Contamination of a hospital environment byClostridium difficile

Clostridium difficile was recovered from a variety of environmental sites in three hospital rooms occupied by a patient who had colitis due to this organism.C. difficile was detected for 40 days after the patient was moved from one of these rooms. These findings suggest that the contaminated hospital environment may be a clinically significant reservoir forC. difficile and that this organism may be a nosocomial pathogen. Isolation of patients and adequate decontamination of rooms may be needed to minimize risk to other patients.     

Interplay of Protein Binding Interactions,DNA Mechanics,and Entropy in?DNA Looping Kinetics

DNA looping plays a key role in many fundamental biological processes, including gene regulation, recombination, and chromosomal organization. The looping of DNA is often mediated by proteins whose structural features and physical interactions can alter the length scale at which the looping occurs. Looping and unlooping processes are controlled by thermodynamic contributions associated with mechanical deformation of the DNA strand and entropy arising from thermal fluctuations of the conformation. To determine how these confounding effects influence DNA looping and unlooping kinetics, we present a theoretical model that incorporates the role of the protein interactions, DNA mechanics, and conformational entropy. We show that for shorter DNA strands the interaction distance affects the transition state, resulting in a complex relationship between the looped and unlooped state lifetimes and the physical properties of the looped DNA. We explore the range of behaviors that arise with varying interaction distance and DNA length. These results demonstrate how DNA deformation and entropy dictate the scaling of the looping and unlooping kinetics versus the J-factor, establishing the connection between kinetic and equilibrium behaviors. Our results show how the twist-and-bend elasticity of the DNA chain modulates the kinetics and how the influence of the interaction distance fades away at intermediate to longer chain lengths, in agreement with previous scaling predictions.     

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid‐mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.     

Solvent accessibility changes in a Na+-dependent C4-dicarboxylate transporter suggest differential substrate effects in a multistep mechanism

The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyze the Na⁺-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na⁺ and succinate. We also observe separable effects of Na⁺ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na⁺ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site–associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.     

Production of diverse brGDGTs by Acidobacterium Solibacter usitatus in response to temperature,pH, and O2 provides a culturing perspective on brGDGT proxies and biosynthesis

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane lipids that are frequently employed as paleoenvironmental proxies because of the strong empirical correlations between their relative abundances and environmental temperature and pH. Despite the ubiquity of brGDGTs in modern and paleoenvironments, the source organisms of these enigmatic compounds have remained elusive, requiring paleoenvironmental applications to rely solely on observed environmental correlations. Previous laboratory and environmental studies have suggested that the globally abundant bacterial phylum of the Acidobacteria may be an important brGDGT producer in nature. Here, we report on experiments with a cultured Acidobacterium, Solibacter usitatus, that makes a large portion of its cellular membrane (24 ± 9% across all experiments) out of a structurally diverse set of tetraethers including the common brGDGTs Ia, IIa, IIIa, Ib, and IIb. Solibacter usitatus was grown across a range of conditions including temperatures from 15 to 30°C, pH from 5.0 to 6.5, and O₂ from 1% to 21%, and demonstrated pronounced shifts in the degree of brGDGT methylation across these growth conditions. The temperature response in culture was in close agreement with trends observed in published environmental datasets, supporting a physiological basis for the empirical relationship between brGDGT methylation number and temperature. However, brGDGT methylation at lower temperatures (15 and 20°C) was modulated by culture pH with higher pH systematically increasing the degree of methylation. In contrast, pH had little effect on brGDGT cyclization, supporting the hypothesis that changes in bacterial community composition may underlie the link between cyclization number and pH observed in environmental samples. Oxygen concentration likewise affected brGDGT methylation highlighting the potential for this environmental parameter to impact paleotemperature reconstruction. Low O₂ culture conditions further resulted in the production of uncommon brGDGT isomers that could be indicators of O₂ limitation. Finally, the production of brGTGTs (trialkyl tetraethers) in addition to the previously discovered iso-C15-based mono- and diethers in S. usitatus suggests a potential biosynthetic pathway for brGDGTs that uses homologs of the archaeal tetraether synthase (Tes) enzyme for tetraether synthesis from diethers.     

Microbial cell responses to a non-ionic surfactant. II. Effects as assessed by fluorescein diacetate uptake

Summary The effects of a non-ionic surfactant, Pluronic F-68, on uptake of fluorescein diacetate (FDA) into yeast cells as measured by increase in fluorescence atca. 500 nm have been studied. The rate of FDA uptake was increased almost 2.5 fold by incubating cells with up to 5% commercial grade pluronic but this depended on source and degree of purity. Dye uptake was reduced by pre-purification of pluronic but this again depended on source of material. None of the pluronic preparations had any significant effects on the rate of enzymemediated FDA hydrolysis by cell-free extracts.     

Strontium marking of hatchery-reared coho salmon (Oncorhynchm kisutch, Walbaum)

In the management of saimonid populations it is often necessary to distinguish hatchery-reared from wild stocks. This study examines the feasability of marking fish tissue by substituting a biologically rare element, strontium, for calcium.
Sixteen-month-old hatchery-reared coho salmon ( Oncorhynchus kisutch , Walbaum 1792) were fed a diet, to which 10000 ppm stable strontium was added, for 60 days. Prior to their seaward migration in June 1976, the treated coho contained 32 times as much strontium in their vertebrae as did the control coho.
Sixty-eight precocious males'jacks' returned to the hatchery in the fall of 1976 and 1200 adults in the fall of 1977. Strontium-treated 'jacks' contained 1·4 times as much strontium as did control 'jacks' however, treated and untreated fish could not be distinguished as adults. The reduction in the strontium concentration of the treated fish could be accounted for by growth.