Effects of altered TatC proteins on protein secretion efficiency via the twin-arginine translocation pathway of Bacillus subtilis

Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Bacillus subtilis Tat machinery lacks TatB and comprises two separate TatAC complexes with distinct substrate specificities: PhoD is secreted by the TatAdCd complex, whereas YwbN is secreted by the TatAyCy complex. To study the role of the Gram-positive TatC proteins in Tat-dependent protein secretion efficiency, we applied several genetic engineering approaches to modify and analyse the B. subtilis TatCd and TatCy proteins. Cytoplasmic and transmembrane domain exchange between TatCd and TatCy resulted in stable chimeric proteins that were unable to secrete both known substrates of the B. subtilis Tat system. Site-directed mutagenesis of conserved residues in the N-terminal part of both TatC proteins revealed significant differences in the degree of importance of these residues between TatCd, TatCy and Escherichia coli TatC. In addition, two small C-terminal deletions in TatCy completely abolished YwbN translocation, indicating that this terminus is essential for Tat translocation activity. Important differences from previous observations for E. coli TatC and implications for substrate binding and translocation are discussed.

Digital standardization of lean manufacturing tools according to Industry 4.0 concept

Standardization is a key element in the effective use of lean manufacturing methodologies and tools for achieving process sustainability. Their combination is conducive to eliminating waste and improving the efficiency of production processes and guarantees the company that employees use the most efficient tools and do not waste time on unnecessary activities. These activities can be further improved by using digital solutions, in accordance with the concept of Industry 4.0. Therefore, the authors have developed the e-Lean system, whose task is to digitize selected lean manufacturing tools. The subject of this work is analysis of the functionality and effectiveness of the essential part of the e-Lean system in the form of specialized TPM (Total Productive Maintenance) software as an application. During implementation in a construction production company, the TPM application was tested by lean manufacturing and maintenance specialists. The research consisted of assessing the functionality and efficiency of processes in relation to conventional TPM solutions. Additional functionalities of the e-Lean system have been confirmed, such as systemic approval of machinery inspection, which requires passing all necessary steps at individual inspection points, direct access for supervisors to the results of inspection activities and their status, direct and easy access to photographic documentation of machines added during inspection both in optimization of working time and its course (e.g., the optimal number of steps taken by the employee during the inspection), as well as an efficient system of motivating employees (collecting points). The improvement in the effectiveness of processes was determined by measuring the control times for three control points (polymerization furnace, packing area, and defibering machines). The average control time was reduced from 16,200 to 13,923 s. Thus, thanks to the use of the application, it was found that the efficiency of using the TPM tool was increased by approx. 15% compared to previously used non-digital solutions.The authors are grateful to FCT (Fundação para a Ciência e Tecnologia, Portugal) who partially financially supported this work through the RD Units Project Scope UIDP/04077/2020 and UIDB/04077/2020

Speläologische Zeitschriften in der Forschungsperspektive der kontrastiven Textlinguistik. Eine exemplarische Analyse am Beispiel von Zeitschriften „Die Höhle“ und „Jaskinie“

Niniejszy artykuł dotyczy tekstologii kontrastywnej, przy czym sama kontrastywność jest rozumiana w szerszym znaczeniu. Autorka poszukuje odpowiedzi na pytanie, na ile artykuły z dwóch czasopism „Jakinie” i „Die Höhle” mogą stanowić punkt wyjścia do badań kontrastywnych, lecz nie opisuje konkretnych rodzajów tekstu, natomiast zajmuje się porównaniem (w różnych aspektach) zawartości obu czasopism. Celem określenia, na ile czasopisma są porównywalne, zostały wzięte pod uwagę następujące aspekty: objętość, layout, główne informacje, cykliczność, wydawca, reklama, dostępność, ograniczoność, język, tematyka, krąg czytelników jak również stopień fachowości. Analiza ma na celu stwierdzenie podobieństw i różnic, które mogą posłużyć do dalszych badań na płaszczyźnie tekstologii kontrastywnej

