Evidence for a human-specific Escherichia coli clone

Escherichia coli is a widespread commensal of the vertebrate intestinal tract. Until recently, no strong association between a particular clone and a given host species has been found. However, members of the B2 subgroup VIII clone with an O81 serotype appear to be human host specific. To determine the degree of host specificity exhibited by this clone, a PCR-based assay was used to screen 723 faecal and clinical isolates from humans, and 904 faecal isolates from animals. This clone was not detected among the animal isolates, but was discovered in people living in Africa, Europe and South America. The clone is rarely isolated from people suffering from intestinal or extraintestinal disease and is avirulent in a mouse model of extraintestinal infection. Fine-scale epidemiological analysis suggests that this clone is competitively dominant relative to other members of the B2 phylogenetic group and that it has increased in frequency over the past 20 years. This clone appears to be a good candidate for use as a probiotic, and may be suitable as an indicator of human faecal contamination in microbial source tracking studies.     

A mutation (R826W) in nucleotide-binding domain 1 of ABCC8 reduces ATPase activity and causes transient neonatal diabetes

Activating mutations in the pore-forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor SUR1 (ABCC8) subunits of the K(ATP) channel are a common cause of transient neonatal diabetes mellitus (TNDM). We identified a new TNDM mutation (R826W) in the first nucleotide-binding domain (NBD1) of SUR1. The mutation was found in a region that heterodimerizes with NBD2 to form catalytic site 2. Functional analysis showed that this mutation decreases MgATP hydrolysis by purified maltose-binding protein MBP-NBD1 fusion proteins. Inhibition of ATP hydrolysis by MgADP or BeF was not changed. The results indicate that the ATPase cycle lingers in the post-hydrolytic MgADP.P(i)-bound state, which is associated with channel activation. The extent of MgADP-dependent activation of K(ATP) channel activity was unaffected by the R826W mutation, but the time course of deactivation was slowed. Channel inhibition by MgATP was reduced, leading to an increase in resting whole-cell currents. In pancreatic beta cells, this would lead to less insulin secretion and thereby diabetes.     

SHOC1, an XPF endonuclease-related protein, is essential for the formation of class I meiotic crossovers

Crossovers (COs) are essential for the completion of meiosis in most species and lead to new allelic combinations in gametes. Two pathways of meiotic crossover formation have been distinguished. Class I COs, which are the major class of CO in budding yeast, mammals, Caenorhabditis elegans, and Arabidopsis, depend on a group of proteins called ZMM and rely on specific DNA structure intermediates that are processed to form COs. We identified a novel gene, SHOC1, involved in meiosis in Arabidopsis. Shoc1 mutants showed a striking reduction in the number of COs produced, a similar phenotype to the previously described Arabidopsis zmm mutants. The early steps of recombination, revealed by DMC1 foci, and completion of synapsis are not affected in shoc1 mutants. Double mutant analysis showed that SHOC1 acts in the same pathway as AtMSH5, a conserved member of the ZMM group. SHOC1 is thus a novel gene required for class I CO formation in Arabidopsis. Sequence similarity studies detected putative SHOC1 homologs in a large range of eukaryotes including human. SHOC1 appears to be related to the XPF endonuclease protein family, which suggests that it is directly involved in the maturation of DNA intermediates that lead to COs.     

New subunits NDH-M, -N, and -O, encoded by nuclear genes, are essential for plastid Ndh complex functioning in higher plants

In higher plants, the Ndh complex reduces plastoquinones and is involved in cyclic electron flow around photosystem I, supplying extra-ATP for photosynthesis, particularly under environmental stress conditions. Based on plastid genome sequences, the Ndh complex would contain 11 subunits (NDH-A to -K), but homologies with bacterial complex indicate the probable existence of additional subunits. To identify missing subunits, tobacco (Nicotiana tabacum) NDH-H was His tagged at its N terminus using plastid transformation. A functional Ndh subcomplex was purified by Ni(2+) affinity chromatography and its subunit composition analyzed by mass spectrometry. Five plastid encoded subunits (NDH-A, -H, -I, -J, and -K) were identified as well as three new subunits (NDH-M, -N, and -O) homologous to cyanobacterial and higher plant proteins. Arabidopsis thaliana mutants missing one of these new subunits lack a functional Ndh complex, and NDH-M and NDH-N are not detected in a tobacco transformant lacking the Ndh complex. We discuss the involvement of these three nuclear-encoded subunits in the functional integrity of the plastidial complex.     

Pathogen-responsive expression of glycosyltransferase genes UGT73B3 and UGT73B5 is necessary for resistance to Pseudomonas syringae pv tomato in Arabidopsis

The genome sequencing of Arabidopsis (Arabidopsis thaliana) has revealed that secondary metabolism plant glycosyltransferases (UGTs) are encoded by an unexpectedly large multigenic family of 120 members. Very little is known about their actual function in planta, in particular during plant pathogen interactions. Among them, members of the group D are of particular interest since they are related to UGTs involved in stress-inducible responses in other plant species. We provide here a detailed analysis of the expression profiles of this group of Arabidopsis UGTs following infection with Pseudomonas syringae pv tomato or after treatment with salicylic acid, methyljasmonate, and hydrogen peroxide. Members of the group D displayed distinct induction profiles, indicating potential roles in stress or defense responses notably for UGT73B3 and UGT73B5. Analysis of UGT expression in Arabidopsis defense-signaling mutants further revealed that their induction is methyljasmonate independent, but partially salicylic acid dependent. T-DNA tagged mutants (ugt73b3 and ugt73b5) exhibited decreased resistance to P. syringae pv tomato-AvrRpm1, indicating that expression of the corresponding UGT genes is necessary during the hypersensitive response. These results emphasize the importance of plant secondary metabolite UGTs in plant-pathogen interactions and provide foundation for future understanding of the exact role of UGTs during the hypersensitive response.     

Functional dissection of the eukaryotic-specific tRNA-interacting factor of lysyl-tRNA synthetase

In the cytoplasm of higher eukaryotic cells, aminoacyl-tRNA synthetases (aaRSs) have polypeptide chain extensions appended to conventional prokaryotic-like synthetase domains. The supplementary domains, referred to as tRNA-interacting factors (tIFs), provide the core synthetases with potent tRNA-binding capacities, a functional requirement related to the low concentration of free tRNA prevailing in the cytoplasm of eukaryotic cells. Lysyl-tRNA synthetase is a component of the multi-tRNA synthetase complex. It exhibits a lysine-rich N-terminal polypeptide extension that increases its catalytic efficiency. The functional characterization of this new type of tRNA-interacting factor has been conducted. Here we describe the systematic substitution of the 13 lysine or arginine residues located within the general RNA-binding domain of hamster LysRS made of 70 residues. Our data show that three lysine and one arginine residues are major building blocks of the tRNA-binding site. Their mutation into alanine led to a reduced affinity for tRNA(3)(Lys) or minimalized tRNA mimicking the acceptor-TPsiC stem-loop of tRNA(3)(Lys) and a decrease in catalytic efficiency similar to that observed after a complete deletion of the N-terminal domain. Moreover, covalent continuity between the tRNA-binding and core domain is a prerequisite for providing LysRS with a tRNA binding capacity. Thus, our results suggest that the ability of LysRS to promote tRNA(Lys) networking during translation or to convey tRNA(3)(Lys) into the human immunodeficiency virus type 1 viral particles rests on the addition in evolution of this tRNA-interacting factor.