Sewage reflects the distribution of human faecal Lachnospiraceae

Faecal pollution contains a rich and diverse community of bacteria derived from animals and humans, many of which might serve as alternatives to the traditional enterococci and Escherichia coli faecal indicators. We used massively parallel sequencing (MPS) of the 16S rRNA gene to characterize microbial communities from wastewater treatment plant (WWTP) influent sewage from 12 cities geographically distributed across the USA. We examined members of the Clostridiales, which included the families Clostridiaceae, Lachnospiraceae and Ruminococcaceae for their potential as sewage indicators. Lachnospiraceae was one of the most abundant groups of faecal bacteria in sewage, and several Lachnospiraceae high‐abundance sewage pyrotags occurred in at least 46 of 48 human faecal samples. Clone libraries targeting Clostridium coccoides (C. coccoides) in sewage samples demonstrated that Lachnospiraceae‐annotated V6 pyrotags encompassed the previously reported C. coccoides group. We used oligotyping to profile the genus Blautia within Lachnospiraceae and found oligotypes comprised of 24 entropy components that showed patterns of host specificity. These findings suggest that indicators based on Blautia might have the capacity to discriminate between different faecal pollution sources. Development of source‐specific alternative indicators would enhance water quality assessments, which leads to improved ecosystem health and reduced human health risk due to waterborne disease.     

Prion induction by the short-lived, stress-induced protein Lsb2 is regulated by ubiquitination and association with the actin cytoskeleton

Yeast prions are self-perpetuating, QN-rich amyloids that control heritable traits and serve as a model for mammalian amyloidoses. De novo prion formation by overproduced prion protein is facilitated by other aggregated QN-rich protein(s) and is influenced by alterations of protein homeostasis. Here we explore the mechanism by which the Las17-binding protein Lsb2 (Pin3) promotes conversion of the translation termination factor Sup35 into its prion form, [PSI(+)]. We show that Lsb2 localizes with some Sup35 aggregates and that Lsb2 is a short-lived protein whose levels are controlled via the ubiquitin-proteasome system and are dramatically increased by stress. Loss of Lsb2 decreases stability of [PSI(+)] after brief heat shock. Mutations interfering with Lsb2 ubiquitination increase prion induction, while a mutation eliminating association of Lsb2 with the actin cytoskeleton blocks its aggregation and prion-inducing ability. These findings directly implicate the UPS and actin cytoskeleton in regulating prions via a stress-inducible QN-rich protein.     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26Aa和cry28Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26Aa和cry28Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26Aa在仅消除cry26Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26Aa所在质粒有关。     

Comparison of Sewage and Animal Fecal Microbiomes by Using Oligotyping Reveals Potential Human Fecal Indicators in Multiple Taxonomic Groups

Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales, but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.     

Systems metabolic engineering design: Fatty acid production as an emerging case study

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C₆ to C₁₆ are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel‐like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high‐yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high‐yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain‐length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain‐lengths and functionalities. Biotechnol. Biotechnol. Bioeng. 2014;111: 849–857. © 2014 Wiley Periodicals, Inc.     

Age-Related Shifts in the Density and Distribution of Genetic Marker Water Quality Indicators in Cow and Calf Feces

Calves make up about 16% of the current bovine population in the United States and can excrete high levels of human pathogens in their feces. We describe the density and distribution of genetic markers from 9 PCR- and real-time quantitative PCR-based assays, including CF128, CF193, CowM2, CowM3, GenBac3, Entero1, EC23S857, CampF2, and ttr-6, commonly used to help assess ambient surface water quality. Each assay was tested against a collection of 381 individual bovine fecal samples representing 31 mother and calf pairings collected over a 10-month time period from time of birth through weaning. Genetic markers reported to be associated with ruminant and/or bovine fecal pollution were virtually undetected in calves for up to 115 days from birth, suggesting that physiological changes in calf ruminant function impact host-associated genetic marker shedding. In addition, general fecal indicator markers for Bacteroidales, Escherichia coli, and Enterococcus spp. exhibited three separate trends across time, indicating that these bacteria respond differently to age-related physiological and dietary changes during calf development. The results of this study suggest that currently available PCR-based water quality indicator technologies can under- or overestimate fecal pollution originating from calves and identify a need for novel calf-associated source identification methods.     

Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.     

Effects of Piper hispidinervum on spermatogenesis and histochemistry of ovarioles of Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3^rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest.     

Improvements in metabolic flux analysis using carbon bond labeling experiments: bondomer balancing and Boolean function mapping

The biosynthetically directed fractional (13)C labeling method for metabolic flux evaluation relies on performing a 2-D [(13)C, (1)H] NMR experiment on extracts from organisms cultured on a uniformly labeled carbon substrate. This article focuses on improvements in the interpretation of data obtained from such an experiment by employing the concept of bondomers. Bondomers take into account the natural abundance of (13)C; therefore many bondomers in a real network are zero, and can be precluded a priori--thus resulting in fewer balances. Using this method, we obtained a set of linear equations which can be solved to obtain analytical formulas for NMR-measurable quantities in terms of fluxes in glycolysis and the pentose phosphate pathways. For a specific case of this network with four degrees of freedom, a priori identifiability of the fluxes was shown possible for any set of fluxes. For a more general case with five degrees of freedom, the fluxes were shown identifiable for a representative set of fluxes. Minimal sets of measurements which best identify the fluxes are listed. Furthermore, we have delineated Boolean function mapping, a new method to iteratively simulate bondomer abundances or efficiently convert carbon skeleton rearrangement information to mapping matrices. The efficiency of this method is expected to be valuable while analyzing metabolic networks which are not completely known (such as in plant metabolism) or while implementing iterative bondomer balancing methods.