Membrane orientation of small multidrug resistance proteins

Oriëntatie eiwitten van de Small Multidrug Resistance familie in het membraan Het proefschrift van Mariska Kolbusz beschrijft de oriëntatie van eiwitten van de Small Multidrug Resistance (SMR) familie in het membraan en de correlatie tussen oriëntatie en de verdeling van positief geladen aminozuur residuen over de intra- en extracellulaire loops van de eiwitten. De ‘positive-inside’ regel stelt dat de meeste positief geladen residuen zich aan de cytoplasmatische kant bevinden en dat dit de oriëntatie bepaalt. De SMR-familie bevat twee typen eiwitten: ‘singles’ en ‘pairs’, die respectievelijk gecodeerd worden door een enkel gen of door een paar genen. Het EmrE-eiwit, een ‘single’, inserteert in het membraan in beide oriëntaties (zogenaamde ‘dual topology’). Om te bestuderen hoe representatief EmrE is voor de SMR eiwitten, bepaalde Kolbusz de oriëntatie van negen ‘singles’ en negen ‘pairs’ uit deze familie. Alle ‘singles’ inserteerden zoals EmrE met ‘dualtopology’. De analyse van de verdeling van de positief geladen residuen over de loops gaf aan dat er een goede correlatie met de oriëtatie was, maar er waren ook uitzonderingen. De twee eiwitten van de ‘pairs’ inserteerden weliswaar met een vaste oriëntatie, maar die was omgekeerd dan voorspeld door de positieve ladingsverdeling. Een gedetailleerde analyse van de rol van positief geladen residuen op de oriëntatie liet zien dat verschillende residuen de orientatie met verschillende sterkten beïnvloeden en dat andere, onbekende factoren de oriëntatie mede bepalen. Een genoom-analyse van negenentwintig Escherichia coli stammen toonde aan dat in tien stammen een langere versie van EmrE aanwezig is. De langere versie bevat vijf membraanspannende segmenten in plaats van vier en inserteert in één oriëntatie in het membraan in tegenstelling tot de normale versie. De functie van de lange versie is onbekend en moet verder onderzocht worden. The thesis of Magdalena Anna Kolbusz focuses on membrane orientation of members of the Small Multidrug Resistance (SMR) protein family and its correlation with distribution of positively charged amino acid residues over the membrane. As described in the positive-inside rule the positively charged residues are located mostly in cytoplasmic loops and seem to bethe key orientation determinant. In the SMR family there are two types of proteins: singlesandpairs encoded by single and paired genes respectively. The EmrE protein, which belongs to the singles, inserts into the membrane in two opposite orientations (dual topology). To verify the orientation of other SMR proteins membrane orientation of 9 singles and 9 pairs was established. It has been shown that singlesacquire dual topology as EmrE. Analysis of distribution of positively charged amino acids showed that there is a good correlation between the orientation and the distribution of positively charged residues, but also exceptions violating the positive-inside rule have been found. Among pairs, proteins inserted into the membrane in fixed, opposite orientations in agreement with the distribution of positively charged amino acids. A detailed analysis ofthe role of charged residues on orientation showed that different residues influence orientation with different strength and probably there are other factors involved in orientation determination. Analysis of 29 Escherichia colii strains revealed that in 10 strains a longer version of EmrE is present. The elongated protein contains 5 TMSs and acquires a single membrane orientation. Its function remains unknown and requires further research.

Reorganizacja stanowiska montażowego w procesie produkcji podłogi przesuwnej do ramp przeładunkowych w kontekście poprawy jakości gotowego produktu

