Summary The translation of many heat shock and virulence genes is controlled by RNA thermometers. Usually, they are located in the 5′‐untranslated region (5′‐UTR) block Shine‐Dalgarno (SD) sequence base pairing. Destabilization structure at elevated temperature permits ribosome binding initiation. We have identified a new type thermometer 5′‐UTR Salmonella agsA gene, which codes for small protein. Transcription gene alternative sigma factor σ 32 . Additional translational control depends on...

The method of chromatin immunoprecipitation combined with microarrays (ChIP-Chip) is a powerful tool for genome-wide analysis protein binding. However, high background signal common phenomenon. Reinvestigation the procedure led us to discover four causes background: i) non-unique sequences, ii) incomplete reversion crosslinks, iii) retention in spin-columns and iv) insufficient RNase treatment. was modified applied analyze binding SeqA σ32 Escherichia coli. False positive findings...

Abstract Expression of many rhizobial small heat-shock genes is controlled by the ROSE element, a thermoresponsive structure in 5′-untranslated region corresponding mRNAs. Using bioinformatics approach, we found more than 20 new potential ROSE-like RNA thermometers upstream wide variety α- and γ-proteobacteria. Northern blot analyses revealed heat-inducible transcripts representative candidate Caulobacter crescentus CC2258 , Escherichia coli ibpA Salmonella typhimurium genes. Typical σ 32...

Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used estimate the approximate numbers of heat-inducible Clp molecules within single cell. According these numbers, different ATPases do not seem compete for proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE ClpX are rapidly degraded wild-type...

Structured RNAs with fundamental sensory and regulatory potential have been discovered in all kingdoms of life. Bacterial RNA thermometers are located the 5'-untranslated region certain heat shock virulence genes. They regulate translation by masking Shine-Dalgarno sequence a temperature-dependent manner. To engineer RNA-based thermosensors, we used combination computer-based rational design vivo screening. After only two rounds selection, several that at least as efficient natural were...

The Escherichia coli ibpAB operon encodes two small heat-shock proteins, the inclusion-body-binding proteins IbpA and IbpB. Here, we report that expression of is a complex process involving at least four different layers control, namely transcriptional RNA processing, translation control protein stability. As typical member regulon, transcription controlled by alternative sigma factor σ(32) (RpoH). Heat-induced bicistronic followed RNase E-mediated processing events, resulting in...

Translation of many small heat shock genes in α- and γ-proteobacteria is controlled by the ROSE (Repression Of Shock gene Expression) element, a thermo-responsive RNA structure 5'-untranslated region. ROSEibpA regulates translation Escherichia coli ibpA coding for an inclusion body-associated protein. We present first structural insights into full-length element examining temperature-induced conformational changes using detailed enzymatic lead probing experiments between 20 50 °C. The...

In Escherichia coli , the SeqA protein binds specifically to GATC sequences which are methylated on A of old strand but not new strand. Such hemimethylated DNA is produced by progression replication forks and lasts until Dam methyltransferase methylates It therefore believed that a region covered follows fork. We show this is, indeed, case using global ChIP Chip analysis in cells synchronized regarding replication. To assess hemimethylation, we developed first genome-wide method for...

Vibrio cholerae, the causative agent of cholera disease, is commonly used as a model organism for study bacteria with multipartite genomes. Its two chromosomes different sizes initiate their DNA replication at distinct time points in cell cycle and terminate synchrony. In this study, time-delayed start Chr2 was verified synchronized population. This pattern suggests possible regulation mechanisms other species sized secondary chromosomes: Either all fixed delay after Chr1 initiation, or...

We have investigated the replication patterns of two chromosomes bacterium Vibrio cholerae grown in four different media. By combining flow cytometry and quantitative real-time PCR with computer simulations, we show that rich media, V. cells grow overlapping cycles both large chromosome (ChrI) small (ChrII). In Luria-Bertani (LB) medium, initiation occurs at copies ChrI origin ChrII origin. Replication was found to occur end period all growth conditions. Novel cell-sorting experiments marker...

Recent developments in DNA-assembly methods make the synthesis of synthetic chromosomes a reachable goal. However, redesign primary bears high risks and still requires enormous resources. An alternative approach is addition to cell. The natural secondary chromosome Vibrio cholerae could potentially serve as template for Escherichia coli. To test this assumption we constructed replicon named synVicII based on replication module V. II (oriII). A new assay assessment stability was developed...

Chromosomal inheritance in bacteria usually entails bidirectional replication of a single chromosome from origin into two copies and subsequent partitioning one copy each daughter cells upon cell division. However, the human pathogen Vibrio cholerae other Vibrionaceae harbor chromosomes, large Chr1 small Chr2. Chr2 have different origins, an oriC-type P1 plasmid-type origin, respectively, driving respective chromosomes. Recently, we described naturally occurring exceptions to two-chromosome...

The genetic make-up of most bacteria is encoded in a single chromosome while about 10% have more than one chromosome. Among these, Vibrio cholerae , with two chromosomes, has served as model system to study various aspects maintenance, mainly replication, and faithful partitioning multipartite genomes. Here, we describe the genomic characterization strains that are an exception rules: naturally occurring single-chromosome V. . Whole genome sequence analyses NSCV1 NSCV2 (natural vibrio)...

Learning by building is one of the core ideas synthetic biology research. Consequently, way to fully understand what a chromosome needs build more and chromosomes. The last years have seen exciting synthetic-chromosome studies. We had previously introduced secondary synVicII in E. coli. It based on replication mechanism Vibrio cholerae. Here we present detailed analysis its genetic characteristics selection approach optimize replicon stability. probe origin diversity chromosomes from...

Short DNA motifs are involved in a multitude of functions such as for example chromosome segregation, replication or mismatch repair. Distribution is often not random and the specific chromosomal pattern relates to respective motif function. Computational approaches which quantitatively assess patterns necessary. Here we present new computer tool DistAMo (Distribution Analysis Motifs). The algorithm uses codon redundancy calculate relative abundance short from single genes entire...

The standard outline of bacterial genomes is a single circular chromosome with replication origin. From the bioengineering perspective, it appears attractive to extend this basic setup. Bacteria split chromosomes or multiple origins have been successfully constructed in last few years. characteristics these engineered strains will largely depend on respective DNA patterns. However, has not investigated systematically bacteria replicons. Here we fill gap by studying set consisting (i) E. coli...

Escherichia coli cells with a point mutation in the dnaN gene causing amino acid change Gly157 to Cys, were found underinitiate replication and grow reduced origin DNA concentration. The mutant β clamp also caused excessive conversion of ATP-DnaA ADP-DnaA. DnaA protein was, however, not element limiting initiation replication. Overproduction protein, which wild-type leads over-replication, had no effect dnaN(G157C) mutant. Origins already opened by seemed remain open for prolonged period,...