Poultry-Associated Salmonella enterica subsp. enterica Serovar 4,12:d:? Reveals High Clonality and a Distinct Pathogenicity Gene Repertoire

A European baseline survey during the years 2005 and 2006 has revealed that the monophasic Salmonella enterica subsp. enterica serovar 4,12:d:− was, with a prevalence of 23.6%, the most frequently isolated serovar in German broiler flocks. In Denmark and the United Kingdom, its serovar prevalences were 15.15% and 2.8%, respectively. Although poultry is a major source of human salmonellosis, serovar 4,12:d:− is rarely isolated in humans (approximately 0.09% per year). Molecular typing studies using pulsed-field gel electrophoresis and DNA microarray analysis show that the serovar is highly clonal and lacks genes with known contributions to pathogenicity. In contrast to other poultry-associated serovars, all strains were susceptible to 17 antimicrobial agents tested and did not encode any resistance determinant. Furthermore, serovar 4,12:d:− lacked the genes involved in galactonate metabolism and in the glycolysis and glyconeogenesis important for energy production in the cells. The conclusion of the study is that serovar 4,12:d:− seems to be primarily adapted to broilers and therefore causes only rare infections in humans.Salmonella spp. are major zoonotic food-borne pathogens which cause outbreaks and sporadic cases of gastroenteritis in humans worldwide (12). Depending on the serovar, cases of salmonellosis can differ substantially in severity (13). The primary sources of salmonellosis are food-producing animals, such as poultry, pigs, and cattle (30). The pathogen is spread by trade in animals and nonheated animal food products (10).A European baseline survey on the prevalence of Salmonella in commercial broiler flocks of Gallus gallus in 2005 and 2006 showed that in the European Union (EU), 23.7% of the broiler flocks were Salmonella positive (8). However, the Salmonella prevalences and serovar distribution varied widely among the EU member states. The five most frequently isolated Salmonella enterica serovars in Europe were those classically observed, like serovar Enteritidis (33.8%), serovar Infantis (22.0%), serovar Mbandaka (8.1%), serovar Hadar (3.7%), and serovar Typhimurium (3.0%). In Germany, the flock prevalence of Salmonella was 15.0% among the 377 broiler flocks investigated. In contrast to the well-known serovars described above, the predominating serovar was monophasic serovar 4,12:d:−, with a prevalence of 23.6%. This serovar was also isolated in Denmark and the United Kingdom, with prevalences of 15.2% and 2.8%, respectively.The German Salmonella National Reference Laboratory (NRL-Salmonella) has received 818 isolates of this serovar between 1998 and 2007, with peaks in 2001 (240 isolates) and 2004 (160 isolates), for diagnosis. Since 2005, the number has doubled annually, and the serovar obviously established itself well in poultry production lines. These isolates were found mostly in broilers (78%), occasionally in turkeys (11.6%) and feedstuff (8.4%), and rarely in pigs (1.3%) and cattle (0.6%). In contrast, infections in humans are only sporadic. During the last 10 years (1998 to 2007), the National Reference Centre for Salmonellae and Other Enterics located at the Robert-Koch Institute, Wernigerode branch, has received 55 strains of this serovar from sporadic human cases of salmonellosis and carriers in Germany (W. Rabsch, personal communication). Similarly, in Denmark, only two isolates in 1993 and in 2002 were isolated from humans (E. Møller Nielsen, Statens Serum Institut, Copenhagen, Denmark, personal communication).Subtyping food-borne pathogens is an approach often applied to facilitate the epidemiological investigation of outbreaks of gastrointestinal disease and to identify the source of entry into the food chain. Several molecular-based tools have been developed to type bacteria genotypically. Pulsed-field gel electrophoresis (PFGE) is currently the method of choice for the molecular subtyping of Salmonella serovars. It has been proven to be a useful discriminatory method which was standardized by the PulseNet Consortium (9). However, although this approach is certainly valuable, it does not reveal data on the gene repertoire and biological properties of a strain. To overcome this weakness, whole-genome DNA microarrays have successfully been applied in comparative genomic hybridizations for Salmonella (7, 24, 25). However, whole-genome arrays reflect only one genome of one strain. Because of many serovar or strain genome variations described for Salmonella, thematic arrays were developed, such as arrays specially targeting genes involved in resistance profiles (2, 17, 32), phage types (23), or serovars (33, 35). A condensed selection of 109 various Salmonella genetic markers comprising the detection of flagellar and somatic antigen-encoding genes, important virulence genes, phage-associated genes, and antibiotic resistance determinants have been used to show the usefulness of DNA microarrays for the discriminative characterization of Salmonella serovars (18).In this study, we elucidate the contradicting situation between the high prevalence in broilers and source attribution of broiler meat for humans and the low infection rates in humans of the serovar 4,12:d:− by genotypic characterization using PFGE and DNA microarray to determine the clonality, the pathogenic gene repertoire, and resistance determinants. These data give basic information to discuss the hazard potential of this serovar for humans. For that purpose, a new prototype of a Salmonella DNA microarray comprising 281 60-mer oligonucleotide probes was developed and validated in house.     

