Microbiological indicators of water quality in submerged karst caves of Wakulla Springs

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A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo

Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads al- lowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an ob- servation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bac- terial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.Comment: 28 pages, 13 figure

The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the endoplasmic reticulum (ER) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show here that the proteasome has a more complex role in ricin intoxication than previously recognised, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. Our results implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated

Selection of Clostridium spp. in biological sand filters neutralizing synthetic acid mine drainage

In this study, three biological sand filter (BSF) were contaminated with a synthetic iron- [1500 mg L-1 Fe(II), 500 mg L-1 Fe(III)] and sulphate-rich (6000 mg L-1 SO2/4-) acid mine drainage (AMD) (pH = 2), for 24 days, to assess the remediation capacity and the evolution of autochthonous bacterial communities (monitored by T-RFLP and 16S rRNA gene clone libraries). To stimulate BSF bioremediation involving sulphate-reducing bacteria, a readily degradable carbon source (glucose, 8000 mg L-1) was incorporated into the influent AMD. Complete neutralization and average removal efficiencies of 81.5 (±5.6)%, 95.8 (±1.2)% and 32.8 (±14.0)% for Fe(II), Fe(III) and sulphate were observed, respectively. Our results suggest that microbial iron reduction and sulphate reduction associated with iron precipitation were the main processes contributing to AMD neutralization. The effect of AMD on BSF sediment bacterial communities was highly reproducible. There was a decrease in diversity, and notably a single dominant operational taxonomic unit (OTU), closely related to Clostridium beijerinckii, which represented up to 65% of the total community at the end of the study period.Web of Scienc

When are bacteria dead? A step towards interpreting flow cytometry profiles after chlorine disinfection and membrane integrity staining

Flow cytometry is increasingly employed by drinking water providers. Its use with appropriate fluorescent stains allows the distinction between intact and membrane-damaged bacteria, which makes it ideally suited for assessment of disinfection efficiency. In contrast to plate counting, the technology allows the visualization of the gradual loss of membrane integrity. Although this sensitivity per se is very positive, it creates the problem of how this detailed viability information compares with binary plate counts where a colony is either formed or not. Guidelines are therefore needed to facilitate interpretation of flow cytometry results and to determine a degree of membrane damage where bacteria can be considered ‘dead’. In this study we subjected Escherichia coli and environmental microorganisms in real water to increasing chlorine concentrations. Resulting flow cytometric patterns after membrane integrity staining were compared with culturability and in part with redox activity. For laboratory-grown bacteria, culturability was lost at lower disinfectant concentrations than membrane integrity making the latter a conservative viability parameter. No recovery from chlorine was observed for four days. For real water, loss of membrane integrity had to be much more substantial to completely suppress colony formation, probably due to the heterogenic composition of the natural microbial community with different members having different susceptibilities to the disinfectant

Growing Apples for Hard Cider Production in the United States-Trends and Research Opportunities

Hard cider, made by fermenting apple (Malus x domestica) juice, was at one time the most widely consumed alcoholic beverage in America. Largely abandoned after Prohibition, within the past 2 decades the rise in popularity of craft beverages has led to the reemergence of hard cider as an alternative to beer, wine, and spirits. Today, hard cider represents one of the fastest growing sectors within the craft beverage industry. The recent interest in cider presents additional marketing opportunities for apple growers and businesses currently involved in, or considering entering, the apple cider or craft beverages industries. However, the lack of a strong history or experience in selecting, producing, and using cider apples poses a significant challenge to this emerging market. This article reviews the current state of research in cider apple production, including economic feasibility, mechanized management, and cultivar evaluation and improvement

Biodiversitätsmonitoring im Südtiroler Kräuteranbau = Biodiversity surveys in medicinal and aromatic plant fields in South Tyrol

