The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• bakteriální proteiny * metabolismus   MeSH
• nukleotidy metabolismus   MeSH
• proteiny SecA metabolismus   MeSH
• proteiny z Escherichia coli * metabolismus   MeSH
• translokační kanály SEC chemie   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In Synechocystis 6803, the DNA methyltransferase M.Ssp6803II modifies the first cytosine in the GGCC motif, forming N4-methylcytosine (GGm4CC). Deletion of the sll0729 gene encoding M.Ssp6803II (∆sll0729) caused a bluish phenotype due to reduced chlorophyll levels, which was reversed by suppressor mutations. Re-sequencing of 7 suppressor clones revealed a common GGCC to GGTC mutation in the slr1790 promoter's discriminator sequence, encoding protoporphyrinogen IX oxidase, HemJ, crucial for tetrapyrrole biosynthesis. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in ∆sll0729 mutants. This aberration led to the accumulation of coproporphyrin III and protoporphyrin IX, indicative of impaired HemJ activity. To confirm the importance of DNA methylation in hemJ expression, hemJ promoter variants with varying discriminator sequences were introduced into the wild type, followed by sll0729 deletion. The sll0729 deletion segregated in strains with the GGTC discriminator motif, resulting in wild-type-like pigmentation, whereas freshly prepared ∆sll0729 mutants with the native hemJ promoter exhibited the bluish phenotype. These findings demonstrate that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression. Thus, cytosine N4-methylation is a relevant epigenetic marker in Synechocystis and likely other cyanobacteria.

• MeSH
• bakteriální proteiny metabolismus   genetika   MeSH
• epigeneze genetická * MeSH
• metylace DNA * MeSH
• mutace MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u bakterií MeSH
• Synechocystis * genetika   metabolismus   MeSH
• tetrapyrroly * metabolismus   biosyntéza   MeSH
• Publikační typ
• časopisecké články MeSH

The uptake of insecticidal Cry1Ab from genetically engineered (GE) maize, via herbivore Rhopalosiphum padi, to a predator Harmonia axyridis and its potential intergenerational transfer were investigated. Cry1Ab concentration was found to be 400-fold lower in R. padi compared to GE maize, and more than two-fold lower in H. axyridis. For 62% of H. axyridis samples, Cry1Ab was under the limit of detection (LOD), for another 13% were under the limit of quantification (LOQ). The concentration of Cry1Ab was similar between H. axyridis exposed short-term and long-term with the exception of adults after long-term. There was no correlation between Cry1Ab in females and eggs and neonates. The performance of H. axyridis was comparable between Cry1Ab and control. Histological investigation did not show any pathological changes in the digestive and reproductive systems. The detected route of exposure is unlikely to be important for functional biological control by H. axyridis in agroecosystem.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• brouci * metabolismus   MeSH
• endotoxiny * MeSH
• geneticky modifikované rostliny metabolismus   MeSH
• hemolyziny genetika   metabolismus   MeSH
• kukuřice setá genetika   metabolismus   MeSH
• larva metabolismus   MeSH
• lidé MeSH
• novorozenec MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Exploring the structure and function of protein complexes requires their isolation in the native state-a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism for the physiology of oxygenic phototrophs, and the biogenesis of membrane-bound photosynthetic complexes has traditionally been studied using this cyanobacterium. In a typical approach, the protein complexes are purified with a combination of His-affinity chromatography and a size-based fractionation method such as gradient ultracentrifugation and/or native electrophoresis. However, His-affinity purification harbors prominent contaminants and the levels of many proteins are too low for a feasible multi-step purification. Here, we have developed a purification method for the isolation of 3x FLAG-tagged proteins from the membrane and soluble fractions of Synechocystis. Soluble proteins or solubilized thylakoids are subjected to a single affinity purification step that utilizes the highly specific binding of FLAG-affinity resin. After an intensive wash, the captured proteins are released from the resin under native conditions using an excess of synthetic 3x FLAG peptide. The protocol allows fast isolation of low abundant protein complexes with a superb purity.

• Publikační typ
• časopisecké články MeSH