofilm Formation.","authors":"Aggeliki Skagia, Eleni Vezyri, Konstantinos Grados, Anastasia Venieraki, Michael Karpusas, Panagiotis Katinakis, Maria Dimou","doi":"10.1159/000478858","DOIUrl":"https://doi.org/10.1159/000478858","url":null,"abstract":"<p><p>The presence of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8) in all domains of life indicates their biological importance. Cyclophilin PpiA, present in the periplasm of gram-negative bacteria, possesses PPIase activity but its physiological functions are still not clearly defined. Here, we demonstrate that the ΔppiA deletion strain from Escherichia coli exhibits an increased ability for biofilm formation and enhanced swimming motility compared to the wild-type strain. To identify structural features of PpiA which are necessary for the negative modulation of biofilm formation, we constructed a series of mutant PpiA proteins using a combination of error-prone and site-directed mutagenesis approaches. We show that the negative effect of PpiA on biofilm formation is not dependent on its PPIase activity, since PpiA mutants with a reduced PPIase activity are able to complement the ΔppiA strain during biofilm growth.</p>","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000478858","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35391709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance.","authors":"Yao Zhai, Zhao Zhang, Zhanwei Wang, Yusheng Chen, Qi Wang, Yuan Lv, Jingping Yang, Tong Zhao, Yatao Guo, Zhancheng Gao","doi":"10.1159/000458708","DOIUrl":"https://doi.org/10.1159/000458708","url":null,"abstract":"<p><strong>Aims: </strong>This work investigated the relative strengths of different blaSHV promoter-associated sequences and their regulation function in blaSHV expression and β-lactam resistance.</p><p><strong>Methods: </strong>Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), tac promoter, and combined fragments of promoter and blaSHV were separately constructed and transformed into Escherichia coli DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of blaSHV were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated.</p><p><strong>Results: </strong>The relative strengths were ranked as P-tac > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of gfp and blaSHV mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain blaSHV groups. In relation to the different promoter-associated sequences, blaSHV-ESBLs displayed significantly different change modes of β-lactam resistance compared with blaSHV-non-ESBLs.</p><p><strong>Conclusion: </strong>The mRNA expression and β-lactam resistance of the blaSHV showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.</p>","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000458708","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34844139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Adam Kuzdraliński, Anna Kot, Hubert Szczerba, Michał Nowak, Marta Muszyńska
{"title":"A Review of Conventional PCR Assays for the Detection of Selected Phytopathogens of Wheat.","authors":"Adam Kuzdraliński, Anna Kot, Hubert Szczerba, Michał Nowak, Marta Muszyńska","doi":"10.1159/000477544","DOIUrl":"https://doi.org/10.1159/000477544","url":null,"abstract":"<p><p>Infection of phyllosphere (stems, leaves, husks, and grains) by pathogenic fungi reduces the wheat yield and grain quality. Detection of the main wheat pathogenic fungi provides information about species composition and allows effective and targeted plant treatment. Since conventional procedures for the detection of these organisms are unreliable and time consuming, diagnostic DNA-based methods are required. Nucleic acid amplification technologies are independent of the morphological and biochemical characteristics of fungi. Microorganisms do not need to be cultured. Therefore, a number of PCR-based methodologies have been developed for the identification of key pathogenic fungi, such as Fusarium spp., Puccinia spp., Zymoseptoria tritici, Parastagonospora nodorum, Blumeria graminis f. sp. tritici, and Pyrenophora tritici-repentis. This article reviews frequently used DNA regions for fungus identification and discusses already known PCR assays for detection of the aforementioned wheat pathogens. We demonstrate that PCR-based wheat pathogen identification assays require further research. In particular, the number of diagnostic tests for Fusarium graminearum, Puccinia spp., and P. tritici-repentis are insufficient.</p>","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000477544","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35379718","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Adam Kuzdraliński, Anna Kot, Hubert Szczerba, Agnieszka Ostrowska, Michał Nowak, Marta Muszyńska, Michał Lechowski, Paweł Muzyka
