A novel method for the adaptation of target gene codon usage to most sequenced prokaryotes and selected eukaryotic gene expression hosts was developed to improve heterologous protein production. In contrast to existing tools, JCat (Java Codon Adaptation Tool) does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators. The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the pasted sequence and the newly adapted sequence. Additionally, a list of genes in FASTA-format can be uploaded to calculate CAI values. In one example, all genes of the genome of Caenorhabditis elegans were adapted to Escherichia coli codon usage and further optimized to avoid commonly used restriction sites. In a second example, the Pseudomonas aeruginosa exbD gene codon usage was adapted to E.coli codon usage with parallel avoidance of the same restriction sites. For both, the degree of introduced changes was documented and evaluated. JCat is integrated into the PRODORIC database that hosts all required information on the various organisms to fulfill the requested calculations. JCat is freely accessible at http://www.prodoric.de/JCat .

One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B₁₂ (cobalamin), which was originally discovered as the anti pernicious anemia factor and whose enigmatic complex structure is matched only by the beguiling chemistry that it mediates. The biosynthesis of this essential nutrient is intricate, involved and, remarkably, confined to certain members of the prokaryotic world, seemingly never have to have made the eukaryotic transition. In humans, the vitamin is required in trace amounts (approximately 1 µg/day) to assist the actions of only two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase; yet commercially more than 10 t of B₁₂ are produced each year from a number of bacterial species. The rich scientific history of vitamin B₁₂ research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described. Current strategies for the improvement of vitamin B₁₂ production using modern biotechnological techniques are outlined.

We have developed PrediSi (Prediction of Signal peptides), a new tool for predicting signal peptide sequences and their cleavage positions in bacterial and eukaryotic amino acid sequences. In contrast to previous prediction tools, our new software is especially useful for the analysis of large datasets in real time with high accuracy. PrediSi allows the evaluation of whole proteome datasets, which are currently accumulating as a result of numerous genome projects and proteomics experiments. The method employed is based on a position weight matrix approach improved by a frequency correction which takes in to consideration the amino acid bias present in proteins. The software was trained using sequences extracted from the most recent version of the SwissProt database. PrediSi is accessible via a web interface. An extra Java package was designed for the integration of PrediSi into other software projects. The tool is freely available on the World Wide Web at http://www.predisi.de.

SUMMARY: The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized.

Summary: A new online framework for the accurate and integrative prediction of transcription factor binding sites (TFBSs) in prokaryotes was developed. The system consists of three interconnected modules: (1) The PRODORIC database as a comprehensive data source and extensive collection of TFBSs with corresponding position weight matrices. (2) The pattern matching tool Virtual Footprint for the prediction of genome based regulons and for the analysis of individual promoter regions. (3) The interactive genome browser GBPro for the visualization of TFBS search results in their genomic context and links to gene and regulator-specific information in PRODORIC. The aim of this service is to provide researchers a free and easy to use collection of interconnected tools in the field of molecular microbiology, infection and systems biology. Availability: Contact:d.jahn@tu-bs.de

Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V‐shaped glutamyl‐tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the “Radical SAM enzyme” coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure–function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.