Population Genetics of Streptococcus dysgalactiae Subspecies equisimilis Reveals Widely Dispersed Clones and Extensive Recombination

Background

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen that can colonize and infect humans. Although most SDSE isolates possess the Lancefield group G carbohydrate, a significant minority have the group C carbohydrate. Isolates are further sub-typed on the basis of differences within the emm gene. To gain a better understanding of their molecular epidemiology and evolutionary relationships, multilocus sequence typing (MLST) analysis was performed on SDSE isolates collected from Australia, Europe and North America.

Methodology/Principal Findings

The 178 SDSE isolates, representing 37 emm types, segregate into 80 distinct sequence types (STs) that form 17 clonal complexes (CCs). Eight STs recovered from all three continents account for >50% of the isolates. Thus, a small number of STs are highly prevalent and have a wide geographic distribution. Both ST and CC strongly correlate with group carbohydrate. In contrast, eleven STs were associated with >1 emm type, suggestive of recombinational replacements involving the emm gene; furthermore, 35% of the emm types are associated with genetically distant STs. Data also reveal a history of extensive inter- and intra-species recombination involving the housekeeping genes used for MLST. Sequence analysis of single locus variants identified through goeBURST indicates that genetic change mediated by recombination occurred ∼4.4 times more frequently than by point mutation.

Conclusions/Significance

A few genetic lineages with an intercontinental distribution dominate among SDSE causing infections in humans. The distinction between group C and G isolates reflects recent evolution, and no long-term genetic isolation between them was found. Lateral gene transfer and recombination involving housekeeping genes and the emm gene are important mechanisms driving genetic variability in the SDSE population.     

New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase,Emerging from Nonconventional Codon Usage and Directed Mutation

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k_cat/K_m) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes'' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.β-Fructofuranosidases (EC 3.2.1.26) are enzymes of biotechnological interest that catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. In general, they exhibit a high degree of sequence homology, and based on their amino acid sequences, they fall into family 32 of the glycosyl-hydrolases (GH), along with invertases, inulinases, and fructosyltransferases (http://www.cazy.org). The GH32 family has been studied intensely, and some three-dimensional structures are now available, such as that of inulinase from Aspergillus awamorii (26), fructan-exohydrolase from Cichorium intybus (CiFEH) (34, 36), or invertase from Thermotoga maritima (2, 3) and Arabidopsis thaliana (35). These proteins contain a five-blade β-propeller N-terminal catalytic module and a C-terminal β-sandwich domain (19). Multiple-sequence alignment of GH32 proteins, which are included in the GH-J clan together with the GH68 proteins of the inulosucrase family, reveals the presence of three conserved motifs, each containing a key acidic residue (in boldface) implicated in substrate binding and hydrolysis: Asn-Asp-Pro-Asn-Gly (NDPNG), Arg-Asp-Pro (RDP), and Glu-Cys (EC) (28). These conserved residues are implicated in a double-displacement reaction in which a covalent glycosyl-enzyme intermediate is formed. Thus, the catalytic mechanism proposed for the Saccharomyces cerevisiae invertase implies that Asp23 (NDPNG) acts as a nucleophile and Glu204 (EC) acts as the acid/base catalyst (29), whereas Asp309 (RDP) of Acetobacter diazotropicus levansucrase influences the efficiency of sucrose hydrolysis (7) and Arg188 and Asp189 of the latter motif define the substrate binding and specificity of exoinulinase from A. awamorii toward fructopyranosyl residues (26).As well as hydrolyzing sucrose, β-fructofuranosidases may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds, depending on the source of the enzyme (12, 21, 31). FOS act as prebiotics, and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, and osteoporosis (16). Currently, FOS are mainly produced by Aspergillus fructosyltransferase in industry (10, 31), providing a mixture of FOS with an inulin-type structure that contains β-(2→1)-linked fructose oligomers (¹F-FOS: 1-kestose or nystose). Curiously, when the link between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose) involves a β-(2→6) link, the prebiotic properties of the FOS may be enhanced beyond that of commercial FOS (23).The yeast Schwanniomyces occidentalis (also called Debaryomyces occidentalis) produces a number of extracellular enzymes that make it of interest in biotechnology. Several of its amylolytic enzymes have been characterized, including amylases and glucoamylase (1, 9), as well as an invertase (17). In addition, we also characterized an extracellular β-fructofuranosidase (Ffase) from this yeast that hydrolyzes sucrose, 1-kestose, and nystose (5). This enzyme exhibited a transfructosylating activity that efficiently produces the trisaccharides 6-kestose and 1-kestose in the ratio 3:1, generating the highest 6-kestose yield yet reported, as far as we know. The Ffase three-dimensional structure has recently been solved (6) and represented as a homodimer, each modular subunit arranged like other GH32 enzymes. The Asp50 (NDPNG) and Glu230 (EC) located at the center of the propeller are the catalytic residues implicated in substrate binding and hydrolysis, whereas Arg178 and Asp179 form the RDP motif (6).The genetic codes of some yeasts incorporate certain variations. For example, while CUG was believed to be a universal codon for leucine, in the cytoplasm of certain species of the genus Candida (15) it encodes a serine, as in Pichia farinosa (33). The reassignment of this codon is mediated by a novel serine-tRNA that acquired a leucine 5′-CAG-3′ anticodon (25).Here, we show that deviation from the standard use of the CUG leucine codon to encode serine was correlated with the transferase capacity and specificity of the Ffase enzyme. Indeed, the S196L substitution enhanced the transferase activity of the enzyme 3-fold. Several site-directed mutants were generated and characterized to study their transferase capacities. These results are considered on the basis of the enzymes'' three-dimensional structure, which enables a novel putative binding site of sucrose that serves as a water substitute donor in the hydrolytic reaction yielding the tranglycosylation product 6-kestose to be identified.     