{"title":"Novel PCR Assays for the Detection of Biological Agents Responsible for Wheat Rust Diseases: Puccinia triticina and Puccinia striiformis f. sp. tritici.","authors":"Adam Kuzdraliński, Anna Kot, Hubert Szczerba, Agnieszka Ostrowska, Michał Nowak, Marta Muszyńska, Michał Lechowski, Paweł Muzyka","doi":"10.1159/000481799","DOIUrl":"https://doi.org/10.1159/000481799","url":null,"abstract":"<p><p>The species Puccinia triticina (Pt) and Puccinia striiformis f. sp. tritici (Pst) are devastating cereal pathogens that cause leaf and stripe rust diseases. We developed PCR assays for the species-specific detection of Pt and Pst, 2 biological agents that cause wheat rust disease. For each pathogen, we validated 3 primer sets that target the second largest subunits of the RNA polymerase II (rpb2) and β-tubulin 1 (tub1) genes. The specificities of the primers were verified using naturally infected plant materials with visual symptoms of disease. All primer sets amplified a single DNA fragment of the expected length. The primer sets LidPr15/16, LidPr1/2, and LidPs13/14 were able to detect small amounts of pure fungal DNA with sensitivities of 0.1, 1, and 10 pg/μL, respectively. A sufficient detection limit (1 pg/μL to 5 ng/μL) was observed for all assays when the sensitivity test was performed with host plant DNA. The study also evaluated the simultaneous detection of both rust pathogens, and the multiplex PCR assay generated amplicons of 240 and 144 bp in length for Pts (LidPs9/10) and Pt (LidPr1/2), respectively.</p>","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000481799","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35596537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Heterologous Expression of Spinosyn Biosynthetic Gene Cluster in Streptomyces Species Is Dependent on the Expression of Rhamnose Biosynthesis Genes.","authors":"Chen Zhao, Ying Huang, Chao Guo, Bolei Yang, Yan Zhang, Zhou Lan, Xiong Guan, Yuan Song, Xiaolin Zhang","doi":"10.1159/000477543","DOIUrl":"https://doi.org/10.1159/000477543","url":null,"abstract":"<p><p>Spinosyns are a group of macrolide insecticides produced by Saccharopolyspora spinosa. Although S. spinosa can be used for industrial-scale production of spinosyns, this might suffer from several limitations, mainly related to its long growth cycle, low fermentation biomass, and inefficient utilization of starch. It is crucial to generate a robust strain for further spinosyn production and the development of spinosyn derivatives. A BAC vector, containing the whole biosynthetic gene cluster for spinosyn (74 kb) and the elements required for conjugal transfer and site-specific integration, was introduced into different Streptomyces hosts in order to obtain heterologous spinosyn-producing strains. The exconjugants of different Streptomyces strains did not show spinosyn production unless the rhamnose biosynthesis genes from S. spinosa genomic DNA were present and expressed under the control of a strong constitutive ermE*p promoter. Using this heterologous expression system resulted in yields of 1 μg/mL and 1.5 μg/mL spinosyns in Streptomyces coelicolor and Streptomyces lividans, respectively. This report demonstrates spinosyn production in 2 Streptomyces strains and stresses the essential role of rhamnose in this process. This work also provides a potential alternative route for producing spinosyn analogs by means of genetic manipulation in the heterologous hosts.</p>","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000477543","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35451657","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vic Norris, Sergey N Krylov, Pratul K Agarwal, Glenn J White
{"title":"Synthetic, Switchable Enzymes.","authors":"Vic Norris, Sergey N Krylov, Pratul K Agarwal, Glenn J White","doi":"10.1159/000464443","DOIUrl":"https://doi.org/10.1159/000464443","url":null,"abstract":"<p><p>The construction of switchable, radiation-controlled, aptameric enzymes - \"swenzymes\" - is, in principle, feasible. We propose a strategy to make such catalysts from 2 (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a 2-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker, thus bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low-intensity, nonionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate product-capturing aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis.</p>","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000464443","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34947221","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sofía García, N. Flores, R. de Anda, G. Hernández, G. Gosset, F. Bolivar, A. Escalante
{"title":"The Role of the ydiB Gene, Which Encodes Quinate/Shikimate Dehydrogenase, in the Production of Quinic, Dehydroshikimic and Shikimic Acids in a PTS- Strain of Escherichia coli","authors":"Sofía García, N. Flores, R. de Anda, G. Hernández, G. Gosset, F. Bolivar, A. Escalante","doi":"10.1159/000450611","DOIUrl":"https://doi.org/10.1159/000450611","url":null,"abstract":"The culture of engineered Escherichia coli for shikimic acid (SA) production results in the synthesis of quinic acid (QA) and dehydroshikimic acid (DHS), reducing SA yield and impairing downstream processes. The synthesis of QA by quinate/shikimate dehydrogenase (YdiB, ydiB) has been previously proposed; however, the precise role for this enzyme in the production of QA in engineered strains of E. coli for SA production remains unclear. We report the