Transposon-Mutagenese in Streptomyceten

Recent whole genome sequencing programs have revealed that the biosynthetic potential of Actinomycetales has been even underexplored with traditional approaches. With the advent of next-generation DNA sequencing techniques, we can access the huge amount of genetic information, which awaits development into new chemical and biological entities. Therefore efficient methods for the genes characterization are of great importance. In vivo transposon-based strategy is a valuable tool to identify functions of a number of genes and to construct random mutant libraries for diverse applications. Despite the wide availability of transposon systems, few options exist for use in actinomycetes. The aim of this project is to establish a system for random transposon mutagenesis in streptomycetes. According to this aim the nucleotide content of Himar1 gene was adapted to the high GC-content of streptomycetes. Set of plasmids for transposon mutagenesis had been constructed and transposon mutant libraries of streptomycetes species had been obtained (S. coelicolor M145, S. albus J1074 and S. lividans 1326). The system was used for identification of novel regulatory genes of actinorhodin biosynthesis in S. coelicolor and for random integration of gusA reporter gene and antibiotic biosynthetic cluster into chromosome of S. albus J1074 with further investigation of position effect in this strain. Also the secondary attB site, discovered in the genome of S. albus J1074, was characterized.Die Methoden der Next-Generation DNA-Sequenzierung erlauben uns Zugang zu einer großen Menge an genetischen Informationen, die intensiv in den Bereichen der Biologie und Chemie genutzt werden sollten. Deswegen ist die Entwicklung von effektiven Methoden für eine funktionelle Gen-Charakterisierung heutzutage sehr wichtig. Die in vivo-Transposon-basierte Strategie ist ein wertvolles Instrument, das man für die Identifizierung der Funktionen zahlreicher Gene und die Konstruktion von Random-Mutanten-Bibliotheken für vielfältige Anwendungen nutzen kann. Trotz der breiten Verfügbarkeit von Transposon-Systemen sind nur wenige davon zuverlässig auf Streptomyceten anwendbar. Das Ziel dieser Arbeit ist es, ein System für Random-Transposon-Mutagenese in Streptomyceten zu entwickeln. Hierfür wurde der Codongebrauch des Himar1 Gens an den hohen GC-Gehalt der Streptomyceten angepasst. Basierend auf diesem Gen wurden die Plasmide für die Transposon-Mutagenese konstruiert und die Bibliotheken der Mutanten von verschiedenen Streptomyceten erzeugt. Das System wurde in S. coelicolor für die Identifizierung von neuen regulatorischen Genen in der Actinorhodin Biosynthese verwendet. Außerdem wurden mit Hilfe unseres Systems das Reporter-Gen gusA und biosynthetische Antibiotika-Cluster zufällig in das S. albus-Chromosom integriert. Damit wurde der Positions-Effekt in diesem Stamm erforscht. Es wurde auch eine sekundäre attB-Stelle von fC31-basierten Plasmiden entdeckt und charakterisiert