The role of high-dose-rate brachytherapy boost in breast-conserving therapy: Long-term results of the Hungarian National Institute of Oncology

AimTo report the long-term results of high-dose-rate (HDR) brachytherapy (BT) boost for breast cancer patients treated with conservative surgery and radiotherapy.Materials and methodsBetween 1995 and 2007, 100 early-stage breast cancer patients received an HDR BT boost after conservative surgery and whole breast irradiation. Ten patients (10%) received a single-fraction HDR boost of 8–10.35 Gy using rigid needles, while 90 (90%) were treated with a fractionated multi-catheter HDR BT boost. The latter consisted of 3 × 4 Gy (n = 19), 3 × 4.75 Gy (n = 70), and 2 × 6.4 Gy (n = 1). Breast cancer related events, cosmetic results and side effects were assessed.ResultsAt a median follow-up time of 94 months (range: 8–152) only 7 (7%) ipsilateral breast failures were observed for a 5- and 8-year actuarial rate of 4.5 and 7.0%, respectively. The 8-year disease-free, cancer-specific, and overall survival was 76.1, 82.8, and 80.4%, respectively. Cosmetic outcome was rated excellent in 17%, good in 39%, fair in 33%, and poor in 11%. Data on late radiation side effects were available for 91 patients (91%). Grade 3 fibrosis and grade 3 telangiectasia occurred in 6 (6.6%) and 2 (2.2%) patients, respectively. In univariate analysis only positive margin status had a significant negative effect on local control.ConclusionsHDR BT boost using multi-catheter implants produce excellent long-term local tumour control with acceptable cosmetic outcome and low rate of grade 3 late radiation side effects.     

Subunit arrangement in beef heart complex III

Beef heart mitochondrial complex III was separated into 12 polypeptide bands representing 11 different subunits by using the electrophoresis conditions described by Sch?gger et al. [(1986) Methods Enzymol. 126, 224-237]. Eight of the 12 polypeptide bands were identified from their NH2-terminal sequences as obtained by electroblotting directly from the NaDodSO4-polyacrylamide gel onto a solid support. The topology of the subunits in complex III was explored by three different approaches. (1) Protease digestion experiments of submitochrondrial particles in the presence and absence of detergent showed that subunits II and VI are on the M side of the inner membrane and subunits V and XI on the C side. (2) Labeling experiments with the membrane-intercalated probes [125I]TID and arylazidoPE indicated that cytochrome b is the predominant bilayer embedded subunit of complex III, while the non-heme iron protein appears to be peripherally located. (3) Cross-linking studies with carbodiimides and homobifunctional cleavable reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and VI+VII. The cytochrome c binding site was found to include subunits IV, VIII, and X. The combined data are used to provide an updated model for the topology of beef heart complex III.     

Heterobifunctional photoaffinity probes for cytochrome P450 2B.

Three heterobifunctional photoaffinity probes, N-(p-azidobenzyl)-N-methyl-p-aminobenzylamine (I), N-(p-azidobenzyl)-N-methyl-p-aminophenethylamine (II), and N-(p-azidophenethyl)-N-methyl-p-aminophenethylamine (III), were synthesized and characterized. These probes, containing a photolabile azido-group and an amino-group on opposite sides of the molecule, were designed for photoaffinty labeling of the cytochrome P450 (CYP) 2B active site cavity differing in distance from the heme iron. Spectroscopic studies proved that probes I and II coordinated with the heme iron via their amino-group in the enzyme active center, whereas probe III did not. This result in conjunction with data from kinetic studies suggests probes I and II are appropriate for photoaffinity labeling of the CYP 2B active center. Thus, probe II was used to identify amino acid residues within a distance of the probe length (about 16.5 A) from the heme. Analysis of a Lys-C digest of the probe II-labeled CYP 2B4 revealed a single labeled hexapeptide corresponding to position 192-197 of the CYP 2B4 sequence. Using postsource decay/matrix-assisted laser desorption ionization-time of flight, Arg197 was identified as a probe II target. The location of the labeled site in three-dimensional structures of bacterial CYPs and in CYP 2B homology models is discussed.     

Population genomics of the raccoon dog (<Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis>) in Denmark: insights into invasion history and population development

The raccoon dog (Nyctereutes procyonoides) has a wide distribution in Europe and is a prominent example of a highly adaptable alien species. It has been recorded sporadically in Denmark since 1980 but observations since 2008 suggested that the species had established a free-ranging, self-sustaining population. To elucidate the origin and genetic patterns of Danish raccoon dogs, we studied the population genomics of 190 individuals collected in Denmark (n = 141) together with reference captive individuals from Poland (n = 21) and feral individuals from different European localities (Germany, Poland, Estonia and Finland, n = 28). We used a novel genotyping-by-sequencing approach simultaneously identifying and genotyping a large panel of single nucleotide polymorphisms (n = 4526). Overall, there was significant indication for contemporary genetic structuring of the analysed raccoon dog populations, into at least four different clusters, in spite of the existence of long distance gene flow and secondary admixture from different population sources. The Danish population was characterized by a high level of genetic admixture with neighbouring feral European ancestries and the presence of private clusters, non-retrieved in any other feral or captive populations sampled. These results suggested that the raccoon dog population in Denmark was founded by escapees from genetically unidentified Danish captive stocks, followed by a recent admixture with individuals migrating from neighbouring Germany.     

An evaluation of semi‐automated methods for collecting ecosystem‐level data in temperate marine systems

Historically, marine ecologists have lacked efficient tools that are capable of capturing detailed species distribution data over large areas. Emerging technologies such as high‐resolution imaging and associated machine‐learning image‐scoring software are providing new tools to map species over large areas in the ocean. Here, we combine a novel diver propulsion vehicle (DPV) imaging system with free‐to‐use machine‐learning software to semi‐automatically generate dense and widespread abundance records of a habitat‐forming algae over ~5,000 m² of temperate reef. We employ replicable spatial techniques to test the effectiveness of traditional diver‐based sampling, and better understand the distribution and spatial arrangement of one key algal species. We found that the effectiveness of a traditional survey depended on the level of spatial structuring, and generally 10–20 transects (50 × 1 m) were required to obtain reliable results. This represents 2–20 times greater replication than have been collected in previous studies. Furthermore, we demonstrate the usefulness of fine‐resolution distribution modeling for understanding patterns in canopy algae cover at multiple spatial scales, and discuss applications to other marine habitats. Our analyses demonstrate that semi‐automated methods of data gathering and processing provide more accurate results than traditional methods for describing habitat structure at seascape scales, and therefore represent vastly improved techniques for understanding and managing marine seascapes.     

Shifts in aquatic macroinvertebrate biodiversity associated with the presence and size of an alien crayfish

To investigate the effects of Procambarus clarkii on macroinvertebrate diversity, we conducted a mesocosm experiment simulating small pools in rice field pads after the rice season. We hypothesized that crayfish predation would negatively impact macroinvertebrate diversity, and the magnitude of this impact should vary with the size of P. clarkii. We conducted a short-term mesocosm experiment to determine macroinvertebrate diversity in the presence of three size classes and in the absence of crayfish, as well as the diet composition of crayfish from the three size classes. At the end of the experiments, the diet of crayfish was composed of the most available taxa (Culicidae, Chironomus, Tanytarsini and Orthocladinae). These results also show evidence that, in confined areas, crayfish are important predators of major rice pests such as rice Chironominae larvae. Macroinvertebrate diversity was negatively affected by crayfish presence, but the effect was inversely proportional to crayfish size. The highest diversity index was obtained in the absence of P. clarkii, and juvenile crayfish significantly reduced macroinvertebrate diversity. Thus, the impact of P. clarkii on aquatic macroinvertebrates is size dependent and may be relevant in small pools formed in rice field pads from early autumn to late winter. Overall, our findings suggest that the negative effects of P. clarkii on macroinvertebrate diversity may be particularly strong in local natural assemblages confined to puddles of water or small ponds in wetland areas.     

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration [1]. In bilaterians, there are at least 12 subfamilies of Wnt genes [2]. Wnt3 and Wnt8 are required for somitogenesis in vertebrates [3-7] and are thought to be involved in posterior specification in deuterostomes in general [8]. Although TCF and beta-catenin have been implicated in the posterior patterning of some short-germ insects [9, 10], the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. Here we investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum[11]. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. Our results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, we propose that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes.