Staphylococcus epidermidis small basic protein (Sbp) forms amyloid fibrils, consistent with its function as a scaffolding protein in biofilms.

Biofilms are communities of microbes embedded in a microbial extracellular matrix. Their formation is considered the main virulence mechanism enabling the opportunistic bacterial pathogen Staphylococcus epidermidis to cause devastating nosocomial, implant-associated infections. Biofilms often contain proteins, and an 18-kDa protein called small basic protein (Sbp) recently was discovered in the S. epidermidis biofilm matrix and may serve as a scaffolding protein in both polysaccharide intercellular adhesin (PIA)-dependent and accumulation-associated protein (Aap)-dependent biofilm formations. In Aap-mediated biofilm formation, Sbp colocalizes with Domain-B of Aap, implying that Sbp directly interacts with Aap's Domain-B. However, the structure of Sbp and its interaction with Aap, as well as the molecular mechanism underlying Sbp's roles in biofilm formation, are incompletely understood. In this work, we used small-angle X-ray scattering (SAXS), NMR, analytical size-exclusion chromatography, and isothermal titration calorimetry analyses to determine the Sbp structure and characterize its interaction with Aap's Domain-B. We found that Sbp is monomeric and partially folded in solution, and, unexpectedly, we observed no direct interactions between Sbp and Aap Domain-B. Instead, we noted that Sbp forms amyloid fibrils both in vitro and in vivo Atomic force, transmission electron, and confocal fluorescence microscopy methods confirmed the formation of Sbp amyloid fibrils and revealed their morphology. Taken together, the Sbp amyloid fibril structures identified here may account for Sbp's role as a scaffolding protein in the S. epidermidis biofilm matrix.

Infrared Probe Technique Reveals a Millipede-like Structure for Aß(8-28) Amyloid Fibril.

Amyloid fibrils are unique fibrous polypeptide aggregates. They have been associated with more than 20 serious human diseases including Alzheimer's disease and Parkinson's disease. Besides their pathological significance, amyloid fibrils are also gaining increasing attention as emerging nanomaterials with novel functions. Structural characterization of amyloid fibril is no doubt fundamentally important for the development of therapeutics for amyloid-related diseases and for the rational design of amyloid-based materials. In this study, we explored to use side-chain-based infrared (IR) probe to gain detailed structural insights into the amyloid fibril by a 21-residue model amyloidogenic peptide, Aß(8-28). We first proposed an approach to incorporate thiocyanate (SCN) IR probe in a site-specific manner into amyloidogenic peptide using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate as cyanylating agent. Using this approach, we obtained three Aß(8-28) variants, labeled with SCN probe at three different positions. We then showed with thioflavin T fluorescence assay, Congo red assay, and atomic force microscopy that the three labeled Aß(8-28) peptides can quickly form amyloid fibrils under high concentration and high salt conditions. Finally, we performed a detailed IR spectral analysis of the Aß(8-28) fibril in both amide I and probe regions and proposed a millipede-like structure for the Aß(8-28) fibril.

Genome-wide characterization of the xyloglucan endotransglucosylase/hydrolase family genes and their response to plant hormone in sugar beet.

Xyloglucan endotransglucosylase/hydrolases (XTHs) play a crucial role in plant growth and development. However, their functional response to phytohormone in sugar beet still remains obscure. In this study, we identified 30 putative BvXTH genes in the sugar beet genome. Phylogenetic and evolutionary relationship analysis revealed that they were clustered into three groups and have gone through eight tandem duplication events under purifying selection. Gene structure and motif composition analysis demonstrated that they were highly conserved and all contained one conserved glycoside hydrolase family 16 domain (Glyco_hydro_16) and one xyloglucan endotransglycosylase C-terminus (XET_C) domain. Transcriptional expression analysis exhibited that all BvXTHs were ubiquitously expressed in leaves, root hairs and tuberous roots, and most of them were up-regulated by brassinolide (BR), jasmonic acid (JA), abscisic acid (ABA) and gibberellic acid (GA3). Further mutant complementary experiment demonstrated that expression of BvXTH17 rescued the retarded growth phenotype of xth22, an Arabidopsis knock out mutant of AtXTH22. The findings in our work provide fundamental information on the structure and evolutionary relationship of the XTH family genes in sugar beet, and reveal the potential function of BvXTH17 in plant growth and hormone response.

Characterization and Expression of KT/HAK/KUP Transporter Family Genes in Willow under Potassium Deficiency, Drought, and Salt Stresses.

The K+ transporter/high-affinity K+/K+ uptake (KT/HAK/KUP) transporters dominate K+ uptake, transport, and allocation that play a pivotal role in mineral homeostasis and plant adaptation to adverse abiotic stresses. However, molecular mechanisms towards K+ nutrition in forest trees are extremely rare, especially in willow. In this study, we identified 22 KT/HAK/KUP transporter genes in purple osier willow (designated as SpuHAK1 to SpuHAK22) and examined their expression under K+ deficiency, drought, and salt stress conditions. Both transcriptomic and quantitative real-time PCR (qRT-PCR) analyses demonstrated that SpuHAKs were predominantly expressed in stems, and the expression levels of SpuHAK1, SpuHAK2, SpuHAK3, SpuHAK7, and SpuHAK8 were higher at the whole plant level, whereas SpuHAK9, SpuHAK11, SpuHAK20, and SpuHAK22 were hardly detected in tested tissues. In addition, both K+ deficiency and salt stress decreased the tissue K+ content, while drought increased the tissue K+ content in purple osier plant. Moreover, SpuHAK genes were differentially responsive to K+ deficiency, drought, and salt stresses in roots. K+ deficiency and salt stress mainly enhanced the expression level of responsive SpuHAK genes. Fifteen putative cis-acting regulatory elements, including the stress response, hormone response, circadian regulation, and nutrition and development, were identified in the promoter region of SpuHAK genes. Our findings provide a foundation for further functional characterization of KT/HAK/KUP transporters in forest trees and may be useful for breeding willow rootstocks that utilize potassium more efficiently.

New insight into amyloid fibril formation of hen egg white lysozyme using a two-step temperature-dependent FTIR approach.

Hen egg white lysozyme (HEWL) is widely used in the mechanistic study of amyloid fibril formation. Yet, the fibrillation mechanism of HEWL is not well understood. In particular, in situ structural evidence for the on-pathway oligomeric intermediate has never been captured. Such evidence is crucial for confirming nucleated conformational conversion mechanism. Herein, we attempt to use a two-step temperature-dependent Fourier transform infrared (FTIR) approach to capture the in situ evidence for the on-pathway oligomeric intermediate and the oligomer-to-fibril transition during HEWL fibrillation. Key features of this approach include using lower temperature to generate the on-pathway oligomeric intermediate, using elevated temperature to eliminate the interference from the off-pathway oligomer and to facilitate the oligomer-to-fibril transition, and using FTIR difference spectroscopy and atomic force microscopy to tackle structure and morphology. Using such an approach, we reveal that the on-pathway oligomeric intermediate is in parallel ß-sheet configuration featuring a frequency at 1622 cm(-1) and the oligomer-to-fibril transition is accompanied by a spectral transition from 1622 to 1618 cm(-1). We also discover the beneficial role of the off-pathway oligomer in the capturing of the transient on-pathway oligomeric intermediate by serving as a monomer-releasing reservoir. This approach should also be useful in other amyloidogenic systems.