Broad spectrum and narrow spectrum mercury resistance encoded by different plasmids of a Pseudomonas putida strain

Abstract Pseudomonas putida strain H harbours two plasmids of different sizes. It was demonstrated that the large plasmid pPGH1 confers a broad spectrum resistance to mercurials, whereas the small plasmid pPGH2 confers a narrow spectrum one. Under the influence of the small plasmid the resistance of cells against poisoning with 2,4-di-chlorophenol or o -cresol increases in comparison to cells without this plasmid. Both plasmids proved to be not self-transmissible, but pPGH1 is transferable by mobilisation by means of the IncP-1 vectors R68.45 or RP1.     

What makes a type IIA topoisomerase a gyrase or a Topo IV?

Type IIA topoisomerases catalyze a variety of different reactions: eukaryotic topoisomerase II relaxes DNA in an ATP-dependent reaction, whereas the bacterial representatives gyrase and topoisomerase IV (Topo IV) preferentially introduce negative supercoils into DNA (gyrase) or decatenate DNA (Topo IV). Gyrase and Topo IV perform separate, dedicated tasks during replication: gyrase removes positive supercoils in front, Topo IV removes pre-catenanes behind the replication fork. Despite their well-separated cellular functions, gyrase and Topo IV have an overlapping activity spectrum: gyrase is also able to catalyze DNA decatenation, although less efficiently than Topo IV. The balance between supercoiling and decatenation activities is different for gyrases from different organisms. Both enzymes consist of a conserved topoisomerase core and structurally divergent C-terminal domains (CTDs). Deletion of the entire CTD, mutation of a conserved motif and even by just a single point mutation within the CTD converts gyrase into a Topo IV-like enzyme, implicating the CTDs as the major determinant for function. Here, we summarize the structural and mechanistic features that make a type IIA topoisomerase a gyrase or a Topo IV, and discuss the implications for type IIA topoisomerase evolution.     

Crystal structures of Thermotoga maritima reverse gyrase: inferences for the mechanism of positive DNA supercoiling

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presented.     

Identification of a clonally expanding haematopoietic compartment in bone marrow

In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self‐renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high‐resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48? putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48? cells. Our results identify a previously unrecognized, vessel‐associated BM compartment with a specific localization and properties distinct from the marrow cavity.     

Synergism between Medihoney and Rifampicin against Methicillin-Resistant Staphylococcus aureus (MRSA)

Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.     

Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions

It is generally expected that 2-pore domain K⁺ (K_2P) channels are open or outward rectifiers in asymmetric physiological K⁺ gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K_2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K_2P subfamilies in transfected Chinese hamster ovary cells with patch-clamp technique, and examine whether their I-V relationships are described by the GHK current equation. K_2P channels display 2 distinct types of I-V curves in asymmetric physiological K⁺ gradients. Two K_2P isoforms in the TWIK subfamily conduct large inward K⁺ currents and have a nearly linear I-V curve. Ten isoforms from 5 other K_2P subfamilies conduct small inward K⁺ currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K_2P channels. The Rectification Index, a ratio of limiting I-V slopes for outward and inward currents, is used to quantitatively describe open rectification of each K_2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K_2P channels and suggest that TWIK K⁺ channels have a unique feature in regulating cellular function.     

Evaluation of 41 Candidate Gene Variants for Obesity in the EPIC-Potsdam Cohort by Multi-Locus Stepwise Regression

Objective

Obesity has become a leading preventable cause of morbidity and mortality in many parts of the world. It is thought to originate from multiple genetic and environmental determinants. The aim of the current study was to introduce haplotype-based multi-locus stepwise regression (MSR) as a method to investigate combinations of unlinked single nucleotide polymorphisms (SNPs) for obesity phenotypes.

Methods

In 2,122 healthy randomly selected men and women of the EPIC-Potsdam cohort, the association between 41 SNPs from 18 obesity-candidate genes and either body mass index (BMI, mean = 25.9 kg/m², SD = 4.1) or waist circumference (WC, mean = 85.2 cm, SD = 12.6) was assessed. Single SNP analyses were done by using linear regression adjusted for age, sex, and other covariates. Subsequently, MSR was applied to search for the ‘best’ SNP combinations. Combinations were selected according to specific AIC_c and p-value criteria. Model uncertainty was accounted for by a permutation test.

Results

The strongest single SNP effects on BMI were found for TBC1D1 rs637797 (β = −0.33, SE = 0.13), FTO rs9939609 (β = 0.28, SE = 0.13), MC4R rs17700144 (β = 0.41, SE = 0.15), and MC4R rs10871777 (β = 0.34, SE = 0.14). All these SNPs showed similar effects on waist circumference. The two ‘best’ six-SNP combinations for BMI (global p-value = 3.45⋅10^–6 and 6.82⋅10^–6) showed effects ranging from −1.70 (SE = 0.34) to 0.74 kg/m² (SE = 0.21) per allele combination. We selected two six-SNP combinations on waist circumference (global p-value = 7.80⋅10^–6 and 9.76⋅10^–6) with an allele combination effect of −2.96 cm (SE = 0.76) at maximum. Additional adjustment for BMI revealed 15 three-SNP combinations (global p-values ranged from 3.09⋅10^–4 to 1.02⋅10^–2). However, after carrying out the permutation test all SNP combinations lost significance indicating that the statistical associations might have occurred by chance.

