Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed

MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis

Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses and fungi

Microbial identification by mass cataloging

BACKGROUND: The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using hybridization and other molecular approaches. In their usual format, such assays are based on the presence of unique subsequences in the target RNA and require a prior knowledge of what organisms are likely to be in a sample. They are thus limited in generality when analyzing an unknown sample. Herein, we demonstrate the utility of catalogs of masses to characterize the bacterial 16S rRNA(s) in any sample. Sample nucleic acids are digested with a nuclease of known specificity and the products characterized using mass spectrometry. The resulting catalogs of masses can subsequently be compared to the masses known to occur in previously-sequenced 16S rRNAs allowing organism identification. Alternatively, if the organism is not in the existing database, it will still be possible to determine its genetic affinity relative to the known organisms. RESULTS: Ribonuclease T(1 )and ribonuclease A digestion patterns were calculated for 1,921 complete 16S rRNAs. Oligoribonucleotides generated by RNase T(1 )of length 9 and longer produce sufficient diversity of masses to be informative. In addition, individual fragments or combinations thereof can be used to recognize the presence of specific organisms in a complex sample. In this regard, 140 strains out of 1,921 organisms (7.3%) could be identified by the presence of a unique RNase T(1)-generated oligoribonucleotide mass. Combinations of just two and three oligoribonucleotide masses allowed 54% and 72% of the specific strains to be identified, respectively. An initial algorithm for recovering likely organisms present in complex samples is also described. CONCLUSION: The use of catalogs of compositions (masses) of characteristic oligoribonucleotides for microbial identification appears extremely promising. RNase T(1 )is more useful than ribonuclease A in generating characteristic masses, though RNase A produces oligomers which are more readily distinguished due to the large mass difference between A and G. Identification of multiple species in mixtures is also feasible. Practical applicability of the method depends on high performance mass spectrometric determination, and/or use of methods that increase the one dalton (Da) mass difference between uracil and cytosine

MICROBIAL CONTRIBUTIONS TO DISEASE PHENOTYPES

The unseen world of microbes has a profound affect on everyday life. Complex microbial communities play a role in everything from climate regulation to human health and disease pathogenesis. Advancements in the field of Metagenomics are providing a window into the world of microbial communities with an unprecedented resolution. Next-generation sequencing technology is allowing researchers to describe the relationships between these complex microbial communities and their host environments. The research in this dissertation investigates these complex microbial host relationships and the various tools and techniques needed to conduct metagenomic research. Chapter 1 presents a current overview of techniques at the disposal of researchers conducting metagenomics experiments. Topics discussed include qualitative DNA fingerprinting techniques, comparison between Next-generation sequencing platforms, and how to handle statistical analysis of large metagenomic datasets. Chapter 2 deals with the development of Peak Studio, a platform independent graphical user interface, intended to be a pre-processing tool for researchers conducting DNA fingerprinting experiments. Chapter 3 explores how time and microenvironment influence the structure of gut microbial communities in a mouse model. Two experimental cohorts of mice are analyzed through the use of Illumina HiSeq sequencing of the 16S rRNA targeted V6 hypervariable region. Also considered are the effects over time of inoculating mice with a founder microbial community. In total, this dissertation emphasizes the importance of experimental design and the development and use of technology in the exploration of complex microbial communities

Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, also known as Johne’s disease, a chronic intestinal infection in cattle and other ruminants. Paratuberculosis is characterised by diarrhea and weight loss that occurs after a period of a few months up to several years without any clinical signs. The considerable economic losses to dairy and beef cattle producers are caused by reduced milk production and poor reproduction performance in subclinically infected animals. Early diagnosis of infected cattle is essential to prevent the spread of the disease. Efforts have been made to eradicate paratuberculosis by using a detection and cull strategy, but eradication is hampered by the lack of suitable and sensitive diagnostic methods. This thesis, based on five scientific investigations, describes the development of different DNA amplification strategies for detection and characterisation of M. paratuberculosis. Various ways to pre-treat bacterial cultures, tissue specimens and fecal samples prior to PCR analysis were investigated. Internal positive PCR control molecules were developed and used in PCR analyses to improve the reliability and to facilitate the interpretation of the results. The sensitivity of the ultimate methods was found to be approximate that of culture and allowed detection of low numbers of M. paratuberculosis expected to be found in subclinically infected animals. Genomic DNA of a Swedish mycobacterial isolate, incorrectly identified by PCR as M. paratuberculosis was characterised. The isolate was closely related to M. cookii and harboured one copy of a DNA segment with 94% similarity to IS900, the target sequence used in diagnostic PCR for detection of M. paratuberculosis. This finding highlighted the urgency of developing or evaluating PCR systems based on genes other than IS900. A PCR-based fingerprinting method using primers targeting the enterobacterial intergenic consensus sequence (ERIC) and the IS900 sequence was developed and successfully used to distinguish M. paratuberculosis from closely related mycobacteria, including the above mentioned mycobacterial isolate. In conclusion, the molecular biology techniques developed in these studies have proved useful for accelerating the diagnostic detection and characterisation of M. paratuberculosis

