Mainstreams of Horizontal Gene Exchange in Enterobacteria: Consideration of the Outbreak of Enterohemorrhagic E. coli O104:H4 in Germany in 2011

Escherichia coli O104:H4 caused a severe outbreak in Europe in 2011. The strain TY-2482 sequenced from this outbreak allowed the discovery of its closest relatives but failed to resolve ways in which it originated and evolved. On account of the previous statement, may we expect similar upcoming outbreaks to occur recurrently or spontaneously in the future? The inability to answer these questions shows limitations of the current comparative and evolutionary genomics methods.status: publishe

Mainstreams of horizontal gene exchange in enterobacteria : consideration of the outbreak of enterohemorrhagic E. coli O104:H4 in Germany in 2011

BACKGROUND: Escherichia coli O104:H4 caused a severe outbreak in Europe in 2011. The strain TY-2482 sequenced from this outbreak allowed the discovery of its closest relatives but failed to resolve ways in which it originated and evolved. On account of the previous statement, may we expect similar upcoming outbreaks to occur recurrently or spontaneously in the future? The inability to answer these questions shows limitations of the current comparative and evolutionary genomics methods. PRINCIPAL FINDINGS: The study revealed oscillations of gene exchange in enterobacteria, which originated from marine c- Proteobacteria. These mobile genetic elements have become recombination hotspots and effective ‘vehicles’ ensuring a wide distribution of successful combinations of fitness and virulence genes among enterobacteria. Two remarkable peculiarities of the strain TY-2482 and its relatives were observed: i) retaining the genetic primitiveness by these strains as they somehow avoided the main fluxes of horizontal gene transfer which effectively penetrated other enetrobacteria; ii) acquisition of antibiotic resistance genes in a plasmid genomic island of b-Proteobacteria origin which ontologically is unrelated to the predominant genomic islands of enterobacteria. CONCLUSIONS: Oscillations of horizontal gene exchange activity were reported which result from a counterbalance between the acquired resistance of bacteria towards existing mobile vectors and the generation of new vectors in the environmental microflora. We hypothesized that TY-2482 may originate from a genetically primitive lineage of E. coli that has evolved in confined geographical areas and brought by human migration or cattle trade onto an intersection of several independent streams of horizontal gene exchange. Development of a system for monitoring the new and most active gene exchange events was proposed.This work was funded by the National Research Foundation (South Africa) grant #71261 for National Bioinformatics and Functional Genomics Programme.http://www.plosone.or

Isolierung und Charakterisierung von neuen Actinobakterien und Myxobakterien aus hauptsächlich marinem Habitat

This thesis is divided into 5 parts. The first part is dedicated to the isolation of marine myxobacteria from the “Marine Myxobacteria Cluster” (MMC). Therefore surface sediment was sampled from the Wadden Sea from northern Germany, the specific sequence for marine myxobacteria (MMC) was detected via specific PCR and diverse isolation approaches were started. Finally, 12 halotolerant Myxococcus strains were isolated which were closely related to terrestrial strains and do not harbour the MMC sequence. From these strains no new bioactive secondary metabolites could be detected. Two parts focus on the taxonomical analysis as well as the screening for novel bioactive secondary metabolites from marine actinobacteria. These were isolated from living marine sponges or sediment from Guam and the rhizosphere sediment from mangrove plants from India. Taxonomically, strains ICN19 and 21 seemed to present a new species. For the screening of novel secondary metabolites a new multi resistant test panel could be established, consisting of Gram - positive and -negative bacteria as well as fungi including same multi resistant clinical relevant strains like methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VREF) and different antibiotic resistant E .coli strains. Rakicidins A, B and E as well as the anthraqiunones aloesaponarin II and 5-hydroxyaloesaponarin II were isolated. All five compounds exhibited bioactivity against Gram-positive bacteria, including the MRSA and VREF strains, which was never described before. Furthermore, 5-hydroxyaloesaponarin II was described to be produced by a bacterial wild type strain for the first time. Moreover the bioactive compound staurosporine was isolated from the marine Streptomyces strain ICN21. Out of deep sea samples from the North Atlantic Ocean 4 marine Streptomyces strains were isolated. Using 16S rRNA PCR, MALDI-TOF, RiboPrinter® and DNA-DNA analyses as well as physiological and morphological approaches, the strain ASO4 wet was described as a new species. In the last part of this thesis strain Streptomyces davawensis (roseoflavin producer) and the cinnabaramid producer JS360 were characterized by polyphasic taxonomy and identified as novel species of the genus Streptomyces.Diese Arbeit ist in fünf Teile gegliedert. Der erste Teil beschäftigt sich mit der Isolierung von marinen Myxobakterien aus dem „Marinen Myxobakterien Cluster“ (MMC). Hierzu wurde Oberflächensediment vom Wattenmeer (Norddeutschland) beprobt, die MMC-Sequenz durch eine spezifische PCR detektiert und diverse Isolationsansätze gestartet. Letztendlich konnten 12 halotolerante Myxococcus Stämme isoliert werden. Diese Stämme waren phylogenetisch eng verwandt mit terrestrischen Myxobakterien. Die MMC-Sequenz konnte in keinem der Bakteriengenome detektiert werden. Zwei Teile der Arbeit fokussieren die taxonomische Analyse und das Screening für neue bioaktive Sekundärmetabolite von marinen Actinobakterien. Diese Bakterien wurden von lebenden marinen Schwämmen bzw. marinem Sediment aus Guam und vom rhizophären Sediment von Mangroven Pflanzen in Indien isoliert. Aus taxonomischer Sicht, sind die Stämme ICN 19 und ICN 21 wahrscheinlich neue Arten. Für das Screening von neuen Sekundärmetaboliten wurde ein Test-Panel aus zum Teil multiresistenten Bakterien etabliert. Dieses Panel besteht aus verschiedenen Grampositiven und –negativen Bakterien sowie Pilzen und beinhaltet zusätzlich einige multiresistente, klinisch relevante Bakterienstämme wie einen Methicillin-resistenten Staphylococcus aureus (MRSA), einen Vancomycin-resistenten Enterococcus faecium (VREF) und verschiedene Antibiotika-resistente E .coli Stämme. Im Laufe dieser Arbeite konnten die Substanzen Rakicidin A, B und E sowie die Anthraqiunone Aloesaponarin II and 5-hydroxyaloesaponarin II isoliert werden. Alle fünf Substanzen zeigten eine Bioaktivität gegen Grampositive Bakterien inklusive dem MRSA und VREF Stamm. Diese Aktivitäten wurden zuvor noch nie beschrieben. Außerdem wurde in dieser Arbeit die Produktion der Substanz 5-hydroxyaloesaponarin II erstmalig von einem bakteriellen Wildtyp-Stamm beschrieben und das bioaktive Staurosporin von einem marinen Streptomyceten (ICN 21) isoliert. Im vierten Teil der Arbeit konnten aus Tiefseeproben aus dem Nordatlantischen Ozean vier marine Streptomyceten Stämme isoliert werden. Mit der Hilfe von 16S rRNA PCR, MALDI-TOF, RiboPrinter® und DNA-DNA Analysen sowie physiologischen und morphologischen Untersuchungen konnte der Stamm ASO4 wet als neue Art beschrieben werden. Im letzten Teil wurden die Streptomyceten Stämme S. davawensis (Produzent vom Roseoflavin) und der Cinnabaramid-Produzent JS360 durch polyphasische Taxonomie charakterisiert und als neue Arten der Gattung Streptomyces identifiziert

Phylogeographyj kinship and molecular ecology of sperm whales (Physeter macrocephalus)

The molecular ecology for sperm whales (Physeter macrocephalus) in the northern Gulf of Mexico was investigated in detail using a suite of molecular markers. In addition, several genetic related aspects for the Mediterranean Sea, North Sea and the North Atlantic Ocean putative sperm whale populations were described. These analyses have provided new insights requiring proper management to ensure the survival of the northern Gulf of Mexico sperm whale stock m an area of increasing industrial activity. The majority of surface behavioural reactions witnessed after biopsy darting were mild and short-term. No significant differences were determined between males and females and repeat sampling events on the same individual did not lead to an increase in the response level. Population structuring between the four putative populations, with respect to mtDNA, was highly significant and warrants the classification of each putative population as a unique stock for management purposes. The majority of Gulf of Mexico samples were from females and young males believed to be sexually immature based on rough size estimates. Incidental resampling of a few individuals over periods of days, months and years adds support for site-fidelity to the northern Gulf of Mexico exhibited by at least some whales. Although our sample set compares a more restricted geographic area than previous studies, the lack of significant nuclear differentiation between neighbouring populations suggests that sexually mature males disperse from their natal populations and spread their genes to the more philopatric females. The genetic composition of Gulf of Mexico sperm whale groups fits the mixed sex and bachelor group type so common in other areas of the world, while the two all-male North Sea stranding groups fit the bachelor group scenario. Relatedness within the Gulf of Mexico female-dominated groups was significantly greater than that found between groups, but still surprisingly low and composed of both single and multiple matrilines. Highly related whales (i.e. parent-offspring) were present within groups, but infrequent. The most common relationship found was that of half- siblings. The all-male bachelor groups were comprised of multiple matrilines and members were generally unrelated, although cases for half-sibling pairs were present

Computational Methods for the Analysis of Genomic Data and Biological Processes

In recent decades, new technologies have made remarkable progress in helping to understand biological systems. Rapid advances in genomic profiling techniques such as microarrays or high-performance sequencing have brought new opportunities and challenges in the fields of computational biology and bioinformatics. Such genetic sequencing techniques allow large amounts of data to be produced, whose analysis and cross-integration could provide a complete view of organisms. As a result, it is necessary to develop new techniques and algorithms that carry out an analysis of these data with reliability and efficiency. This Special Issue collected the latest advances in the field of computational methods for the analysis of gene expression data, and, in particular, the modeling of biological processes. Here we present eleven works selected to be published in this Special Issue due to their interest, quality, and originality

Evolutionary genetics of neutral and immune related loci in North American thinhorn sheep (Ovis dalli spp)

The regulation of genetic variation in natural populations is via both neutral and selective processes. Signatures of the neutral processes of drift and gene flow can be found within non-coding regions of the genome, while natural selection acts on variation within coding genes that confer changes in fitness. The affects of neutral and selective processes are examined in thinhorn sheep (Ovis daW spp), a rare example of a North American large mammal that occupies most of its native range. There are currently two recognised thinhorn subspecies (0. d. daW and 0. d. stone i), the validity of which remains uncertain. Microsatellites reveal significant genetic structure throughout the thinhorn species range (FsT=0.16). At least eight regional genetic groups can be defined, the limits of which are delineated by mountain range topology. Strong isolation-by-distance is evident (r=0.75, P<O.OOI), suggesting limited dispersal and philopatry within the species. Analysis of mitochondrial DNA reveals that sheep survived Pleistocene glaciations in four refugia. In addition to the well known refugia of eastern Beringia and areas south of the Laurentide and Cordilleran ice sheets I show evidence of two smaller refugia, providing support for the presence of the disputed 'ice-free corridor' through eastern British Columbia. MtDNA also reveals a genetic signal confirming the past hybridization of thinhorn and bighorn sheep. Patterns of variation in linked microsatellite loci show no evidence of natural selection acting on three genes involved in the thinhorn immune region. Although Watterson tests suggest balancing selection is acting in all genes, evidence for selection is confounded by popUlation structure. Concordantly, the translated coding sequences of thinhorn interferon gamma, natural resistance associated macrophage protein and prion protein have low genetic diversity. In contrast, the major histocompatibility complex locus DRB3 shows significant evidence of overdominance through both an excess of nonsynonymous substitution and transspecies polymorphism

Fuzzy classification of DNA sequences

Práce se zabývá Fuzzy klasifikací sekvencí DNA. V první části práce jsou teoreticky shrnuty informace o fuzzy logice a metodách jejího využití v klasifikaci biologických sekvenčních dat. Druhá část se již prakticky zabývá řešením klasifikačního algoritmu pro posouzení podobnosti sekvencí. Konkrétně pro rozdělení kódujících a nekódujících částí sekvence a využití fuzzy klasifikace v DNA barcodingu.The work deals with the fuzzy classification of DNA sequences. In the first part the theory summarized information about Fuzzy logic and methods of its use in the classification of biological sequence data. The second part is practically deal with the classification algorithm for assessing the similarity of sequences. Specifically, the dividing of coding and non-coding parts of the sequence and the use of fuzzy classification in DNA barcoding.