The paper presents the definitions of the term quality quoted in the literature on the subject. FMEA (Failure Mode and Effects Analysis) methodology was presented. A reloading ramp was characterized as the subject of the study. A cause-and-effect analysis of non-compliance in the finished product was performed using the Pareto-Lorenz diagram, Ishikawa diagram and brainstorming. The FMEA analysis of the assembly table indicated the elements of the workstation causing the most frequently occurring non-conformities. Based on the obtained results, corrective actions were proposed to reorganize the assembly station. The implemented activities made it possible to reduce the critical RPN coefficients for the elements of the assembly table and to shorten the time necessary to make one piece of the finished product.W artykule przedstawiono definicje pojęcia jakość przytaczane w literaturze przedmiotu. Zaprezentowano metodykę FMEA (Failure Mode and Effects Analysis). Scharakteryzowano rampę przeładunkową jako przedmiot badań. Dokonano analizy przyczynowo-skutkowej powstawania niezgodności w wyrobie gotowym z wykorzystaniem diagramu Pareto-Lorenza oraz diagramu Ishikawy i burzy mózgów. Dzięki przeprowadzonej analizie FMEA stołu montażowego wskazano elementy stanowiska powodujące najczęściej powstające niezgodności. Na podstawie uzyskanych wyników zaproponowano działania korygujące, mające na celu reorganizacje stanowiska montażu. Wdrożone działania naprawcze umożliwiły obniżenie krytycznych współczynników RPN w elementach stołu montażowego oraz skrócenie czasu niezbędnego do wykonania jednej sztuki wyrobu gotowego na stanowisku montażu

Role of Individual Positive Charges in the Membrane Orientation and Activity of Transporters of the Small Multidrug Resistance Family

The effect of individual positively charged residues on the orientation in the membrane was analyzed in three dual-topology transporters of the small. multidrug resistance (SMR) family: AAVE4701aave of Acidovorax avenae, EMREecol of Escherichia coli, and RRUA0272rrub of Rhodospirillum rubrum. It is shown that (i) individual positive charges have different impacts on the orientation, (ii) positive charges that are conserved in the three different proteins do not have the same impact on the orientation, (iii) positive charges in odd- and even-numbered loops have different impacts, (iv) for some, but not all, the impact depends on the presence of other positive charges, and (v) proteins from which all positive charges are removed in some cases are dual-topology proteins and in other cases have a single orientation. A small number of positive charges placed in the loops of the latter proteins results in the violation of the so-called positive-inside rule that has been reported previously [Kolbusz, M. A., et al. (2010) J. Mol. Biol. 402, 127-138]. We conclude that each positive charge shifts the distribution between the two orientations toward the state that has the positive charge in the cytoplasm but that intrinsic factors other than positive charges determine the orientation as well. The ability of the mutants of AAVE4701aave and EMREecol to confer resistance against ethidium bromide revealed an essential role in catalysis for a conserved pair of positive charges in the second loop. No significant relation between activity and the relative orientation of the monomeric subunits in the dimer could be demonstrated

Orientation of Small Multidrug Resistance Transporter Subunits in the Membrane:Correlation with the Positive-Inside Rule

Small multidrug resistance (SMR) transport proteins provide a model for the evolution of larger two-domain transport proteins. The orientation in the membrane of 27 proteins from the SMR family was determined using the reporter fusion technique. Nine members were encoded monocistronically (singles) and shown to insert in both orientations (dual topology). Eighteen members were encoded in pairs on the chromosome and shown to insert in fixed orientations; the two proteins in each pair invariably had opposite orientations in the membrane. Interaction between the two proteins in pairs was demonstrated by copurification. The orientation in the membrane of either protein in the pair was affected only marginally by the presence of the other protein. For the proteins in pairs, the orientation in the membrane correlated well with the distribution of positively charges residues (R + K) over the cytoplasmic and extracellular loops (positive-inside rule). In contrast, dual-topology insertion of the singles was predicted less well by the positive-inside rule. Three singles were predicted to insert in a single orientation with the N-terminus and the C-terminus at the extracellular side of the membrane. Analysis of charge distributions suggests the requirement of a threshold number of charges in the cytoplasmic loops for the positive-inside rule to be of predictive value. It is concluded that a combined analysis of gene organization on the chromosome and phylogeny is sufficient to distinguish between fixed or dual topology of SMR members and, probably, similar types of membrane proteins. The positive-inside rule can be used to predict the orientation of members in pairs, but is not suitable as a sole predictor of dual topology. (C) 2010 Elsevier Ltd. All rights reserved