Transposon mutagenesis in streptomycetes

Recent whole genome sequencing programs have revealed that the biosynthetic potential of Actinomycetales has been even underexplored with traditional approaches. With the advent of next-generation DNA sequencing techniques, we can access the huge amount of genetic information, which awaits development into new chemical and biological entities. Therefore efficient methods for the genes characterization are of great importance. In vivo transposon-based strategy is a valuable tool to identify functions of a number of genes and to construct random mutant libraries for diverse applications. Despite the wide availability of transposon systems, few options exist for use in actinomycetes. The aim of this project is to establish a system for random transposon mutagenesis in streptomycetes. According to this aim the nucleotide content of Himar1 gene was adapted to the high GC-content of streptomycetes. Set of plasmids for transposon mutagenesis had been constructed and transposon mutant libraries of streptomycetes species had been obtained (S. coelicolor M145, S. albus J1074 and S. lividans 1326). The system was used for identification of novel regulatory genes of actinorhodin biosynthesis in S. coelicolor and for random integration of gusA reporter gene and antibiotic biosynthetic cluster into chromosome of S. albus J1074 with further investigation of position effect in this strain. Also the secondary attB site, discovered in the genome of S. albus J1074, was characterized.Die Methoden der Next-Generation DNA-Sequenzierung erlauben uns Zugang zu einer großen Menge an genetischen Informationen, die intensiv in den Bereichen der Biologie und Chemie genutzt werden sollten. Deswegen ist die Entwicklung von effektiven Methoden für eine funktionelle Gen-Charakterisierung heutzutage sehr wichtig. Die in vivo-Transposon-basierte Strategie ist ein wertvolles Instrument, das man für die Identifizierung der Funktionen zahlreicher Gene und die Konstruktion von Random-Mutanten-Bibliotheken für vielfältige Anwendungen nutzen kann. Trotz der breiten Verfügbarkeit von Transposon-Systemen sind nur wenige davon zuverlässig auf Streptomyceten anwendbar. Das Ziel dieser Arbeit ist es, ein System für Random-Transposon-Mutagenese in Streptomyceten zu entwickeln. Hierfür wurde der Codongebrauch des Himar1 Gens an den hohen GC-Gehalt der Streptomyceten angepasst. Basierend auf diesem Gen wurden die Plasmide für die Transposon-Mutagenese konstruiert und die Bibliotheken der Mutanten von verschiedenen Streptomyceten erzeugt. Das System wurde in S. coelicolor für die Identifizierung von neuen regulatorischen Genen in der Actinorhodin Biosynthese verwendet. Außerdem wurden mit Hilfe unseres Systems das Reporter-Gen gusA und biosynthetische Antibiotika-Cluster zufällig in das S. albus-Chromosom integriert. Damit wurde der Positions-Effekt in diesem Stamm erforscht. Es wurde auch eine sekundäre attB-Stelle von fC31-basierten Plasmiden entdeckt und charakterisiert

Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins

Fluorescent proteins are a major cell biology tool to analyze protein sub-cellular topology. Here we have applied this technology to study protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, a widely used host for heterologous protein secretion biotechnology. Green and monomeric red fluorescent proteins were fused behind Sec (SPSec) or Tat (SPTat) signal peptides to direct them through the respective export pathway. Significant secretion of fluorescent eGFP and mRFP was observed exclusively through the Tat and Sec pathways, respectively. Plasmid over-expression was compared to a chromosomally integrated spSec-mRFP gene to allow monitoring secretion under high and low level synthesis in various media. Fluorimetric detection of SPSec-mRFP recorded folded states, while immuno-staining detected even non-folded topological intermediates. Secretion of SPSec-mRFP is unexpectedly complex, is regulated independently of cell growth phase and is influenced by the growth regime. At low level synthesis, highly efficient secretion occurs until it is turned off and secretory preforms accumulate. At high level synthesis, the secretory pathway overflows and proteins are driven to folding and subsequent degradation. High-level synthesis of heterologous secretory proteins, whether secretion competent or not, has a drastic effect on the endogenous secretome, depending on their secretion efficiency. These findings lay the foundations of dissecting how protein targeting and secretion are regulated by the interplay between the metabolome, secretion factors and stress responses in the S. lividans model

Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions

Alternative sigma factors control numerous aspects of bacterial life, including adaptation to physiological stresses, morphological development, persistence states and virulence. This is especially true for the physiologically complex actinobacteria. Here we report the development of a robust gene deletions system for Streptomyces lividans TK24 based on a BAC library combined with the λ-Red recombination technique. The developed system was validated by systematically deleting the most highly expressed genes encoding alternative sigma factors and several other regulatory genes within the chromosome of S. lividans TK24. To demonstrate the possibility of large scale genomic manipulations, the major part of the undecylprodigiosin gene cluster was deleted as well. The resulting mutant strains were characterized in terms of morphology, growth parameters, secondary metabolites production and response to thiol-oxidation and cell-wall stresses. Deletion of SLIV_12645 gene encoding S. coelicolor SigR1 ortholog has the most prominent phenotypic effect, resulted in overproduction of actinorhodin and coelichelin P1 and increased sensitivity to diamide. The secreted proteome analysis of SLIV_12645 mutant revealed SigR1 influence on trafficking of proteins involved in cell wall biogenesis and refactoring. The reported here gene deletion system will further facilitate work on S. lividans strain improvement as a host for either secondary metabolites or protein production and will contribute to basic research in streptomycetes physiology, morphological development, secondary metabolism. On the other hand, the systematic deletion of sigma factors encoding genes demonstrates the complexity and conservation of regulatory processes conducted by sigma factors in streptomycetes

