Bacillus subtilis Marburg has only one intrinsic restriction and modification system BsuM that recognizes the CTCGAG (XhoI site) sequence. It consists of two operons, BsuMM operon for two cytosine DNA methyltransferases, and BsuMR operon for a restriction nuclease and two associated proteins of unknown function. In this communication, we analyzed the BsuM system by utilizing phage SP10 that possesses more than twenty BsuM target sequences on the phage genome. SP10 phages grown in the restriction and modification-deficient strain could not make plaques on the restriction-proficient BsuMR(+) indicator strain. An enforced expression of the wild type BsuMM operon in the BsuMR(+) indicator strain, however, allowed more than thousand times more plaques. DNA extracted from SP10 phages, thus, propagated became more but not completely refractory to XhoI digestion in vitro. Thus, the SP10 phage genome DNA is able to be nearly full-methylated but some BsuM sites are considered to be unmethylated.
Glucolipids in Bacillus subtilis are synthesized by UgtP processively transferring glucose from UDP-glucose to diacylglycerol. Here we conclude that the abnormal morphology of a ugtP mutant is caused by lack of glucolipids, since the same morphology arises after abolition of glucolipid production by disruption of pgcA and gtaB, which are involved in UDP-glucose synthesis. Conversely, expression of a monoglucosyldiacylglycerol (MGlcDG) produced by 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii (alMGS) almost completely suppressed the ugtP disruptant phenotype. Activation of extracytoplasmic function (ECF) sigmas (SigM, SigV, and SigX) in the ugtP mutant was decreased by alMGS expression, and was suppressed to low levels by MgSO4 addition. When alMGS and alDGS (A. laidlawii 1,2-diacylglycerol-3-glucose (1-2)-glucosyltransferase producing diglucosyldiacylglycerol (DGlcDG)) were simultaneously expressed, SigX activation was repressed to wild type level. These observations suggest that MGlcDG molecules are required for maintenance of B. subtilis cell shape and regulation of ECF sigmas, and DGlcDG regulates SigX activity.
Extracytoplasmic function (ECF) σ factors respond to environmental stresses and regulate numerous genes required for adaptation. Under normal growth conditions, the ECF σ factors are sequestered by transmembrane anti-σ factor proteins, from which they are released under stress conditions. In Bacillus subtilis ugtP null mutant cells, which lack glucolipids, three of the seven ECF σ factors, σM, σV and σX, are activated. The Escherichia coli cell membrane does not contain glucolipids. When the genes for these three ECF σ and anti-σ factors were introduced into E. coli cells, expression of lacZ fused to the ECF σ factor-regulated promoters indicated ECF σ factor activity. Additional expression of the ugtP gene in these E. coli cells led to the synthesis of small amounts of glucolipids, and the activities of σM and σV were repressed, but the activity of σX was unaffected. It is likely that glucolipids directly influence anti-σM and anti-σV factors by stabilizing conformations that sequester the respective ECF σ factors.
SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.
Abstract Eukaryotic in vitro translation systems require large numbers of protein and RNA components and thereby rely on the use of cell extracts. Here we established a new in vitro translation system based on rice callus extract (RCE). We confirmed that RCE maintains its initial activity even after five freeze-thaw cycles and that the optimum temperature for translation is around 20°C. We demonstrated that the RCE system allows the synthesis of hERG, a large membrane protein, in the presence of liposomes. We also showed that the introduction of a bicistronic mRNA based on 2A peptide to RCE allowed the production of two distinct proteins from a single mRNA. Our new method thus facilitates laboratory-scale production of cell extracts, making it a useful tool for the in vitro synthesis of proteins for biochemical studies.
The outer membrane lipoprotein RcsF is an essential component of the Rcs phosphorelay signal transduction system in Escherichia coli. It senses stresses imposed on the cell envelope and conveys the information to histidine kinase RcsC in the cytoplasmic membrane. Mislocalization of RcsF to the periplasm, effected by fusing it to the periplasmic maltose-binding protein, or to the cytoplasmic membrane, brought about by changing the lipoprotein sorting signal, leads to high activation of the Rcs system, suggesting that RcsF functions as a ligand for RcsC in activating the system. Here, we focus on the proline-rich region (PRR) in the N-terminal half of RcsF, a region which also contains many basic amino acid residues. Deletion of the PRR in the mislocalized RcsF resulted in even higher activation of the Rcs system. The same deletion in wild-type RcsF lipoprotein that is correctly localized to the outer membrane, however, blocked activation of the system under stresses that normally should activate it. It is highly likely that the PRR plays an important role in the regulation of the function of RcsF in activating the Rcs system.
