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.

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.

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

BACKGROUND: The Great East Japan Earthquake of 2011 triggered large tsunami waves, which flooded broad areas of land along the Pacific coast of eastern Japan and changed the soil environment drastically. However, the microbial characteristics of tsunami-affected soil at the genomic level remain largely unknown. In this study, we isolated microbes from a soil sample using general low-nutrient and seawater-based media to investigate microbial characteristics in tsunami-affected soil. RESULTS: As expected, a greater proportion of strains isolated from the tsunami-affected soil than the unaffected soil grew in the seawater-based medium. Cultivable strains in both the general low-nutrient and seawater-based media were distributed in the genus Arthrobacter. Most importantly, whole-genome sequencing of four of the isolated Arthrobacter strains revealed independent losses of siderophore-synthesis genes from their genomes. Siderophores are low-molecular-weight, iron-chelating compounds that are secreted for iron uptake; thus, the loss of siderophore-synthesis genes indicates that these strains have adapted to environments with high-iron concentrations. Indeed, chemical analysis confirmed the investigated soil samples to be rich in iron, and culture experiments confirmed weak cultivability of some of these strains in iron-limited media. Furthermore, metagenomic analyses demonstrated over-representation of denitrification-related genes in the tsunami-affected soil sample, as well as the presence of pathogenic and marine-living genera and genes related to salt-tolerance. CONCLUSIONS: Collectively, the present results would provide an example of microbial characteristics of soil disturbed by the tsunami, which may give an insight into microbial adaptation to drastic environmental changes. Further analyses on microbial ecology after a tsunami are envisioned to develop a deeper understanding of the recovery processes of terrestrial microbial ecosystems.

Functional equilibrium of the KcsA structure revealed by NMR.

KcsA is a tetrameric K(+) channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (p(perm)) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1-3% 2,2,2-trifluoroethanol increases p(perm) and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.

Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay.

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1-Ccr4 and Pan2-Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2-PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3' end of poly(A).

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.

Quantitative characterization of Tob interactions provides the thermodynamic basis for translation termination-coupled deadenylase regulation.

Translation termination-coupled deadenylation is the first and often the rate-limiting step of eukaryotic mRNA decay in which two deadenylases, Ccr4-Caf1 and Pan2, play key roles. One of the deadenylases, Caf1, associates with Tob, which recruits Caf1 to the poly(A) tail through interactions with a cytoplasmic poly(A)-binding protein 1 (PABPC1). We previously proposed that the competition between Tob and eRF3 (a translation termination factor that interacts with PABPC1) is responsible for the regulation of deadenylase activity. However, the molecular mechanism of the regulation should be addressed by investigating the binding affinity and the cellular levels of these proteins. In this work, we characterized the human Tob interactions with Caf1 and a C-terminal domain of PABPC1 (PABC). Nuclear magnetic resonance (NMR) and Western blot analyses revealed that Tob consists of a structured N-terminal BTG-Tob domain and an unstructured C-terminal region with two conserved PAM2 (PABPC1-interacting motif 2) motifs. The BTG-TOB domain associates with Caf1, whereas the C-terminal PAM2 motif binds to PABC, with a K(d) value of 20 microM. Furthermore, we demonstrated that the levels of eRF3 and Tob in HeLa cells are 4-5 microM and less than 0.2 microM, respectively. On the basis of these results, we propose a thermodynamic mechanism for the translation termination-coupled deadenylation mediated by the Tob-Caf1 complex.