Current status of transcriptional regulation systems.

Many attempts have been undertaken to control transgene activity in mammalian cells. This is of importance for both applied biotechnology and basic research activities. State of the art regulatory systems use elements for transgene regulation which are unrelated to host regulatory networks and thus do not interfere with endogenous activities.

Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli.

During production in recombinant Escherichia coli, the human basic fibroblast growth factor (hFGF-2) partly aggregates into stable cytoplasmic inclusion bodies. These inclusion bodies additionally contain significant amounts of the heat-shock chaperone DnaK, and putative DnaK substrates such as the elongation factor Tu (ET-Tu) and the metabolic enzymes dihydrolipoamide dehydrogenase (LpdA), tryptophanase (TnaA), and d-tagatose-1,6-bisphosphate aldolase (GatY). Guanidinium hydrochloride induced disaggregation studies carried out in vitro on artificial aggregates generated through thermal aggregation of purified hFGF-2 revealed identical disaggregation profiles as hFGF-2 inclusion bodies indicating that the heterogenic composition of inclusion bodies did not influence the strength of interactions of hFGF-2 in aggregates formed in vivo as inclusion bodies compared to those generated in vitro from native and pure hFGF-2 through thermal aggregation.

Proteomic characterization of transient expression and secretion of a stress-related metalloprotease in high cell density culture of Bacillus megaterium.

Intracellular and extracellular proteome analysis was carried out by combined two-dimensional gel electrophoresis and mass spectrometric analysis (2DE/MS) for high cell density fed-batch culture of recombinant Bacillus megaterium strains. In the early feeding phase with a constant growth rate of 0.12h(-1) under glucose limitation, high expression and secretion of a metalloprotease (referred as Bmg1465) was detected.

The positive aspects of stress: strain initiates domain decondensation (SIDD).

The conventional string-based bioinformatic methods of genomic sequence analysis are often insufficient to identify DNA regulatory elements, since many of these do not have a recognizable motif. Even in case a sequence pattern is known to be associated with an element it may only partially mediate its function. This suggests that properties not correlated with the details of base sequence contribute to regulation.

Discovery and investigation of a new, second triose phosphate isomerase in Klebsiella pneumoniae.

In this study, a tpi1 gene encoding for the enzyme triose phosphate isomerase in Klebsiella pneumoniae DSM2026 was knocked out in an effort to metabolically engineer this strain as a model system for the production of 1,3-propanediol. Investigations of the tpi1 knockout mutant led to the discovery of a second tpi gene (tpi2) in this organism. The new tpi2 gene was cloned and sequenced.

A protein database constructed from low-coverage genomic sequence of Bacillus megaterium and its use for accelerated proteomic analysis.

Peptide mass fingerprint (PMF) matching is a high-throughput method used for protein spot identification in connection with two-dimensional gel electrophoresis (2DE). However, the success of PMF matching largely depends on whether the proteins to be identified exist in the database searched. Consequently, it is often necessary to apply other more sophisticated but also time-consuming technologies to generate sequence-tags for definitive protein identification.

Composition and dynamics of bacterial communities of a drinking water supply system as assessed by RNA- and DNA-based 16S rRNA gene fingerprinting.

Bacterial community dynamics of a whole drinking water supply system (DWSS) were studied from source to tap. Raw water for this DWSS is provided by two reservoirs with different water characteristics in the Harz mountains of Northern Germany. Samples were taken after different steps of treatment of raw water (i.

In search of functional association from time-series microarray data based on the change trend and level of gene expression.

Background: The increasing availability of time-series expression data opens up new possibilities to study functional linkages of genes. Present methods used to infer functional linkages between genes from expression data are mainly based on a point-to-point comparison. Change trends between consecutive time points in time-series data have been so far not well explored.

Identification of a Thiomicrospira denitrificans-like epsilonproteobacterium as a catalyst for autotrophic denitrification in the central Baltic Sea.

Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon.

Signal-theoretical DNA similarity measure revealing unexpected similarities of E. coli promoters.

We present an implementation of the signal theory based approach for detection of novel types of DNA similarity which are based on physical properties of DNA. Systematic study of the sensitivity of the new similarity measure revealed qualitative differences to letter-based similarity. A variety of physical parameters of DNA double strands, which in a straightforward way reflect different kinds of information hidden behind the primary structure of DNA, showed a wide range of recognition power of the signal similarity measure.

Identification of StiR, the first regulator of secondary metabolite formation in the myxobacterium Cystobacter fuscus Cb f17.1.

Myxobacteria are well established as proficient producers of natural products with numerous biological activities. Although some knowledge has been gained regarding the biosynthesis of secondary metabolites in this class of bacteria, almost nothing is known about the underlying regulatory mechanisms. In order to identify regulatory elements, we submitted the argyrin and stigmatellin producer Cystobacter fuscus to a random transposon mutagenesis strategy and screened 1,000 mutants for the occurrence of strains showing remarkably increased or decreased production of these compounds.

Vaccination equally enables both genetically susceptible and resistant mice to control infection with group A streptococci.

There is substantial evidence that host genetic factors are important in determining susceptibility to infection with group A streptococci (GAS). Several studies have revealed that, similarly to humans, a genetic component may be important in determining susceptibility to GAS infection in mice. Thus, C3H/HeN mice are much more susceptible to streptococcal infection than BALB/c mice.

