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Characteristic and role of chromosomal type II toxin-antitoxin systems locus in Enterococcus faecalis ATCC29212.
Li, Z, Shi, C, Gao, S, Zhang, X, Lu, D, Liu, G
Journal of microbiology (Seoul, Korea). 2020;(12):1027-1036
Abstract
The Gram-positive bacterium Enterococcus faecalis is currently one of the major pathogens of nosocomial infections. The lifestyle of E. faecalis relies primarily on its remarkable capacity to face and survive in harsh environmental conditions. Toxin-antitoxin (TA) systems have been linked to the growth control of bacteria in response to adverse environments but have rarely been reported in Enterococcus. Three functional type II TA systems were identified among the 10 putative TA systems encoded by E. faecalis ATCC29212. These toxin genes have conserved domains homologous to MazF (DR75_1948) and ImmA/IrrE family metallo-endopeptidases (DR75_1673 and DR75_2160). Overexpression of toxin genes could inhibit the growth of Escherichia coli. However, the toxin DR75_1673 could not inhibit bacterial growth, and the bacteriostatic effect occurred only when it was coexpressed with the antitoxin DR75_1672. DR75_1948-DR75_1949 and DR75_160-DR75_2161 could maintain the stable inheritance of the unstable plasmid pLMO12102 in E. coli. Moreover, the transcription levels of these TAs showed significant differences when cultivated under normal conditions and with different temperatures, antibiotics, anaerobic agents and H2O2. When DR75_2161 was knocked out, the growth of the mutant strain at high temperature and oxidative stress was limited. The experimental characterization of these TAs loci might be helpful to investigate the key roles of type II TA systems in the physiology and environmental stress responses of Enterococcus.
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[In silico Screening of Flavones and its Derivatives as Potential Inhibitors of Quorum-Sensing Regulator LasR of Pseudomonas aeruginosa].
Abelyan, N, Grabski, H, Tiratsuyan, S
Molekuliarnaia biologiia. 2020;(1):153-163
Abstract
Antibiotic resistance is a global problem nowadays and in 2017 the World Health Organization published the list of bacteria for which treatment are urgently needed, where Pseudomonas aeruginosa is of critical priority. Current therapies lack efficacy because this organism creates biofilms conferring increased resistance to antibiotics and host immune responses. The strategy is to "not kill, but disarm" the pathogen and resistance will be developed slowly. It has been shown that LasI/LasR system is the main component of the quorum sensing system in P. aeruginosa. LasR is activated by the interaction with its native autoinducer. A lot flavones and their derivatives are used as antibacterial drug compounds. The purpose is to search compounds that will inhibit LasR. This leads to the inhibition of the synthesis of virulence factors thus the bacteria will be vulnerable and not virulent. We performed virtual screening using AutoDock Vina, rDock, LeDock for obtaining consensus predictions. The results of virtual screening suggest benzamides which are synthetical derivatives of flavones as potential inhibitors of transcriptional regulator LasR. These are consistent with recently published experimental data, which demonstrate the high antibacterial activity of benzamides. The compounds interact with the ligand binding domain of LasR with higher binding affinity than with DNA binding domain. Among the selected compounds, by conformational analysis, it was found that there are compounds that bind to the same amino acids of ligand binding domain as the native autoinducer. This could indicate the possibility of competitive interaction of these compounds. A number of compounds that bind to other conservative amino acids ligand binding domain have also been discovered, which will be of interest for further study. Selected compounds meet the criteria necessary for their consideration as drugs and can serve as a basis for conducting further in vitro/in vivo experiments. It could be used for the development of modern anti-infective therapy based on the quorum sensing system of P. aeruginosa.
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Coupling Ion Specificity of the Flagellar Stator Proteins MotA1/MotB1 of Paenibacillus sp. TCA20.
