CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zinc- and imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics.     

Purification of a plant cell wall fibronectin-like adhesion protein involved in plant response to salt stress

The structural role of extracellular-matrix (ECM) has been recognized in both plants and animals as a support and anchorage-inducing cell behavior. Unlike the animal ECM proteins, the proteins that have been identified in plant ECM have not yet been purified from whole plants and cell wall. As several immunological data indicate the presence of animal ECM-like proteins in plants cell wall, especially under salt stress or water deficit, we propose a protocol to purify a fibronectin-like protein from the cell wall of epicotyls of young germinating peas. The process consists of a combination of gelatin and heparin affinity chromatography, close to the classical one used for human blood plasma fibronectin purification. Proteins with affinity for gelatin and heparin, immunologically related to human fibronectin, are found in the cell wall of epicotyls grown under salt stress or not. Total amount of purified proteins is 3-4 times more enriched in salt stressed epicotyls. SDS-PAGE and Western blot with antibodies directed against human blood plasma fibronectin give evidence that the cell wall proteins purified by gelatin/heparin affinity chromatography are closely related to human fibronectin. The present protocol leads us to purify 17 (control) or 65 (salt stress) micrograms of protein per g of fresh starting material. Our results suggest that plant cell wall proteins can provide better anchorage of the cell to its cell-wall during salt stress or water deficit and could be considered not only as cell adhesion but also as signaling molecules.     

Molecular detection and characterization of nodavirus in several marine fish species from the northeastern Atlantic

Nodaviruses (NNV) are responsible for causing disease outbreaks mainly in hatchery-reared larvae and juveniles of a wide variety of fishes throughout the world. This disease has seriously limited the culture of marine fishes over the last decade. In the Atlantic provinces of Canada, disease caused by a nodavirus was first reported in juvenile Atlantic cod being reared in Nova Scotia, in 1999. More recently, disease outbreaks caused by nodavirus have been identified in hatchery-reared Atlantic cod and haddock in Newfoundland and New Brunswick, respectively, and along the east coast of the USA. The presence of NNV in wild Atlantic cod adults and wild winter flounder has also been reported. Nodaviruses were isolated from cultured Atlantic cod and haddock, as well as from wild winter flounder from a variety of geographical localities, and their virus coat (capsid) protein genes were partially sequenced. An analysis of the data indicates that all of the nodaviruses isolated from eastern North America were closely related to one another, but that they were distinct from the European isolates already sequenced. Regardless of host species, isolates from close geographical localities were more similar than those from distant geographical areas. At the protein level, differences in coat protein sequences were seen only for strains isolated from Atlantic cod originating from Newfoundland. Our results suggest that NNV may have been present in the Atlantic off Canada and on the east coast of the USA for some time, and has evolved to form a monophyletic group, distinct from other isolates found in cold-water species. Non-lethal methods for detection of NNV are necessary to develop management strategies for this disease, and would be an asset to diagnosticians and producers. Based on the results of this study, new primers were designed and developed for an improved RT-PCR assay able to detect North Atlantic nodaviruses in ovarian fluids, eggs and other tissues. The application of this test to field samples is discussed.     

Bioaugmented sulfur-oxidizing denitrification system with <Emphasis Type="Italic">Alcaligenes defragrans</Emphasis> B21 for high nitrate containing wastewater treatment

The performance of enriched sludge augmented with the B21 strain of Alcaligenes defragrans was compared with that of enriched sludge, as well as with pure Alcaligenes defragrans B21, in the context of a sulfur-oxidizing denitrification (SOD) process. In synthetic wastewater treatment containing 100–1,000 mg NO₃⁻-N/L, the single strain-seeded system exhibited superior performance, featuring higher efficiency and a shorter startup period, provided nitrate loading rate was less than 0.2 kg NO₃⁻-N/m³ per day. At nitrate loading rate of more than 0.5 kg NO₃⁻-N/m³ per day, the bioaugmented sludge system showed higher resistance to shock loading than two other systems. However, no advantage of the bioaugmented system over the enriched sludge system without B21 strain was observed in overall efficiency of denitrification. Both the bioaugmented sludge and enriched sludge systems obtained stable denitrification performance of more than 80% at nitrate loading rate of up to 2 kg NO₃⁻-N/m³ per day.     

Computational study of coagulation factor VIIa’s affinity for phospholipid membranes