Medicinal and aromatic plants in mountain regions such as South Tyrol are cultivated on small-scale farms, which are characterized by a high diversity of cultivated crop species grown on a relatively small area. This small-scale cultivation of medicinal and aromatic plants suggests that MAP fields are of high ecological value. However, research on this topic is generally lacking. In this study flower-visiting arthropods were recorded with pan traps in three herb fields during three survey events conducted in 2021. Our results indicate that medicinal and aromatic plant fields are valuable habitats for several taxa. In total 12.570 individuals were collected. Wild bees were particularly species-rich, accounting for 10 % of the regional wild bee species pool. Next to beneficial arthropods, potential pests, such as aphids were also highly abundant. However, natural enemies possibly counteracting pests were also numerous. Overall, we conclude that medicinal and aromatic plant cultivation may act as resource-rich oases for several arthropod groups, thereby promoting biodiversity also on a broader scale.Der Anbau von Arznei- und Gewürzpflanzen zeichnet sich in der Regel durch vielfältige Anbaukulturen auf relativ kleinen Flächen aus. Dies gilt insbesondere für Südtirol, wo diese Kulturen hauptsächlich von kleinen Betrieben im Berggebiet angebaut werden. Dieser kleinflächige Anbau von Arznei- und Gewürzpflanzen lässt vermuten, dass die Betriebe einen hohen ökologischen Wert haben. Es gibt wenige Studien zur Erfassung der Biodiversität im Anbau von Arznei- und Gewürzpflanzen. Daher wurden in dieser Arbeit Kräuteranbau-Betriebe als Lebensraum für blütenbesuchende Arthropoden untersucht. An drei Untersuchungsstandorten wurden im Jahr 2021 jeweils an drei Terminen Farbschalen zur Sammlung von Arthropoden verwendet. Kräuteranbau-Betriebe stellten sich als ein wertvoller Lebensraum für verschiedene Arthropoden heraus. Insgesamt wurden 12.570 Individuen mit den Farbschalen gesammelt. Insbesondere Wildbienen waren mit 10 % des regionalen Artenpools sehr artenreich. Auch potenzielle Schädlinge, wie zum Beispiel Blattläuse, waren sehr häufig anzutreffen, wobei natürliche Feinde, wie zum Beispiel Parasitoide, ebenfalls zahlreich vertreten waren. Insgesamt können Kräuteranbaubetriebe als strukturreiche Oasen für Arthropoden fungieren und sich somit auf einer breiteren Skala positiv auf die Biodiversität auswirken

Disruption of cells in biosolids affects E. coli dynamics in storage

Achieving microbial compliance during biosolids storage can be complicated by the unpredictable increase of Escherichia coli. Thermal treatment during anaerobic digestion (AD) and the effects of dewatering may be a significant factor contributing to indicator survival. Shear forces present during dewatering may promote cell damage, releasing nutrient for E. coli growth. The effect of cell damage on E. coli survival was assessed in laboratory-scale thermal and physical disruption experiments. E. coli growth curves for disrupted treatments were compared with control conditions and quantified using flow cytometry and membrane filtration techniques. A significant difference (p < 0.05) in the level of damaged cells between control and disrupted conditions was observed. For thermal and physical disruption treatments, the peak of E. coli concentration increased significantly by 1.8 Log and 2.4 Log (CFU (colony forming units) g−1 DS), respectively, compared with control treatments. Research findings contribute to the understanding of bacterial growth and death dynamics in biosolid

Regulation of proteasome assembly and activity in health and disease

The proteasome degrades most cellular proteins in a controlled and tightly regulated manner and thereby controls many processes, including cell cycle, transcription, signalling, trafficking and protein quality control. Proteasomal degradation is vital in all cells and organisms, and dysfunction or failure of proteasomal degradation is associated with diverse human diseases, including cancer and neurodegeneration. Target selection is an important and well-established way to control protein degradation. In addition, mounting evidence indicates that cells adjust proteasome-mediated degradation to their needs by regulating proteasome abundance through the coordinated expression of proteasome subunits and assembly chaperones. Central to the regulation of proteasome assembly is TOR complex 1 (TORC1), which is the master regulator of cell growth and stress. This Review discusses how proteasome assembly and the regulation of proteasomal degradation are integrated with cellular physiology, including the interplay between the proteasome and autophagy pathways. Understanding these mechanisms has potential implications for disease therapy, as the misregulation of proteasome function contributes to human diseases such as cancer and neurodegeneration.</p