Antimicrobial potential of Clostridium and closely related species derived from farm environmental samples : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand

The exploration of antimicrobial compounds from natural sources such as bacteria, has been fast tracked by the development of antimicrobial resistance to existing antimicrobials and the increasing consumer demand for natural food preservatives. So far, antimicrobial discovery has been biased towards aerobic and facultative anaerobic bacteria and fungi. Strict anaerobes such as Clostridium species have not been thoroughly investigated for their antimicrobial potential. The objective of the current study was to evaluate the antimicrobial potential of Clostridium and closely related species against bacteria associated with food spoilage, food safety, and human health. Tests on culture media inoculated with Clostridium and closely related species from farm samples (conditioned media/CMs) showed various degrees of antimicrobial activity. Farm 4 soil conditioned medium (F4SCM) showed potential for further investigation in the search for potent antimicrobials with its promising antimicrobial activity. Bacterial isolates (FS01, FS2.2, FS03, and FS04) belonging to Clostridium and closely related spp. associated with F4SCM showed antimicrobial potential as evident by culture-based and genome-based methods. F4SCM and FS03CM (CM prepared from FS03) metabolomes showed the presence of several putative antimicrobial metabolites. Among them, 2-hydroxyisocaproic acid (HICA) showed antimicrobial activity against a wide range of bacteria associated with food spoilage and safety indicating its potential as a bio-preservative agent in food products. The cell cytoplasmic membrane is a likely target of the HICA’s antimicrobial activity. Overall, this study demonstrates that anaerobic bacterial species, Clostridium, and closely related species can produce antimicrobial metabolites, that have potential applications in food preservation and human health. The knowledge obtained in this study will help future investigations to identify and characterize antimicrobials from these Clostridium and closely related bacteria and expands the understanding of the potential to produce antimicrobial compounds from the genus Clostridium and closely related species

Novel bioinformatics programs for taxonomical classification and functional analysis of the whole genome sequencing data of arbuscular mycorrhizal fungi

Résumé [TITRE] Classification taxonomique et analyse fonctionnelle spécifique àla position des séquences génomique des champignons mycorhiziens arbusculaires et les microorganismes qui leurs sont associés [PROBLÉMATIQUE ET CADRE CONCEPTUEL] Les champignons mycorhiziens arbusculaires (CMA) sont des symbiotes obligatoires des racines de la majoritédes plantes vasculaires. Les CMA appartiennent au phylum Glomeromycota et ils sont considérés comme une lignée fongique primitive qui a conservé la structure coenocytique des hyphes et la production des spores asexuées multinucléées. De nombeuses études ont démontréque plusieurs microorganismes sont associés avec les mycélia des CMA soit àla surface des hyphes et des spores mais aussi àl'intérieurs de celles-ci. Le séquençage des génomes des CMA cultivés in-vivo représente un défi considérable car il s’agit d’un métagénome constituédu génome du CMA lui-même et les génomes des microbes qui lui sont associés. Par conséquence, l’identification de l'origine taxonomique de chaque séquence représente une tâche extrêmement ardue. Dans mon projet, j’ai développédeux nouveaux programmes bioinformatiques qui permettent de classer les séquences selon groupe taxonomique et d’identifier les fonctions de celles-ci. J’ai crééune base de données avec 444 génomes d'espèces appartenant à54 genres. Le choix de ces espèces des bactéries et des champignons a étébasésur leur abondance dans les sols). [MÉTHODOLOGIE] Le programme bioinformatique utilise le tableau des références des microorganismes et des méthodes statistiques pour la classification taxonomique des séquences. Par la suite, des tableaux des codons synonymes étaient créés àpartir des structures secondaires (SS) des bases de données de protéines (PDB) pour les séquences codantes (SC) et des motifs de composition pour les séquences non-codantes (SNC). Chaque tableau est composéde 3 niveaux - les caractéristiques d'acides aminés; l'utilisation des acides aminés synonymes correspondants, et l'utilisation des codons synonymes correspondants. En comparant les méthodes existantes qui utilisent les taux de substitution moyenne globale quelle que soit les spécificités des acides aminés dans diverses structures, mon programme fournit une classification àhaute résolution pour des séquences courtes (150-300 pb) parce que les biais dans l'utilisation des codons synonymes àpartir d'environ 8000 trimères d'acides aminés spécifiques des sous-unités de structure secondaire, ont étéextraits avec des substitutions d'acides aminés pris en considération dans chaque trimère spécifique. Pour l'analyse fonctionnelle, le programme crée dynamiquement des données comparatives de 54 genres microbiens basés sur leurs biais dans l'utilisation des codons synonymes d'appariement de trois codons d’ADN (9-mères) identifiés dans une séquence de requête. Le programme applique une analyse en composantes principales basée sur la matrice de corrélation en association avec le partitionnement en k-moyennes aux données comparatives. [RETOMBÉES] Les taux de prédiction correcte de la CDS et les non-CDS étaient de 50 à71% pour les bactéries, et 65 à73% pour les champignons, respectivement. Pour les CMA, 49% des CDS et 72% des non-CDS ont étécorrectement classés. Ce programme nous permet d'estimer les abondances approximatives des communautés microbiennes associées au CMA. Les résultats de l'analyse fonctionnelle peuvent fournir des informations sur des sites d'interaction moléculaire importants impliqués dans la diversification des séquences et l’évolution des gènes. Les programmes sont disponibles gratuitement sur www.fungalsesame.org. Mots-clés: sesame, sesame PS function, les caractéristiques d'acides aminés, trois codons ADN 9-mères, structure secondaire, classification taxonomique, analyse fonctionnelle spécifique àla position; Code génétique; Étude Comparative; Génome MitochondrialAbstract Arbuscular Mycorrhizal Fungi (AMF) are obligate plant-root symbionts belonging to the phylum Glomeromycota. They form coenocytic hyphae and reproduce through large multinucleated asexual spores. Numerous studies have shown that AMF interact closely or loosely with a myriad of microorganisms, particularly bacteria and fungi that live on the surface of or inside of their mycelia and spores. Whole genome sequencing (WGS) data of the AMF grown in-vivo (typically grown in root of a host plant in pot filled with soil) contain a large amount of sequences from microorganisms inhabiting in their spore along with their own genome sequences, resulting in a metagenome. The goal of my study was to develop bioinformatics programs for taxonomical classification and for functional analysis of the WGS data of the AMF. In the area of metagenomics, there are mainly two approaches for taxonomical classification: similarity-based (i.e., homology search) and composition-based (i.e., k-mers) methods. Similarity-based method solely depends on bioinformatics sequence databases and homology search programs such as BLAST program. The similarity-based method may not be suitable for ancient fungi AMF, because bioinformatics databases represent only a small fraction of the diversity of existing microorganisms, and gene prediction programs are highly biased towards intensively studied microorganisms. Considering that AMF have high inter/ intra genome variations, in addition to coenocytic and multi-genomic characteristics, probably due to their adaptation via various kinds of symbioses, composition-based method alone is not an effective solution for AMF, because it relies on base composition biases and focuses on taxonomical classification for prokaryotic organisms. In the first project, I a developed novel bioinformatics program, called SeSaMe (Spore associated Symbiotic Microbes), for taxonomical classification of the WGS data of the AMF. I selected microorganisms that were dominant in soil environment and grouped them into 54 genera which were used as references. I created a reference sequence database with a variable called Three codon DNA 9-mer. They were created based on a large number of structure files from Protein Data Bank (PDB): approx. 224,000 Three codon DNA 9-mers encoding for subunits of protein secondary structures. Based on the reference sequence database, I created genus specific usage databases containing codon usage and amino acid usage per taxonomic rank- genus. The program distinguishes between coding sequence (CDS) and non-CDS, detects an open reading frame, and classifies a query sequence into a genus group out of 54 genera used as reference. The developed program enables us to estimate relative abundances of taxonomic groups and to assess symbiotic roles of taxonomic groups associated with AMF. The program can be applied to other microorganisms as well as soil metagenome data. The program has applications in applied environmental microbiology. The developed program is available for free of charge at www.fungalsesame.org. In the second project, I developed another bioinformatics program, called SeSaMe PS Function, for position specific functional analysis of the WGS data of the AMF. AMF may contain a large portion of genes with unknown functions for which we may not be able to find homologues in existing sequence databases. While existing motif annotation programs rely on sequence alignment and have limitations for inferring functionality of novel genes, the developed program identifies potentially important interaction sites that are structurally and functionally distinctive from other subsequences, within a query sequence with exploratory data analysis. The program identifies matching Three codon DNA 9-mers in a query sequence, and dynamically creates comparative dataset of 54 genera, based on codon usage bias information retrieved from the genus specific usage databases. The program applies correlation Principal Component Analysis in conjunction with K-means clustering method to the comparative dataset. The program identifies outliers; Three codon DNA 9-mers, assigned into a cluster with a single member or with only a few members, are often outliers with important structures that may play roles in molecular interaction. In the third project, I developed a novel bioinformatics program called Posts (POsition Specific genetic code Tables) that assigns a codon into an amino acid group according to the codon position. The standard genetic code table may be more readily applicable to the genes whose genetic codes comply with the standard biological coding rules obtained from model organisms grown under laboratory condition. However, it may be insufficient for studying evolutions of genetic codes that may provide important information about codon properties. The mainstream hypotheses of genetic code origin suggested that codon position played important roles in the evolution of genetic codes. As a case study, we investigated irregular codons in 187 mitochondrial genomes of plants, lichen-forming fungi, endophytic fungi, and AMF. Each column of the Post contains 16 codons and the amino acids encoded by these are called an amino acid characteristics group (A.A. Char Group). Based on A.A. Char Group, an irregular codon can be classified into within-column type or trans-column type. The majority of the identified irregular codons belonged to the within-column type. The Post may offer new perspectives on codon property and codon assignment. The developed program is freely available at www.codon.kr. Taken together, the developed programs, the SeSaMe, the SeSaMe PS Function, and the Post, provide important research tools for advancing our knowledge of AMF genomics and for studying their symbiotic relations with associated microorganisms. Keywords: Sesame; Spore associated Symbiotic Microbes; Symbiosis; Sesame PS function; Arbuscular mycorrhizal fungi; Three codon DNA 9-mer; Amino acid characteristics; Secondary structure; Taxonomical classification; Position specific functional analysis; Position specific genetic code tables; Post; Comparative study; Mitochondrial genom

