Tracking the evolution of function in diverse enzyme superfamilies

Tracking the evolution of function in enzyme superfamilies is key in understanding how important biological functions and mechanisms have evolved. New genes are being sequenced at a rate that far surpasses the ability of characterization by wet-lab techniques. Moreover, bioinformatics allows for the use of methods not amenable to wet lab experimentation. We now face a situation in which we are aware of the existence of many gene families but are ignorant of what they do and how they function. Even for families with many structurally and functionally characterized members, the prediction of function of ancestral sequences can be used to elucidate past patterns of evolution and highlight likely future trajectories. In this thesis, we apply in silico structure and function methods to predict the functions of protein sequences from two diverse superfamily case studies. In the first, the metallo-β-lactamase superfamily, many members have been structurally and functionally characterised. In this work, we asked how many times the same function has independently evolved in the same superfamily using ancestral sequence reconstruction, homology modelling and alignment to catalytic templates. We found that in only 5% of evolutionary scenarios assessed, was there evidence of a lactam hydrolysing ancestor. This could be taken as strong evidence that metallo-β-lactamase function has evolved independently on multiple occasions. This finding has important implications for predicting the evolution of antibiotic resistance in this protein fold. However, as discussed, the interpretation of this statistic is not clear-cut. In the second case study, we analysed protein sequences of the DUF-62 superfamily. In contrast to the metallo-β-lactmase superfamily, very few members of this superfamily have been structurally and functionally characterised. We used the analysis of alignment, gene context, species tree reconciliation and comparison of the rates of evolution to ask if other functions or cellular roles might exist in this family other than the ones already established. We find that multiple lines of evidence present a compelling case for the evolution of different functions within the Archaea, and propose possible cellular interactions and roles for members of this enzyme family

Untersuchungen genetischer Antibiotikaresistenz-Determinanten durch Transkriptomsequenzierungen von klinischen Pseudomonas aeruginosa Isolaten