Regulation of antibiotic production in Actinobacteria: new perspectives from the post-genomic era

The antimicrobial activity of many of their natural products has brought prominence to the Streptomycetaceae, a family of Gram-positive bacteria that inhabit both soil and aquatic sediments. In the natural environment, antimicrobial compounds are likely to limit the growth of competitors, thereby offering a selective advantage to the producer, in particular when nutrients become limited and the developmental programme leading to spores commences. The study of the control of this secondary metabolism continues to offer insights into its integration with a complex lifecycle that takes multiple cues from the environment and primary metabolism. Such information can then be harnessed to devise laboratory screening conditions to discover compounds with new or improved clinical value. Here we provide an update of the review we published in NPR in 2011. Besides providing the essential background, we focus on recent developments in our understanding of the underlying regulatory networks, ecological triggers of natural product biosynthesis, contributions from comparative genomics and approaches to awaken the biosynthesis of otherwise silent or cryptic natural products. In addition, we highlight recent discoveries on the control of antibiotic production in other Actinobacteria, which have gained considerable attention since the start of the genomics revolution. New technologies that have the potential to produce a step change in our understanding of the regulation of secondary metabolism are also described

The role of the incentive system in shaping employer internal branding

Employer branding stanowi pojęcie stosunkowo nowe, jednakże realia kształtowania wizerunku pracodawcy stanowią jeden z obszarów, który funkcjonuje od bardzo dawna w przedsiębiorstwach. Podmioty gospodarcze są bowiem świadome, że tworzenie wartościowej marki, jako pracodawcy warunkuje sukces całej organizacji. To właśnie za sprawą pozytywnego postrzegania danego pracodawcy przez obecnych i potencjalnych zatrudnionych możliwe jest ograniczenie fluktuacji kadr, a co najważniejsze zatrzymanie i pozyskanie utalentowanych jednostek. W związku z powyższym podjęto temat pracy, którego celem była ocena systemu motywacyjnego w aspekcie kształtowania employer brandingu wewnętrznego. Nieodzownym elementem kreowania marki pracodawcy jest zadowolenie i wysoki poziom satysfakcji osobistej pracowników. Orężem w tym względzie jest właśnie system motywacyjny, jako element wspierający budowanie wysokiego poziomu zaangażowania zatrudnionych oraz ich satysfakcji z pracy. W niniejszym opracowaniu dokonano analizy literatury przedmiotu w aspekcie poruszanego problemu, ale również przeprowadzono autorski sondaż diagnostyczny wśród pracowników wybranego podmiotu, aby ukazać realia związane z procesem kształtowania employer brandingu w praktyce. W toku przeprowadzonej analizy badawczej zdiagnozowano funkcjonujący w wybranym przedsiębiorstwie – firmie Burger King, system motywacyjny, wraz ze wskazaniem wykorzystywanych narzędzi i instrumentów motywacyjnych. Zweryfikowano także postrzeganie pracodawcy z perspektywy samych zatrudnionych, aby zgłębić płaszczyznę employer brandingu, jaki jest kształtowany w wybranej firmie. W ten sposób starano się kompleksowo ukazać wpływ systemu motywacyjnego na proces budowania marki pracodawcy. Przeprowadzona analiza empiryczna ujawniła, iż firma Burger King wykazuje dbałość o zatrudnionych poprzez wdrożony system motywacyjny, jak również dbałość, aby pracownicy byli współkreatorami pozytywnego wizerunku firmy. Burger King podejmuje liczne inicjatywy na rzecz empower brandingu, czego wyrazem są takie działania jak: organizowanie wewnętrznych konkursów motywacyjnych, wydawanie magazynu dla pracowników przez firmę oraz regularne badanie poziomu satysfakcji zatrudnionych. Wyniki sondażu diagnostycznego ujawniły także, iż konieczne jest przeprowadzenie pewnych modyfikacji w kwestii kreowania zaangażowania pracowników, a co najważniejsze budowania ich lojalności wobec przedsiębiorstwa. W toku analizy badawczej ujawniono bowiem, iż mimo, że ankietowani są zadowolenia z pracy w firmie Burger King to jednak nie wiążą z nią poważnych planów na przyszłość traktując tę pracę jako chwilowy epizod zawodowy. To przedkłada się na niski poziom zaangażowania osobistego, a także niski współczynnik rekomendacji firmy, jako miejsca zatrudnienia dla znajomych.Employer branding is a relatively new concept, but the realities of shaping the employer's image are one of the areas that has been functioning for a very long time in enterprises. Business entities are aware that creating a valuable brand as an employer determines the success of the entire organization. It is thanks to the positive perception of a given employer by current and potential employees that it is possible to reduce staff fluctuations, and most importantly to retain and to acquire talented individuals. In connection with the above, the this thesis has been written the topic of which aims to assess the incentive system in the aspect of shaping employer internal branding. An indispensable element of creating an employer brand is satisfaction and a high level of personal satisfaction of employees. The most important aspect in this respect is the incentive system, as an element supporting building a high level of employees engagement and job satisfaction. This study analyzes the literature on the subject in the aspect of the problem discussed, but also conducts an original diagnostic survey among employees of a selected entity to show the realities associated with the process of shaping employer branding in practice. In the course of the research analysis, the incentive system functioning in the selected company – Burger King – was diagnosed, along with an indication of the tools and incentive instruments used. The perception of the employer from the perspective of the employees themselves was also verified in order to explore the level of employer branding that is shaped in the selected company. In this way, attempts were made to comprehensively show the impact of the incentive system on the process of building the employer's brand.The empirical analysis revealed that Burger King shows care for employees through the implemented incentive system, as well as care that employees are co-creators of a positive image of the company. Burger King undertakes numerous initiatives for the empowerment of branding, which is reflected in such activities as: organizing internal incentive competitions, publishing a magazine for employees by the company and regularly surveying the level of employee satisfaction. The results of the diagnostic survey also revealed that it is necessary to carry out some modifications in terms of creating employee engagement and, most importantly, building their loyalty to the company. In the course of the research analysis, it was revealed that although the respondents are satisfied with their work at Burger King, they do not see their future there, and treat this job as a temporary professional episode. This account for a low level of personal engagement, as well as a low recommendation rate for the company as a place of employment for friends

Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074.

Efforts to construct the Streptomyces host strain with enhanced yields of heterologous product have focussed mostly on engineering of primary metabolism and/or the deletion of endogenous biosynthetic gene clusters. However, other factors, such as chromosome compactization, have been shown to have a significant influence on gene expression levels in bacteria and fungi. The expression of genes and biosynthetic gene clusters may vary significantly depending on their location within the chromosome. Little is known about the position effect in actinomycetes, which are important producers of various industrially relevant bioactive molecules

An influence of the copy number of biosynthetic gene clusters on the production level of antibiotics in a heterologous host

Manderscheid N, Bilyk B, Busche T, et al. An influence of the copy number of biosynthetic gene clusters on the production level of antibiotics in a heterologous host. JOURNAL OF BIOTECHNOLOGY. 2016;232:110-117.Streptomyces albus J1074 is a well-known host for heterologous expression of secondary metabolites. To further increase its potential and to study the influence of cluster multiplication, additional phi C31 attachment site was integrated into its genome using a system for transposon mutagenesis. Four secondary metabolite clusters were expressed in strains with different numbers of attachment sites, ranging from one to three copies of the site. Secondary metabolite production was examined and a new compound could be detected, purified and its structure was elucidated. (C) 2016 Elsevier B.V. All rights reserved

Копир-взрыхлитель автомата вождения корнеуборочной машины

Копір-розпушувач автомата водіння коренезбиральної машини, який складається зі стійки, культиваторної лапи з центральним кутом атаки, правого і лівого пер, кронштейнів кріплення і кріпильних елементів, який відрізняється тим, що культиваторна лапа з двох сторін є видовженої форми леза з кутом відслідковування, який є меншим кута атаки культиваторної лапи, а до кронштейнів кріплення жорстко закріплені лапи правого і лівого пер з ребрами жорсткості, причому крайні ліві кріпильні отвори від культиваторної лапи виконані циліндричної форми, а всі інші - у вигляді радіусного паза з радіусом осьової лінії паза, рівним міжосьовій віддалі між сусідніми кріпильними отворами, крім цього міжосьова віддаль t між сусідніми пазами рівна відстані між центром крайнього лівого циліндричного отвору і центром сусіднього паза