Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria

A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.     

Native faunal communities depend on habitat from non-native plants in novel but not in natural ecosystems

Invasive non-native plants are a major driver of native biodiversity loss, yet native biodiversity can sometimes benefit from non-native species. Depending on habitat context, even the same non-native species can have positive and negative effects on biodiversity. Blackberry (Rubus fruticosus aggregate) is a useful model organism to better understand a non-native plant with conflicting impacts on biodiversity. We used a replicated capture-mark-recapture study across 11 consecutive seasons to examine the response of small mammal diversity and abundance to vegetation structure and density associated with non-native blackberry (R. anglocandicans) in native, hybrid and blackberry-dominated novel ecosystems in Australia. Across the three habitat types, increasing blackberry dominance had a positive influence on mammal diversity, while the strength and direction of this influence varied for abundance. At a microhabitat scale within hybrid and native habitat there were no significant differences in diversity, or the abundance of most species, between microhabitats where blackberry was absent versus dominant. In contrast, in novel ecosystems diversity and abundances were very low without blackberry, yet high (comparable to native ecosystems) within blackberry as it provided functionally-analogous vegetation structure and density to the lost native understory. Our results indicate the ecological functions of non-native plant species vary depending on habitat and need to be considered for management. Comparative studies such as ours that apply a standardized approach across a broad range of conditions at the landscape and habitat scale are crucial for guiding land managers on control options for non-native species (remove, reduce or retain and contain) that are context-sensitive and scale-dependent.     

Application of cation-exchange membranes for characterisation and imaging ammonia-oxidising bacteria in soils

A new approach, in which ammonia-oxidizing bacteria (AOB) are entrapped from soil onto cation-exchange membranes, was applied to identify terrestrial AOB by fluorescence in situ hybridization (FISH). An experimental hot spot of ammonia oxidation was developed by establishing a gradient of ammonium substrate (200 to <20 mg NH4+-N l(-1)) diffused through the cation-exchange membranes incubated in soil for 6 months. By this approach we were able to characterise and image indigenous AOB populations growing in heavily oil-polluted soil using FISH and sequence analysis of PCR-amplified 16S rRNA genes, respectively. The FISH results revealed that Nitrosospira-like AOB were dominant on the ammonium-enriched membranes incubated in the soil. Fourteen unique Nitrosospira-like 16S rRNA gene sequences belonging to clusters 2 and 3 were recovered from the soil-incubated membranes and from the soil, suggesting the importance of Nitrosospira-like AOB in the oil-polluted landfarming soil.     

Mitotic phosphorylation of the anaphase-promoting complex inhibitory subunit Mnd2 is necessary for efficient progression through meiosis i

The yeast anaphase-promoting complex (APC) subunit Mnd2 is necessary for maintaining sister chromatid cohesion in prophase I of meiosis by inhibiting premature ubiquitination and subsequent degradation of substrates by the APC(Ama1) ubiquitin ligase. In a proteomics screen for post-translational modifications on the APC, we discovered that Mnd2 is phosphorylated during mitosis in a cell cycle-dependent manner. We identified and characterized the sites of mitotic Mnd2 phosphorylation during the cell cycle. Collective mutation of Mnd2 phosphorylation sites to alanine had no effect on vegetative growth but a striking effect (>85% reduction) on the percentage of tetrad-forming cells compared with the wild type strain. Similar to the MND2 deletion strain, cells harboring the alanine mutant that did not form spores arrested after premeiotic S phase with a single undivided nucleus and low levels of the APC(Ama1) meiotic substrate, Clb5, relative to wild type cells. In contrast, collective mutation of Mnd2 phosphorylation sites to aspartic acid resulted in partial suppression of the sporulation defect. No differences were observed in the binding between each Mnd2 isoform and the APC in vitro. However, in vivo, we observed a gradient in the abundance of APC-associated Mnd2 in each strain that was proportional to the observed differences in sporulation and Clb5 levels. Taken together, these data suggest that mitotic phosphorylation of Mnd2 is necessary for APC-mediated progression beyond the first meiotic nuclear division.     

METANNOGEN: compiling features of biochemical reactions needed for the reconstruction of metabolic networks

Background  

One central goal of computational systems biology is the mathematical modelling of complex metabolic reaction networks. The first and most time-consuming step in the development of such models consists in the stoichiometric reconstruction of the network, i. e. compilation of all metabolites, reactions and transport processes relevant to the considered network and their assignment to the various cellular compartments. Therefore an information system is required to collect and manage data from different databases and scientific literature in order to generate a metabolic network of biochemical reactions that can be subjected to further computational analyses.     

