Novel phenotypes of Escherichia coli tat mutants revealed by global gene expression and phenotypic analysis

The Tat protein export system serves to export folded proteins harboring an N-terminal twin arginine signal peptide across the cytoplasmic membrane. In this study, we have used gene expression profiling of Escherichia coli supported by phenotypic analysis to investigate how cells respond to a defect in the Tat pathway. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are defective in the integrity of their cell envelope because of the mislocalization of two amidases involved in cell wall metabolism (Ize, B., Stanley, N. R., Buchanan, G., and Palmer, T. (2003) Mol. Microbiol. 48, 1183-1193). To distinguish between genes that are differentially expressed specifically because of the cell envelope defect and those that result from other effects of the tatC deletion, we also analyzed two different transposon mutants of the DeltatatC strain that have their outer membrane integrity restored. Approximately 50% of the genes that were differentially expressed in the tatC mutant are linked to the envelope defect, with the products of many of these genes involved in self-defense or protection mechanisms, including the production of exopolysaccharide. Among the changes that were not explicitly linked to envelope integrity, we characterized a role for the Tat system in iron acquisition and copper homeostasis. Finally, we have demonstrated that overproduction of the Tat substrate SufI saturates the Tat translocon and produces effects on global gene expression that are similar to those resulting from the DeltatatC mutation.     

Structural consequences of cysteine substitutions C1977Y and C1977R in calcium-binding epidermal growth factor-like domain 30 of human fibrillin-1

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.     

Biofilm development and cell death in the marine bacterium Pseudoalteromonas tunicata

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A Delta alpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.     

Extracellular matrix distribution and islet morphology in the early postnatal pancreas: anomalies in the non-obese diabetic mouse

Previously, we reported elevated numbers of macrophages in the pancreas of NOD mice, a spontaneous animal model for T1D, during the early postnatal period. Extracellular matrix plays an important role in the tissue trafficking and retention of macrophages as well as in postnatal pancreas development. Therefore, we have examined the expression and distribution of laminin and fibronectin, two major extracellular matrix proteins and their corresponding integrin receptors, in the pre-weaning pancreases of NOD mice and control mouse strains. In addition, we have characterized the pancreas morphology during this period, since the morphology of the pre-weaning pancreas before the onset of lymphocytic peri-insulitis, when the pancreas is still subject to developmental changes, has been poorly documented. We show that laminin labeling is mainly associated with exocrine tissue, whereas fibronectin labeling was mostly localized at the islet-ductal pole, islet periphery and in intralobular septa. Moreover, the protein expression level of fibronectin was increased in NOD pancreases at the early stage of postnatal development, as compared to pancreases of C57BL/6 and BALB/c mouse strains. Interestingly, pancreatic macrophages were essentially found at sites of intense fibronectin labeling. The increased fibronectin content in NOD neonatal pancreas coincided with altered islet morphology, histologically reflected by enlarged and irregular shaped islets and increased percentages of total endocrine area as compared to that of control strains. In conclusion, increased levels of the extracellular matrix protein fibronectin were found in the early postnatal NOD pancreas, and this is associated with an enhanced accumulation of macrophages and altered islet morphology.This work was supported by the Centre Nationale de la Recherche Scientifique, Université Paris V and grants from the 5th PCRD MONODIAB.     

Proteome analysis of the rice etioplast: metabolic and regulatory networks and novel protein functions

We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.     

Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells

Nef-specific CD8⁺ T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive nef variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef_165–173IW9 epitope revealed highly restricted evolution. A common acute escape variant, T₁₇₀I, unexpectedly and uniquely degraded Nef''s major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication.     

A novel case of ACOX2 deficiency leads to recognition of a third human peroxisomal acyl-CoA oxidase

Peroxisomal acyl-CoA oxidases catalyze the first step of beta-oxidation of a variety of substrates broken down in the peroxisome. These include the CoA-esters of very long-chain fatty acids, branched-chain fatty acids and the C27-bile acid intermediates. In rat, three peroxisomal acyl-CoA oxidases with different substrate specificities are known, whereas in humans it is believed that only two peroxisomal acyl-CoA oxidases are expressed under normal circumstances. Only three patients with ACOX2 deficiency, including two siblings, have been identified so far, showing accumulation of the C27-bile acid intermediates. Here, we performed biochemical studies in material from a novel ACOX2-deficient patient with increased levels of C27-bile acids in plasma, a complete loss of ACOX2 protein expression on immunoblot, but normal pristanic acid oxidation activity in fibroblasts. Since pristanoyl-CoA is presumed to be handled by ACOX2 specifically, these findings prompted us to re-investigate the expression of the human peroxisomal acyl-CoA oxidases. We report for the first time expression of ACOX3 in normal human tissues at the mRNA and protein level. Substrate specificity studies were done for ACOX1, 2 and 3 which revealed that ACOX1 is responsible for the oxidation of straight-chain fatty acids with different chain lengths, ACOX2 is the only human acyl-CoA oxidase involved in bile acid biosynthesis, and both ACOX2 and ACOX3 are involved in the degradation of the branched-chain fatty acids. Our studies provide new insights both into ACOX2 deficiency and into the role of the different acyl-CoA oxidases in peroxisomal metabolism.     

Facilitating the restoration of aquatic plant communities in a Ramsar wetland

Human activities such as land clearing and intensive land use around water bodies, particularly wetlands, have a detrimental impact on water quality and quantity, aquatic plant communities, and associated wetland fauna. Lake Alexandrina and Lake Albert are internationally significant Ramsar wetlands located at the terminus of the Murray River, Australia's longest river system. Agriculture, water regulation, and extraction and droughts have had a detrimental impact on native plant communities in the lakes. We studied the influence of young (<1–3 years) and old (8–11 years) plantings of a native sedge (bulrush), Schoenoplectus tabernaemontani, to facilitate the establishment of aquatic plant communities in comparison with remnant and control sites. We also measured how planting structure (height, stand width, and stem density) changed with age in comparison with remnant sites. Results suggest that as plantings age they get substantially wider and have a greater maximum height, although do not reach similar stand widths by 11 years when compared to remnant areas. However, old plantings do not differ from remnant habitats in relation to aquatic plant species richness, counts of aquatic plants, and community composition. Young plantings have substantially less abundant and diverse plant communities, but are developing on a similar trajectory to old plantings. It is likely that planting sedges along lake shorelines causes a breakwater effect that facilitates the recolonization of wetland plants between the planted area and the water's edge. Management agencies should consider restoring native sedges to increase aquatic biodiversity, and potentially reduce erosion.     

Regret and the rationality of choices

Regret helps to optimize decision behaviour. It can be defined as a rational emotion. Several recent neurobiological studies have confirmed the interface between emotion and cognition at which regret is located and documented its role in decision behaviour. These data give credibility to the incorporation of regret in decision theory that had been proposed by economists in the 1980s. However, finer distinctions are required in order to get a better grasp of how regret and behaviour influence each other. Regret can be defined as a predictive error signal but this signal does not necessarily transpose into a decision-weight influencing behaviour. Clinical studies on several types of patients show that the processing of an error signal and its influence on subsequent behaviour can be dissociated. We propose a general understanding of how regret and decision-making are connected in terms of regret being modulated by rational antecedents of choice. Regret and the modification of behaviour on its basis will depend on the criteria of rationality involved in decision-making. We indicate current and prospective lines of research in order to refine our views on how regret contributes to optimal decision-making.