During this work, a Pseudomonas aeruginosa optimized, strand-specific RNA-sequencing method was developed to allow for multiplexed transcriptome data acquisition on Illumina next generation sequencing systems. This technique was used to obtain global sequence and expression information from 149 clinical isolates of different sample origins and with diverse antibiotic resistance profiles (most of them multidrug resistant). The received fraction of up to 75 % mapped mRNA reads per sample, resulting in up to 90 % total genome coverage, was sufficient for in depth data analysis at the single nucleotide level. This data was successfully used to determine high resolution taxonomic relatedness of the isolates and screen for genetic determinants correlating with ß-lactam resistance phenotypes of phylogenetically unrelated strains. A combination of de novo assembly and coverage determination was used to search for acquired resistance genes and led to the identification of nine different ß-lactamases in overall 19 isolates. Furthermore, statistical comparisons were employed and integrated in a publically available database for the analysis of core genetic alteration enrichments (SNPs, stops, gene expression) in phenotypically related groups of isolates. Thereby, six genes of significance in ceftazidime and two in meropenem non-susceptible isolates were identified. Single gene investigation revealed that more than 95 % of the isolates harbored known, dominant genetic resistance determinants which, apart from acquired enzymes, were displayed by constitutive intrinsic ampC overexpression or lack of OprD porin. Furthermore, the effects of low-level sub-inhibitory concentrations of bactericidal antibiotics on the transcriptome of PA14 wild type cells were investigated, revealing a variety of specific responses (DNA repair, cell envelope modification, ribosome regulation), but also common ones as SOS response and the induction of denitrification pathways. These results could be connected to global metabolic reactions as a consequence of intracellular reactive oxygen stress. Using isotope labelling studies, we detected an enhanced biosynthesis of amino acids which are directly derived from tricarboxylic acid cycle dependent or closely related NAD+ and NADP+ dependent pathways. This suggests common impacts of low-level bactericidal antimicrobials on bacterial cellular systems, regardless of their chemical structure, with a likely connection to respiration hyperactivation.Im Verlauf dieser Arbeit wurde eine für Pseudomonas aeruginosa optimierte, Strang-spezifische Sequenziermethode genutzt, um globale Sequenz- und Expressionsinformationen von 149 klinischen Isolaten unterschiedlicher geografischer Herkunft und variierender Antibiotikaresistenzprofile aufzunehmen. Mit einem Anteil von bis zu 75 % mRNA pro Probe konnte eine Gesamtgenomabdeckung von bis zu 90 % erzielt werden, welche ausreichend für detaillierte, sequenzspezifische Analysen auf Einzelnukleotidebene war. Diese Daten wurden dazu verwendet, die Verwandtschaftsbeziehungen der verschiedenen Isolate hoch-auflösend darzustellen und nach genetischen Markern zu suchen, welche in phylogenetisch nicht verwandten Stämmen in Zusammenhang mit ß-Lactam-Resistenz standen. Durch eine Kombination aus de novo assembly und Analyse der relativen Genabdeckung konnten insgesamt neun verschiedene erworbene ß-Lactamasen in zusammen 19 Isolaten detektiert werden. Des Weiteren wurden datenbankintegrierte Hilfsmittel für statistische Vergleiche entwickelt um die Anreicherungen genetischer Marker (SNPs, Stopps, Expressionsdifferenzen) in phänotypisch ähnlichen Gruppen von Isolaten zu ermöglichen. Hiermit konnten sechs signifikante Gene in Ceftazidim und zwei in Meropenem resistenten Isolaten identifiziert werden. Eine detaillierte Untersuchung ausgewählter Gene zeigte zudem, dass in über 95 % die jeweilige erworbene ß-Lactam-Resistenz auf bekannte Hauptmarker wie erworbenen Resistenzenzyme, eine Überexpression der intrinsischen ß-Lactamase ampC oder ein Fehlen des OprD porins zurückzuführen war. Weiterhin wurde der Effekt von geringen, sub-inhibitorischen Konzentrationen bakterizider Antibiotika auf Wildtyp Zellen untersucht. Hierbei konnten neben spezifischen transkriptionellen Anpassungen eine Reihe von globalen Stressadaptionen wie die Induktion der SOS Antwort und des Denitrifikationsstoffwechsels beobachtet werden, die vermutlich aufgrund erhöhter intrazellulärer Sauerstoffradikalkonzentrationen auftraten. Mit Hilfe von Isotopenmarkierungen konnte eine Verbindung zu globalen metabolischen Veränderungen aufgezeigt werden. Diese führten unabhängig vom eingesetzten Antibiotikum zu einer erhöhten Biosynthese von Aminosäuren, die in direkter Abhängigkeit zum Zitratzyklus oder nahe verwandten NAD+ und NADP+ bedingten Prozessen standen. Somit lässt sich eine Überstimulation der Atmungskette als unabhängige Stressantwort auf geringe Antibiotikakonzentrationen vermuten

Complete Genome Sequence of the N2-Fixing Broad Host Range Endophyte Klebsiella pneumoniae 342 and Virulence Predictions Verified in Mice

We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels

Interactions of insertion sequences targetting integron associated recombination sites

Bacteria of genus Pseudomonas are known for crossing ecological barriers and establishing in clinical settings. Their ability to withstand antibiotic selection in the clinical environment is largely due to interactions between different mobile genetic elements (MGEs) and the presence of multidrug resistance integrons (MRIs). It is difficult to predict resistance gene maintenance within a bacterial population, as the source of these genes is unknown, and the biological processes governing their flow is difficult to quantify in vivo. This study explores the IS1111-attC subgroup of insertion sequences as a model for this process in Pseudomonads. In this study, IS1111-attC elements were found to be overrepresented within non-clinical Pseudomonas isolates relative to clinical Pseudomonads, as well as an enteric outgroup. The observed IS1111-attC distribution suggests that all instances of IS1111-attC elements in class 1 integrons represent recent invasions of attC sites occurring when class 1 integrons were present in the same cells as chromosomal integrons. Target site preferences and transposition mechanisms of the IS1111-attC elements distribution patterns were investigated using in vitro and in vivo models. These elements were shown to specifically recognize the attC recombination sites of integrons in binding assays and to specifically target the attC in mobility assays. Factors affecting the rate of movement between environmental and clinical Pseudomonads were also examined. Significantly, the IS1111-attC transposase binds preferentially to the single strand forms of the top strand of the attC site, rather than the bottom strand attC site which is the target of the integron integrase. This is the first evidence for IS1111-attC mobility in Pseudomonas cells occurring via a similar mechanism to integron gene mobilization, illustrating a way for these elements both to move between chromosomal and plasmid borne integrons, and to facilitate interactions between them

