Genomic Characterization of Non-Mucus-Adherent Derivatives of Lactobacillus rhamnosus GG Reveals Genes Affecting Pilus Biogenesis

Lactobacillus rhamnosus GG is one of the best-characterized lactic acid bacteria and can be considered a probiotic paradigm. Comparative and functional genome analysis showed that L. rhamnosus GG harbors a genomic island including the spaCBA-srtC1 gene cluster, encoding the cell surface-decorating host-interacting pili. Here, induced mutagenesis was used to study pilus biogenesis in L. rhamnosus GG. A combination of two powerful approaches, mutation selection and next-generation sequencing, was applied to L. rhamnosus GG for the selection of pilus-deficient mutants from an enriched population. The isolated mutants were first screened by immuno-dot blot analysis using antiserum against pilin proteins. Relevant mutants were selected, and the lack of pili was confirmed by immunoelectron microscopy. The pilosotype of 10 mutant strains was further characterized by analyzing pilin expression using Western blot, dot blot, and immunofluorescence methods. A mucus binding assay showed that the mutants did not adhere to porcine intestinal mucus. Comparative genome sequence analysis using the Illumina MiSeq platform allowed us to determine the nature of the mutations in the obtained pilus-deficient derivatives. Three major classes of mutants with unique genotypes were observed: class I, with mutations in the srtC1 gene; class II, with a deletion containing the spaCBA-srtC1 gene cluster; and class III, with mutations in the spaA gene. Only a limited number of collateral mutations were observed, and one of the pilus-deficient derivatives with a deficient srtC1 gene contained 24 other mutations. This strain, PB12, can be considered a candidate for human trials addressing the impact of the absence of pili.     

Levels of endogenous abscisic acid and indole-3-acetic acid influence shoot organogenesis in callus cultures of rice subjected to osmotic stress

Osmotic stress and endogenous hormone levels may have a role in shoot organogenesis, but a systematic study has not yet to investigate the links. We evaluated the changes of the endogenous indole-3-acetic acid (IAA) and abscisic acid (ABA) levels in rice (Oryza sativa L. cv. Tainan 5) callus during shoot organogenesis induced by exogenous plant growth regulator treatments or under osmotic stress. Non-regenerable callus showed low levels of endogenous ABA and IAA, with no fluctuation in level during the period evaluated. The addition of 100 μM ABA or 2 mM anthranilic acid (IAA precursor) into Murashige and Skoog basal induction medium containing 10 μM 2,4-D enhanced the regeneration frequency slightly, to 5 and 35%, respectively, and their total cellular ABA or IAA levels were increased significantly, correspondingly to the treatments. However, the regeneration frequency was greatly increased to 80% after treatment with 0.6 M sorbitol or 100 μM ABA and 2 mM anthranilic acid combined. Both treatments produced high levels of total cellular ABA and IAA at the callus stage, which was quickly decreased on the first day after transfer to regeneration medium. Thus, osmotic stress-induced simultaneous accumulation of endogenous ABA and IAA is involved in shoot regeneration in rice callus.     

Evaluation of the catalytic specificity,biochemical properties,and milk clotting abilities of an aspartic peptidase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.     

Allee threshold and extinction threshold for spatially explicit metapopulation dynamics with Allee effects

In this paper, we investigate a spatially explicit metapopulation model with Allee effects. We refer to the patch occupancy model introduced by Levins (Bull Entomol Soc Am 15:237–240, 1969) as a spatially implicit metapopulation model, i.e., each local patch is either occupied or vacant and a vacant patch can be recolonized by a randomly chosen occupied patch from anywhere in the metapopulation. When we transform the model into a spatially explicit one by using a lattice model, the obtained model becomes theoretically equivalent to a “lattice logistic model” or a “basic contact process”. One of the most popular or standard metapopulation models with Allee effects, developed by Amarasekare (Am Nat 152:298–302, 1998), supposes that those effects are introduced formally by means of a logistic equation. However, it is easier to understand the ecological meaning of associating Allee effects with this model if we suppose that only the logistic colonization term directly suffers from Allee effects. The resulting model is also well defined, and therefore we can naturally examine it by Monte Carlo simulation and by doublet and triplet decoupling approximation. We then obtain the following specific features of one-dimensional lattice space: (1) the metapopulation as a whole does not have an Allee threshold for initial population size even when each local population follows the Allee effects; and (2) a metapopulation goes extinct when the extinction rate of a local population is lower than that in the spatially implicit model. The real ecological metapopulation lies between two extremes: completely mixing interactions between patches on the one hand and, on the other, nearest neighboring interactions with only two nearest neighbors. Thus, it is important to identify the metapopulation structure when we consider the problems of invasion species such as establishment or the speed of expansion.     

