Proline biosynthesis regulates proline transport in Staphylococcus aureus.

Staphylococcus aureus is metabolically diverse with the ability to rapidly adapt to a vast array of nutrient sources. This allows the pathogen to colonize a variety of niches in the host. For instance, S. aureus is the leading cause of skin and soft tissue infections, a niche that has been shown to become glucose-depleted over the course of an infection. Previous studies have shown that in niches where glucose is deficient, S. aureus utilizes peptides and free amino acids as nutrient sources. Primarily, these amino acids include glutamate and amino acids that can serve as substrates for glutamate synthesis. While arginine and histidine serve as substrates in glutamate synthesis, proline is the primary source of glutamate. Indeed, S. aureus utilizes proline as a secondary carbon source only when glucose is absent, and it can be synthesized from arginine or acquired via proline transporters from its environment. Although S. aureus encodes two putative pathways for proline biosynthesis, it has been shown that pyrroline-5-carboxylate reductase (encoded by proC) is the sole proline biosynthetic pathway in S. aureus. Studies from our laboratory have revealed that despite encoding five putative proline transporters (B7H15_03660, opuC, opuD, proP, putP), only two of the transporters, PutP and B7H15_03660 are responsible for a majority of proline transport under the laboratory conditions tested. Surprisingly, when we introduced the proC mutation into the B7H15_03660 putP double mutant, we observed proline-dependent growth, even though the primary proline transporters and proline biosynthetic pathway were knocked-out. In contrast, a transporter null ΔproC strain was unable to grow. These data suggest that inhibiting proline biosynthesis alters proline transport, and therefore one or more of the additional transporters, OpuC, OpuD, and/or ProP, are activated under these conditions. After introducing opuC, opuD, and/or proP mutations into the Δ03660 ΔputP ΔproC strain, we found that both OpuC and ProP are important for proline transport. Additionally, we observed proline-dependent growth in a proline transporter null ΔproC strain when high amounts of exogenous proline are added to the media. This growth appears to be due to an acquired mutation and will be studied more in the future. Overall these studies have revealed that proline transport is tightly linked to proline biosynthesis.https://digitalcommons.unmc.edu/surp2021/1021/thumbnail.jp

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased Vmax and decreased the Km for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family member

Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development

Prevalence of Non-O157:H7 Shiga Toxin-Producing \u3ci\u3eEscherichia coli\u3c/i\u3e in Diarrheal Stool Samples from Nebraska

We determined the prevalence of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stool samples from Nebraska by three methods: cefixime-tellurite sorbitol MacConkey (CT-SMAC) culture, enterohemorrhagic E. coli (EHEC) enzyme immunoassay, and stx1,2 polymerase chain reaction (PCR). Fourteen (4.2%) of 335 specimens were positive by at least one method (CT-SMAC culture [6 of 14], EHEC enzyme immunoassay [13 of 14], stx1,2 PCR [14 of 14]). Six contained serogroup 0157, while non-0157 were as prevalent as 0157 serogroups

Why Dose Frequency Affects Spoken Vocabulary in Preschoolers With Down Syndrome

In an earlier randomized clinical trial, daily communication and language therapy resulted in more favorable spoken vocabulary outcomes than weekly therapy sessions in a subgroup of initially nonverbal preschoolers with intellectual disabilities that included only children with Down syndrome (DS). In this reanalysis of the dataset involving only the participants with DS, we found that more therapy led to larger spoken vocabularies at posttreatment because it increased children’s canonical syllabic communication and receptive vocabulary growth early in the treatment phase

First Records of Established Populations of \u3ci\u3eIxodes scapularis\u3c/i\u3e (Acari: Ixodidae) Collected from Three Nebraska Counties

Reported cases of Lyme disease in Nebraska have been assumed to be imported from other endemic areas. Previous surveillance efforts provided no evidence of established populations as only individual specimens of Ixodes scapularis (Say) had been collected. In the winter of 2018, adult I. scapularis were found on a dog at Two Rivers State Recreation Area, Douglas County, prompting tick collection at the site and nearby natural areas. In May 2019, all life stages of host-seeking I. scapularis were collected using dragging and flagging techniques in sites located near the Platte River in Douglas, Sarpy, and Saunders counties. This is the first documentation of established populations of I. scapularis in Nebraska