Contribution of natural killer cells to the pathogenesis of septic shock induced by Streptococcus pyogenes in mice.

Natural killer (NK) cells are critical components of the innate immune system and have been implicated in the pathogenesis of septic shock. In the present study, the relative contribution of NK cells to the development of Streptococcus pyogenes-induced septic shock was investigated in a mouse model of group A streptococcal infection that resembles the development of this condition in humans. C3H/HeN mice were depleted of NK cells by in vivo administration of anti-asialo ganglio-N-tetraosylceramide antibodies and then were examined for their response to infection with S.

Prospects for a group A streptococcal vaccine.

Group A streptococcal (GAS) infections are associated with a number of human diseases, including pharyngitis, impetigo, necrotizing fasciitis, streptococcal toxic shock syndrome and rheumatic heart disease. An increase in the incidence of severe GAS infections in Western countries, and the awareness of the burden of GAS-associated diseases in developing nations, which remains high in spite of the availability of antibiotics, has provided the impetus for development of a safe and efficacious GAS vaccine. This has focused on the M protein, a major GAS virulence factor, however, with the publication of several GAS genomes, a number of non-M vaccine candidates are now under investigation.

Characterization of a B220+ lymphoid cell subpopulation with immune modulatory functions in nasal-associated lymphoid tissues.

Complex mechanisms operate on mucosal tissues to regulate immune responsiveness and tolerance. When the lymphocyte subpopulations from murine nasal-associated lymphoid tissues (NALT) were characterized, we observed an accumulation of B220(low)CD3(low)CD4(-)CD8(-)CD19(-)c-Kit(+) cells. TCR transgenic mice and athymic mice were used for monitoring T cell lineage and the presence of extrathymic T cell precursors.

An extended transcriptional regulatory network of Escherichia coli and analysis of its hierarchical structure and network motifs.

Recent studies of genome-wide transcriptional regulatory network (TRN) revealed several intriguing structural and dynamic features of gene expression at a system level. Unfortunately, the network under study is often far from complete. A critical question is thus how much the network is incomplete and to what extent this would affect the results of analysis.

Hierarchical structure and modules in the Escherichia coli transcriptional regulatory network revealed by a new top-down approach.

Background: Cellular functions are coordinately carried out by groups of genes forming functional modules. Identifying such modules in the transcriptional regulatory network (TRN) of organisms is important for understanding the structure and function of these fundamental cellular networks and essential for the emerging modular biology. So far, the global connectivity structure of TRN has not been well studied and consequently not applied for the identification of functional modules.

The Mycoplasma-derived macrophage-activating 2-kilodalton lipopeptide triggers global immune activation on nasal mucosa-associated lymphoid tissues.

A better knowledge on how immune responses are initiated in mucosal tissues would facilitate the design of new mucosal vaccines, as well as improve our understanding on host defense against infection. We investigated the mechanisms of adjuvanticity of the Mycoplasma-derived macrophage-activating 2-kDa lipopeptide (MALP-2), which binds to the heterodimer formed by the Toll-like receptors 2 and 6 (TLR2 and -6), at the level of the murine nasal mucosa-associated lymphoid tissues (NALT). TLR2 expression analysis demonstrated that several cell types from the nasal cavity were able to overexpress this receptor, either constitutively (such as B cells) or after stimulation (i.

Transcriptional control of SV40 T-antigen expression allows a complete reversion of immortalization.

Conditional proliferation of mouse embryo fibroblasts was achieved with a novel autoregulatory vector for Tet-dependent expression of the SV40 T-antigen. The majority of cell clones that were isolated under induced conditions showed strict regulation of cell growth. Status switches were found to be fully reversible and highly reproducible with respect to gene expression characteristics.

Plasmin(ogen)-binding alpha-enolase from Streptococcus pneumoniae: crystal structure and evaluation of plasmin(ogen)-binding sites.

Alpha-enolases are ubiquitous cytoplasmic, glycolytic enzymes. In pathogenic bacteria, alpha-enolase doubles as a surface-displayed plasmin(ogen)-binder supporting virulence. The plasmin(ogen)-binding site was initially traced to the two C-terminal lysine residues.

Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways.

Background: Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. Recently, it has been proposed that autoinducer-2 (AI-2), a furanosyl borate diester derived from the recycling of S-adenosyl-homocysteine (SAH) to homocysteine, serves as a universal signal for interspecies communication.

Results: In this study, 138 completed genomes were examined for the genes involved in the synthesis and detection of AI-2.

Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins.

Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix- or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure) as well as the presence of buffer additives on the refolding process is elucidated.

Identification and analysis of the core biosynthetic machinery of tubulysin, a potent cytotoxin with potential anticancer activity.

Myxobacteria are well known for their biosynthetic potential, especially for the production of cytotoxic compounds with potential anticancer activities. The tubulysins are currently in preclinical development. They are produced in very low quantities, and genetic manipulation of producing strains has never been accomplished.

IdentiCS--identification of coding sequence and in silico reconstruction of the metabolic network directly from unannotated low-coverage bacterial genome sequence.

Background: A necessary step for a genome level analysis of the cellular metabolism is the in silico reconstruction of the metabolic network from genome sequences. The available methods are mainly based on the annotation of genome sequences including two successive steps, the prediction of coding sequences (CDS) and their function assignment. The annotation process takes time.

Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism.

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation.