Investigation of TGF-α-overexpressing mouse hepatocytes (TAMH) cultured as spheroids for use in hepatotoxicity screening of drug candidates.

The immortalized mouse liver cell line TAMH has been described as a valuable tool for studying hepatotoxic mechanisms, but until now, it has only been reported to grow as a monolayer in culture. However, culturing hepatocytes as three-dimensional (3D) spheroids has been shown to result in improved liver-specific functions (e.g., metabolic capacity) by better mimicking the in vivo environment. This approach may lead to more reliable detection of drug-induced liver injury (DILI) in the early phase of drug discovery, preventing post-marketing drug withdrawals. Here, we investigated the cultivation of TAMH as 3D spheroids, characterizing them with optical and transmission electron microscopy as well as analyzing their gene expression at mRNA level (especially drug-metabolizing enzymes) compared to TAMH monolayer. In addition, comparisons were made with spheroids grown from the human hepatoblastoma cell line HepG2, another current spheroid model. The results indicate that TAMH spheroids express hepatic structures and show elevated levels of some of the key phase I and II drug-metabolizing enzymes, in contrast to TAMH monolayer. The in vitro hepatotoxic potencies of the drugs acetaminophen and flupirtine maleate were found to be very similar between TAMH spheroidal and the monolayer cultures. Both the advantages and disadvantages of TAMH spheroids as an in vitro hepatotoxicity model compared to monolayer model are discussed.

An auto-inducible phosphate-controlled expression system of Bacillus licheniformis.

BACKGROUND: A promoter that drives high-level, long-term expression of the target gene under substrate limited growth conditions in the absence of an artificial inducer would facilitate the efficient production of heterologous proteins at low cost. A novel phosphate-regulated expression system was constructed using the promoter of the phytase encoding gene phyL from Bacillus licheniformis for the overexpression of proteins in this industrially relevant host. RESULTS: It is shown that the phyL promoter enables a strong overexpression of the heterologous genes amyE and xynA in B. licheniformis when cells were subjected to phosphate limitation. Whether B. licheniformis can use phytate as an alternative phosphate source and how this substrate influences the PphyL controlled gene expression under growth conditions with limited inorganic phosphate concentrations were also investigated. It is shown that B. licheniformis cells are able to use sodium phytate as alternative phosphate source. The addition of small amounts of sodium phytate (≤ 5 mM) to the growth medium resulted in a strong induction and overexpression of both model genes in B. licheniformis cells under phosphate limited growth conditions. CONCLUSIONS: The PphyL controlled expression of the investigated heterologous genes in B. licheniformis is strongly auto-induced under phosphate limited conditions. The proposed PphyL expression system enables an overexpression of target genes in B. licheniformis under growth conditions, which can be easily performed in a fed-batch fermentation process.

A Phosphate Starvation-Inducible Ribonuclease of Bacillus licheniformis.

The BLi03719 protein of Bacillus licheniformis DSM13 belongs to the most abundant extracellular proteins under phosphate starvation conditions. In this study, the function of this phosphate starvation inducible protein was determined. An amino-acid sequence analysis of the BLi03719-encoding gene showed a high similarity with genes encoding the barnase of Bacillus amyloliquefaciens FZB42 and binase-like RNase of Bacillus pumilus SARF-032. The comparison of the control strain and a BLi03719-deficient strain revealed a strongly reduced extracellular ribonuclease activity of the mutant. Furthermore, this knockout mutant exhibited delayed growth with yeast RNA as an alternative phosphate and carbon source. These results suggest that BLi03719 is an extracellular ribonuclease expressed in B. licheniformis under phosphate starvation conditions. Finally, a BLi03719 mutant showed an advantageous effect on the overexpression of the heterologous amyE gene under phosphate-limited growth conditions.

A proteomic view of cell physiology of the industrial workhorse Bacillus licheniformis.

