Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1–5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10858-012-9628-3) contains supplementary material, which is available to authorized users

Dynamic domain arrangement of CheA-CheY complex regulates bacterial thermotaxis, as revealed by NMR

Bacteria utilize thermotaxis signal transduction proteins, including CheA, and CheY, to switch the direction of the cell movement. However, the thermally responsive machinery enabling warm-seeking behavior has not been identified. Here we examined the effects of temperature on the structure and dynamics of the full-length CheA and CheY complex, by NMR. Our studies revealed that the CheA-CheY complex exists in equilibrium between multiple states, including one state that is preferable for the autophosphorylation of CheA, and another state that is preferable for the phosphotransfer from CheA to CheY. With increasing temperature, the equilibrium shifts toward the latter state. The temperature-dependent population shift of the dynamic domain arrangement of the CheA-CheY complex induced changes in the concentrations of phosphorylated CheY that are comparable to those induced by chemical attractants or repellents. Therefore, the dynamic domain arrangement of the CheA-CheY complex functions as the primary thermally responsive machinery in warm-seeking behavior.Peer reviewe

Seasonal Analysis of Microbial Communities in Precipitation in the Greater Tokyo Area, Japan

The presence of microbes in the atmosphere and their transport over long distances across the Earth's surface was recently shown. Precipitation is likely a major path by which aerial microbes fall to the ground surface, affecting its microbial ecosystems and introducing pathogenic microbes. Understanding microbial communities in precipitation is of multidisciplinary interest from the perspectives of microbial ecology and public health; however, community-wide and seasonal analyses have not been conducted. Here, we carried out 16S rRNA amplicon sequencing of 30 precipitation samples that were aseptically collected over 1 year in the Greater Tokyo Area, Japan. The precipitation microbial communities were dominated by Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria and were overall consistent with those previously reported in atmospheric aerosols and cloud water. Seasonal variations in composition were observed; specifically, Proteobacteria abundance significantly decreased from summer to winter. Notably, estimated ordinary habitats of precipitation microbes were dominated by animal-associated, soil-related, and marine-related environments, and reasonably consistent with estimated air mass backward trajectories. To our knowledge, this is the first amplicon-sequencing study investigating precipitation microbial communities involving sampling over the duration of a year

Additional file 2 of Genomic and metagenomic analysis of microbes in a soil environment affected by the 2011 Great East Japan Earthquake tsunami

Relative abundance of functional gene categories in the Arthrobacter genomes. The relative abundance of CDSs assigned to each eggNOG functional category is plotted for each Arthrobacter genome. (PDF 166 kb