Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum β-lactamase in clinical populations of the familyEnterobacteriaceaein Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besidesbla_CTX-M-3, it also bears thebla_TEM-1,aacC2, andarmAgenes, as well as integronicaadA2,dfrA12, andsul1, which altogether confer resistance to the majority of β-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks,traandtrb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Onlybla_CTX-M-3, accompanied by an ISEcp1element, is placed separately, in a DNA fragment previously identified as a fragment of theKluyvera ascorbatachromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogenErwinia amylovoraby acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

This survey reviews modern ideas on the structure and functions of mitochondrial and cytosolic aconitase isoenzymes in eukaryotes. Cumulative experimental evidence about mitochondrial aconitases (Aco2) as one of the main targets of reactive oxygen and nitrogen species is generalized. The important role of Aco2 in maintenance of homeostasis of the intracellular iron pool and maintenance of the mitochondrial DNA is discussed. The role of Aco2 in the pathogenesis of some neurodegenerative diseases is highlighted. Inactivation or dysfunction of Aco2 as well as mutations found in the ACO2 gene appear to be significant factors in the development and promotion of various types of neurodegenerative diseases. A restoration of efficient mitochondrial functioning as a source of energy for the cell by targeting Aco2 seems to be one of the promising therapeutic directions to minimize progressive neurodegenerative disorders.

ThekfrAgene of the IncP-1 broad-host-range plasmids is the best-studied member of a growing gene family that shows strong linkage to the minimal replicon of many low-copy-number plasmids. KfrA is a DNA binding protein with a long, alpha-helical, coiled-coil tail. Studying IncP-1βplasmid R751, evidence is presented thatkfrAand its downstream genesupf54.8andupf54.4were organized in a tricistronic operon (renamed herekfrA kfrB kfrC), expressed from autoregulatedkfrAp, that was also repressed by KorA and KorB. KfrA, KfrB and KfrC interacted and may have formed a multi-protein complex. Inactivation of eitherkfrAorkfrBin R751 resulted in long-term accumulation of plasmid-negative bacteria, whereas wild-type R751 itself persisted without selection. Immunofluorescence studies showed that KfrA_R751formed plasmid-associated foci, and deletion of the C terminus of KfrA caused plasmid R751ΔC₂kfrAfoci to disperse and mislocalize. Thus, the KfrABC complex may be an important component in the organization and control of the plasmid clusters that seem to form the segregating unit in bacterial cells. The studied operon is therefore part of the set of functions needed for R751 to function as an efficient vehicle for maintenance and spread of genes in Gram-negative bacteria.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein–small molecule interaction, a newly developed protein–protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.