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Background: Bacillus subtilis is widely used for industrial enzyme production due to its capacity to efficiently secrete proteins. However, secretion efficiency of enzymes varies widely, and optimizing secretion is crucial to make production commercially viable. Previously, we have shown that overexpression of the xylanase XynA lowers expression of Clp protein chaperones, and that inactivation of CtsR, which regulates and represses clp transcription, increases the production of XynA. In the current study, we examined whether the same is the case for overexpression of the α-amylase AmyM from Geobacillus stearothermophilus by B. subtilis, and why XynA shows a different timing of secretion compared to AmyM. Results: Transcriptome analyses revealed that B. subtilis cells overexpressing AmyM exhibited a distinct profile compared to XynA overexpressing cells, however there were also similarities and in both cases expression of CtsR controlled genes was downregulated. In contrast to XynA, inactivation of CtsR did not improve AmyM production. Upregulation of other protein chaperones, including GroEL/ES and DnaJ/K, by inactivating their transcriptional repressor HrcA, had almost no effect on XynA yields and in fact considerably lowered that of AmyM. Despite using the same promoter, the production of XynA peaks well before AmyM reaches its optimal secretion rate. Transcriptome and ribosome profiling indicated that this is neither related to transcription nor to translation regulation. We show that the reduced secretion in the stationary phase is partially due to the activity of secreted proteases, but also due to the activity of the intracellular protease LonA. The absence of this protein resulted in a 140% and 20% increased production for XynA and AmyM, respectively. Conclusion: The combination of transcriptome and ribosome profiling offered important information to determine at which cellular level production bottlenecks occurred. This helped us to identify LonA protease as an important factor influencing enzyme production yields in B. subtilis. [ABSTRACT FROM AUTHOR]

Abstract Background The bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq. Results Using the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%. Conclusions Overproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms.

Background: The bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq. Results: Using the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%. Conclusions: Overproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms. [ABSTRACT FROM AUTHOR]

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI, dnaG, and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytvI, dnaG, and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI , dnaG , and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytv I, dnaG , and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium. [ABSTRACT FROM AUTHOR]

In the last few years, the need for Continuous Speech Recognition system in Tamil language has been increased widely. In this research work, efficient Continuous Tamil Speech Recognition (CTSR) technique is proposed under non stationary noisy environments. This research work consists of two stages such as speech enhancement and modelling phase. In this, the modified Modulation Magnitude Estimation based Spectral Subtraction with Chi-Square Distribution based Noise Estimation (SS-NE) algorithm is proposed to enhance the noisy Tamil speech signal under various non-stationary noise environments. In order to extract the speech segments from the continuous speech, further the enhanced speech signal is segmented through the combination of short-time signal energy and spectral centroid features of the signal. In this work, 26 mel frequency cepstral coefficients per frame are found as optimal values and they are considered as acoustic feature vectors for each frame. In this research work, the Fuzzy C-Means (FCM) clustering is used in order to cluster the extracted feature vectors into discrete symbols. From the evaluation results, it is found that the optimal number of clusters 'C' as 5. Finally, Tamil speech from various speakers is recognized using Expectation Maximization Gaussian Mixture Model (EM-GMM) with 16 component densities under continuous measurements of labelled features from FCM clustering techniques in order to reduce the word error rate. From the simulated results, it is observed that the proposed FCM with EM-GMM model for CTSR improves the recognition accuracy from 1.2 to 4.4% when compared to the existing algorithms under different noisy environments by reducing the WER from 1.6 to 5.47%. [ABSTRACT FROM AUTHOR]

Oenococcus oeni is a lactic acid bacterium responsible for malolactic fermentation of wine. While many stress response mechanisms implemented by O. oeni during wine adaptation have been described, little is known about their regulation. CtsR is the only regulator of stress response genes identified to date in O. oeni. Extensively characterized in Bacillus subtilis, the CtsR repressor is active as a dimer at 37°C and degraded at higher temperatures by a proteolytic mechanism involving two adapter proteins, McsA and McsB, together with the ClpCP complex. The O. oeni genome does not encode orthologs of these adapter proteins and the regulation of CtsR activity remains unknown. In this study, we investigate CtsR function in O. oeni by using antisense RNA silencing in vivo to modulate ctsR gene expression. Inhibition of ctsR gene expression by asRNA leads to a significant loss in cultivability after heat shock (58%) and acid shock (59%) highlighting the key role of CtsR in the O. oeni stress response. Regulation of CtsR activity was studied using a heterologous expression system to demonstrate that O. oeni CtsR controls expression and stress induction of the O. oeni hsp18 gene when produced in a ctsR-deficient B. subtilis strain. Under heat stress conditions, O. oeni CtsR acts as a temperature sensor and is inactivated at growth temperatures above 33°C. Finally, using an E. coli bacterial two-hybrid system, we showed that CtsR and ClpL1 interact, suggesting a key role for ClpL1 in controlling CtsR activity in O. oeni.

