1H-NMR metabolite profiles of different strains of Plasmodium falciparum

Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using ¹H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite.     

Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.     

Efficient and direct fermentation of starch to ethanol by sake yeast strains displaying fungal glucoamylases

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.     

Novel metabolite structures from biotransformation of a sesquiterpenoid ketone by selected fungal strains

The sesquiterpenoid ketone, 1,4,4-trimethyltricyclo[5.4.0.0(3.5)]undec-7-en-9-one (1), was subjected to microbial transformation by six fungal strains: Aspergillus niger ATCC 9142, Aspergillus ochraceus DSM 824, Beauveria bassiana ATCC 7159, Cunninghamella echinulata ATCC 9244, Rhizopus arrhizus ATCC 11.145, and Absidia blakesleeana ATCC 10.148. Four main metabolites were formed from 1: 10(R)- and 10(S)-hydroxy-1,4,4-trimethyltricyclo-[5.4.0.0(3.5)]undec-7- en- 9-one (2 and 3, respectively), besides 4(R)- and 4(S)-(hydroxymethyl)-1,4-dimethyltricyclo[5.4.0.0(3.5)]undec -7-en-9-one (4 and 5, respectively). Compounds 2-5 were isolated with varying percentages from the respective transformations, and their structures established unequivocally by a combination of spectroscopic methods. Metabolites 2 and 3 are products of hydroxylation at C-10, in either R- or S-position; in 4 and 5, one geminal CH3 group each on the cyclopropane ring has been transformed into a CH2OH function.     

Plasma cortisol and metabolite level profiles in two isogenic strains of common carp during confinement

A rapid increase in common carp Cyprinus carpio plasma cortisol levels was noted, in two experiments, after 30 mins of a 3 h net confinement, which was sustained while the fish were held in the nets. After release from the nets, cortisol levels returned to control values in 1 h. Plasma glucose and free fatty acid levels were elevated by the confinement. Glucose was increased after 30 min but returned to basal levels after 22 h of recovery while free fatty acids were not elevated until 3 h of confinement and remained high for the duration of the recovery period. After confinement for 3 h, plasma lactate levels were reduced and remained low for a further 1 h. No change in either plasma triglyceride or cholesterol levels were found during the study. Confinement had no effect on haematocrit levels but blood haemoglobin levels were reduced. In both experiments hypochloraemia occurred in response to confinement. However, values returned to pre-confinement levels 22 h after confinement. These results show that rearing isogenic carp strains, under identical conditions, results in a reproducable response to an acute stressor and that these carp respond in a similar manner to other teleost species.     

Differences in proteolytic activity and gene profiles of fungal strains isolated from the total parenteral nutrition patients

Fungal infections constitute a serious clinical problem in the group of patients receiving total parenteral nutrition. The majority of species isolated from infections of the total parenteral nutrition patients belong to Candida genus. The most important factors of Candida spp. virulence are the phenomenon of “phenotypic switching,” adhesins, dimorphism of fungal cells and the secretion of hydrolytic enzymes such as proteinases and lipases, including aspartyl proteinases. We determined the proteolytic activity of yeast-like fungal strains cultured from the clinical materials of patients receiving total parenteral nutrition and detected genes encoding aspartyl proteinases in predominant species Candida glabrata—YPS2, YPS4, and YPS6, and Candida albicans—SAP1–3, SAP4, SAP5, and SAP6. C. albicans released proteinases on the various activity levels. All C. glabrata strains obtained from the clinical materials of examined and control groups exhibited secretion of the proteinases. All 13 isolates of C. albicans possessed genes SAP1–3. Gene SAP4 was detected in genome of 11 C. albicans strains, SAP5 in 6, and SAP6 in 11. Twenty-six among 31 of C. glabrata isolates contained YPS2 gene, 21 the YPS4 gene, and 28 the YPS6 gene. We observed that clinical isolates of C. albicans and C. glabrata differed in SAPs and YPSs gene profiles, respectively, and displayed differentiated proteolytic activity. We suppose that different sets of aspartyl proteinases genes as well as various proteinase-activity levels would have the influence on strains virulence.     

Integrating phenotypic and expression profiles to map arsenic-response networks

Background  

Arsenic is a nonmutagenic carcinogen affecting millions of people. The cellular impact of this metalloid in Saccharomyces cerevisiae was determined by profiling global gene expression and sensitivity phenotypes. These data were then mapped to a metabolic network composed of all known biochemical reactions in yeast, as well as the yeast network of 20,985 protein-protein/protein-DNA interactions.     

A method for the selection of production media for actinomycete strains based on their metabolite HPLC profiles

The manipulation of growth conditions of microorganisms is a common strategy used by pharmaceutical companies to improve the quantities and spectra of secondary metabolites with potential therapeutic interest. In this work, the effects of fermentation media on secondary metabolite production from a set of Actinomycetes was statistically compared. For this purpose, we created an automated method for comparing the ability of microorganisms to produce different secondary metabolites. HPLC analyses guided the selection of those media in which a wider chemical diversity was obtained from microorganisms inoculated in a wide spectrum of production media. Fermented media yielding a better secondary metabolite profile were included in subsequent drug discovery screening.     