The interaction between the γ-carboxyglutamic acid-rich domain of coagulation factor VIIa (FVIIa), a vitamin-K-dependent enzyme, and phospholipid membranes plays a major role in initiation of blood coagulation. However, despite a high sequence and structural similarity to the Gla domain of other vitamin-K-dependent enzymes with a high membrane affinity, its affinity for negatively charged phospholipids is poor. A few amino acid differences are responsible for this observation. Based on the X-ray structure of lysophosphatidylserine (lysoPS) bound to the Gla domain of bovine prothrombin (Prth), models of the Gla domain of wildtype FVIIa and mutated FVIIa Gla domains in complex with lysoPS were built. Molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on the complexes were applied to investigate the significant difference in the binding affinity. The MD simulation approach provides a structural and dynamic support to the role of P10Q and K32E mutations in the improvement of the membrane contact. Hence, rotation of the Gly11 main chain generated during the MD simulation results in a hydrogen bond with Q10 side chain as well as the appearance of a hydrogen bond between E32 and Q10 forcing the loop harbouring Arg9 and Arg15 to shrink and thereby enhances the accessibility of the phospholipids to the calcium ions. Furthermore, the application of the SMD simulation method to dissociate C6-lysoPS from a series of Gla domain models exhibits a ranking of the rupture force that can be useful in the interpretation of the PS interaction with Gla domains. Finally, adiabatic mapping of Gla6 residue in FVIIa with or without insertion of Tyr4 confirms the critical role of the insertion on the conformation of the side chain Gla6 in FVIIa and the corresponding Gla7 in Prth.     

Adding value to cocoa (Theobroma cacao L.) germplasm information with domestication history and admixture mapping

A sound understanding of crop history can provide the basis for deriving novel genetic information through admixture mapping. We confirmed this, by using characterization data from an international collection of cocoa, collected 25 years ago, and from a contemporary plantation. We focus on the trees derived from three centuries of admixture between Meso-American Criollo and South American Forastero genomes. In both cacao sets of individuals, linkage disequilibrium extended over long genetic distances along chromosome regions, as expected in populations derived from recent admixture. Based on loose genome scans, genomic regions involved in useful traits were identified. Fifteen genomic regions involved in seed and fruit weight variation were highlighted. They correspond to ten previously identified QTLs and five novel ones. Admixture mapping can help to add value to genetic resources and thus, help to encourage investment in their conservation. Maria Marcano and Tatiana Pugh contributed equally to this work.     

Iron does not cause arrhythmias in the guinea pig model of transfusional iron overload

Cardiac events, including heart failure and arrhythmias, are the leading cause of death in patients with beta thalassemia. Although cardiac arrhythmias in humans are believed to result from iron overload, excluding confounding factors in the human population is difficult. The goal of the current study was to determine whether cardiac arrhythmias occurred in the guinea pig model of secondary iron overload. Electrocardiograms were recorded by using surgically implanted telemetry devices in guinea pigs loaded intraperitoneally with iron dextran (test animals) or dextran alone (controls). Loading occurred over approximately 6 wk. Electrocardiograms were recorded for 1 wk prior to loading, throughout loading, and for approximately 4 wk after loading was complete. Cardiac and liver iron concentrations were significantly increased in the iron-loaded animals compared with controls and were in the range of those reported for humans with thalassemia. Arrhythmias were rare in both iron-loaded and control guinea pigs. No life-threatening arrhythmias were detected in either group. These data suggest that iron alone may be insufficient to cause cardiac arrhythmias in the iron-loaded guinea pig model and that arrhythmias detected in human patients with iron overload may be the result of a complex interplay of factors.     

Low Inbreeding and High Pollen Dispersal Distances in Populations of Two Amazonian Forest Tree Species

Recent studies suggest that tropical tree species exhibit low inbreeding and high gene dispersal levels despite the typically low density of conspecifics in tropical forests. To examine this, we undertook a study of pollen gene dispersal and mating system of two Amazonian tree species. We analyzed 341 seeds from 33 trees at four microsatellite loci in a Carapa guianensis population from Brazil, and 212 seeds from 22 trees at four microsatellite loci in a Sextonia rubra population from French Guiana. Differentiation of allele frequencies among the pollen pool of individual trees was Φ_FT= 0.053 (95% CI: 0.027–0.074) for C. guianensis and Φ_FT= 0.064 (95% CI: 0.017–0.088) for S. rubra. The mean pollen dispersal distances were estimated at 69–355 m for C. guianensis , and 86–303 m for S. rubra , depending on the pollen dispersal model and the estimate of reproductive tree density used. The multi-locus outcrossing rate was estimated at 0.918 and 0.945, and the correlation of paternity at 0.089 and 0.096, for C. guianensis and S. rubra , respectively, while no significant levels of biparental inbreeding were detected. Comparing trees with high and low local density of conspecifics, we found no evidence for differences in inbreeding levels. The results are discussed within the framework of the emerging picture of the reproductive biology of tropical forest trees.     

Pax‐6 is expressed early in the differentiation of a corneal epithelial model system

Pax‐6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1‐(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax‐6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax‐6‐positive cells were also K3 keratin‐positive, suggesting that Pax‐6 is expressed in differentiating cells. Pax‐6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT‐qPCR. The raise in Pax‐6 expression preceded for 9 h the increase in LDH‐H and LDH‐M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full‐length mRNAs encoding for the two major Pax‐6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax‐6 mRNA was 2‐ to 2.5‐fold more abundant than Pax‐6(5a) mRNA. The ectopic expression of Pax‐6 or Pax‐6(5a) decreased proliferative ability leading to the formation of abortive, non‐proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax‐6. Collectively, results show that Pax‐6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax‐6 as the main driver of the differentiation of corneal epithelial cells. J. Cell. Physiol. 220: 348–356, 2009. © 2009 Wiley‐Liss, Inc.