Molybdenum X-ray absorption edges from 200 to 20,000 eV: The benefits of soft X-ray spectroscopy for chemical speciation

We have surveyed the chemical utility of the near-edge structure of molybdenum X-ray absorption edges from the hard X-ray K-edge at 20,000 eV down to the soft X-ray M_4,5-edges at ∼230 eV. We compared, for each edge, the spectra of two tetrahedral anions, and . We used three criteria for assessing near-edge structure of each edge: (i) the ratio of the observed chemical shift between and and the linewidth, (ii) the chemical information from analysis of the near-edge structure and (iii) the ease of measurement using fluorescence detection. Not surprisingly, the K-edge was by far the easiest to measure, but it contained the least information. The L_2,3-edges, although harder to measure, had benefits with regard to selection rules and chemical speciation in that they had both a greater chemical shift as well as detailed lineshapes which could be theoretically analyzed in terms of Mo ligand field, symmetry, and covalency. The soft X-ray M_2,3-edges were perhaps the least useful, in that they were difficult to measure using fluorescence detection and had very similar information content to the corresponding L_2,3-edges.Interestingly, the soft X-ray, low energy (∼230 eV) M_4,5-edges had greatest potential chemical sensitivity and using our high-resolution superconducting tunnel junction (STJ) fluorescence detector they appear to be straightforward to measure. The spectra were amenable to analysis using both the TT-multiplet approach and FEFF. The results using FEFF indicate that the sharp near-edge peaks arise from 3d → 5p transitions, while the broad edge structure has predominately 3d → 4f character. A proper understanding of the dependence of these soft X-ray spectra on ligand field and site geometry is necessary before a complete assessment of the utility of the Mo M_4,5-edges can be made. This work includes crystallographic characterization of sodium tetrathiomolybdate.     

UV-light-induced conversion and aggregation of prion proteins

Increasing evidence suggests a central role for oxidative stress in the pathology of prion diseases, a group of fatal neurodegenerative disorders associated with structural conversion of the prion protein (PrP). Because UV-light-induced protein damage is mediated by direct photo-oxidation and radical reactions, we investigated the structural consequences of UVB radiation on recombinant murine and human prion proteins at pH 7.4 and pH 5.0. As revealed by circular dichroism and dynamic light scattering measurements, the observed PrP aggregation follows two independent pathways: (i) complete unfolding of the protein structure associated with rapid precipitation or (ii) specific structural conversion into distinct soluble β-oligomers. The choice of pathway was directly attributed to the chromophoric properties of the PrP species and the susceptibility to oxidation. Regarding size, the oligomers characterized in this study share a high degree of identity with oligomeric species formed after structural destabilization induced by other triggers, which significantly strengthens the theory that partly unfolded intermediates represent initial precursor molecules directing the pathway of PrP aggregation. Moreover, we identified the first suitable photo-trigger capable of inducing refolding of PrP, which has an important biotechnological impact in terms of analyzing the conversion process on small time scales.     

Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii , like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae.     

Dipeptidyl nitrile inhibitors of Cathepsin L

A series of potent Cathepsin L inhibitors with good selectivity with respect to other cysteine Cathepsins is described and SAR is discussed with reference to the crystal structure of a protein-ligand complex.     

Designed Human Serum Hyaluronidase 1 Variant, HYAL1��L, Exhibits Activity up to pH 5.9