The understanding of the heat shock response (HSR) in lactobacilli from a regulatory point of view is still limited, though an increased knowledge on the regulation of this central stress response can lead to improvements in the exploitation of these health promoting microorganisms. Therefore the aim of this in silico study, that is the first to be carried out for members of the Lactobacillus genus, was predicting how HSR influences cell functions in the food associated and probiotic species Lactobacillus casei and Lactobacillus rhamnosus . To this purpose, thirteen whole genomes of these bacteria were analyzed to identify which genes involved in HSR are present. It was found that all the genomes share 25 HSR related genes, including those encoding protein repair systems, HSR repressors, HrcA and CtsR, and the positive regulators of HSR, alternative σ factors σ 32 and σ 24 . Two genes encoding a σ 70 /σ 24 factor and a Lon protease, respectively, were found only in some genomes. The localization of the HSR regulators binding sites in genomes was analyzed in order to identify regulatory relationships driving HSR in these lactobacilli. It was observed that the binding site for the HrcA repressor is found upstream of the hrc A- grp E- dna K- dna J and gro ES- gro EL gene clusters, of two hsp genes, clp E, clp L and clp P, while the CtsR repressor binding site precedes the cts R- clp C operon, clp B, clp E and clp P. Therefore the ClpE-ClpP protease complex is dually regulated by HrcA and CtsR. Consensus sequences for the promoters recognized by the HSR alternative σ factors were defined for L. casei and L. rhamnosus and were used in whole genome searches to identify the genes that are possibly regulated by these transcription factors and whose expression level is expected to increases in HSR. The results were validated by applying the same procedure of promoter consensus generation and whole genome search to an additional 11 species representative of the main Lactobacillus lineages. The composition of the resulting regulons highlighted the existence of relationships between HSR and relevant cell functions, including nutrient utilization, DNA repair, protein synthesis and export of toxic substances. In fact, some of the predicted members of the σ 32 regulon are central regulators ccp A, spx A, cad A, and functional proteins brn Q, ldh , cho S, pox L and nag B involved in the tolerance to different stress factors. The analysis of the expression level of these molecular markers of cell protective mechanisms can be used to select the heat shock exposure times and temperatures that maximize the tolerance of L. casei and L. rhamnosus to technological and environmental stress factors. [ABSTRACT FROM AUTHOR]