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

BACKGROUND: The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP). PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS) lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1-69; however, this targeting sequence has not been experimentally examined or validated. RESULTS: Using a site-directed mutagenesis approach, we found that lysines 19 and 20, located within a previously described bipartite NLS, are not required for nuclear localization of PARP-2. In contrast, lysine 36, which is located within a predicted classical monopartite NLS, was required for PARP-2 nuclear localization. While wild type PARP-2 interacted with importin alpha3 and to a very weak extent with importin alpha1 and importin alpha5, the mutant PARP-2 (K36R) did not interact with importin alpha3, providing a molecular explanation why PARP-2 (K36R) is not targeted to the nucleus. CONCLUSION: Our results provide strong evidence that lysine 36 of PARP-2 is a critical residue for proper nuclear targeting of PARP-2 and consequently for the execution of its biological functions

Cooperative Lewis pairs based on late transition metals: activation of small molecules by platinum(0) and B(C6F5)3

A Lewis basic platinum(0)–CO complex supported by a diphosphine ligand and B(C6F5)3 act cooperatively, in a manner reminiscent of a frustrated Lewis pair, to activate small molecules such as hydrogen, CO2, and ethene. This cooperative Lewis pair facilitates the coupling of CO and ethene in a new way

Alkalic-Type Epithermal Gold Deposit Model: Chapter R of Mineral Deposit Models for Resource Assessment

This report summarizes the primary characteristics of alkalic-type epithermal gold (Au) deposits and provides an updated descriptive model. These deposits, primarily of Mesozoic to Neogene age, are among the largest epithermal gold deposits in the world. Considered a subset of low-sulfidation epithermal deposits, they are spatially and genetically linked to small stocks or clusters of intrusions containing high alkali-element contents. Deposits occur as disseminations, breccia-fillings, and veins and may be spatially and genetically related to skarns and low-grade porphyry copper (Cu) or molybdenum (Mo) systems. Gold commonly occurs as native gold, precious metal tellurides, and as sub-micron gold in arsenian pyrite. Quartz, carbonate, fluorite, adularia, and vanadian muscovite/roscoelite are the most common gangue minerals. Alkalic-type gold deposits form in a variety of geological settings including continent-arc collision zones and back-arc or post-subduction rifts that are invariably characterized by a transition from convergent to extensional or transpressive tectonics. The geochemical compositions of alkaline igneous rocks spatially linked with these deposits span the alkaline-subalkaline transition. Their alkali enrichment may be masked by potassic alteration, but the unaltered or least altered rocks (1) have chondrite normalized patterns that are commonly light rare earth element (LREE) enriched, (2) are heavy rare earth element (HREE) depleted, and (3) have high large ion lithophile contents and variable enrichment of high-field strength elements. Radiogenic isotopes suggest a mantle derivation for the alkalic magmas but allow crustal contamination. Oxygen and hydrogen isotope compositions show that the fluids responsible for deposit formation are dominantly magmatic, although meteoric or other external fluids (seawater, evolved groundwater) also contributed to the ore-forming fluids responsible for these deposits. Carbon and sulfur isotope compositions in vein-hosted carbonates and sulfide gangue minerals, respectively, coincide with magmatic values, although a sedimentary source of carbon and sulfur is evident in several deposits. Deep-seated structures are critical for the upwelling of hydrous alkalic magmas and for focusing magmatic-hydrothermal fluids to the site of precious metal deposition. The source of gold, silver (Ag), tellurium (Te), vanadium (V), and fluorine (F) was probably the alkalic igneous rocks themselves, and the coexistence of native gold, gold tellurides, and roscoelite in several deposits is primarily a function of similar physicochemical conditions during deposition (for example, overlapping pH and oxygen fugacity (fO2). Potential environmental impacts related to the mining and processing of alkalic-type epithermal gold deposits include acid mine drainage with high levels of metals, especially zinc (Zn), copper, lead (Pb), and arsenic. However, because alkalic-type gold deposits typically contain carbonates, which contribute calcium and magnesium ions that increase water hardness, aquatic life may be afforded some protection. Impacts vary widely as a function of host rocks, climate, topography, and mining methods. Geologic mapping to (1) highlight the distribution of potassic alteration; (2) define fault density and orientation of structures; (3) determine the distribution of alkaline rocks and hydrothermal breccias; and (4) identify uniquely colored gangue minerals, such as fluorite and roscoelite, will be critical to exploration and future discoveries. Geophysical techniques that identify potassium (K) anomalies (for example, radiometric and spectroscopic surveys), as well as magnetic, resistivity, aeromagnetic, and gravity surveys, may help locate zones of high-permeability that control advecting hydrothermal fluids. Geochemical surveys that include analyses for Au, Ag, barium, Te, K, F, V, Mo, and mercury, which are key elements in these deposits, should be undertaken along with the measurement of other pathfinder elements such as arsenic, bismuth, Cu, iron, nickel, Pb, antimony, selenium, and Zn