Onoe, S, Yoshida, M, Terahara, N, Sowa, Y
Biomolecules. 2020;(7)
Abstract
The bacterial flagellar motor is a reversible rotary molecular nanomachine, which couples ion flux across the cytoplasmic membrane to torque generation. It comprises a rotor and multiple stator complexes, and each stator complex functions as an ion channel and determines the ion specificity of the motor. Although coupling ions for the motor rotation were presumed to be only monovalent cations, such as H+ and Na+, the stator complex MotA1/MotB1 of Paenibacillus sp. TCA20 (MotA1TCA/MotB1TCA) was reported to use divalent cations as coupling ions, such as Ca2+ and Mg2+. In this study, we initially aimed to measure the motor torque generated by MotA1TCA/MotB1TCA under the control of divalent cation motive force; however, we identified that the coupling ion of MotA1TCAMotB1TCA is very likely to be a monovalent ion. We engineered a series of functional chimeric stator proteins between MotB1TCA and Escherichia coli MotB. E. coli ΔmotAB cells expressing MotA1TCA and the chimeric MotB presented significant motility in the absence of divalent cations. Moreover, we confirmed that MotA1TCA/MotB1TCA in Bacillus subtilis ΔmotABΔmotPS cells generates torque without divalent cations. Based on two independent experimental results, we conclude that the MotA1TCA/MotB1TCA complex directly converts the energy released from monovalent cation flux to motor rotation.
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Proteomic profile of extracellular vesicles released by Lactiplantibacillus plantarum BGAN8 and their internalization by non-polarized HT29 cell line.
Bajic, SS, Cañas, MA, Tolinacki, M, Badia, J, Sánchez, B, Golic, N, Margolles, A, Baldomá, L, Ruas-Madiedo, P
Scientific reports. 2020;(1):21829
Abstract
In recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The aim of this study was to characterize the EVs released by the dairy isolate Lactiplantibacillus plantarum BGAN8 and to gain an insight into the putative mechanism of EVs uptake by intestinal epithelial cells. The cryo-TEM observation undoubtedly demonstrated the release of EVs (20 to 140 nm) from the surface of BGAN8, with exopolysaccharides seems to be part of EVs surface. The proteomic analysis revealed that the EVs are enriched in enzymes involved in central metabolic pathways, such as glycolysis, and in membrane components with the most abundant proteins belonging to amino acid/peptide ABC transporters. Putative internalization pathways were evaluated in time-course internalization experiments with non-polarized HT29 cells in the presence of inhibitors of endocytic pathways: chlorpromazine and dynasore (inhibitors of clathrin-mediated endocytosis-CME) and filipin III and nystatin (disrupting lipid rafts). For the first time, our results revealed that the internalization was specifically inhibited by dynasore and chlorpromazine but not by filipin III and nystatin implying that one of the entries of L. plantarum vesicles was through CME pathway.
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Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein.
Faulkner, M, Szabó, I, Weetman, SL, Sicard, F, Huber, RG, Bond, PJ, Rosta, E, Liu, LN
Scientific reports. 2020;(1):17501
Abstract
Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intent of engineering BMC-based metabolic modules for new functions in synthetic biology.
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Evidence for Pentapeptide-Dependent and Independent CheB Methylesterases.
Velando, F, Gavira, JA, Rico-Jiménez, M, Matilla, MA, Krell, T
International journal of molecular sciences. 2020;(22)
Abstract
Many bacteria possess multiple chemosensory pathways that are composed of homologous signaling proteins. These pathways appear to be functionally insulated from each other, but little information is available on the corresponding molecular basis. We report here a novel mechanism that contributes to pathway insulation. We show that, of the four CheB paralogs of Pseudomonas aeruginosa PAO1, only CheB2 recognizes a pentapeptide at the C-terminal extension of the McpB (Aer2) chemoreceptor (KD = 93 µM). McpB is the sole chemoreceptor that stimulates the Che2 pathway, and CheB2 is the methylesterase of this pathway. Pectobacterium atrosepticum SCRI1043 has a single CheB, CheB_Pec, and 19 of its 36 chemoreceptors contain a C-terminal pentapeptide. The deletion of cheB_Pec abolished chemotaxis, but, surprisingly, none of the pentapeptides bound to CheB_Pec. To determine the corresponding structural basis, we solved the 3D structure of CheB_Pec. Its structure aligned well with that of the pentapeptide-dependent enzyme from Salmonella enterica. However, no electron density was observed in the CheB_Pec region corresponding to the pentapeptide-binding site in the Escherichia coli CheB. We hypothesize that this structural disorder is associated with the failure to bind pentapeptides. Combined data show that CheB methylesterases can be divided into pentapeptide-dependent and independent enzymes.
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Comparative Analysis of Aggregation of Thermus thermophilus Ribosomal Protein bS1 and Its Stable Fragment.
