Journal of Molecular Microbiology and Biotechnology最新文献

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.

Aims: This work investigated the relative strengths of different blaSHV promoter-associated sequences and their regulation function in blaSHV expression and β-lactam resistance.

Methods: 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.

Results: 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.

Conclusion: 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.

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.

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.

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.

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.