Bacillus licheniformis is known for its high protein secretion capacity and is being applied extensively as a host for the industrial production of enzymes such as proteases and amylases. In its natural environment as well as in fermentation processes the bacterium is often facing adverse conditions such as oxidative or osmotic stress or starvation for nutrients. During the last years detailed proteome and transcriptome analyses have been performed to study the adaptation of B. licheniformis cells to various stresses (heat, ethanol, oxidative or salt stress) and starvation conditions (glucose, nitrogen or phosphate starvation). A common feature of the response to all tested conditions is the downregulation of many genes encoding house-keeping proteins and, consequently, a reduced synthesis of the corresponding proteins. Induction of the general stress response (σ(B) regulon) is only observed in cells subjected to heat, ethanol or salt stress. This paper summarizes our current knowledge on general and specific stress and starvation responses of this important industrial bacterium. The importance of selected marker genes and proteins for the monitoring and optimization of B. licheniformis based fermentation processes is discussed.

Bacillus pumilus reveals a remarkably high resistance to hydrogen peroxide provoked oxidative stress.

Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.

Stress responses of the industrial workhorse Bacillus licheniformis to osmotic challenges.

The Gram-positive endospore-forming bacterium Bacillus licheniformis can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, B. licheniformis cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of B. licheniformis. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in B. licheniformis. The data set focusing on the transcriptional profile of B. licheniformis was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of B. licheniformis to both sudden and sustained osmotic stress.

The response of Bacillus licheniformis to heat and ethanol stress and the role of the SigB regulon.

The heat and ethanol stress response of Bacillus licheniformis DSM13 was analyzed at the transcriptional and/or translational level. During heat shock, regulons known to be heat-induced in Bacillus subtilis 168 are upregulated in B. licheniformis, such as the HrcA, SigB, CtsR, and CssRS regulon. Upregulation of the SigY regulon and of genes controlled by other extracytoplasmic function (ECF) sigma factors indicates a cell-wall stress triggered by the heat shock. Furthermore, tryptophan synthesis enzymes were upregulated in heat stressed cells as well as regulons involved in usage of alternative carbon and nitrogen sources. Ethanol stress led to an induction of the SigB, HrcA, and CtsR regulons. As indicated by the upregulation of a SigM-dependent protein, ethanol also triggered a cell wall stress. To characterize the SigB regulon of B. licheniformis, we analyzed the heat stress response of a sigB mutant. It is shown that the B. licheniformis SigB regulon comprises additional genes, some of which do not exist in B. subtilis, such as BLi03885, encoding a hypothetical protein, the Na/solute symporter gene BLi02212, the arginase homolog-encoding gene BLi00198 and mcrA, encoding a protein with endonuclease activity.

Sensitive detection of idiotypic platelet-reactive alloantibodies by an electrical protein chip.

To prevent and treat immune-mediated platelet disorders (e.g. neonatal allo-immune thrombocytopenia and platelet transfusion refractoriness) the causative idiotypic platelet-reactive antibodies have to be detected with high sensitivity and specificity. The "Monoclonal Antibody Immobilization Platelet Assay" (MAIPA) is the diagnostic gold standard for immunotyping sera with respect to alloantibodies against human platelet antigens (HPA). However, it is labor-intensive and time-consuming. In this work, an automated protein chip assay (enzyme-linked sandwich immunoassay) based on interdigitated gold microelectrodes in combination with an electrical read-out system was developed and optimized. For this purpose, specific capture antibodies were immobilized on the gold electrodes. The binding of the target is detected via an enzyme-labeled detection antibody by a redox-recycling process that corresponds to the amount of bound target molecule. With this electrical chip assay it is possible to detect antibodies against HPA-1a, HPA-5b and HLA with high sensitivity and specificity in less than half the duration of the MAIPA protocol with similar intra- and interassay variance.

The peroxide stress response of Bacillus licheniformis.