Hyaluronidases from diverse species and sources have different pH optima. Distinct mechanisms with regard to dynamic structural changes, which control hyaluronidase activity at varying pH, are unknown. Human serum hyaluronidase 1 (HYAL1) is active solely below pH 5.1. Here we report the design of a HYAL1 variant that degrades hyaluronan up to pH 5.9. Besides highly conserved residues in close proximity of the active site of most hyaluronidases, we identified a bulky loop formation located at the end of the substrate binding crevice of HYAL1 to be crucial for substrate hydrolysis. The stretch between cysteine residues 207 and 221, which normally contains 13 amino acids, could be replaced by a tetrapeptide sequence of alternating glycine serine residues, thereby yielding an active enzyme with an extended binding cleft. This variant exhibited hyaluronan degradation at elevated pH. This is indicative for appropriate substrate binding and proper positioning being decisively affected by sites far off from the active center.Hyaluronan (HA),³ a linear polysaccharide found in the extracellular matrix of most tissues and body fluids of vertebrates, is enzymatically degraded by hyaluronidases (1). Mammalian-type hyaluronidases are grouped into EC 3.2.1.35 (2, 3) or the glycoside hydrolase family 56 (4). Members of this enzyme family hydrolyze the 1,4-β-glycosidic linkage between N-acetyl-d-glucosamine and d-glucuronate within HA polymers (5). In mammalians, hyaluronidases have been found in testis, liver lysosomes, and serum. They are involved in controlling HA levels and are thus implicated in various diseases related to defects of HA metabolism (6).The crystal structures of hyaluronidase from bee (7), wasp (8), and only recently that of human serum hyaluronidase 1 (HYAL1) (9) have been deciphered. In addition to the N-terminal catalytic domain of the insect enzymes, which resembles a distorted (β/α)₈ barrel, HYAL1 contains yet another domain. HA hydrolysis is achieved by a pair of acidic amino acids via a retaining double displacement mechanism and a substrate-assisted catalysis, in which the carbonyl oxygen of the N-acetyl group of the cleaved HA subunit acts as the catalytic nucleophile (7).Mammalian-type hyaluronidases display different pH optima. HYAL1 (10) and hyaluronidase 2 (HYAL2) (11) exhibit highest activities at acidic conditions, whereas the hyaluronidase found in Xenopus laevis kidney is only active at neutral pH (12). Bee venom hyaluronidase (13), as well as sperm hyaluronidase, PH20 (SPAM1) (14), are capable of degrading HA over a broad pH range. Up to three PH20 isoforms with greatly different pH optima could be found in protein preparations from bovine testis (15). Extensive analysis of hyaluronidase structures did not bring forward any insights as to what residues or regions of the enzymes specify a specific pH optimum.Profiles of pH-dependent activities can be assigned by computing the electrostatic interactions of the enzyme, which are primarily determined by the ionization states of its amino acid side chains. The pK_a values of titratable groups of the enzyme reflect pH-dependent properties such as stability, enzymatic interaction, and substrate interactions (16). Here we present computational and experimental data on the replacement of a loop region located at the end of the substrate binding groove yielding a variant hyaluronidase with an altered pH profile.     

Classification of hybrid and altered Vibrio cholerae strains by CTX prophage and RS1 element structure

Analysis of the CTX prophage and RS1 element in hybrid and altered Vibrio cholera O1 strains showed two classifiable groups. Group I strains contain a tandem repeat of classical CTX prophage on the small chromosome. Strains in this group either contain no element(s) or an additional CTX prophage or RS1 element(s) on the large chromosome. Group II strains harbor RS1 and CTX prophage, which has an E1 Tor type rstR and classical ctxB on the large chromosome.     

Environmental correlates for tropical tree diversity and distribution patterns in Borneo

Aim Identify environmental correlates for tropical tree diversity and composition. Location Borneo, Southeast Asia. Methods A GIS‐environmental database with 5 arc minute (c. 10 × 10 km) resolution was combined with tree inventory data. Tree diversity, phylogenetic diversity (PD) and the two main compositional gradients were determined for 46 tree inventories. Akaike's information criterion and a data jackknifing procedure were used to select 50 explanatory models for diversity and composition gradients. The average of these models was used as our final diversity and compositional model. We applied Moran's I to detect spatial autocorrelation of residuals. Results Tree diversity, PD and the two main compositional gradients in Borneo were all significantly correlated with the environment. Tree diversity correlated negatively with elevation, soil depth, soil coarseness (texture) and organic carbon content, whereas it correlated positively with soil C:N ratio, soil pH, moisture storage capacity and annual rainfall. Tree PD was correlated positively with elevation and temperature seasonality and was largely determined by gymnosperms. However, angiosperm PD also correlated positive with elevation. Compositional patterns were strongly correlated with elevation but soil texture, cation‐exchange‐capacity, C:N ratio, C and N content and drainage were also important next to rainfall seasonality and El Niño Southern Oscillation drought impact. Main conclusions Although elevation is the most important correlate for diversity and compositional gradients in Borneo, significant additional variability is explained by soil characteristics (texture, carbon content, pH, depth, drainage and nutrient status) and climate (annual rainfall, rainfall seasonality and droughts). The identified environmental correlates for diversity and composition gradients correspond to those found in other tropical regions of the world. Differences between the regions are mainly formed by differences in the relative importance of the environmental variables in explaining diversity and compositional gradients.