Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Use of an artificial root to examine the influence of 8-hydroxyquinoline on soil microbial activity and bacterial community structure

Invasive plant species have been shown to alter the microbial community composition of the soils they invade and it is suggested that this below-ground perturbation of potential pathogens, decomposers or symbionts may feedback positively to allow invasive success. Whether these perturbations are mediated through specific components of root exudation are not understood. We focussed on 8-hydroxyquinoline, a putative allelochemical of Centaurea diffusa (diffuse knapweed) and used an artificial root system to differentiate the effects of 8-hydroxyquinoline against a background of total rhizodeposition as mimicked through supply of a synthetic exudate solution. In soil proximal (0-10 cm) to the artificial root, synthetic exudates had a highly significant (P < 0.001) influence on dehydrogenase, fluorescein diacetate hydrolysis and urease activity. in addition, 8-hydroxyquinoline was significant (p = 0.003) as a main effect on dehydrogenase activity and interacted with synthetic exudates to affect urease activity (p = 0.09). Hierarchical cluster analysis of 16S rDNA-based DGGE band patterns also identified a primary affect of synthetic exudates and a secondary affect of 8-hydroxyquinoline on bacterial community structure. Thus, we show that the artificial rhizosphere produced by the synthetic exudates was the predominant effect, but, that the influence of the 8-hydroxyquinoline signal on the activity and structure of soil microbial communities could also be detected. (C) 2009 Elsevier Ltd. All rights reserved.

Influences of space, soil, nematodes and plants on microbial community composition of chalk grassland soils

Microbial communities respond to a variety of environmental factors related to resources (e.g. plant and soil organic matter), habitat (e.g. soil characteristics) and predation (e.g. nematodes, protozoa and viruses). However, the relative contribution of these factors on microbial community composition is poorly understood. Here, we sampled soils from 30 chalk grassland fields located in three different chalk hill ridges of Southern England, using a spatially explicit sampling scheme. We assessed microbial communities via phospholipid fatty acid (PLFA) analyses and PCR-denaturing gradient gel electrophoresis (DGGE) and measured soil characteristics, as well as nematode and plant community composition. The relative influences of space, soil, vegetation and nematodes on soil microorganisms were contrasted using variation partitioning and path analysis. Results indicate that soil characteristics and plant community composition, representing habitat and resources, shape soil microbial community composition, whereas the influence of nematodes, a potential predation factor, appears to be relatively small. Spatial variation in microbial community structure was detected at broad (between fields) and fine (within fields) scales, suggesting that microbial communities exhibit biogeographic patterns at different scales. Although our analysis included several relevant explanatory data sets, a large part of the variation in microbial communities remained unexplained (up to 92% in some analyses). However, in several analyses, significant parts of the variation in microbial community structure could be explained. The results of this study contribute to our understanding of the relative importance of different environmental and spatial factors in driving the composition of soil-borne microbial communities.

Gene conversion and evolution of Xq28 duplicons involved in recurring inversions causing severe hemophilia A

Inversions breaking the 1041 bp int1h-1 or the 9.5-kb int22h-1 sequence of the F8 gene cause hemophilia A in 1/30,000 males. These inversions are due to homologous recombination between the above sequences and their inverted copies on the same DNA molecule, respectively, int1h-2 and int22h-2 or int22h-3. We find that (1) int1h and int22h duplicated more than 25 million years ago; (2) the identity of the copies (>99%) of these sequences in humans and other primates is due to gene conversion; (3) gene conversion is most frequent in the internal regions of int22h; (4) breakpoints of int22h-related inversions also tend to involve the internal regions of int22h; (5) sequence variations in a sample of human X chromosomes defined eight haplotypes of int22h-1 and 27 of int22h-2 plus int22h-3; (6) the latter two sequences, which lie, respectively, 500 and 600 kb telomeric to int22h-1 are five-fold more identical when in cis than when in trans, thus suggesting that gene conversion may be predominantly intrachromosomal; (7) int1h, int22h, and flanking sequences evolved at a rate of about 0.1% substitutions per million years during the divergence between humans and other primates, except for int1h during the human-chimpanzee divergence, when its rate of evolution was significantly lower. This is reminiscent of the slower evolution of palindrome arms in the male specific regions of the Y chromosome and we propose, as an explanation, that intrachromosomal gene conversion and cosegregation of the duplicated regions favors retention of the ancestral sequence and thus reduces the evolution rate.

Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function

Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.

Microbial relatives of the seed storage proteins of higher plants: conservation of structure and diversification of function during evolution of the cupin superfamily.

This review summarizes the recent discovery of the cupin superfamily (from the Latin term "cupa," a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein. Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic beta-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids. In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing. This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase. In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors. As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1, 2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis. Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation. Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications.

Microbial relatives of seed storage proteins: conservation of motifs in a functionally diverse superfamily of enzymes

Plant storage proteins comprise a major part of the human diet. Sequence analysis has revealed that these proteins probably share a common ancestor with a fungal oxalate decarboxylase and/or related bacterial genes. Additionally, all these proteins share a central core sequence with several other functionally diverse enzymes and binding proteins, many of which are associated with synthesis of the extracellular matrix during sporulation/encystment. A possible prokaryotic relative of this sequence is a bacterial protein (SASP) known to bind to DNA and thereby protect spores from extreme environmental conditions. This ability to maintain cell viability during periods of dehydration in spores and seeds may relate to absolute conservation of residues involved in structure determination.

CD40 Triggering of Heterodimeric IL-12 p70 Production by Dendritic Cells In Vivo Requires a Microbial Priming Signal

CD40 ligation triggers IL-12 production by dendritic cells (DC) in vitro. Here, we demonstrate that CD40 cross-linking alone is not sufficient to induce IL-12 production by DC in vivo. Indeed, resting DC make neither the IL-12 p35 nor IL-12 p40 subunits and express only low levels of CD40. Nevertheless, after DC activation by microbial stimuli that primarily upregulate IL-12 p40 and augment CD40 expression, CD40 ligation induces a significant increase in IL-12 p35 and IL-12 p70 heterodimer production. Similarly, IL-12 p70 is produced during T cell activation in the presence but not in the absence of microbial stimuli. Thus, production of bioactive IL-12 by DC can be amplified by T cell–derived signals but must be initiated by innate signals.

Systemic gut microbial modulation of bile acid metabolism in host tissue compartments

We elucidate the detailed effects of gut microbial depletion on the bile acid sub-metabolome of multiple body compartments (liver, kidney, heart, and blood plasma) in rats. We use a targeted ultraperformance liquid chromatography with time of flight mass-spectrometry assay to characterize the differential primary and secondary bile acid profiles in each tissue and show a major increase in the proportion of taurine-conjugated bile acids in germ-free (GF) and antibiotic (streptomycin/penicillin)-treated rats.Although conjugated bile acids dominate the hepatic profile (97.0 ± 1.5%) of conventional animals, unconjugated bile acids comprise the largest proportion of the total measured bile acid profile in kidney (60.0±10.4%) andheart (53.0 ± 18.5%) tissues. In contrast, in the GF animal, taurine-conjugated bile acids (especially taurocholic acid and tauro-β-muricholic acid) dominated the bile acid profiles (liver: 96.0 ± 14.5%; kidney: 96 ± 1%; heart: 93 ± 1%; plasma: 93.0 ± 2.3%), with unconjugated and glycine-conjugated species representing a small proportion of the profile. Higher free taurine levels were found in GF livers compared with the conventional liver (5.1-fold; P < 0.001). Bile acid diversity was also lower in GF and antibiotic-treated tissues compared with conventional animals. Because bile acids perform important signaling functions, it is clear that these chemical communication networks are strongly influencedbymicrobial activitiesormodulation, as evidenced by farnesoid X receptor-regulated pathway transcripts. The presence of specific microbial bile acid co-metabolite patterns in peripheral tissues (including heart and kidney) implies a broader signaling role for these compounds and emphasizes the extent of symbiotic microbial influences in mammalian homeostasis.

Plant species and functional group effects on abiotic and microbial soil properties and plant-soil feedback responses in two grasslands

1 Plant species differ in their capacity to influence soil organic matter, soil nutrient availability and the composition of soil microbial communities. Their influences on soil properties result in net positive or negative feedback effects, which influence plant performance and plant community composition. 2 For two grassland systems, one on a sandy soil in the Netherlands and one on a chalk soil in the United Kingdom, we investigated how individual plant species grown in monocultures changed abiotic and biotic soil conditions. Then, we determined feedback effects of these soils to plants of the same or different species. Feedback effects were analysed at the level of plant species and plant taxonomic groups (grasses vs. forbs). 3 In the sandy soils, plant species differed in their effects on soil chemical properties, in particular potassium levels, but PLFA (phospholipid fatty acid) signatures of the soil microbial community did not differ between plant species. The effects of soil chemical properties were even greater when grasses and forbs were compared, especially because potassium levels were lower in grass monocultures. 4 In the chalk soil, there were no effects of plant species on soil chemical properties, but PLFA profiles differed significantly between soils from different monocultures. PLFA profiles differed between species, rather than between grasses and forbs. 5 In the feedback experiment, all plant species in sandy soils grew less vigorously in soils conditioned by grasses than in soils conditioned by forbs. These effects correlated significantly with soil chemical properties. None of the seven plant species showed significant differences between performance in soil conditioned by the same vs. other plant species. 6 In the chalk soil, Sanguisorba minor and in particular Briza media performed best in soil collected from conspecifics, while Bromus erectus performed best in soil from heterospecifics. There was no distinctive pattern between soils collected from forb and grass monocultures, and plant performance could not be related to soil chemical properties or PLFA signatures. 7 Our study shows that mechanisms of plant-soil feedback can depend on plant species, plant taxonomic (or functional) groups and site-specific differences in abiotic and biotic soil properties. Understanding how plant species can influence their rhizosphere, and how other plant species respond to these changes, will greatly enhance our understanding of the functioning and stability of ecosystems.

