Tetrapodal amidoxime ligands I. Coordination isomerism due to self-complementary dimerization of a pyramidal cobalt(III) coordination module

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.A bis-μ-amidoximato-bridged cobalt(III) dimer obtained with a new tetrapodal ligand possesses interesting structural parameters as a consequence of intramolecular hydrogen bonding intentionally built into the complex. Its synthesis and properties are described. The new ligand type combines attributes of two previously described ligand classes: It binds a metal ion in a tetrapodal pentadentate fashion and forms a pseudomacrocycle through hydrogen bonds, characteristic of chelating oxime ligands. Coordination isomerism, which is a consequence of dimer formation, has been analyzed by means of X-ray crystallography and carbon-13 nuclear magnetic resonance spectroscopy

Regulation of Gram-Positive Conjugation

Type IV Secretion Systems (T4SSs) are membrane-spanning multiprotein complexes dedicated to protein secretion or conjugative DNA transport (conjugation systems) in bacteria. The prototype and best-characterized T4SS is that of the Gram-negative soil bacterium Agrobacterium tumefaciens. For Gram-positive bacteria, only conjugative T4SSs have been characterized in some biochemical, structural, and mechanistic details. These conjugation systems are predominantly encoded by self-transmissible plasmids but are also increasingly detected on integrative and conjugative elements (ICEs) and transposons. Here, we report regulatory details of conjugation systems from Enterococcus model plasmids pIP501 and pCF10, Bacillus plasmid pLS1, Clostridium plasmid pCW3, and staphylococcal plasmid pSK41. In addition, regulation of conjugative processes of ICEs (ICEBs1, ICESt1, ICESt3) by master regulators belonging to diverse repressor families will be discussed. A special focus of this review lies on the comparison of regulatory mechanisms executed by proteins belonging to the RRNPP family. These regulators share a common fold and govern several essential bacterial processes, including conjugative transfer

Graphene-Based Bacterial Filtration via Electrostatic Adsorption

Flexible graphene oxide (GO) microsheets with attached positively charged polymers, termed GOX microsheets, are efficient at bacterial adsorption, as they bind electrostatically to bacterial membranes’ negative surface charge. The authors explore an antimicrobial water filter application for GOX's extremely high surface area and its previously described efficient bacterial adsorption.Cellulose-fiber carrier material is functionalized with GOX microsheets to create an adsorption-based bacteria filtration material. The morphology and charge density (7.8 × 1019 g–1) of the prepared GOX fibers are determined by scanning electron microscopy and dye adsorption assay, and widefield fluorescence microscopy is used to visualize the adsorption of stained Escherichia coli bacterial cells on the fibers. GOX fibers are tested in filtration setups to investigate their bacteria removal performance. The experimental results, with 100 mg of GOX fibers filtering 2.4 × 109 colony-forming units (CFU) from an E. coli bacterial culture with 99.5% bacterial reduction, demonstrate the fibers’ high bacteria loading capacity. The electrostatic adsorption-based filtration mechanism allows the filter to be operated at higher flow rates than micropore membrane filters, while maintaining 3-log bacterial reduction. GOX filter materials removing bacteria via adsorption are a high flow rate alternative to current water filtration processes that rely on size-exclusion

Biofilm Forming Antibiotic Resistant Gram-Positive Pathogens Isolated From Surfaces on the International Space Station

The International Space Station (ISS) is a closed habitat in a uniquely extreme and hostile environment. Due to these special conditions, the human microflora can undergo unusual changes and may represent health risks for the crew. To address this problem, we investigated the antimicrobial activity of AGXX®, a novel surface coating consisting of micro-galvanic elements of silver and ruthenium along with examining the activity of a conventional silver coating. The antimicrobial materials were exposed on the ISS for 6, 12, and 19 months each at a place frequently visited by the crew. Bacteria that survived on the antimicrobial coatings [AGXX® and silver (Ag)] or the uncoated stainless steel carrier (V2A, control material) were recovered, phylogenetically affiliated and characterized in terms of antibiotic resistance (phenotype and genotype), plasmid content, biofilm formation capacity and antibiotic resistance transferability. On all three materials, surviving bacteria were dominated by Gram-positive bacteria and among those by Staphylococcus, Bacillus and Enterococcus spp. The novel antimicrobial surface coating proved to be highly effective. The conventional Ag coating showed only little antimicrobial activity. Microbial diversity increased with increasing exposure time on all three materials. The number of recovered bacteria decreased significantly from V2A to V2A-Ag to AGXX®. After 6 months exposure on the ISS no bacteria were recovered from AGXX®, after 12 months nine and after 19 months three isolates were obtained. Most Gram-positive pathogenic isolates were multidrug resistant (resistant to more than three antibiotics). Sulfamethoxazole, erythromycin and ampicillin resistance were most prevalent. An Enterococcus faecalis strain recovered from V2A steel after 12 months exposure exhibited the highest number of resistances (n = 9). The most prevalent resistance genes were ermC (erythromycin resistance) and tetK (tetracycline resistance). Average transfer frequency of erythromycin, tetracycline and gentamicin resistance from selected ISS isolates was 10−5 transconjugants/recipient. Most importantly, no serious human pathogens such as methicillin resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococci (VRE) were found on any surface. Thus, the infection risk for the crew is low, especially when antimicrobial surfaces such as AGXX® are applied to surfaces prone to microbial contamination

