Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.     

Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.     

Ferrisiderophore reductase activity associated with an aromatic biosynthetic enzyme complex in Bacillus subtilis

The cytoplasmic fractions obtained from Bacillus subtilis strains W168 and WB2802 catalyzed reductive release of iron from the ferric chelate of 2,3-dihydroxybenzoic acid (ferri-DHB), the ferrisiderophore produced by B. subtilis. Ferrisiderophore reductase activity may insert iron into metabolism. This activity required a reductant (reduced nicotinamide adenine dinucleotide phosphate was preferred), was oxygen sensitive, and was stimulated by flavin mononucleotide plus certain divalent cations. The cytoplasmic fractions also reduced 2,6-dichlorophenolindophenol; this reaction was stimulated by flavin mononucleotide plus a divalent cation. Ferri-DHB and 2,6-dichlorophenolindophenol reductase activities were copurified by phosphocellulose and diethylaminoethyl-cellulose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the purified material revealed that both ferri-DHB and 2,6-dichlorophenolindophenol reductase activities were located in a protein band at Rf 0.75. The chromatographic procedures purified a reductase known to be associated with two aromatic biosynthetic enzymes, chorismate synthase and dehydroquinate synthase. Therefore, a portion of the ferrisiderophore reductase activity in B. subtilis may be catalyzed by a reductase that also is essential for aromatic biosynthesis.     

Species specificity and intraspecific variation in the chemical profiles of Heliconius butterflies across a large geographic range

In many animals, mate choice is important for the maintenance of reproductive isolation between species. Traits important for mate choice and behavioral isolation are predicted to be under strong stabilizing selection within species; however, such traits can also exhibit variation at the population level driven by neutral and adaptive evolutionary processes. Here, we describe patterns of divergence among androconial and genital chemical profiles at inter‐ and intraspecific levels in mimetic Heliconius butterflies. Most variation in chemical bouquets was found between species, but there were also quantitative differences at the population level. We found a strong correlation between interspecific chemical and genetic divergence, but this correlation varied in intraspecific comparisons. We identified “indicator” compounds characteristic of particular species that included compounds already known to elicit a behavioral response, suggesting an approach for identification of candidate compounds for future behavioral studies in novel systems. Overall, the strong signal of species identity suggests a role for these compounds in species recognition, but with additional potentially neutral variation at the population level.     

A single amino acid deletion in the α2(I) chain of type I collagen produces osteogenesis imperfecta type III

RNase A protection analysis was used in the search for the cause of a non-lethal osteogenesis imperfecta (OI) phenotype (Sillence type III). Cleavage of the hybrid formed between a normal 2(I) sequence and RNA isolated from the patient indicated the presence of a mismatch. The position of the mismatch was determined and the corresponding area of COL1A2 was amplified using the polymerase chain reaction. Sequencing of cloned amplified DNA revealed the deletion, which was not present in either parent, of the final three bases of exon 19 in one of the patient's two COL1A2 alleles. The deletion results in the loss of amino acid 255 (a valine encoded by the last codon of exon 19) of the triple helical region of half of the 2(I) collagen chains but does not disrupt the splicing of the heterogeneous nuclear RNA (hnRNA). This provides further evidence that OI type III may result from autosomal dominant mutations rather than only from autosomal recessive mutations as had previously been believed.     

A comparison of diversity estimators applied to a database of host–parasite associations

Understanding the drivers of biodiversity is important for forecasting changes in the distribution of life on earth. However, most studies of biodiversity are limited by uneven sampling effort, with some regions or taxa better sampled than others. Numerous methods have been developed to account for differences in sampling effort, but most methods were developed for systematic surveys in which all study units are sampled using the same design and assemblages are sampled randomly. Databases compiled from multiple sources, such as from the literature, often violate these assumptions because they are composed of studies that vary widely in their goals and methods. Here, we compared the performance of several popular methods for estimating parasite diversity based on a large and widely used parasite database, the Global Mammal Parasite Database (GMPD). We created artificial datasets of host–parasite interactions based on the structure of the GMPD, then used these datasets to evaluate which methods best control for differential sampling effort. We evaluated the precision and bias of seven methods, including species accumulation and nonparametric diversity estimators, compared to analyzing the raw data without controlling for sampling variation. We find that nonparametric estimators, and particularly the Chao2 and second-order jackknife estimators, perform better than other methods. However, these estimators still perform poorly relative to systematic sampling, and effect sizes should be interpreted with caution because they tend to be lower than actual effect sizes. Overall, these estimators are more effective in comparative studies than for producing true estimates of diversity. We make recommendations for future sampling strategies and statistical methods that would improve estimates of global parasite diversity.     

Quantitative Imaging of Protein Secretions from Single Cells in Real Time

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.     

The macroecology of infectious diseases: a new perspective on global‐scale drivers of pathogen distributions and impacts

Identifying drivers of infectious disease patterns and impacts at the broadest scales of organisation is one of the most crucial challenges for modern science, yet answers to many fundamental questions remain elusive. These include what factors commonly facilitate transmission of pathogens to novel host species, what drives variation in immune investment among host species, and more generally what drives global patterns of parasite diversity and distribution? Here we consider how the perspectives and tools of macroecology, a field that investigates patterns and processes at broad spatial, temporal and taxonomic scales, are expanding scientific understanding of global infectious disease ecology. In particular, emerging approaches are providing new insights about scaling properties across all living taxa, and new strategies for mapping pathogen biodiversity and infection risk. Ultimately, macroecology is establishing a framework to more accurately predict global patterns of infectious disease distribution and emergence.