Grishin, SY, Dzhus, UF, Selivanova, OM, Balobanov, VA, Surin, AK, Galzitskaya, OV
Biochemistry. Biokhimiia. 2020;(3):344-354
Abstract
Functionally important multidomain bacterial protein bS1 is the largest ribosomal protein of subunit 30S. It interacts with both mRNA and proteins and is prone to aggregation, although this process has not been studied in detail. Here, we obtained bacterial strains overproducing ribosomal bS1 protein from Thermus thermophilus and its stable fragment bS1(49) and purified these proteins. Using fluorescence spectroscopy, dynamic light scattering, and high-performance liquid chromatography combined with mass spectrometric analysis of products of protein limited proteolysis, we demonstrated that disordered regions at the N- and C-termini of bS1 can play a key role in the aggregation of this protein. The truncated fragment bS1(49) was less prone to aggregation compared to the full-size bS1. The revealed properties of the studied proteins can be used to obtain protein crystals for elucidating the structure of the bS1 stable fragment.
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Iron-sulfur cluster signaling: The common thread in fungal iron regulation.
Gupta, M, Outten, CE
Current opinion in chemical biology. 2020;:189-201
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Abstract
Iron homeostasis in fungi involves balancing iron uptake and storage with iron utilization to achieve adequate, nontoxic levels of this essential nutrient. Extensive work in the nonpathogenic yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe has uncovered unique iron regulation networks for each organism that control iron metabolism via distinct molecular mechanisms. However, common themes have emerged from these studies. The activities of all fungal iron-sensing transcription factors characterized to date are regulated via iron-sulfur cluster signaling. Furthermore, glutaredoxins often play a key role in relaying the intracellular iron status to these DNA-binding proteins. Recent work with fungal pathogens, including Candida and Aspergillus species and Cryptococcus neoformans, has revealed novel iron regulation mechanisms, yet similar roles for iron-sulfur clusters and glutaredoxins in iron signaling have been confirmed. This review will focus on these recent discoveries regarding iron regulation pathways in both pathogenic and nonpathogenic fungi.
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Mechanosensitive channels of Corynebacterium glutamicum functioning as exporters of l-glutamate and other valuable metabolites.
Kawasaki, H, Martinac, B
Current opinion in chemical biology. 2020;:77-83
Abstract
In the industrial l-glutamate production established on the use of Corynebacterium glutamicum, l-glutamate synthesized intracellularly is exported through mechanosensitive transmembrane channel proteins (MscCG and MscCG2) activated by the force-from-lipids. The involvement of MscCG2 in l-glutamate export by C. glutamicum was demonstrated in 2018; however, MscCG was previously found to be the major exporter of l-glutamate. Recent advances in research methods, such as development of the microbial patch clamp, revealed unique characteristics of MscCG, including its conductance, opening and closing thresholds, and gating hysteresis, as well as the significant effect of membrane lipids on the channel properties. In addition, the cryoelectron microscopic structure of Escherichia coli MscS, the canonical representative of the mechanosensitive channel family to which MscCG and MscCG2 belong, revealed its new membrane-interacting region, new position within the lipid bilayer, and hook lipids in a newly defined cavity between subunits. In this short review, the applications of bacterial mechanosensitive channels in the development of effective microbial cell factories, which will contribute to sustainable development, are discussed.
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Efficient Sortase-Mediated Ligation Using a Common C-Terminal Fusion Tag.
Reed, SA, Brzovic, DA, Takasaki, SS, Boyko, KV, Antos, JM
Bioconjugate chemistry. 2020;(5):1463-1473
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Abstract
Sortase-mediated ligation is a powerful method for generating site-specifically modified proteins. However, this process is limited by the inherent reversibility of the ligation reaction. To address this, here we report the continued development and optimization of an experimentally facile strategy for blocking reaction reversibility. This approach, which we have termed metal-assisted sortase-mediated ligation (MA-SML), relies on the use of a solution additive (Ni2+) and a C-terminal tag (LPXTGGHH5) that is widely used for converting protein targets into sortase substrates. In a series of model systems utilizing a 1:1 molar ratio of sortase substrate and glycine amine nucleophile, we find that MA-SML consistently improves the extent of ligation. This enables the modification of proteins with fluorophores, PEG, and a bioorthogonal cyclooctyne moiety without the need to use precious reagents in excess. Overall, these results demonstrate the potential of MA-SML as a general strategy for improving reaction efficiency in a broad range of sortase-based protein engineering applications.