Microbiology of water and wastewater: Discovery of a new genus numerically dominant in municipal wastewater and antimicrobial resistances in numerically dominant bacteria from Oklahoma lakes.

Three bacterial isolates from municipal wastewater were investigated for their phenotypic, biochemical, and molecular characters. All strains were isolated from municipal wastewater, and the type strain, NRS1T, was isolated as a numerically dominant heterotrophic bacterium. The isolates NRS1T, NRS30, and NRS32 were Gram-negative, catalase and oxidase positive, nonmotile, non spore-forming, yellow to orange pigmented rods. Pigments were of the carotenoid-type, and no flexirubin-type pigments were detected. All strains degraded gelatin, starch, esculin, casein, and weakly degraded DNA. None degraded cellulose, chitin, agar, urea, pectin, alginate, uric acid, xanthine, or hypoxanthine. Indole was produced by all strains. All strains were methyl red and Voges-Proskauer negative, and nitrate was not reduced. Pyruvate was observed as an end-product from growth on unbuffered glucose. The predominant respiratory quinone was menaquinone MK-6. The DNA base composition was 31 mole % G+C. 16S rRNA gene sequence analysis indicated that these three strains, as well as two uncharacterized strains, MRS7 and MRS14, were members of the Chryseobacterium-Bergeyella-Riemerella branch of the family Flavobacteriaceae. This bacterium could not be ascribed to any known genus by cellular fatty acid analysis or BIOLOG analysis. All strains were phylogenetically unaffiliated with any described genus from the 16S ribosomal gene sequence analysis. It is therefore proposed that the unknown bacterium be classified in a new genus Cloacibacterium gen. nov., as Cloacibacterium normanensis gen. nov. sp. nov. The type species of the genus Cloacibacterium is Cloacibacterium normanensis strain NRS1T (=ATCC BAA-825T) (=DSM 15886T) (=CCUG 46293T) (=CIP 108613 T). The GenBank accession number for the 16S gene sequence of strain NRS1T is AJ575430. The habitat for Cloacibacterium is municipal wastewater, where it was discovered as a numerically dominant species

On the Importance of Atomic Fluctuations, Protein Flexibility, and Solvent in Ion Permeation

Proteins, including ion channels, often are described in terms of some average structure and pictured as rigid entities immersed in a featureless solvent continuum. This simplified view, which provides for a convenient representation of the protein's overall structure, incurs the risk of deemphasizing important features underlying protein function, such as thermal fluctuations in the atom positions and the discreteness of the solvent molecules. These factors become particularly important in the case of ion movement through narrow pores, where the magnitude of the thermal fluctuations may be comparable to the ion pore atom separations, such that the strength of the ion channel interactions may vary dramatically as a function of the instantaneous configuration of the ion and the surrounding protein and pore water. Descriptions of ion permeation through narrow pores, which employ static protein structures and a macroscopic continuum dielectric solvent, thus face fundamental difficulties. We illustrate this using simple model calculations based on the gramicidin A and KcsA potassium channels, which show that thermal atomic fluctuations lead to energy profiles that vary by tens of kcal/mol. Consequently, within the framework of a rigid pore model, ion-channel energetics is extremely sensitive to the choice of experimental structure and how the space-dependent dielectric constant is assigned. Given these observations, the significance of any description based on a rigid structure appears limited. Creating a conducting channel model from one single structure requires substantial and arbitrary engineering of the model parameters, making it difficult for such approaches to contribute to our understanding of ion permeation at a microscopic level

The effect of hydrophobic and hydrophilic channel walls on the structure and diffusion of water and ions

Molecular dynamics simulations are carried out to determine the effects of channel wall structure on water and ion properties. We compare hydrophobic (Lennard-Jones 5-3 and atomic) and molecular-hydrophilic cylindrical pores of 2-6 Å in effective radius

Method for Monitoring Quality of Extension Programs: A Dashboard Construction Process

We describe a process for construction and use of a dashboard for monitoring the quality of in-person nonformal education programs. We followed the process to develop a 4-H special interest (SPIN) club dashboard, but the steps are applicable to any education program that includes instruction over multiple sessions. The dashboard construction process comprises selecting performance measures, choosing data collection strategies, designing the content and layout of the dashboard, collecting data, and populating the dashboard with performance metrics. Although these process steps are intuitive, specific decision options within each step can be complex. Our article includes discussion of these complexities in the context of our SPIN club application

The role of membrane thickness in charged protein–lipid interactions

AbstractCharged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26kcal/mol for 18 carbons to 6kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pKa shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein–lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function

Ball-and-chain inactivation in a calcium-gated potassium channel

Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a ‘ball-and-chain’ mechanism3–7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum—a purely calcium-gated and inactivating channel—in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism