Selecting representative model micro-organisms

Background

The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system.

Results

The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci.

Conclusion

This archived set of Gateway^® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.     

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB(1) receptor density.     

Nitrogen starvation-induced chlorosis in Synechococcus PCC 7942. Low-level photosynthesis as a mechanism of long-term survival

Cells of the non-diazotrophic cyanobacterium Synechococcus sp. strain PCC 7942 acclimate to nitrogen deprivation by differentiating into non-pigmented resting cells, which are able to survive prolonged periods of starvation. In this study, the physiological properties of the long-term nitrogen-starved cells are investigated in an attempt to elucidate the mechanisms of maintenance of viability. Preservation of energetic homeostasis is based on a low level of residual photosynthesis; activities of photosystem II and photosystem I were approximately 0.1% of activities of vegetatively growing cells. The low levels of photosystem I activity were measured by a novel colorimetric assay developed from the activity staining of ferredoxin:NADP+ oxidoreductase. Photosystem II reaction centers, as determined by chlorophyll fluorescence measurements, exhibited normal properties, although the efficiency of light harvesting was significantly reduced compared with that of control cells. Long-term chlorotic cells carried out protein synthesis at a very low, but detectable level, as revealed by in vivo [35S]methionine labeling and two-dimensional gel electrophoresis. In conjunction with the very low levels of total cellular protein contents, this implies a continuous protein turnover during chlorosis. Synthesis of components of the photosynthetic apparatus could be detected, whereas factors of the translational machinery were stringently down-regulated. Beyond the massive loss of protein during acclimation to nitrogen deprivation, two proteins that were identified as SomA and SomB accumulated due to an induced expression following nitrogen reduction.     

Recent advances in the design and performance of research assistants

Every laboratory needs personnel who work 24 hours a day 7 days a week, are efficient, obedient, capable of showing initiative, insightful and affable. Our experience of laboratory workers tells us that there is an immense way to go to achieve these goals. Having tired of the normal, Darwinian approach, a project has been initiated to achieve these ends through genetic technologies.     

ChIC and ChEC; genomic mapping of chromatin proteins

To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100-200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.     

Optimal re-design of primary metabolism in Escherichia coli using linlog kinetics

This paper examines the validity of the linlog approach, which was recently developed in our laboratory, by comparison of two different kinetic models for the metabolic network of Escherichia coli. The first model is a complete mechanistic model; the second is an approximative model in which linlog kinetics are applied. The parameters of the linlog model (elasticities) are derived from the mechanistic model. Three different optimization cases are examined. In all cases, the objective is to calculate the enzyme levels that maximize a certain flux while keeping the total amount of enzyme constant and preventing large changes of metabolite concentrations. For an average variation of metabolite levels of 10% and individual changes of a factor 2, the predicted enzyme levels, metabolite concentrations and fluxes of both models are highly similar. This similarity holds for changes in enzyme level of a factor 4-6 and for changes in fluxes up to a factor 6. In all three cases, the predicted optimal enzyme levels could neither have been found by intuition-based approaches, nor on basis of flux control coefficients. This demonstrates that kinetic models are essential tools in Metabolic Engineering. In this respect, the linlog approach is a valuable extension of MCA, since it allows construction of kinetic models, based on MCA parameters, that can be used for constrained optimization problems and are valid for large changes of metabolite and enzyme levels.     

The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions

The epoxide hydrolases and haloalkane dehalogenases database (EH/HD) integrates sequence and structure of a highly diverse protein family, including mainly the Asp-hydrolases of EHs and HDs but also proteins, such as Ser-hydrolases non-heme peroxidases, prolyl iminopetidases and 2-hydroxymuconic semialdehyde hydrolases. These proteins have a highly conserved structure, but display a remarkable diversity in sequence and function. A total of 305 protein entries were assigned to 14 homologous families, forming two superfamilies. Annotated multisequence alignments and phylogenetic trees are provided for each homologous family and superfamily. Experimentally derived structures of 19 proteins are superposed and consistently annotated. Sequence and structure of all 305 proteins were systematically analysed. Thus, deeper insight is gained into the role of a highly conserved sequence motifs and structural elements. AVAILABILITY: The EH/HD database is available at http://www.led.uni-stuttgart.de     

Toward non-toxic antifouling: synthesis of hydroxy-, cinnamic acid-, sulfate-, and zosteric acid-labeled poly[3-hydroxyalkanoates

The side-chain double bonds of bacterial poly[3-hydroxyalkanoate-co-3-hydroxyalkenoate] (PHAE, 1) were transformed into thioether bonds (derivative 2) via the radical addition reaction of 11-mercapto-1-undecanol. The terminal hydroxy functionalities of derivative 2 were subsequently esterified with cinnamic acid (derivative 3), sulfatized with ClSO(3)H (derivative 4), or coupled with tert-butyldimethylsilyl-protected coumaric acid, to give, after deprotection with tetrabutylammonium fluoride (derivative 5) followed by sulfatization, p-(sulfooxy) cinnamic acid- (zosteric acid) labeled PHAE (derivative 6). The reactions proceeded with good yields and little side reactions, which was confirmed with (1)H NMR and GPC experiments. These functionalized polyesters are currently investigated as environmentally friendly coatings to protect surfaces from biofouling.     

Proteome comparison of Vibrio cholerae cultured in aerobic and anaerobic conditions

The pathogen Vibrio cholerae causes severe diarrheal disease in humans. This environmental inhabitant has two distinct life cycles, in the environment and in the human small intestine, in which it differs in its multiplication behavior and virulence expression. Anaerobiosis, limitation of some nutrient elements, and excess burden from host metabolism reactants are the major stresses for V. cholerae living in intestine, in comparison to conditions in the environment and laboratory medium. For an insight into the response of V. cholerae to different microenvironments, we cultured the bacteria in aerobic and anaerobic conditions, and compared the whole cell proteome by two-dimensional electrophoresis. Among the protein spots identified, some protein species involved in aerobic respiration and the nutrient carbohydrate transporters were found to be more abundant in aerobic conditions, and some enzymes for anaerobic respiration and some stress response proteins were found more abundant in anaerobic culture. One spot corresponding to flagellin B subunit was decreased in anaerobic conditions, which suggests correlation with the meticulous regulation of bacterial motility during infection in the host intestine. This proteome analysis is the starting point for in-depth understanding of V. cholerae behavior in different environments.