effect of the inactivation or the overexpression of ydiB in E. coli strain PB12.SA22 on SA, QA, and DHS production in batch fermentor cultures. The results showed that the inactivation of ydiB resulted in a 75% decrease in the molar yield of QA and a 6.17% reduction in the yield of QA (mol/mol) relative to SA with respect to the parental strain. The overexpression of ydiB caused a 500% increase in the molar yield of QA and resulted in a 152% increase in QA (mol/mol) relative to SA, with a sharp decrease in SA production. Production of SA, QA, and DHS in parental and derivative ydiB strains suggests that the synthesis of QA results from the reduction of 3-dehydroquinate by YdiB before its conversion to DHS.","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2016-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000450611","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"65135098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
J. Rodríguez-Mejía, Abigail Roldán-Salgado, J. Osuna, E. Merino, P. Gaytán
{"title":"A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression","authors":"J. Rodríguez-Mejía, Abigail Roldán-Salgado, J. Osuna, E. Merino, P. Gaytán","doi":"10.1159/000448786","DOIUrl":"https://doi.org/10.1159/000448786","url":null,"abstract":"Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2016-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000448786","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"65106000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Molecular Epidemiology of Human Metapneumovirus in Riyadh Province, Saudi Arabia","authors":"H. Amer","doi":"10.1159/000448374","DOIUrl":"https://doi.org/10.1159/000448374","url":null,"abstract":"Human metapneumovirus (HMPV) is an important cause of respiratory tract illness in children. Two HMPV subgroups, A and B, and four genotypes, A1, A2, B1 and B2, have been identified. Concurrent circulation of the different genotypes in yearly epidemics has been recorded globally, but not in Saudi Arabia. The current report was designed to study HMPV epidemiology in Saudi children and to analyze the genetic diversity and circulation patterns. Nasopharyngeal aspirates (n = 174) were collected from hospitalized children in Riyadh (2008-2009). The screening of samples using real-time RT-PCR identified 19 HMPV strains. The majority of the strains belonged to subgroup B, while all strains of subgroup A were members of genotype A2. In 2008, only subgroup B was recognized, whereas in 2009 both subgroups were identified to be cocirculating at similar rates. The full-length attachment (G) gene and a partial sequence of the fusion (F) gene of positive samples were sequenced. The G gene showed a high degree of genetic diversity and exhibited a variable number of positively selected sites in different lineages. In contrast, the F gene demonstrated an extensive genetic stability with a higher tendency toward purifying selection. This is the first report on HMPV genotype circulation in Saudi Arabia; however, the exact circulation kinetics requires further retrospective and prospective study.","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2016-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000448374","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"65097598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
H. Amer, X. Jiao, U. Kwon, M. Saier, Harikrishnan Kuppusamykrishnan, Larry M. Chau, G. Moreno-Hagelsieb, Hongqin Song, X. Kang, Yun Yang, Yang Jiao, Li Song, D. Xiong, Lili Wu, Z. Pan, Junrui Wang, Junli Zhang, Q. Fu, Sufang Guo, La Ta, Peng Sun, M. Douraghi, S. Jasemi, M. Kodori, M. Rahbar, M. Boroumand, Naveen Kumar, S. Barua, Riyesh Thachamvally, B. N. Tripathi, Thu Trang Thi Vu, B. Jeong, M. Krupa, Jung-A song, Bich Hang Do, M. Nguyen, T. Seo, A. N. Nguyen, C. Joo, H. Choe, Abdelhamid Mokhtari, Q. M. M. Pham, P. Joyet, J. Deutscher, Takfarinas Kentache, E. Milohanic, Thanh Nguyen Cao, F. Aké, Satz Mengensatzproduktion, Druckerei Stückle
{"title":"Contents Vol. 26, 2016","authors":"H. Amer, X. Jiao, U. Kwon, M. Saier, Harikrishnan Kuppusamykrishnan, Larry M. Chau, G. Moreno-Hagelsieb, Hongqin Song, X. Kang, Yun Yang, Yang Jiao, Li Song, D. Xiong, Lili Wu, Z. Pan, Junrui Wang, Junli Zhang, Q. Fu, Sufang Guo, La Ta, Peng Sun, M. Douraghi, S. Jasemi, M. Kodori, M. Rahbar, M. Boroumand, Naveen Kumar, S. Barua, Riyesh Thachamvally, B. N. Tripathi, Thu Trang Thi Vu, B. Jeong, M. Krupa, Jung-A song, Bich Hang Do, M. Nguyen, T. Seo, A. N. Nguyen, C. Joo, H. Choe, Abdelhamid Mokhtari, Q. M. M. Pham, P. Joyet, J. Deutscher, Takfarinas Kentache, E. Milohanic, Thanh Nguyen Cao, F. Aké, Satz Mengensatzproduktion, Druckerei Stückle","doi":"10.1159/000452182","DOIUrl":"https://doi.org/10.1159/000452182","url":null,"abstract":"","PeriodicalId":16370,"journal":{"name":"Journal of Molecular Microbiology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2016-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000452182","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"65160611","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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