Molecular adaptation mechanisms employed by ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds

Current international interest in finding alternative sources of energy to the diminishing supplies of fossil fuels has encouraged research efforts in improving biofuel production technologies. In countries which lack sufficient food, the use of sustainable lignocellulosic feedstocks, for the production of bioethanol, is an attractive option. In the pre-treatment of lignocellulosic feedstocks for ethanol production, various chemicals and/or enzymatic processes are employed. These methods generally result in a range of fermentable sugars, which are subjected to microbial fermentation and distillation to produce bioethanol. However, these methods also produce compounds that are inhibitory to the microbial fermentation process. These compounds include products of sugar dehydration and lignin depolymerisation, such as organic acids, derivatised furaldehydes and phenolic acids. These compounds are known to have a severe negative impact on the ethanologenic microorganisms involved in the fermentation process by compromising the integrity of their cell membranes, inhibiting essential enzymes and negatively interact with their DNA/RNA. It is therefore important to understand the molecular mechanisms of these inhibitions, and the mechanisms by which these microorganisms show increased adaptation to such inhibitors. Presented here is a concise overview of the molecular adaptation mechanisms of ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds. These include general stress response and tolerance mechanisms, which are typically those that maintain intracellular pH homeostasis and cell membrane integrity, activation/regulation of global stress responses and inhibitor substrate-specific degradation pathways. We anticipate that understanding these adaptation responses will be essential in the design of ‘intelligent’ metabolic engineering strategies for the generation of hyper-tolerant fermentation bacteria strains.IS

Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings

Sorghum bicolor, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. In this study, seeds of a sweet sorghumvariety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination with MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p<0.05) to salt stress treatments. Of the 118 spots, 79 were selected for tandem mass spectrometric identification, owing to their good resolution and abundance levels, and of these, 55 were positively identified. Identified proteins were divided into six functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of our proteins of interest are currently under investigation via bioinformatic and molecular biology approaches.Web of Scienc

An integrated and comparative approach towards identification, characterization and functional annotation of candidate genes for drought tolerance in sorghum (Sorghum bicolor (L.) Moench)

BACKGROUND: Drought is the most disastrous abiotic stress that severely affects agricultural productivity worldwide. Understanding the biological basis of drought-regulated traits, requires identification and an in-depth characterization of genetic determinants using model organisms and high-throughput technologies. However, studies on drought tolerance have generally been limited to traditional candidate gene approach that targets only a single gene in a pathway that is related to a trait. In this study, we used sorghum, one of the model crops that is well adapted to arid regions, to mine genes and define determinants for drought tolerance using drought expression libraries and RNA-seq data. RESULTS: We provide an integrated and comparative in silico candidate gene identification, characterization and annotation approach, with an emphasis on genes playing a prominent role in conferring drought tolerance in sorghum. A total of 470 non-redundant functionally annotated drought responsive genes (DRGs) were identified using experimental data from drought responses by employing pairwise sequence similarity searches, pathway and interprodomain analysis, expression profiling and orthology relation. Comparison of the genomic locations between these genes and sorghum quantitative trait loci (QTLs) showed that 40% of these genes were co-localized with QTLs known for drought tolerance. The genome reannotation conducted using the Program to Assemble Spliced Alignment (PASA), resulted in 9.6% of existing single gene models being updated. In addition, 210 putative novel genes were identified using AUGUSTUS and PASA based analysis on expression dataset. Among these, 50% were single exonic, 69.5% represented drought responsive and 5.7% were complete gene structure models. Analysis of biochemical metabolism revealed 14 metabolic pathways that are related to drought tolerance and also had a strong biological network, among categories of genes involved. Identification of these pathways, signifies the interplay of biochemical reactions that make up the metabolic network, constituting fundamental interface for sorghum defence mechanism against drought stress. CONCLUSIONS: This study suggests untapped natural variability in sorghum that could be used for developing drought tolerance. The data presented here, may be regarded as an initial reference point in functional and comparative genomics in the Gramineae family

INPPO actions and recognition as a driving force for progress in plant proteomics: Change of guard, INPPO update, and upcoming activities

The International Plant Proteomics Organization (INPPO) is a non-profit organization whose members are scientists involved or interested in plant proteomics. Since the publication of the first INPPO highlights in 2012, continued progress on many of the organization’s mandates/goals has been achieved. Two major events are emphasized in this second INPPO highlights. First, the change of guard at the top, passing of the baton from Dominique Job, INPPO founding President to Ganesh Kumar Agrawal as the incoming President. Ganesh K. Agrawal, along with Dominique Job and Randeep Rakwal initiated the INPPO. Second, the most recent INPPO achievements and future targets, mainly the organization of first the INPPO World Congress in 2014, tentatively planned for Hamburg (Germany), are mentioned.Web of Scienc

Synthesis of Al doped ZnO nanoparticles by aqueous coprecipitation

International audienceAl-doped ZnO particles were obtained by a simple route: soda addition in aqueous cationic solution. The effects of temperature, hydrolysis duration, reagent concentration and time were investigated. A non-topotactic reaction mechanism, involving firstly the precipitation of various hydroxide compounds depending on the route (low or high pH), followed by the dissolution-recrystallization of the hydroxide species into ZnO was demonstrated. The Al concentration in the final ZnO nanopowders did not exceed 0.3 at.% which correspond to the solubility limit of Al in ZnO. The different experimental conditions allow the morphology of ZnO particles to be controlled from isotropic nanoparticles of several tens of nanometers, platelets of several hundreds of nanometers or agglomerates of needle like particles

A comparison of faecal microbial populations of South African Windsnyer-type indigenous pigs (SAWIPs) and Large White × Landrace (LW × LR) crosses fed diets containing ensiled maize cobs

