Not so black,not so white:differences in microorganism load of contiguous feathers from white stork chicks

Many organisms are characterized by strikingly contrasting black and white coloration, but the function of such contrasts has been inadequately studied. In this article, we tested the function of black and white contrasting plumage in white stork Ciconia ciconia chicks. We found greater abundance and diversity of microorganisms on black compared with adjacent white feathers. In addition, nest size was positively correlated with the abundance and diversity of microorganisms on white feathers. Flight initiation distance (FID), defined as the distance at which adult white storks took flight when approached by a human, was negatively correlated with most measurements of microorganism abundance. Breeding success was generally positively correlated with the abundance and diversity of microorganisms on black feathers. The feather growth rate was positively correlated with some and negatively correlated with other measurements of microbial abundance and diversity. Finally, chick growth was negatively correlated with the number of microbial species on black feathers and positively with the abundance and diversity of microorganisms on white feathers. These findings are consistent not only with the role of microorganisms in the maintenance of a benign microbial environment which differs between black and white feathers, but also with the hypothesis that several taxa of microorganisms found in black and white plumage are virulent, with negative effects on the fitness of their hosts.     

Cluster Roots in Casuarinaceae: Role and Relationship to Soil Nutrient Factors

N₂-fixing actinorhizal trees in the family Casuarinaceae areeconomically of great interest in tropical and sub-tropicalzones because they are used for many purposes including protectionagainst wind, stabilization of sand dunes and the productionof firewood and charcoal. They are usually able to grow in sandysoils with low fertility by virtue of their ability to fix N₂.The objective of this review is to discuss briefly the roleof mycorrhizas and, more extensively, that of cluster (proteoid)roots, developed by a number of species of Casuarinaceae toimprove the absorption of nutrients other than N from soil,especially those needed for N₂fixation and growth. After evaluatingthe actual relationships between mycorrhizas and the Casuarinaceae,we highlight the possible role of cluster roots as an effectivealternative to mycorrhizas, and as a means of improvement ofgrowth of the trees in nutrient-deficient soils. This raisesthe question of what triggers the formation of cluster rootsin the Casuarinaceae. In addition to phosphorus deficiency,iron deficiency seems to be a major factor inducing the formationof cluster roots in Casuarina glauca and C. cunninghamiana.The number of cluster roots and the precocity of their formationare directly related to plant chlorosis due to Fe deficiency,as expressed by the critical concentration of chlorophyll inthe shoot (0.60 mg g ^{- 1}shoot f.wt). The effect of the nitrogensource on cluster root formation is discussed in relation topH values in the plant culture solution. The number of clusterroots formed in nitrate-fed plants increases with pH in therange of 5 to 9. Experiments carried out with alkaline and acidicsoils show that cluster roots are only produced when they areneeded to overcome soil nutrient deficiency due to the immobilizationof nutrient elements (P and Fe) by soil alkalinity. The possibleinvolvement of ethylene in the initiation and/or the morphogenesisof cluster roots is discussed. Copyright 2000 Annals of BotanyCompany Casuarinaceae, Casuarina cunninghamiana, Casuarina glauca, cluster roots, ethylene, iron deficiency, phosphorus deficiency, proteoid roots     

Localization of the voltage-dependent anion channel-1 Ca2+-binding sites

Photoreactive azido ruthenium (AzRu) has been recently shown to specifically interact with Ca(2+)-binding proteins and to strongly inhibit their Ca(2+)-dependent activities. Upon UV irradiation, AzRu can bind covalently to such proteins. In this study, AzRu was used to localize and characterize Ca(2+)-binding sites in the voltage-dependent anion channel (VDAC). AzRu decreased the conductance of VDAC reconstituted into a bilayer while Ca(2+), in the presence of 1M NaCl, but not Mg(2+), prevented this effect. AzRu had no effect on mutated E72Q- or E202Q-VDAC1 conductance, and [(103)Ru]AzRu labeled native but not E72Q-VDAC1, suggesting that these residues are required for AzRu interaction with the VDAC Ca(2+)-binding site(s). AzRu protected against apoptosis induced by over-expression of native but not E72Q- or E202Q- murine VDAC1 in T-REx-293 cells depleted of endogenous hVDAC1. Chymotrypsin and trypsin digestion of AzRu-labeled VDAC followed by MALDI-TOF analysis revealed two AzRu-bound peptides corresponding to E72- and E202-containing sequences. These results suggest that the VDAC Ca(2+)-binding site includes E72 and E202, located, according to a proposed VDAC1 topology model, on two distinct cytosolic loops. Furthermore, AzRu protection against apoptosis involves interaction with these residues. Photoreactive AzRu represents an important tool for identifying novel Ca(2+)-binding proteins and localizing their Ca(2+)-binding sites.     

