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Author(s): John W. Trauger [1]; Rahul M. Kohli [1]; Henning D. Mootz [2]; Mohamed A. Marahiel [2]; Christopher T. Walsh (corresponding author) [1] In the biosynthesis of many macrocyclic natural [...]

The role of the condensation domain in preventing misinitiation and timing of epimerization has been studied with focus on control of directionality in nonribosomal peptide synthesis. Epimerization domains of elongation modules were seen to be able to, in principle, epimerize aminoacyl S-Ppant and peptidyl-S-Ppant substrates, but efficiency for epimerizing the noncognate aminoacyl-S-Ppant substrates seems to be lowered. Natural peptides of pharmacological interest are often synthesized from bacteria/fungi by nonribosomal peptide synthetases.

The article discusses the mechanisms to understand Bacillus subtlis cold shock survival. The role of isoleucine in the synthesis of fatty acids and prevention of cell membrane disruption are examined.

A putative lichenysin biosynthesis operon from Bacillus licheniformis ATCC 10716 was identified and characterized. Three genes, namely, licA, licB and licC, with structural patterns common to peptide synthetases and a gene designated licTE were revealed after the cloning and sequencing of the entire lic operon. It is proposed that the identified genes are involved in the nonribosomal synthesis of the portion of the lichenysin peptide with the primary sequence L-Gln-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Ile.

Members of a family of cold-shock proteins (CSPs) are found throughout the eubacterial domain and appear to function as RNA-chaperones. They have been implicated in various cellular processes, including adaptation to low temperatures, cellular growth, nutrient stress and stationary phase. The discovery of a domain - the cold-shock domain - that shows strikingly high homology and similar RNA-binding properties to CSPs in a growing number of eukaryotic nucleic-acid-binding proteins suggests that these proteins have an ancient origin.

An experiment is made to determine if the biosynthetic genes for the lipopeptide calcium-dependent antibiotic of Streptomyces coelicolor A3(2) can be cloned. In this study, a PCR-based technique was used to isolate peptide-synthetase-encoding DNA fragments from the bacteria's chromosome. Results confirm the cloning of the genes as seen from disruptions in calcium-dependent antibiotic production.

A study was conducted on the cloning and sequencing of the entire tyc operon of Bacillus brevis and on the expression of active internal enzyme fragments. The results provide strong support for the functional integrities of single domains within multifunctional peptide synthetases. Moreover, two tandem ABC transporters, which may play a key role in conferring resistance against tyrocidine, and a putative thioesterase were also identified.

Understanding of the structures and synthesis of bioactive peptides produced via ribosomal and nonribosomal mechanisms has progressed. The nonribosomal peptide synthetic route is now generally accepted as an alternative way of manufacturing highly specialized polypeptides. The functional domains of modular peptide synthetases are described.

Two dimensional gel electrophoresis reveals that bacteria respond to a decrease in temperature with the introduction of a class of proteins called cold-induced proteins (CIPs). Three small acidic proteins show highest relative induction after a cold shock and belong to a class of proteins having CspB as one of its members. CspB affects the induction level of many CIPs. The CIPs present in B. subtilis have a broad spectrum of functional applications in cellular physiology.

The peptidyl-prolyl cis-trans isomerase (PPIase) from Bacillus subtilis was examined for the role of its residues, V52, H90 and H109, in binding cyclosporin A (CsA) and their catalytic efficiencies. Results of residue substitution showed H90 and H109 to be responsible for CsA affinity with V52 governing the conversion rate/catalytic efficiency. Substitutions were made to simulate PPIases from human and E. coli.

Site directed mutagenesis of the core sequence in the D-Phe activating region of the E. coli tyrocidine synthetase I (TycA) reveals the presence of a4'-phosphopantetheine binding site that labels the tycA gene. The site is a beta-(3H)alanine, indicating that a cofactor containing beta-alanine modifies TycA.

Lipopeptide antibiotic surfactin production is enhanced by the gsp gene-induced trans-complemented defect in sfp gene. The sequence and functions of the sfp and gsp genes are identical to EntD protein. The three proteins are essential for peptide secretion. Lipopeptide antibiotic surfactin is a cyclic heptapeptide and retards fibrin clotting.

The structure of CspB, one of the major cold-shock proteins, obtained by a sudden drop in growth temperature in both Escherichia coli and Bacillus subtilis, is ascertained on the basis of two crystal forms. CspB exists in both the crystal forms as an antiparallel five stranded beta-barrel. In the three consecutive beta-strands, the central strand which contains the RNP1 motif produces a rich surface of aromatic and basic residues that may be participating in nucleic acid binding.

The cold shock-inducible cspB gene encoding the Bacillus subtilis cold shock protein CspB was cloned, sequenced and characterized. The gene encodes an acidic protein with a molecular mass of 7,365 Da. Deduced amino acid sequence comparisons revealed that the protein is similar to the Escherichia coli cold shock protein CS7.4. It was shown that the cspB transcript is present in a single copy and is rapidly induced upon shift from 37 degrees C to 10 degrees C. Moreover, cell viability of a strain containing the disrupted cspB gene was strongly affected at freezing temperatures, supporting a role for CspB protein in the acquired cold tolerance of B. subtilis.

Iron deprivation in bacteria causes the derepression of genes controlled by the ferric uptake regulator (Fur). The present microarray analysis of iron-starved Bacillus subtilis cells grown in minimal medium unveils additional physiological effects on a large number of genes linked to stringent-response regulation and to genes involved in amino acid biosynthesis associated with pathways essential for bacillibactin production.