Conclusion

MSR provides a tool to search for risk-related SNP combinations of common traits or diseases. However, the search process does not always find meaningful SNP combinations in a dataset.     

Aminopeptidase N (APN)/CD13-dependent CXCR4 downregulation is associated with diminished cell migration,proliferation and invasion

Aminopeptidase N (APN/CD13) is a 150 kDa membrane-bound ubiquitously expressed protease with a broad functional repertoire. It hydrolyzes small peptide mediators, modulates cell motility and adhesion to extracellular matrix and also acts as a viral receptor. In order to dissect the function of enzymatically active and inactive APN/CD13, substitutions of different enzymatic active amino acid residues were generated by site-directed mutagenesis and stably transfected into human embryonic kidney cells. All APN variants analyzed exhibited a complete loss of enzymatic activity, whereas wild type APN transfectants exerted a strong aminopeptidase-specific activity. Furthermore, wild type APN expression was associated with a significant decrease in proliferation, migration and also reduced anchorage-independent growth when compared to enzymatically inactive APN variants and controls. This appeared to be due to a downregulated mRNA and protein expression of the chemokine receptor CXCR4 and an inhibition of the stromal cell-derived factor (SDF)-1α/CXCL12-mediated migration. Thus, high APN enzyme activity may antagonize the cellular properties regulated by the CXCR4/SDF-1α system in embryonic kidney cells.     

The invasion history of the exotic freshwater zooplankter Daphnia lumholtzi (Cladocera, Crustacea) in North America: a genetic analysis

Daphnia lumholtzi is a planktonic crustacean native to subtropical regions in Africa, Asia and Australia. Since its invasion to the southern USA in ~1990 it has spread across North America as far north as the Laurentian Great Lakes. We assessed invasion history using microsatellite makers and to explore the influence of mean annual temperature on the genetic structure along a latitudinal gradient in North America. Genotypic data were obtained from 9 microsatellite markers for 178 individuals from 13 populations (eight populations introduced to North America and five populations in the native range). Pairwise F_st values as well as Bayesian clustering showed a strong subdivision between native and introduced populations. Bayesian clustering identified multiple genetic clusters in recently invaded locations, suggestive of multiple invasions from various sources, including Asia and Africa. Using variation partitioning, we determined the amount of variation for genetic clusters of populations in the invaded range due to mean annual air temperature and the year of first detection. The results point to a primary introduction into the southern range of North America, with a subsequent northward expansion, and multiple introductions possibly from both the native range and by secondary spread from previously-invaded locations. Separate analysis of genetic clusters within the invaded range suggests additional effects of temperature conditions on geographic genetic structure, possibly as a consequence of D. lumholtzi’s tropical origin.     

The conformational flexibility of the helicase-like domain from Thermotoga maritima reverse gyrase is restricted by the topoisomerase domain

Reverse gyrase is the only enzyme known to introduce positive supercoils into DNA. Positive supercoiling is achieved by the functional cooperation of a helicase-like and a topoisomerase domain. The isolated helicase-like domain is a DNA-stimulated ATPase, and the isolated topoisomerase domain can relax supercoiled DNA. In the context of reverse gyrase, these individual activities are suppressed or attenuated. The helicase-like domain of Thermotoga maritima reverse gyrase is a nucleotide-dependent conformational switch that binds DNA and ATP cooperatively. It provides a nucleotide-dependent DNA-binding site to reverse gyrase and thus serves as a valuable model for the investigation of the effect of nucleotides on DNA processing by reverse gyrase that is key to its supercoiling activity. To improve our understanding of the structural basis for the functional cooperation of a helicase domain with a DNA topoisomerase, we have determined the structures of the isolated helicase-like domain of T. maritima reverse gyrase in five different conformations. Comparison of these structures reveals extensive domain flexibility in the absence of conformational restrictions by the topoisomerase that is consistent with single-molecule Fo?rster resonance energy transfer experiments presented here. The structure of the first ADP-bound form provides novel details about nucleotide binding to reverse gyrase. It demonstrates that reverse gyrases use the canonical nucleotide binding mode common to superfamily 2 helicases despite large deviations in the conserved motifs. A characteristic insert region adopts drastically different structures in different reverse gyrases. Counterparts of this insert region are located at very different positions in other DNA-processing enzymes but may point toward a general role in DNA strand separation.