Faecal microbial communities in South African Windsnyer-type indigenous pigs (SAWIPs) and Large White × Landrace (LW × LR) crosses were investigated using high-throughput sequencing of the 16S rDNA genes. The faecal microbial communities in LW × LR crosses and SAWIPs fed control (CON) and high maize cob (HMC) diets were evaluated through parallel sequencing of 16S rDNA genes. Butrivibrio, Faecalibacterium and Desulfovibrio, although present in LW × LR pigs, were absent from the SAWIP microbial community. Bacteroides, Succiniclasticum, Peptococcus and Akkermansia were found in SAWIPs but not in LW × LR crosses. The ratios of Bacteroidia to Clostridia on the CON and HMC diets were similar (0.37 versus 0.39) in SAWIPs but different (0.24 versus 0.1) in LW × LR crosses. The faecal microbial profiles determined were different between the LW × LR and SAWIP breeds but not between pigs fed the CON and HMC diets. The composition of faecal bacterial communities in SAWIPs was determined for the first time. The differences in microbial communities detected may explain the enhanced ability of SAWIPs to digest fibrous diets compared with the LW × LR crosse

Identification and characterisation of the Arabidopsis thaliana cell wall proteome: unravelling novel cell wall proteins and new potential functions of the plant extracellular matrix

The application of the proteomic approach has facilitated efforts directed toward the mapping of the entire Arabidopsis cell wall proteome. Proteins were sequentially extracted from purified cell walls using 0.2 M CaC1(_2) followed by a urea buffer. The extracts were resolved via large format two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were visualised via Coomassie brilliant blue staining. The aim was to identify and characterise as many cell wall proteins as possible, with the hope of identifying novel cell wall proteins. Out of 325 spots visualised on the 2-D polyacrylamide gel, 144 gave a positive protein identification representing 104 different proteins. The identified proteins were divided into 3 categories. The first category included proteins that have been previously identified as plant cell wall proteins. The second category was designated to include novel cell wall proteins (hypothetical proteins) and the third category was made up of proteins, which had recognised functions, but had never hitherto been known to be secreted to the extracellular matrix. Among the identified novel cell wall proteins there were several that shared high homology with protein kinases. These proteins possessed all the characteristics of secreted polypeptides, such as the cleavable N-terminal signal peptide, and were found to lack both the transmembrane domain and the endoplasmic reticulum retention tetrapeptides (HDEL and KDEL). These observations suggested that, as in animal cells, plant cells had extracellular protein kinase activity (phosphorylation). This was supported by the recent discovery that plant cells secrete ATP to the extracellular matrix (Thomas et al., 2000). Verification of the occurrence of extracellular protein kinase activity was further strengthened by the identification of phosphorylated bona fide cell wall proteins and stress responses caused by the depletion extracellular ATP

Mapping and characterisation of the sorghum cell suspension culture secretome

Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step towards understanding their functions during plant growth and development. Proteins secreted into the culture medium of 10-day old sorghum cell suspension cultures termed culture filtrate (CF) proteins were separated by two dimensional gel electrophoresis (2DE) and visualised using Coomassie brilliant blue (CBB) R-250 staining. Of the 25 visualised CBB stainable spots, 15 abundant, well resolved and reproducible spots amongst the three biological replicates used were selected for identification using matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI-TOF-TOF MS). Of these spots, 14 were positively identified, representing four different protein classes: Peroxidases, germin proteins, oxalate oxidases and α-galactosidases. All the identified proteins are known secretory proteins, with predicted signal peptides, which target proteins for the secretory pathway. The identified proteins have known functions in signalling processes, defence mechanisms and cell wall metabolism which is consistent with their location outside the cell. Western blotting analysis of the CF protein extracts using an antibody against β-tubulin, a cytoplasmic protein, indicated that our CF protein preparations are free from any detectable amounts of this marker protein. Therefore, our sorghum cell culture system is ideal for use in the proteomic analysis of secreted proteins. The findings of this study are a step in the process of bridging the gap that currently exists in sorghum proteomics and also provides a foundation for future studies on understanding the roles played by secreted proteins during plant growth and development of the same crop.Keywords: Sorghum, cell suspension cultures, culture filtrate, secreted proteins, proteomics analysis, two-dimensional gel electrophoresis, β-tubuli

Characterisation of Thiol-releasing and Lower Volatile Acidityforming Intra-genus Hybrid Yeast Strains for Sauvignon blanc Wine

A single Saccharomyces cerevisiae wine yeast strain produces a range of aroma and flavour metabolites (e.g. volatile thiols), as well as unfavourable metabolites (e.g. volatile acidity [VA]) during the alcoholic fermentation of white wine, especially Sauvignon blanc. The former contributes to the organoleptic quality of the final wine. Previous research showed that yeast derived enzymes (proteins) are involved in the release of wine quality enhancing or reducing metabolites during fermentation. Small-scale winemaking trials were initiated to evaluate protein expression and metabolite release of tropical fruit aroma wine producing S. cerevisiae hybrid yeasts. Commercial ‘thiol-releasing’ wine yeasts (TRWY) were included in winemaking trials as references. Improved hybrids were identified which showed enhanced thiol-releasing, specifically 3-mercaptohexanol (3MH), and lower VA formation during the production of Sauvignon blanc wines compared to some commercial TRWY references. It is noteworthy that the hybrid NH 56 produced wines with the second highest 3MH levels after hybrid NH 84, and lowest acetic acid of all strains included in this study. This yeast was also the only strain to have down-regulated proteins linked to amino acid biosynthesis, pentose phosphate pathway, glycolysis and fructose and galactose metabolism during the lag phase. Furthermore, differences in protein expression were reflected in the variation of metabolite release by different strains, thereby confirming that enzymes (proteins) are the final effectors for metabolite release.