Nocardia vulneris sp. nov., isolated from wounds of human patients in North America

Nocardia species are ubiquitous in the environment with an increasing number of species isolated from clinical sources. From 2005 to 2009, eight isolates (W9042, W9247, W9290, W9319, W9846, W9851^T, W9865, and W9908) were obtained from eight patients from three states in the United States and Canada; all were from males ranging in age from 47 to 81 years old; and all were obtained from finger (n = 5) or leg (n = 3) wounds. Isolates were characterized by polyphasic analysis using molecular, phenotypic, morphologic and chemotaxonomic methods. Sequence analysis of 16S rRNA gene sequences showed the eight isolates are 100 % identical to each other and belong in the genus Nocardia. The nearest phylogenetically related neighbours were found to be the type strains for Nocardia altamirensis (99.33 % sequence similarity), Nocardia brasiliensis (99.37 %), Nocardia iowensis (98.95 %) and Nocardia tenerifensis (98.44 %). The G+C content of isolate W9851^T was determined to be 68.4 mol %. The DNA–DNA relatedness between strain W9851^T and the N. brasiliensis type strain was 72.8 % and 65.8 % when measured in the laboratory and in silico from genome sequences, respectively, and 95.6 % ANI. Whole-cell peptidoglycan was found to contain meso-diaminopimelic acid; MK-8-(H₄)_ω-cyc was identified as the major menaquinone; the major fatty acids were identified as C_16:0, 10 Me C_18:0, and C_{18:1 w9c}, the predominant phospholipids were found to include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides; whole-cell sugars detected were arabinose and galactose; and mycolic acids ranging from 38 to 60 carbon atoms were found to be present. These chemotaxonomic analyses are consistent with assignment of the isolates to the genus Nocardia. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra of the clinical isolates showed genus and species level profiles that were different from other Nocardia species. All isolates were resistant to ciprofloxacin, clarithromycin and imipenem but were susceptible to amikacin, amoxicillin/clavulanate, linezolid and trimethoprim/sulfamethoxazole. The results of our polyphasic analysis suggest the new isolates obtained from wound infections represent a novel species within the genus Nocardia, for which the name Nocardia vulneris sp. nov. is proposed, with strain W9851^T (= DSM 45737^T = CCUG 62683^T = NBRC 108936^T) as the type strain.     

Alpha-actinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and rapidly degraded cytoskeletal protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant alpha-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant alpha-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a "knockin" mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant alpha-actinin-4, mediated, at least in part, by the ubiquitin-proteasome pathway. We correlate these findings with studies of alpha-actinin-4 expression in human samples. "Knockin" mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 "knockin" and "knockout" mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.     

Linkage disequilibrium and founder effect analysis of the NF1 gene in French Canadians from the Quebec population

We genotyped 19 neurofibromatosis type 1 (NF1) families from French Canadians of the Quebec population with four intragenic microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Linkage analysis of the four microsatellite markers among the 19 NF1 families indicates that the four microsatellites are strongly linked with NF1 disease (LOD = 2.76-3.64). The four markers are associated (P = 0-0.077) except marker pair IVS26-2.3/IVS27AC33.1 (P = 0.18 or 0.17). However, perhaps due to the high mutation rate of the NF1 gene, no founder effect for NF1 was detected in the Quebec French Canadians.     

ABA and polyamines act independently in primary leaves of cold-stressed tomato (Lycopersicon esculentum)

The effects of ABA and putrescine, a polyamine, on cold-induced membrane leakage were investigated using primary leaves of wild-type and an ABA-deficient mutant, flacca , of tomato ( Lycopersicon esculentum Mill.). The amount of chilling-induced electrolyte leakage from flacca leaves was much higher than that from the wild-type leaves. When applied exogenously ABA reduced cold-induced electrolyte leakage from leaves of both wild-type and the flacca mutant. However, the cold-induced electrolyte leakage from flacca leaves was not as pronounced as in the wild-type indicating that ABA is an important mediator in response to cold stress in the leaves. Putrescine reduced cold-induced electrolyte leakage from both wild-type and flacca leaves. Synthesis of putrescine in the leaves was increased by cold treatment. DFMO, a biosynthetic inhibitor of the polyamine, increased electrolyte leakage from cold-treated leaves, and exogenously applied putrescine decreased the enhanced leakage to the control level. Therefore, this polyamine is thought also to be involved in the response to cold stress of tomato leaves. Both ABA and putrescine were protective against cold stress, but exogenously applied ABA decreased the endogenous level of putrescine in the leaves. Furthermore, the DMFO-increased electrolyte leakage in cold-stressed leaves was completely abolished by the application of ABA. These results suggest that ABA is a major regulator in the response to cold stress in tomato leaves and that it does not exert its role via putrescine in the response to cold stress.     

Phosphorus compartmentation in Pinus serotina Michx. (pond pine); observations from in vivo nuclear magnetic resonance spectroscopy

³¹P nuclear magnetic resonance (NMR) spectroscopy was used to estimate the amount of inorganic phosphate (Pi) present in the cytoplasm and vacuole of root tips and subapical root segments of pond pine ( Pinus serotina Michx.). In root tips of seedlings grown with 100 mmol m^–3P (HP) the cytoplasmic Pi content, on a root volume basis, was ≈ 1·5 μ mol cm^–3 and the vacuolar Pi content, on a root volume basis, was ≈ 3·4 μ mol cm^–3. In root tips from Pi starved seedlings the cytoplasmic Pi content, on a root volume basis, was ≈ 0·75 μ mol cm^–3; vacuolar Pi was too low to be reliably estimated. Similar results were obtained with subapical root segments; the Pi concentration in the cytoplasm was maintained at around 2 mol m^–3 while that in the vacuole varied with Pi supply. This work demonstrates for the first time that quantitative measurements of the subcellular compartmentation of Pi can be made in young tissues of a woody species. The results indicate that cytoplasmic Pi levels are maintained across a range of external Pi supplies probably by withdrawing Pi stored in the vacuole.     

Molecular architecture of a multifunctional MCM complex

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.     

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.     

Closely related plasmids from Staphylococcus aureus and soil bacilli

Antibiotic resistance plasmids from staphylococci and soil bacilli have been isolated and compared. A tetracycline resistance (Tc^r) plasmid, indistinguishable from pT181, which is typical of Tc^r plasmids that are widely dispersed among human clinical isolates of S. aureus, has been found also in bovine mastitis isolates. This plasmid, however, shows no detectable homology to a family of related Tc^r plasmids, typified by pBC16, that is widely dispersed among aerobic spore-forming bacilli. However, and rather unexpectedly, pBC16 is highly homologous to and incompatible with pUB110, an S. aureus plasmid specifying kanamycin resistance. The two plasmids are homologous except for the region occupied by their resistance determinants, which has the appearance of a heterologous substitution. These results suggest the occurrence of natural plasmid transfer between staphylococci and soil bacilli.