The composition of the gut microbiota shapes the colon mucus barrier

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria—which is comparable to what we observed in free-living wild mice—whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Highly sensitive systems for experimental insertional mutagenesis in repair-deficient genetic environment in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: New opportunities for studying postreplication repair of double-stranded DNA breaks and mechanisms of transposable element migration

Spontaneous mutations in Drosophila melanogaster are related mainly to transposable elements (TEs). They are caused by both migration of TEs over the genome (transpositions) and the ability of TEs to induce chromosomal mutations. Migration of DNA transposons is accompanied by formation of double-strand DNA breaks (DSBs), which are repaired by host repair systems encoded by genes for recombination repair. We relied on this notion to develop a combined approach to the investigation of the type of DNA breaks accompanying transpositions; investigation of systems involved in DSB repair; and detection of repair genes, whose products were involved in repair of DNA breaks induced by TE transposition. The approach is based on the combination of experimental insertional mutagenesis systems and genetic environment deficient for enzymes of the repair system in a single genome. The main advantages of this approach are versatility, wide applicability, and simple design.     

Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells

Background  

Pertussis toxin (PT) is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.     

The Fsp4HI restriction-modification system: Gene cloning, comparison of protein structures, and biochemical properties of recombinant DNA methyltransferase M.Fsp4HI

Genes coding for the Flavobacterium sp. 4H restriction-modification (RM) system, which recognizes the sequence 5′-GCNGC-3′, were cloned in Escherichia coli ER2267 and sequenced. The Fsp4HI RM system includes two genes: one for DNA methyltransferase (M.) and the other for restriction endonuclease (R.), immediately following the former in the same direction. The genes partly overlap. According to the deduced amino acid sequences, M.Fsp4HI belongs to C5 DNA methyltransferases, whereas R.Fsp4HI is only slightly similar to some restriction enzymes recognizing similar sequences. M.Fsp4HI was purified by column chromatography. The optimal conditions for the enzyme are 30°C and pH 7.5. M.Fsp4HI modifies the first cytosine in 5′-GCNGC-3′.     

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

Background  

MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species.     

Quantitative estimation of gibberellic acid by paper chromatography

Метод для количественного определения от gibberellic кислоты в процессе брожения средства массовой информации, с использованием бумаги по убыванию хроматографии в butylacetate воды описана. Образца корректируется, чтобы рН 2.5-3.0, добыто с н-бутанола, и 0,05 мл. органического слоя пятнами на Хроматографический бумагу. После equilibration от Атмосфера в банке, chromatogram Разработана в butylacetate насыщенных с водой, за 7 часов, и растворитель разрешено покинуть капельного нижней части листа. Обнаружение осуществляется путем опрыскивания с 3% раствор серной кислоты в метаноле и после сушки бумаги, пятна с синий u.v. флуоресценции наблюдается. Определенный артикль площадь пятна оценивается с помощью калибровочной кривой, заговор с ценностей, стандартов, соответствующих 20, 60 и 120 μ g. gibberellic кислоты. Погрешность оценки составляет ± 10-15% когда оценки выполняются тщательно. Низкий предел чувствительности 5 μg.