Asparaginase-based antibody Drug Conjugates

L-Asparaginase (ASNase, EC 3.5.1.1) is a key component of the established combined chemotherapy used for the treatment of pediatric acute lymphoblastic leukemia, having significantly increased the percentage of complete remissions in patients since its introduction in 1970s. The benefit of ASNase treatment is supported by extensive clinical data, while resistance to asparaginase is correlated with poor prognosis. ASNase is an amidohydrolase which shows a prevalent asparaginolytic and a secondary glutaminolytic activity; its therapeutic benefit comes from the depletion of asparagine from the blood stream, on which leukemic cells depends, given their absent or compromised capability to express asparagine synthetase (EC 6.3.5.4) under stress conditions. The ASNase molecules currently used in the clinics are derived from either E. coli (EcAII) or E. chrysanthemi, with the first line drug being PEG-Asparaginase (Oncaspar ®). However, most of the available ASNase products lack optimal pharmaceutical features, in particular because of ASNase high toxicity, due to its untargeted activity; high immunogenicity, due to its bacterial origin and large size; short blood serum half-life, and poor efficacy in specific sub-classes of patients (high risk). The aim of this work was to address these limitations, in order to improve EcAII efficacy, and in particular its high toxicity, on one side, by targeting the drug onto leukemic cells, and its high immunogenicity, on the other side, by miniaturizing the drug. It is expected that tackling these two points should also help to increase the drug efficacy in the treatment of high-risk patients. The adopted strategy consisted in the design of a radically new, anti-CD19 Asparaginase-based Antibody Drug Conjugate (ADC), which was conceived by our research group after the successful engineering of a single domain antibody (sdAb) with asparagynolitic activity, obtained through the rational transfer of E. coli type II asparaginase catalytic residues onto a camelid sdAb backbone (sdASNase) (PATENT# E0115946). The addition of a targeting domain nanobody to the catalytic sdASNase lead to the new concept of Targeted Catalytic Nanobodies (TCANs).In particular, the molecule designed in this work (TCAN3) was composed by a newly selected anti-CD19 nanobody (targeting domain) and by the catalytic sdASNase nanobody (catalytic domain). In order to produce such molecule, several steps were followed. Firstly, the extracellular domain of CD19 was expressed as a C-terminal fusion with the human Fc fragment. For the selection of targeting nanobodies, the Phage Display technique was set up in house, but, due to obstacles encountered in both recombinant CD19 purification and classical Phage Display selection, the Yeast Two Hybrid system was chosen as an alternative strategy for the screening of anti-CD19 intracellular nanobodies. The collaboration with the group of Prof. Cattaneo (SNS, Pisa) resulted in the successful isolation of a nanobody, whose binding to CD19 was confirmed through ELISA tests. The TCAN3 was then assembled joining the selected anti-CD19 nanobody targeting domain to the available sdASNase nanobody through a linker. The TCAN3 was then expressed and purified, and its binding to CD19 was confirmed through ELISA tests. In the meantime, preliminary tests for co-localization studies of CD19 and lysosomes were set up. In conclusion, in this work, TCAN3, a promising targeted asparaginase-based molecule which could help in leukemia therapy, and especially in high-risk forms, was designed and expressed. TCANs, focusing for the first time on the specificity of the metabolic traits of a given tumor and coupling the correct catalytic activity to the appropriate target specificity,might potentially represent a novel general approach to tackle any type of cancer which shows sensitivity to a specific metabolite deprivation

Microbial ecology of biotechnological processes

The investigation of phylogenetic diversity and functionality of complex microbial communities in relation to changes in the environmental conditions represents a major challenge of microbial ecology research. Nowadays, particular attention is paid to microbial communities occurring at environmental sites contaminated by recalcitrant and toxic organic compounds. Extended research has evidenced that such communities evolve some metabolic abilities leading to the partial degradation or complete mineralization of the contaminants. Determination of such biodegradation potential can be the starting point for the development of cost effective biotechnological processes for the bioremediation of contaminated matrices. This work showed how metagenomics-based microbial ecology investigations supported the choice or the development of three different bioremediation strategies. First, PCR-DGGE and PCR-cloning approaches served the molecular characterization of microbial communities enriched through sequential development stages of an aerobic cometabolic process for the treatment of groundwater contaminated by chlorinated aliphatic hydrocarbons inside an immobilized-biomass packed bed bioreactor (PBR). In this case the analyses revealed homogeneous growth and structure of immobilized communities throughout the PBR and the occurrence of dominant microbial phylotypes of the genera Rhodococcus, Comamonas and Acidovorax, which probably drive the biodegradation process. The same molecular approaches were employed to characterize sludge microbial communities selected and enriched during the treatment of municipal wastewater coupled with the production of polyhydroxyalkanoates (PHA). Known PHA-accumulating microorganisms identified were affiliated with the genera Zooglea, Acidovorax and Hydrogenophaga. Finally, the molecular investigation concerned communities of polycyclic aromatic hydrocarbon (PAH) contaminated soil subjected to rhizoremediation with willow roots or fertilization-based treatments. The metabolic ability to biodegrade naphthalene, as a representative model for PAH, was assessed by means of stable isotope probing in combination with high-throughput sequencing analysis. The phylogenetic diversity of microbial populations able to derive carbon from naphthalene was evaluated as a function of the type of treatment