Comparison of methane produced by straw fed sheep in open-circuit respiration with methane predicted by fermentation characteristics measured by an in vitro gas procedure

Reducing carbon conversion of ruminally degraded feed into methane increases feed efficiency and reduces emission of this potent greenhouse gas into the environment. Accurate, yet simple, predictions of methane production of ruminants on any feeding regime are important in the nutrition of ruminants, and in modeling methane produced by them. The current work investigated feed intake, digestibility and methane production by open-circuit respiration measurements in sheep fed 15 untreated, sodium hydroxide (NaOH) treated and anhydrous ammonia (NH3) treated wheat, barley and oat straws. In vitro fermentation characteristics of straws were obtained from incubations using the Hohenheim gas production system that measured gas production, true substrate degradability, short-chain fatty acid production and efficiency of microbial production from the ratio of truly degraded substrate to gas volume. In the 15 straws, organic matter (OM) intake and in vivo OM digestibility ranged from 563 to 1201 g and from 0.464 to 0.643, respectively. Total daily methane production ranged from 13.0 to 34.4 l, whereas methane produced/kg OM matter apparently digested in vivo varied from 35.0 to 61.8 l. The OM intake was positively related to total methane production (R2 = 0.81, P<0.0001), and in vivo OM digestibility was also positively associated with methane production (R2 = 0.67, P<0.001), but negatively associated with methane production/kg digestible OM intake (R2 = 0.61, P<0.001). In the in vitro incubations of the 15 straws, the ratio of acetate to propionate ranged from 2.3 to 2.8 (P<0.05) and efficiencies of microbial production ranged from 0.21 to 0.37 (P<0.05) at half asymptotic gas production. Total daily methane production, calculated from in vitro fermentation characteristics (i.e., true degradability, SCFA ratio and efficiency of microbial production) and OM intake, compared well with methane measured in the open-circuit respiration chamber (y = 2.5 + 0.86x, R2 = 0.89, P<0.0001, Sy.x = 2.3). Methane production from forage fed ruminants can be predicted accurately by simple in vitro incubations combining true substrate degradability and gas volume measurements, if feed intake is known.

Microbial control of diamondback moth, Plutella xylostella L. (Lepidoptera:Yponomeutidae) using bacteria (Xenorhabdus nematophila) and its metabolites from the entomopathogenic nematode Steinernema carpocapsae

Cells and cell-free solutions of the culture filtrate of the bacterial symbiont, Xenorhabdus nematophila taken from the entomopathogenic nematode Steinernema carpocapsae in aqueous broth suspensions were lethal to larvae of the diamondback moth Plutella xylostella. Their application on leaves of Chinese cabbage indicated that the cells can penetrate into the insects in the absence of the nematode vector. Cell-free solutions containing metabolites were also proved as effective as bacterial cells suspension. The application of aqueous suspensions of cells of X. nematophila or solutions containing its toxic metabolites to the leaves represents a possible new strategy for controlling insect pests on foliage.

In vitro microbial inoculum: A review of its function and properties

This review considers microbial inocula used in in vitro systems from the perspective of their ability to degrade or ferment a particular substrate, rather than the microbial species that it contains. By necessity, this required an examination of bacterial, protozoal and fungal populations of the rumen and hindgut with respect to factors influencing their activity. The potential to manipulate these populations through diet or sampling time are examined, as is inoculum preparation and level. The main alternatives to fresh rumen fluid (i.e., caecal digesta or faeces) are discussed with respect to end-point degradabilities and fermentation dynamics. Although the potential to use rumen contents obtained from donor animals at slaughter offers possibilities, the requirement to store it and its subsequent loss of activity are limitations. Statistical modelling of data, although still requiring a deal of developmental work, may offer an alternative approach. Finally, with respect to the range of in vitro methodologies and equipment employed, it is suggested that a degree of uniformity could be obtained through generation of a set of guidelines relating to the host animal, sampling technique and inoculum preparation. It was considered unlikely that any particular system would be accepted as the 'standard' procedure. However, before any protocol can be adopted, additional data are required (e.g., a method to assess inoculum 'quality' with respect to its fermentative and/or degradative activity), preparation/inoculation techniques need to be refined and a methodology to store inocula without loss of efficacy developed. (c) 2005 Elsevier B.V. All rights reserved.