The oxidative stress response of Bacillus licheniformis after treatment with hydrogen peroxide was investigated at the transcriptome, proteome and metabolome levels. In this comprehensive study, 84 proteins and 467 transcripts were found to be up or downregulated in response to the stressor. Among the upregulated genes were many that are known to have important functions in the oxidative stress response of other organisms, such as catalase, alkylhydroperoxide reductase or the thioredoxin system. Many of these genes could be grouped into putative regulons by genomic mining. The occurrence of oxidative damage to proteins was analyzed by a 2-DE-based approach. In addition, we report the induction of genes with hitherto unknown functions, which may be important for the specific oxidative stress response of B. licheniformis. The genes BLi04114 and BLi04115, that are located adjacent to the catalase gene, were massively induced during peroxide stress. Furthermore, the genes BLi04207 and BLi04208, which encode proteins homologous to glyoxylate cycle enzymes, were also induced by peroxide. Metabolomic analyses support the induction of the glyoxylate cycle during oxidative stress in B. licheniformis.

Regulation of acetoin and 2,3-butanediol utilization in Bacillus licheniformis.

The acoABCL and acuABC operons of Bacillus licheniformis DSM13 are strongly induced at the transcriptional level during glucose starvation conditions. Primer extension analyses of this study indicate that the acoABCL operon is controlled by a sigmaL-dependent promoter and the acuABC operon by a sigmaA-dependent promoter. Transcription at the acoA promoter is repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The acuA promoter shows a similar induction pattern, but its activity is independent from the presence of acetoin. It is demonstrated that the acoABCL operon is mainly responsible for acetoin and 2,3-butanediol degradation in B. licheniformis.

Quality control of inclusion bodies in Escherichia coli.

BACKGROUND: Bacterial inclusion bodies (IBs) are key intermediates for protein production. Their quality affects the refolding yield and further purification. Recent functional and structural studies have revealed that IBs are not dead-end aggregates but undergo dynamic changes, including aggregation, refunctionalization of the protein and proteolysis. Both, aggregation of the folding intermediates and turnover of IBs are influenced by the cellular situation and a number of well-studied chaperones and proteases are included. IBs mostly contain only minor impurities and are relatively homogenous. RESULTS: IBs of alpha-glucosidase of Saccharomyces cerevisiae after overproduction in Escherichia coli contain a large amount of (at least 12 different) major product fragments, as revealed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight Mass-Spectrometry (MALDI-ToF MS) identification showed that these fragments contain either the N- or the C-terminus of the protein, therefore indicate that these IBs are at least partially created by proteolytic action. Expression of alpha-glucosidase in single knockout mutants for the major proteases ClpP, Lon, OmpT and FtsH which are known to be involved in the heat shock like response to production of recombinant proteins or to the degradation of IB proteins, clpP, lon, ompT, and ftsH did not influence the fragment pattern or the composition of the IBs. The quality of the IBs was also not influenced by the sampling time, cultivation medium (complex and mineral salt medium), production strategy (shake flask, fed-batch fermentation process), production strength (T5-lac or T7 promoter), strain background (K-12 or BL21), or addition of different protease inhibitors during IB preparation. CONCLUSIONS: alpha-glucosidase is fragmented before aggregation, but neither by proteolytic action on the IBs by the common major proteases, nor during downstream IB preparation. Different fragments co-aggregate in the process of IB formation together with the full-length product. Other intracellular proteases than ClpP or Lon must be responsible for fragmentation. Reaggregation of protease-stable alpha-glucosidase fragments during in situ disintegration of the existing IBs does not seem to occur.

Electrical protein array chips for the detection of staphylococcal virulence factors.

A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.

Novel developments for improved detection of specific mRNAs by DNA chips.

Microarrays have revolutionized gene expression analysis as they allow for highly parallel monitoring of mRNA levels of thousands of genes in a single experiment. Since their introduction some 15 years ago, substantial progress has been achieved with regard to, e.g., faster or more sensitive analyses. In this review, interesting new approaches for a more sensitive detection of specific mRNAs will be highlighted. Particularly, the potential of electrical DNA chip formats that allow for faster mRNA analyses will be discussed.

The role of thioredoxin TrxA in Bacillus subtilis: a proteomics and transcriptomics approach.