A systems-biology analysis of feedback inhibition in the Sho1 osmotic-stress-response pathway

BACKGROUND: A common property of signal transduction systems is that they rapidly lose their ability to respond to a given stimulus. For instance in yeast, the mitogen-activated protein (MAP) kinase Hog1 is activated and inactivated within minutes, even when the osmotic-stress stimulus is sustained. RESULTS: Here, we used a combination of experimental and computational analyses to investigate the dynamic behavior of Hog1 activation in vivo. Computational modeling suggested that a negative-feedback loop operates early in the pathway and leads to rapid attenuation of Hog1 signaling. Experimental analysis revealed that the membrane-bound osmosensor Sho1 is phosphorylated by Hog1 and that phosphorylation occurs on Ser-166. Moreover, Sho1 exists in a homo-oligomeric complex, and phosphorylation by Hog1 promotes a transition from the oligomeric to monomeric state. A phosphorylation-site mutation (Sho1(S166E)) diminishes the formation of Sho1-oligomers, dampens activation of the Hog1 kinase, and impairs growth in high-salt or sorbitol conditions. CONCLUSIONS: These findings reveal a novel phosphorylation-dependent feedback loop leading to diminished cellular responses to an osmotic-stress stimulus.     

The antifungal protein PAF interferes with PKC/MPK and cAMP/PKA signalling of Aspergillus nidulans

The Penicillium chrysogenum antifungal protein PAF inhibits polar growth and induces apoptosis in Aspergillus nidulans. We report here that two signalling cascades are implicated in its antifungal activity. PAF activates the cAMP/protein kinase A (Pka) signalling cascade. A pkaA deletion mutant exhibited reduced sensitivity towards PAF. This was substantiated by the use of pharmacological modulators: PAF aggravated the effect of the activator 8‐Br‐cAMP and partially relieved the repressive activity of caffeine. Furthermore, the Pkc/mitogen‐activated protein kinase (Mpk) signalling cascade mediated basal resistance to PAF, which was independent of the small GTPase RhoA. Non‐functional mutations of both genes resulted in hypersensitivity towards PAF. PAF did not increase MpkA phosphorylation or induce enzymes involved in the remodelling of the cell wall, which normally occurs in response to activators of the cell wall integrity pathway. Notably, PAF exposure resulted in actin gene repression and a deregulation of the chitin deposition at hyphal tips of A. nidulans, which offers an explanation for the morphological effects evoked by PAF and which could be attributed to the interconnection of the two signalling pathways. Thus, PAF represents an excellent tool to study signalling pathways in this model organism and to define potential fungal targets to develop new antifungals.     

Stromal upregulation of lateral epithelial adhesions: Gene expression analysis of signalling pathways in prostate epithelium

Background  

Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process.     

The role of osteopontin (OPN/SPP1) haplotypes in the susceptibility to Crohn's disease

Background

Osteopontin represents a multifunctional molecule playing a pivotal role in chronic inflammatory and autoimmune diseases. Its expression is increased in inflammatory bowel disease (IBD). The aim of our study was to analyze the association of osteopontin (OPN/SPP1) gene variants in a large cohort of IBD patients.

Methodology/Principal Findings

Genomic DNA from 2819 Caucasian individuals (n = 841 patients with Crohn''s disease (CD), n = 473 patients with ulcerative colitis (UC), and n = 1505 healthy unrelated controls) was analyzed for nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138). Considering the important role of osteopontin in Th17-mediated diseases, we performed analysis for epistasis with IBD-associated IL23R variants and analyzed serum levels of the Th17 cytokine IL-22. For four OPN SNPs (rs4754, rs1126616, rs1126772 and rs9138), we observed significantly different distributions between male and female CD patients. rs4754 was protective in male CD patients (p = 0.0004, OR = 0.69). None of the other investigated OPN SNPs was associated with CD or UC susceptibility. However, several OPN haplotypes showed significant associations with CD susceptibility. The strongest association was found for a haplotype consisting of the 8 OPN SNPs rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138 (omnibus p-value = 2.07×10⁻⁸). Overall, the mean IL-22 secretion in the combined group of OPN minor allele carriers with CD was significantly lower than that of CD patients with OPN wildtype alleles (p = 3.66×10⁻⁵). There was evidence for weak epistasis between the OPN SNP rs28357094 with the IL23R SNP rs10489629 (p = 4.18×10⁻²) and between OPN SNP rs1126616 and IL23R SNP rs2201841 (p = 4.18×10⁻²) but none of these associations remained significant after Bonferroni correction.

Conclusions/Significance

Our study identified OPN haplotypes as modifiers of CD susceptibility, while the combined effects of certain OPN variants may modulate IL-22 secretion.