Fine structure of the ventral nerve centre and interspecific identification of individual neurons in the enigmatic Chaetognatha

The enigmatic arrow worms (Chaetognatha) are marine carnivores and among the most abundant planktonic organisms. Their phylogenetic position has been heavily debated for a long time. Most recent molecular studies still provide a diverging picture and suggest arrow worms to be some kind of basal protostomes. In an effort to understand the organization of the nervous system in this clade for a broad comparison with other Metazoa we analysed the ultrastructure of the ventral nerve centre in Spadella cephaloptera by transmission electron microscopy. We were able to identify six different types of neurons in the bilateral somata clusters by means of the cytoplasmic composition (regarding the structure of the neurite and soma including the shape and eu-/heterochromatin ratio within the nucleus) as well as the size and position of these neurons. Furthermore, our study provides new insights into the neuropil composition of the ventral nerve centre and several other fine structural features. Our second goal was to examine if individually identifiable neurons are present in the ventral nerve centres of four chaetognath species, Sagitta setosa, Sagitta enflata, Pterosagitta draco, and Spadella cephaloptera. For that purpose, we processed whole mount specimens of these species for immunolocalization of RFamide-related neuropeptides and analysed them with confocal laser-scanning microscopy. Our experiments provide evidence for the interspecific homology of individual neurons in the ventral nerve centres of these four chaetognath species suggesting that the potential to generate serially arranged neurons with individual identities is part of their ground pattern.     

River Red Gum (Eucalyptus camaldulensis Dehnh.) organic matter as a carbon source in the lower Darling River,Australia

The Darling River, in New South Wales, Australia, is a large semi-arid system with a highly variable flow regime, characterised by unpredictable events of flooding and drought. In large lowland rivers like the Darling, lateral (river-floodplain) interactions can greatly influence both physical and biological components of the system. The floodplain and riparian zone of the Darling River is dominated by River Red Gum (RRG), Eucalyptus camaldulensis. The large amount of organic matter they produce accumulates on the floodplain and on benches within the channel, and is subject to alternate periods of flooding and drying as a result of highly variable flows. This paper examines the effect of alternate periods of flooding and drying on the processing of E. camaldulensis organic matter. Results of the 6-month in situ field study, together with results from laboratory experiments comparing dissolved organic carbon (DOC) release from various RRG litter types, suggest that RRG leaves provide the most bio-available source of carbon to the system, while bark may be more important as a habitat for invertebrates and other fauna. Laboratory experiments exploring the effect of drying and re-flooding on litter breakdown and release of DOC suggested that the majority of DOC was released from RRG leaves in the first 24 h of inundation. Also, upon drying and re-flooding of the leaves, a smaller but significant release of DOC occurred. However, an alternative wet/dry cycle did not affect weight loss of the leaf litter. Results of the field and lab experiments suggest that RRG leaves represent an important source of carbon to the Darling River, with inputs being influenced by the highly variable flow regime.     