Further toxic properties of the fungal metabolite dothistromin

The toxicity of the fungal phytotoxin dothistromin (l) to microorganisms, its lysis of human red blood corpuscles and beetroot tissue, and its unexpectedly selective inhibition of radicle elongation for Trigonella foenum-graecum were strongly light-dependent. Dothistromin was also toxic to Artemia salina but without requiring light activation. It was not active as a wilt or necrosis toxin, possible because of its ready adsorption onto external plant tissue.     

Integrating gene expression and metabolic profiles

Recent advances in high throughput technologies have generated an abundance of biological information, such as gene expression, protein-protein interaction, and metabolic data. These various types of data capture different aspects of the cellular response to environmental factors. Integrating data from different measurements enhances the ability of modeling frameworks to predict cellular function more accurately and can lead to a more coherent reconstruction of the underlying regulatory network structure. Different techniques, newly developed and borrowed, have been applied for the purpose of extracting this information from experimental data. In this study, we developed a framework to integrate metabolic and gene expression profiles for a hepatocellular system. Specifically, we applied genetic algorithm and partial least square analysis to identify important genes relevant to a specific cellular function. We identified genes 1) whose expression levels quantitatively predict a metabolic function and 2) that play a part in regulating a hepatocellular function and reconstructed their role in the metabolic network. The framework 1) preprocesses the gene expression data using statistical techniques, 2) selects genes using a genetic algorithm and couples them to a partial least squares analysis to predict cellular function, and 3) reconstructs, with the assistance of a literature search, the pathways that regulate cellular function, namely intracellular triglyceride and urea synthesis. This provides a framework for identifying cellular pathways that are active as a function of the environment and in turn helps to uncover the interplay between gene and metabolic networks.     

The fungal metabolite culmorin and related compounds

This paper reviews the toxicology of culmorins, a family of compounds found in grains contaminated by Fusarium graminearum and related fungi. We include the results of an Ames test and studies based on Quantitative Structure-Activity Relationships. Culmorin has low toxicity in several in vitro assays and in one study in swine and is Ames test negative. Culmorin is moderately antifungal. QSAR analysis suggested that the plant compound longifolene was similar. Longifolene is a GRAS compound used in cosmetics and is also moderately antifungal.     

A rapid technique for screening of lovastatin-producing strains of Aspergillus terreus by agar plug and Neurospora crassa bioassay

The success of strain improvement programme depends on the number of isolates that can be screened after mutagenic treatment. A technique to rapidly screen large number of high-yielding isolates was developed. The 'agar plug' method that utilizes the anti-fungal property of lovastatin to produce a zone of inhibition against Neurospora crassa was not only economical but also less labour-intensive. We were able to isolate a high-yielding strain, the productivity of which increased by 138% as compared to the parent strain in the submerged fermentation process.     

Integrating ecosystem engineering and food webs

Ecosystem engineering, the physical modification of the environment by organisms, is a common and often influential process whose significance to food web structure and dynamics is largely unknown. In the light of recent calls to expand food web studies to include non‐trophic interactions, we explore how we might best integrate ecosystem engineering and food webs. We provide rationales justifying their integration and present a provisional framework identifying how ecosystem engineering can affect the nodes and links of food webs and overall organization; how trophic interactions with the engineer can affect the engineering; and how feedbacks between engineering and trophic interactions can affect food web structure and dynamics. We use a simple integrative food chain model to illustrate how feedbacks between the engineer and the food web can alter 1) engineering effects on food web dynamics, and 2) food web responses to extrinsic environmental perturbations. We identify four general challenges to integration that we argue can readily be met, and call for studies that can achieve this integration and help pave the way to a more general understanding of interaction webs in nature. Synthesis All species are affected by their physical environment. Because ecosystem engineering species modify the physical environment and belong to food webs, such species are potentially one of the most important bridges between the trophic and non‐trophic. We examine how to integrate the so far, largely independent research areas of ecosystem engineering and food webs. We present a conceptual framework for understanding how engineering can affect food webs and vice versa, and how feedbacks between the two alter ecosystem dynamics. With appropriate empirical studies and models, integration is achievable, paving the way to a more general understanding of interaction webs in nature.     

Objective criteria to assess representativity of soil fungal community profiles

Soil fungal community structures are often highly heterogeneous even among samples taken from small field plots. Sample pooling is widely used in order to overcome this heterogeneity, however, no objective criteria have yet been defined on how to determine the number of samples to be pooled for representatively profiling a field plot. In the present study PCR/RFLP and T-RFLP analysis of fungal 18S rDNA in ten soil samples obtained from a grassland plot of 400 m(2) also revealed this known heterogeneity in fungal community structures. Based on these data a three-step approach to assess representativity of fungal community profiles was established. First, soil DNA quantities needed for robust community profiling were determined. Second, profiles of single or multiple samples were theoretically averaged to test for statistically significant clustering in order to determine the minimal number of samples to be pooled to achieve representativity. Third, DNA extracts of single or multiple samples were pooled prior to profiling in order to test for statistically significant clustering. Analyses revealed robust profiles for 50 ng soil DNA but not for 5 ng. Averaged T-RFLP profiles from five or more soil samples and experimental T-RFLP profiles from pools of seven or more samples formed one significant branch. Theoretical averaging and experimental pooling revealed that five to seven samples have to be pooled for robustly representing the field plot. Our results demonstrate that representativity of soil fungal community profiles can objectively be determined for a field plot with only little deviation between theoretical and experimental approaches. This three-step approach will be of assistance for designing sampling and pooling strategies for comparative analyses of soil fungal communities in ecological studies.