BACKGROUND Since extrusion has many desirable characteristics such as high shear, rapid mixing, short residence time, controllable barrel temperature, and adaptability to process modification, the technology has been applied as a viable continuous pretreatment method for empty fruit bunches ( EFB). RESULTS Optimized conditions for a continuous twin-screw-driven reactor ( CTSR) were determined to be 170°C barrel temperature, 1.6% (w/v) sodium hydroxide, 1:8 solid to liquid ratio, and 5 rpm screw rotational speed. Under these operating conditions, 56.55% of lignin and 39.18% of hemicellulose fractions were solubilized from raw EFB, which increases the glucan content in pretreated EFB from 39.25% to 53.60%. After subsequent 48-h enzymatic hydrolysis of pretreated EFB, the highest digestibility of 92.4% was achieved with an enzyme loading of 30 FPU g−1-glucan for cellulase and 70 pNPG g−1-glucan for β-glucosidase. CONCLUSION CTSR processing has great potential for increasing the efficiency of EFB pretreatment compared with batch pretreatments. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Background To cope with environmental challenges bacteria possess sophisticated defense mechanisms that involve stress-induced adaptive responses. The canonical stress regulators CtsR and HrcA play a central role in the adaptations to a plethora of stresses in a variety of organisms. Here, we determined the CtsR and HrcA regulons of the lactic acid bacterium Lactobacillus plantarum WCFS1 grown under reference (28°C) and elevated (40°C) temperatures, using ctsR, hrcA, and ctsR-hrcA deletion mutants. Results While the maximum specific growth rates of the mutants and the parental strain were similar at both temperatures (0.33 ± 0.02 h-1 and 0.34 ± 0.03 h-1, respectively), DNA microarray analyses revealed that the CtsR or HrcA deficient strains displayed altered transcription patterns of genes encoding functions involved in transport and binding of sugars and other compounds, primary metabolism, transcription regulation, capsular polysaccharide biosynthesis, as well as fatty acid metabolism. These transcriptional signatures enabled the refinement of the gene repertoire that is directly or indirectly controlled by CtsR and HrcA of L. plantarum. Deletion of both regulators, elicited transcriptional changes of a large variety of additional genes in a temperature-dependent manner, including genes encoding functions involved in cell-envelope remodeling. Moreover, phenotypic assays revealed that both transcription regulators contribute to regulation of resistance to hydrogen peroxide stress. The integration of these results allowed the reconstruction of CtsR and HrcA regulatory networks in L. plantarum, highlighting the significant intertwinement of class I and III stress regulons. Conclusions Taken together, our results enabled the refinement of the CtsR and HrcA regulatory networks in L. plantarum, illustrating the complex nature of adaptive stress responses in this bacterium. [ABSTRACT FROM AUTHOR]

Background Rare diseases pose many research challenges specific to their scarcity. Advances in potential therapies have made it more important than ever to be able to adequately identify not only patients with particular genotypes (via patient registries) but also the medical professionals who provide care for them at particular specialist centres of expertise and who may be competent to participate in trials. Work within the neuromuscular field provides an example of how this may be achieved. Methods This paper describes the development of the TREAT-NMD Care and Trial Site Registry (CTSR), an initiative of an EU-funded Network of Excellence, and its utility in providing an infrastructure for clinical trial feasibility, recruitment, and other studies. Results 285 CTSR-registered centres, reporting 35,495 neuromuscular patients, are described alongside an analysis of their provision for DMD. Site characteristics vary by country: the average number of DMD patients seen per site in the United States (96) is more than in Germany (25), and paediatric/adult breakdown is also markedly distinct. Over 70% of sites have previous trial experience, with a majority including a Clinical Trials Unit. Most sites also have MLPA diagnostic capability and access to a range of medical specialists. However, in the three countries reporting most sites (US, the UK and Germany), few had access to all core DMD specialists internally. Over 60% of sites did not report any form of transition arrangement. Conclusions Registries of care and trial sites have significant utility for research into rare conditions such as neuromuscular diseases, demonstrated by the significant engagement by industry and other researchers with the CTSR. We suggest that this approach may be applicable to other fields needing to identify centres of expertise with the potential to carry out clinical research and engage in clinical trials. Such registries also lend themselves to the developing context of European Reference Networks (ERNs), which seek to build networks of centres of expertise which fit specific criteria, and which may themselves aid the sustainability of such registries. This is particularly the case given the utility of registries such as the CTSR in enabling networks of best-practice care centres. [ABSTRACT FROM AUTHOR]

Abstract: High pressure treatment is a novel food processing approach for reducing pathogens in foods and food ingredients. However, relatively little is known about the pathogenic potential of organisms that survive the treatment. Twelve previously isolated and characterized variants of Listeria monocytogenes LO28 obtained after a high pressure treatment were assessed for their virulence potential and antibiotic susceptibility. Ten variants showed attenuated virulence while two variants retained full virulence in a mouse model of infection. Seven of the attenuated variants demonstrated a reduction in virulence factor activity. Compared to the wild type, all variants exhibited similar or increased susceptibility to multiple antibiotics commonly used in listeriosis treatment. [Copyright &y& Elsevier]

Prior research suggests that students' understanding of scientific concepts is pre-determined by their reasoning ability. Other efforts suggest that American students' scientific literacy is in decline. One difficulty Bybee (2009) acknowledges is that there are two divergent philosophical models of scientific literacy. The first describes the content knowledge and conceptual understanding that is desirable for future scientists. The second is an application of science to "real life" that is critical for every American citizen. In either case, we propose that the essential subordinate skill required for scientific literacy is scientific thinking ability as defined by Piaget (1964) and Lawson (2002). This article outlines the relation between students' scientific thinking ability and both their conceptual understanding and literacy. It also provides national norms for students in grades 10-12 using a measure of scientific thinking skills, the Classroom Test of Scientific Reasoning (CTSR). These norms can be used by educators when making curricular and policy decisions regarding student achievement. [ABSTRACT FROM AUTHOR]

Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria. [ABSTRACT FROM AUTHOR]

Abstract: Streptococcus thermophilus is an important dairy starter used for the manufacture of fermented milks and cheeses. As for many other lactic acid bacteria, its survival during propagation and preservation of starter cultures and its performance in dairy fermentations are significantly affected by its ability to cope with many environmental stresses. This, in turn, is dependent on the expression of a several genes, which is often controlled by negative response regulators, and has complex relationships with the control of metabolism. In this paper we describe the effect of growth phase, adaptation and inactivation of genes for stress response regulators (hrcA, ctsR and rr01) on the growth, acid production and stress resistance and intracellular protein patterns in the yoghurt strain S. thermophilus Sfi39. Inactivation of stress response regulators significantly affected growth, acid production in milk and stress tolerance. Although mutants showed an increased resistance during the exponential phase compared to the wild type strain, the effect of the mutations was complex and in some cases mutants were unable to mount an acid- or heat-shock response after exposure to mild heat and acid stresses. Significant differences in intracellular protein patterns analyzed by SDS-PAGE and 2-DE/mass spectrometry were found as a function of growth phase, adaptation and mutation, but a detailed transcriptomic and proteomic study would be needed to provide a mechanistic interpretation of the effect of the mutations and to identify a strategy for the construction of food-grade mutants with improved growth and performances. [Copyright &y& Elsevier]

Lactic acid bacteria (LAB) constitute a heterogeneous group of bacteria that are traditionally used to produce fermented foods. The industrialization of food transformations has increased the economical importance of LAB, as they play a crucial role in the development of the organoleptic and hygienic quality of fermented products. However, the strains selected for industrial purposes, should tolerate adverse conditions encountered in industrial processes, either during starter handling and storage (freeze-drying, freezing, or spray-drying) or during food processing in which abiotic stresses such as heat, cold, acidity, and high concentration of NaCl or ethanol are common. Wine LAB have to deal with several stresses including an acidic pH, a high alcoholic content, non optimal growth temperatures, and growth-inhibitory compounds such as fatty acids and tannins, originated from yeast and bacteria metabolism. Wine LAB have developed several mechanisms to escape or to tolerate wine conditions. They carry out a malolactic fermentation in this stressful environment. In addition to the regulation of the expression of specific genes, bacteria have evolved adaptive networks to face the challenges of a changing environment and to survive under conditions of stress. The so called Global Regulatory Systems control the simultaneous expression of a large number of genes in response to a variety of environmental stress factors. CIRCE sequences able to bind the HrcA repressor, σ B dependent promoters and CtsR regulatory elements have been observed in several genes identified from wine LAB. Improved knowledge of regulators and a better understanding of LAB stress responses could constitute a basis of comparison with the well known model microorganisms, Escherichia coli and Bacillus subtilis . Moreover, it can provide an important insight into improving current industrial starter strains. [ABSTRACT FROM AUTHOR]

All organisms respond to a sudden increase in temperature by inducing the synthesis of a set of proteins called heat shock proteins (HSPs). Although the induction of HSPs is a universal response, a diversity of mechanisms control HSP synthesis in different organisms. In Streptomyces, the synthesis of major HSPs, such as the widespread molecular chaperones DnaK, ClpB, GroEL and HSP18, is negatively controlled at the transcriptional level by at least three different repressors. The control of groE gene expression involves an inverted repeat (called the CIRCE element) that is highly conserved among eubacteria, and the HrcA repressor. The dnaK operon and clpB belong to the HspR /HAIR regulon. The HspR repressor-HAIR operator system is used in some bacteria but is not widespread. In particular, it has not been found in gram-positive bacteria with low G+C content. Transcription of hsp18, which encodes a small HSP, is regulated by the RheA repressor. This repressor, which has intrinsic thermosensor activity, has to date been identified only in Streptomyces. [ABSTRACT FROM AUTHOR]