Thiol-disulfide oxidoreductases of the thioredoxin superfamily are crucial for maintaining the thiol redox state in living organisms. For the bacterium Bacillus subtilis thioredoxin A (TrxA) was described as the product of an essential gene indicating a key role during growth. By means of mRNA profiling Smits et al. (J. Bacteriol. 2005, 187, 3921-3930) suggested a critical function for TrxA in sulfur utilization during stationary phase. We extended the analysis of TrxA to exponential growth and characterized a trxA conditional mutant by proteome analysis complemented by transcriptomics. After TrxA-depletion, the growth rate was dramatically decreased. The cells responded at mRNA and protein level by the increased expression of genes involved in the utilization of sulfur, which represents the most obvious response as visualized by gel-based proteomics. Furthermore, several genes of the antioxidant response were found at higher expression levels after TrxA-depletion. When sulfate was replaced by thiosulfate or methionine as sulfur source, the growth inhibition was abolished. In the presence of thiosulfate but in the absence of TrxA, the induction of the sulfur limitation response and the oxidative stress response was not observed. Our results show that the global change of gene expression is primarily caused by the interruption of the sulfate utilization after TrxA depletion. Thus, its function in sulfate assimilation renders TrxA an essential protein in growing B. subtilis cells.

Improved sandwich-hybridization assay for an electrical DNA-chip-based monitoring of bioprocess-relevant marker genes.

Recently, it was shown that electrical DNA-chips in connection with a magnetic bead-based sandwich-hybridization assay can be a suitable alternative for the analysis of gene expression by monitoring the respective mRNA levels. In this study, we established an improved assay which allowed for a significantly shortened but sensitive detection of specific mRNAs. These improvements include the change to a solution-based sandwich-hybridization and the rearrangement of the used oligonucleotide probes. The introduction of a second labeled detection probe further increased the hybridization signals and, in turn, leads to a substantial time reduction of the detection protocol. The presented solution-based sandwich-hybridization protocol for the electrochemical detection of specific mRNAs requires about 60 min and the whole procedure, including sampling, cell disruption, and RNA isolation, approx. 80 min. The assay of this study was verified by nutrient-limited growth experiments and the analysis of selected starvation marker genes of the industrial host Bacillus licheniformis. The expression profiles determined with the electrical chip and the optimized protocol were, in most cases, comparable with the profiles determined by real-time RT-PCR measurements.

The glucose and nitrogen starvation response of Bacillus licheniformis.

The glucose and nitrogen starvation stimulons of Bacillus licheniformis were determined by transcriptome and proteome analyses. Under both starvation conditions, the main response of B. licheniformis was a switch to the usage of alternative nutrient sources. This was indicated by an induction of genes involved in the metabolism of C-2 substrates during glucose limitation. In addition, B. licheniformis seems to be using other organic substances like amino acids and lipids as carbon sources when subjected to glucose starvation. This observation is supported by the induction of a high number of genes coding for proteins involved in amino acid and lipid degradation. During nitrogen starvation, genes for several proteases and peptidases involved in nitrate and nitrite assimilation were induced, which enables this bacterium to recruit nitrogen from alternative sources. Both starvation conditions led to a down-regulation of transcription of most vegetative genes, which was subsequently reflected by a reduced synthesis of the corresponding proteins. A selected set of genes was induced by both starvation conditions. Among them were yvyD, citA and the putative methylcitrate shunt genes mmgD, mmgE and yqiQ. However, both starvation conditions did not induce a general SigmaB-dependent stress response.

An acetoin-regulated expression system of Bacillus subtilis.