Stress response of a clinical Enterococcus faecalis isolate subjected to a novel antimicrobial surface coating

Emerging antibiotic resistance among pathogenic bacteria, paired with their ability to form biofilms on medical and technical devices, represents a serious problem for effective and long-term decontamination in health-care environments and gives rise to an urgent need for new antimicrobial materials. Here we present the impact of AGXX®, a novel broad-spectrum antimicrobial surface coating consisting of micro-galvanic elements formed by silver and ruthenium, on the transcriptome of Enterococcus faecalis. A clinical E. faecalis isolate was subjected to metal stress by growing it for different periods in presence of the antimicrobial coating or silver-coated steel meshes. Subsequently, total RNA was isolated and next-generation RNA sequencing was performed to analyze variations in gene expression in presence of the antimicrobial materials with focus on known stress genes. Exposure to the antimicrobial coating had a large impact on the transcriptome of E. faecalis. After 24min almost 1/5 of the E. faecalis genome displayed differential expression. At each time-point the cop operon was strongly up-regulated, providing indirect evidence for the presence of free Ag+-ions. Moreover, exposure to the antimicrobial coating induced a broad general stress response in E. faecalis. Genes coding for the chaperones GroEL and GroES and the Clp proteases, ClpE and ClpB, were among the top up-regulated heat shock genes. Differential expression of thioredoxin, superoxide dismutase and glutathione synthetase genes indicates a high level of oxidative stress. We postulate a mechanism of action where the combination of Ag+-ions and reactive oxygen species generated by AGXX®results in a synergistic antimicrobial effect, superior to that of conventional silver coatings.</p

Deletion of the glycosyltransferase bgsB of Enterococcus faecalis leads to a complete loss of glycolipids from the cell membrane and to impaired biofilm formation

<p>Abstract</p> <p>Background</p> <p>Deletion of the glycosyltransferase <it>bgsA </it>in <it>Enterococcus faecalis </it>leads to loss of diglucosyldiacylglycerol from the cell membrane and accumulation of its precursor monoglucosyldiacylglycerol, associated with impaired biofilm formation and reduced virulence in vivo. Here we analyzed the function of a putative glucosyltransferase EF2890 designated <it>biofilm-associated glycolipid synthesis B (bgsB) </it>immediately downstream of <it>bgsA</it>.</p> <p>Results</p> <p>A deletion mutant was constructed by targeted mutagenesis in <it>E. faecalis </it>strain 12030. Analysis of cell membrane extracts revealed a complete loss of glycolipids from the cell membrane. Cell walls of 12030Δ<it>bgsB </it>contained approximately fourfold more LTA, and ¹H-nuclear magnetic resonance (NMR) spectroscopy suggested that the higher content of cellular LTA was due to increased length of the glycerol-phosphate polymer of LTA. 12030Δ<it>bgsB </it>was not altered in growth, cell morphology, or autolysis. However, attachment to Caco-2 cells was reduced to 50% of wild-type levels, and biofilm formation on polystyrene was highly impaired. Despite normal resistance to cationic antimicrobial peptides, complement and antibody-mediated opsonophagocytic killing in vitro, 12030Δ<it>bgsB </it>was cleared more rapidly from the bloodstream of mice than wild-type bacteria. Overall, the phenotype resembles the respective deletion mutant in the <it>bgsA </it>gene. Our findings suggest that loss of diglucosyldiacylglycerol or the altered structure of LTA in both mutants account for phenotypic changes observed.</p> <p>Conclusions</p> <p>In summary, BgsB is a glucosyltransferase that synthesizes monoglucosyldiacylglycerol. Its inactivation profoundly affects cell membrane composition and has secondary effects on LTA biosynthesis. Both cell-membrane amphiphiles are critical for biofilm formation and virulence of <it>E. faecalis</it>.</p

Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an indepth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (Fn- Cas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a.We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using singlemolecule FRET measurements and assayed the precrRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site

Multivalent bacteria binding by flexible polycationic microsheets matching their surface charge density

Aiming at the overall negative surface charge of bacteria, a new strategy of antibacterial agents based on large polymer‐modified graphene oxide (GO) sheets is assessed. The presented flexible, polycationic sheets match the size and charge density of the Escherichia coli surface charge density (2 × 1014 cm−2). These matching parameters create an unspecific but very strong bacteria adsorber by multivalent, electrostatic attraction. Their interaction with bacteria is visualized via atomic force and confocal microscopy and shows that they effectively bind and wrap around E. coli cells, and thereby immobilize them. The incubation of Gram‐negative and ‐positive bacteria (E. coli and methicillin‐resistant Staphylococcus aureus , MRSA) with these polycationic sheets leads to the inhibition of proliferation and a reduction of the colony forming bacteria over time. This new type of antibacterial agent acts in a different mode of action than classical biocides and could potentially be employed in medicinal, technical, or agriculture applications. The presented microsheets and their unspecific binding of cell interfaces could further be employed as adsorber material for bacterial filtration or immobilization for imaging, analysis, or sensor technologies