Evaluation of methods for the extraction and purification of DNA from the human microbiome

Background

DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.

Methodology/Principal Findings

In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.

Conclusions/Significance

Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.     

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.     

Nucleotide-binding sites in the voltage-dependent anion channel: characterization and localization

In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [alpha-(32)P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5'-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [alpha-(32)P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of approximately 17- and approximately 14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotide-binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.     

Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9

Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.     

Statistical methods for characterizing diversity of microbial communities by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

The analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes has proven to be a facile means to compare microbial communities and presumptively identify abundant members. The method provides data that can be used to compare different communities based on similarity or distance measures. Once communities have been clustered into groups, clone libraries can be prepared from sample(s) that are representative of each group in order to determine the phylogeny of the numerically abundant populations in a community. In this paper methods are introduced for the statistical analysis of T-RFLP data that include objective methods for (i) determining a baseline so that 'true' peaks in electropherograms can be identified; (ii) a means to compare electropherograms and bin fragments of similar size; (iii) clustering algorithms that can be used to identify communities that are similar to one another; and (iv) a means to select samples that are representative of a cluster that can be used to construct 16S rRNA gene clone libraries. The methods for data analysis were tested using simulated data with assumptions and parameters that corresponded to actual data. The simulation results demonstrated the usefulness of these methods in their ability to recover the true microbial community structure generated under the assumptions made. Software for implementing these methods is available at http://www.ibest.uidaho.edu/tools/trflp_stats/index.php.     

Mapping the ruthenium red-binding site of the voltage-dependent anion channel-1

We have previously shown that ruthenium red (RuR) binds to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, decreasing channel conductance and protecting against apoptotic cell death. In this report, we define the murine and yeast VDAC1 amino acid residues involved in the interaction with RuR. Binding of RuR to bilayer-reconstituted mVDAC1 and the resulting channel closure was inhibited upon mutation of specific VDAC1 residues. RuR protection against cell death, as induced by overexpression of native or mutated mVDAC1, was also diminished upon mutation of these amino acids. Moreover, RuR-mediated inhibition of cytochrome c release normally induced by staurosporine was not observed in cells expressing mutants VDAC1. We found that four glutamate residues, two each located in the first and third mVDAC1 cytosolic loops, are required for the interaction of VDAC1 with RuR and subsequent protection against cell death. Similar results were obtained with Q72E-yeast VDAC1, except that only three glutamate residues, located in two cytosolic loops were required. As a hexavalent reagent, RuR is expected to bind to more than one negatively charged group. Our results thus clearly indicate that RuR protects against cell death via a direct interaction with VDAC1 to inhibit cytochrome c release and subsequent cell death.     

DNA A-tracts bending: polarization effects on electrostatic interactions across their minor groove

Bending by the DNA A-tracts constitutes a contentious issue, suggesting deficiencies in the physics employed so far. Here, we inquire as to the importance in this bending of many-body polarization effects on the electrostatic interactions across their narrow minor groove. We have done this on the basis of the findings of Jarque and Buckingham who developed a procedure based on a Monte Carlo simulation for two charges of the same sign embedded in a polarizable medium. Remarkably, the present analysis reveals that for compact DNA conformations, which result from dynamic effects, an overall attractive interaction operates between the phosphate charges; this interaction is especially strong for the narrow minor groove of the A-tracts, suggesting a tendency for DNA to bend toward this groove. This tendency is in agreement with the conclusions of electrophoretic and NMR solution studies. The present analysis is also consistent with the experimental observations that the minor groove is much more easily compressible than the major groove and the bending propensity of the A-tracts is greatly reduced at “premelting” temperatures. By contrast, the dielectric screening model predicts a repulsion between the phosphate charges and is not consistent with the aforementioned bending tendency or experimental observations.