Phenotypic and genotypic analysis of blaCTX-M encoding plasmids isolated from bovine E. coli samples in the United Kingdom

The purpose of this study was to characterize blaCTX-M plasmids originating from bovine Escherichia coli and investigate their contribution to bacterial host fitness. In this study 52 bovine Escherichia coli strains collected between March and October 2007 encoding blaCTX-M, an extended spectrum β-lactamase (ESBL) gene conferring resistance to 3rd generation cephalosporins, were characterized. The majority of strains belonged to E. coli commensal phylogroups A and B1 expressing a multi-drug resistance (MDR) phenotype and harboured multiple plasmids of which 90% were transferred by conjugation. Transconjugants or transformants were made successfully from all 52 strains when selecting for resistance to cefotaxime. All plasmids were shown by PCR and sequence analysis to harbour blaCTX-M and nearly 80 % encoded multiple resistances. Plasmid sequence analysis of four plasmids encoding blaCTX-M-14b (IncI1-X1), -15 (IncFII-FIA-FIB) and -32 (IncX1 and IncB), identified genes necessary for stable plasmid maintenance and spread. Five representative plasmids encoding blaCTX-M-1, -15, -14b and -32 were assayed for their fitness impact upon the host. Efficiencies of β-lactam hydrolysis using whole cell extracts were determined in the same E. coli BL21 host strain with the most efficient encoded by blaCTX-M-14b and blaTEM-1 ESBL genes and least efficient encoded blaCTX-M-15 only. A 160 kb plasmid encoding 13 resistance genes was grown in the presence of 380 different metabolites and differences in metabolite utilisation between this and the plasmid-free BL21 strain determined. The plasmid-harbouring strain utilized less phosphor-sulphur compounds, suggesting the metabolic cost incurred by acquiring the plasmid may have implications of cellular utilization of alternative phosphate sources. There were no differences in growth was observed in nutrient rich media. The contribution of active efflux to resistance was investigated using L-phenylalanyl-L- 4 arginyl-b-naphthylamide (PAβN) in combination with ampicillin, cefotaxime or ceftazidime. Minimum inhibitory concentration (MIC) values were found to decrease ≥ 2 fold in the presence of the efflux pump inhibitor (EPI), in some cases becoming completely susceptible to ampicillin. This indicates that the possible use of EPIs in combination with previously failed antimicrobial drugs to potentially restore efficacy of treatment

Genome analysis of an entomopathogenic nematode belonging to the genus Oscheius and its insect pathogenic bacterial endosymbiont

A thesis submitted to the Faculty of Science under the school of Molecular and Cell Biology in fulfilment for requirements for Doctor of Philosophy Degree. February 2016The use of synthetic chemical pesticides has several negative implications for the Agricultural industry, which include the development of resistance to the insecticides, crop contamination and the killing of non-target insects. This has brought many scientists in the field of nematology and entomology to investigate biological control agents which can help solve identified challenges and these biocontrol agents have also included entomopathogenic nematodes. The majority of entomopathogenic nematodes species that have been isolated belong to Heterorhabditids and Steinernematids which act as vectors for insect pathogenic bacteria species belonging to the genera, Photorhabdus and Xenorhabdus, respectively. However, other species of nematodes, one of which includes a strain of Caenorhabditis briggsae, have also been shown to act as a vector for an insect pathogenic strain of Serratia marcescens. Oscheius sp. TEL-2014 EPNs have been observed to act as vectors for insect pathogenic bacteria belonging to the genus Serratia. In this study a novel insect pathogenic Serratia sp. strain TEL was isolated from the gut of infective juveniles belonging to a species of Oscheius sp. TEL-2014. Next generation sequencing of the bacteria was conducted by generating genomic DNA paired-end libraries with the Nextera DNA sample preparation kit (Illumina) and indexed using the Nextera index kit (Illumina). Paired-end (2 × 300 bp) sequencing was performed on a MiSeq Illumina using the MiSeq reagent kit v3 at the Agricultural Research Council Biotechnology Platform. Quality control and adapter trimming was performed and the genome was assembled using SPADES. 19 contigs were generated with an average length of 301767 bp and N50 of 200,110 bp. The genome of the Serratia sp. TEL was found to be 5,000,541 bp in size, with a G+C content of 59.1%, which was similar to that of other Serratia species previously identified. Furthermore, the contigs were annotated using NCBI Prokaryotic Genome Automatic Annotation Pipeline. Features of the annotated genome included protein encoding sequence or genes, rRNA encoding genes, tRNA encoding genes, ncRNA sequences and repeat regions. 4,647 genes were found and 4,495 were protein-coding sequences (CDS). The genome contains 36 pseudo genes, 2 CRISPR arrays, 13 rRNA genes with five operons (5S, 16S, 23S), 88 tRNAs genes, 15 ncRNA sequences and 9 frameshifted genes. Several genes involved in virulence, disease, defense, stress response, cell division, motility and chemotaxis were identified. This genome sequence will allow for the investigation of identified genes and that will be critical in furthering the understanding of the insect pathogenicity of Serratia sp. strain TEL. Furthermore, it will provide additional genomic insights about the insect-nematode interactions and thus help us improve their ability to be used as biological control agents in agricultural industries. Oscheius sp. TEL-2014 was tested for its entomopathogenicity and it was found that this species was able to infect and kill two model insects Galleria mellonella and Tenebrio molitor. This new nematode species brought 100% mortality within 72 h post-exposure in G. mellonella and whereas, within 96 hours in T. molitor. Following morphometrics analysis of Oscheius sp. TEL-2014 it was concluded that this nematode is described as a novel entomopathogenic nematode species based on its morphometrics and 18S rRNA gene sequence originality. Whole genome sequencing of Oscheius sp. TEL-2014 inbred lines (7 and 13) was performed using Illumina Hiseq sequencing system and paired ends library preparation protocol. Sequencing reads assembled on Velvet resulted in generation of 75965 contigs (line 7) and 53190 contigs (line 13). Gene prediction tools showed that proteins involved in gene expression and DNA replication are present in Oscheius sp. TEL-2014. The draft genome of Oscheius nematodes will support the improvement and initiation of further studies intended to help us understand the molecular and metabolic processes in this genus

Proteomic analysis of neonatal meningitis-causing Escherichia coli

Meningitis in newborns is a serious infection that causes mortality and neurological injury worldwide. The infection progresses from sepsis, and is dependent on the pathogen being able to cross the blood-brain barrier and invade the spinal fluid. One of the most common gram-negative organisms to cause neonatal meningitis is Escherichia coli. In this work, neonatal meningitis-causing E. coli strains H622 and IHE3034 were grown in clinical blood cultures alongside commensal E. coli K12 derivative J53. The bacteria were purified, lysed, and digested with proteases, before being analysed using mass spectrometry. The mass spectrometry results were quantified in order to create quantitative protein profiles of each bacterium. By using statistical and computational analysis, we compared the strains and identified proteins that were differentially expressed between the pathogenic strains and J53. The results indicate that the pathogenic strains share a number of regulatory mechanism, and demonstrate a higher expression than J53 of virulence factors, motility proteins, and proteins involved in capsule synthesis. In addition to the mass spectrometric analysis, the bacteria were characterised using genetic and phenotypic methods. The results indicate that the pathogenic strains H622 and IHE3034 share a closer evolutionary relationship than either does with J53. The mass spectrometry raw files have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD005779.Masteroppgave i biomedisinBMED39