Microbial boundstone dominated carbonate slope (Upper Carboniferous,N Spain): Microfacies,lithofacies distribution and stratal geometry

Summary The Carboniferous, particularly during the Serpukhovian and Bashkirian time, was a period of scarce shallow-water calcimicrobial-microbialite reef growth. Organic frameworks developed on high-rising platforms are, however, recorded in the Precaspian Basin subsurface, Kazakhstan, Russia, Japan and Spain and represent uncommon occurrences within the general trend of low accumulation rates and scarcity of shallow-water reefs. Sierra del Cuera (Cantabrian Mountains, N Spain) is a well-exposed high-rising carbonate platform of Late Carboniferous (Bashkirian-Moscovian) age with a microbial boundstone-dominated slope dipping from 20° up to 45°. Kilometer-scale continuous exposures allow the detailed documentation of slope geometry and lithofacies spatial distribution. This study aims to develop a depositional model of steep-margined Late Paleozoic platforms built by microbial carbonates and to contribute to the understanding of the controlling factors on lithofacies characteristics, stacking patterns, accumulation rates and evolution of the depositional architecture of systems, which differ from light-dependent coralgal platform margins. From the platform break to depths of nearly 300 m, the slope is dominated by massive cement-rich boundstone, which accumulated through the biologically induced precipitation of micrite. Boundstone facies (type A) with peloidal carbonate mud, fenestellid and fistuliporid bryozoans, sponge-like molds and primary cavities filled by radiaxial fibrous cement occurs all over the slope but dominates the deeper settings. Type B boundstone consists of globose centimeter-scale laminated accretionary structures, which commonly host botryoidal cement in growth cavities. The laminae nucleate around fenestellid bryozoans, sponges, Renalcis and Girvanella-like filaments. Type B boundstone typically occurs at depths between 20–150 m to locally more than 300 m and forms the bulk of the Bashkirian prograding slope. The uppermost slope boundstone (type C; between 0 and 20–100 m depth) includes peloidal micrite, radiaxial fibrous cement, bryozoans, sponge molds, Donezella, Renalcis, Girvanella, Ortonella, calcareous algae and calcitornellid foraminifers. From depths of 80–200 m to 450 m, 1–30 m thick lenses of crinoidal packstone, spiculitic wackestone, and bryozoan biocementstone with red-stained micrite matrix are episodically intercalated with boundstone and breccias. These layers increase in number from the uppermost Bashkirian to the Moscovian in parallel with the change from a rapidly prograding to an aggrading architecture. The red-stained strata share comparable features with Lower Carboniferous deeper-water mud-mound facies and were deposited during relative rises of sea level and pauses in boundstone production. Rapid relative sea-level rises might have been associated with changes in oceanographic conditions not favourable for thecalcimicrobial boundstone growth, such as upwelling of colder, nutrient-rich waters lifting the thermocline to depths of 80–200 m. Downslope of 150–300 m, boundstones interfinger with layers of matrix-free breccias, lenses of matrix-rich breccias, platform- and slope-derived grainstone and crinoidal packstone. Clast-supported breccias bound by radiaxial cement are produced by rock falls and avalanches coeval to boundstone growth. Matrix-rich breccias are debris flow deposits triggered by the accumulation of red-stained layers. Debris flows develop following the relative sea-level rises, which favour the deposition of micrite-rich lithofacies on the slope rather than being related to relative sea-level falls and subaerial exposures. The steep slope angles are the result of in situ growth and rapid stabilization by marine cement in the uppermost part, passing into a detrital talus, which rests at the angle of repose of noncohesive material. In the Moscovian, the aggradational architecture and steeper clinoforms are the result of increased accommodation space due to tectonic subsidence and due to a reduction of slope accumulation rates (from 240±45−605±35 m/My to 130±5 m/My). The increasing number of red-stained layers and the decrease of boundstone productivity are attributed to environmental changes in the adjacent basin, in particular during relative rises of sea level and to possible cooling due to icehouse conditions. The geometry of the depositional system appears to be controlled by boundstone growth rates. During the Bashkirian, the boundstone growth potential is at least 10 times greater than average values for ancient carbonate systems. The slope progradation rates (nearly 400–1000 m/My) are similar to the highest values deduced for the Holocene Bahamian prograding platform margin. The fundamental differences with modern systems are that progradation of the microbial-boundstone dominated steep slope is primarily controlled by boundstone growth rates rather than by highstand shedding from the platform top and that boundstone growth is largely independent from light and controlled by the physicochemical characteristics of seawater.