An expression system, which is based on the promoter of the acoABCL operon of Bacillus subtilis was developed and characterized. The acoABCL operon codes for the acetoin dehydrogenase complex, which is the major enzyme system responsible for the catabolism of acetoin in B. subtilis. Besides weak organic acids, the neutral overflow metabolite acetoin is metabolized by the cells in the early stationary phase. Transcription of reporter gene fusions with the acoA promoter of this operon is strongly repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The co-expression of an additional copy of the regulator gene acoR led to more than twofold higher activity of the acoA promoter. It is demonstrated that the induction of this promoter in growing cells with acetoin is possible with non-phosphotransferase system sugars as carbon and energy source and in a ccpA mutant background. Moreover, it could be shown that the activity of the acoA-directed expression system correlates with the level of acetoin in the medium. During glucose limitation, the utilization of the alternative energy source acetoin keeps the protein synthesis machinery of B. subtilis cells active and thus allows for a long lasting acoA-controlled expression of recombinant genes.

The phosphate-starvation response of Bacillus licheniformis.

The phosphate-starvation stimulon of Bacillus licheniformis was analyzed at the transcriptional and translational level. The comparison of the transcriptome and the proteome demonstrated that this specific starvation response of B. licheniformis is partially similar to that of B. subtilis. However, it is also shown that B. licheniformis has evolved its own strategies to cope with this nutrient limitation. By means of the secretome analysis the phytase was identified as the most abundant protein under phosphate-starvation conditions. Data of this study indicate that, unlike in B. subtilis, phosphate starvation in B. licheniformis does not induce the SigmaB-dependent general stress response.

Application of an electric DNA-chip for the expression analysis of bioprocess-relevant marker genes of Bacillus subtilis.

The knowledge of critical process-relevant genes can be used for an improved control of bioprocesses. So far bioprocess-relevant marker genes can be analyzed by established expression analysis methods only off-line. In this study, an alternative approach for a potential at-line monitoring of gene expression during bioprocesses is suggested. This approach is based on the measurement of specific mRNAs on an electric DNA-chip in connection with a magnetic bead-based sandwich hybridization. In order to allow an at-line measurement of specific mRNAs an improved method for a fast and partially automated isolation of high quality-RNA samples was developed. The expression analysis of the electric DNA-chip was compared with optical DNA micro arrays and the real time RT-PCR for three selected process-relevant genes of Bacillus subtilis. We demonstrate that the mRNA analysis by means of the electric DNA-chip gives similar results compared to the micro array analysis and the real time RT-PCR technique.

Global expression profiling of Bacillus subtilis cells during industrial-close fed-batch fermentations with different nitrogen sources.

A detailed gene expression analysis of industrial-close Bacillus subtilis fed-batch fermentation processes with casamino acids as the only nitrogen source and with a reduced casamino acid concentration but supplemented by ammonia was carried out. Although glutamine and arginine are supposed to be the preferred nitrogen sources of B. subtilis, we demonstrate that a combined feeding of ammonia and casamino acids supports cell growth under fed-batch fermentation conditions. The transcriptome and proteome analyses revealed that the additional feeding of ammonia in combination with a reduced amino acid concentration results in a significantly lower expression level of the glnAR or tnrA genes, coding for proteins, which are mainly involved in the nitrogen metabolism of B. subtilis. However, the mRNA levels of the genes of the ilvBHC-leuABD and hom-thrCB operons were significantly increased, indicating a valine, leucine, isoleucine, and threonine limitation under these fermentation conditions. In contrast, during the fermentation with casamino acids as the only nitrogen source, several genes, which play a crucial role in nitrogen metabolism of B. subtilis (e.g., glnAR, nasCDE, nrgAB, and ureABC), were up-regulated, indicating a nitrogen limitation under these conditions. Furthermore, increased expression of genes, which are involved in motility and chemotaxis (e.g., hag, fliT) and in acetoin metabolism (e.g., acoABCL), was determined during the fermentation with the mixed nitrogen source of casamino acids and ammonia, indicating a carbon limitation under these fermentation conditions. Under high cell density and slow growth rate conditions a weak up-regulation of autolysis genes could be observed as well as the induction of a number of genes involved in motility, chemotaxis and general stress response. Results of this study allowed the selection of marker genes, which could be used for the monitoring of B. subtilis fermentation processes. The data suggest for example acoA as a marker gene for glucose limitation or glnA as an indicator for nitrogen limitation.