The nucleic acid binding cold shock proteins (CSPs) and the cold-induced DEAD box RNA helicases have been proposed separately to act as RNA chaperones, but no experimental evidence has been reported on a direct cooperation. To investigate the possible interaction of the putative RNA helicases CshA and CshB and the CSPs from Bacillus subtilis during cold shock, we performed genetic as well as fluorescence resonance energy transfer (FRET) experiments. Both cshA and cshB genes could be deleted only in the presence of a cshB copy in trans, showing that the presence of one csh gene is essential for viability. The combined gene deletion of cshB and cspD resulted in a cold-sensitive phenotype that was not observed for either helicase or csp single mutants. In addition to the colocalization of the putative helicases CshA and CshB with CspB and the ribosomes in areas surrounding the nucleoid, we detected a strong FRET interaction in vivo between CshB and CspB that depended on active transcription. In contrast, a FRET interaction was not observed for CshB and the ribosomal protein L1. Therefore, we propose a model in which the putative cold-induced helicases and the CSPs work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures in B. subtilis.A sudden decrease in temperature is a serious challenge for all living microorganisms. The cellular reaction required for efficient adaptation to low temperatures is termed cold shock response. The cell has to cope with changes at different physiological levels, such as reduced fluidity of the membrane and slowdown of protein synthesis and protein folding (39). Accordingly, a general decrease of protein synthesis was observed in Escherichia coli and Bacillus subtilis cells following a cold shock (9, 17). The stress arising from a decrease in temperature can in principle be traced back to a reduction in molecular dynamics. The initiation of translation was shown to be a limiting factor for bacterial growth at low temperatures (3, 6). In addition to the delicate interaction of the complex translation machinery, the formation of secondary structures of mRNA prevents efficient initiation of translation (12). The tendency of RNA to fold and become kinetically trapped was described as a general problem and interferes with proper biological function (14). The formation of unfavorable secondary structures is even more intricate at lower temperatures. In analogy to protein folding mechanisms, RNA chaperones have been proposed to resolve and prevent misfolding of RNA molecules by either destabilizing RNA duplexes or binding to singlestranded nucleic acids (21). In the cold shock response, DEAD box RNA helicases have already been identified to destabilize RNA duplexes and the major cold shock proteins (CSPs) to bind RNA. DEAD box RNA helicases belong to the large family of DExD/H-box proteins, which generally have an RNA-dependent ATPase activity in vitro. They are involved in various cellular processes, such as ribosome assembly, i...

The surface charge as well as the electrochemical properties and ligand binding abilities of the Gram‐positive cell wall is controlled by the d‐alanylation of the lipoteichoic acid. The incorporation of d‐Ala into lipoteichoic acid requires the d‐alanine:d‐alanyl carrier protein ligase (DltA) and the carrier protein (DltC). We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DltA with its substrate DltC. We found that apo‐DltC is recognized by both endogenous 4′‐phosphopantetheinyl transferases AcpS and Sfp. After the biochemical characterization of DltA and DltC, we designed an inhibitor (d‐alanylacyl‐sulfamoyl‐adenosine), which is able to block the d‐Ala adenylation by DltA at a Ki value of 232 nmin vitro. We also performed in vivo studies and determined a significant inhibition of growth for different Bacillus subtilis strains when the inhibitor is used in combination with vancomycin.

Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr → Asn) could be correlated to a cytosine to adenine substitution at DNA( desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe → Tyr) to amino acid limitation in the chemically defined fermentation medium.

Although sigma factor-dependent transcriptional regulation was shown to be essential for adaptation to different environmental stimuli, no such sigma factor has been related to the regulation of the cold shock response in Bacillus subtilis. In this study, we present genetic evidence for participation of L ( 54 ) and the two L -dependent transcriptional enhancers BkdR and YplP in the cold shock response of Bacillus subtilis JH642. Single-gene deletion of either sigL, bkdR, or yplP resulted in a cold-sensitive phenotype.

The Bacillus subtilis laboratory strain JH642 shows a cold-sensitive phenotype after a temperature shift from 37 to 15 degrees C in comparison to wild type strain MR168. A mutation in the acetolactate synthase complex IlvBH was found to be partially responsible for this growth defect after cold shock. Via DNA sequencing, genetic and biochemical studies, this defect was characterized, which entails a substitution of two adenines to guanines in the ilvB gene. This results in an amino acid substitution from lysine at position 176 to glycine. As a consequence, the acetolactate synthase efficiency in strain JH642 was found to be reduced by 51-fold.