Using oligonucleotide signatures to build a system for effective detection of pathogenic bacteria in metagenomic samples

Pathogenic bacteria are responsible for millions of deaths every year with an estimated mortality of 70 million people by 2010 for Mycobacterium tuberculosis alone. Novel methods for identification of bacterial species in hosts, urban environments, water sources and food stuffs are required to advance diagnosis and preventative medicine. Detection of bacterial species in environmental samples is a complex task since large numbers of bacteria are present and are resistant to culturing. Therefore, the genetic content of the entire sample has to be analysed simultaneously and this constitutes a metagenomic sample. Commonly-used methods of bacterial identification focus on detection of specific genomic regions to determine species. Currently only one percent of a metagenomic sample can be used for identification employing phylogenetic markers. This method is highly inefficient. The search for more widespread markers within each genome is essential to improve detection methods. Also, modern sequencing technologies used in these environments have short read lengths which prove difficult to assemble e.g. repeats can lead to incorrect assembly. The use of overrepresented oligonucleotides provides a solution to both of these difficulties. Overrepresented oligonucleotides (8-14bp in length) are utilised to differentiate between species based on observed frequency of occurrence rather than presence or absence. They occur throughout the genome thereby increasing genomic coverage. Furthermore, overrepresented oligonucleotides can be easily identified in a raw metagenomic sample, bypassing the need for sequence assembly. Raw oligonucleotide data was filtered, analysed and imported into a structured database. A program, Oligosignatures, allowed for creation of species and phylogenetic lineage specific oligonucleotide markers dependent on the selection of species specified by the user. For the purposes of this study, the context of bacterial identification in an unknown environment was selected. A similarity trial was then executed to determine if strains of the same species can be separated from each other using overrepresented oligonucleotides. Outcomes of this test provided a guideline for the creation of species and lineage specific oligonucleotide markers. Each species and lineage was therefore described by a marker profile which consisted of representative oligonucleotide markers. These marker profiles were then tested against artificial and experimental data to determine their effectivity. Two approaches were used for testing, namely Oligonucleotide frequency analysis and Sequence read analysis. Oligonucleotide frequency analysis focused on the identification of species dependent on the global frequencies of marker oligonucleotides within each marker profile. Sequence read analysis attempted to assign metagenomic reads to a specific species dependent on the number of marker oligonucleotides present within the read. The final database contained 439 bacterial genomes from 22 different phylogenetic lineages. Interpretation of the results obtained after strain similarity testing showed that strains of the same species had highly similar markers and were not separable using this approach. All strains of a species that conformed to this premise were reduced to a single representative member. Similarly, species marker profiles demonstrated that closely related species remained difficult to separate. Twenty-one of the 22 lineages showed sufficient lineage specific markers for use in testing. This provides support for the abundance of overrepresented oligonucleotides and their potential for use as a detection method. In general, metagenomic testing of marker profiles showed that species specific determination was prone to interference, specifically, in closely related species. However, more distantly related species could be separated using both methods. Lineage discrimination generated more reliable results proving that lineage determination was possible in both artificial and experimental datasets. Oligonucleotide frequency analysis, the most sensitive approach, showed the best results for lineage determination but poorer results for species identification. Sequence read analysis provided a more effective method of determining confidence using different thresholds for read classification. In conclusion, the use of overrepresented oligonucleotides holds promise as a novel method for bacterial identification in a metagenomic context. Although several obstacles still prevent optimal utilization of these oligonucleotides, with further research the classification and identification of species and phylogenetic lineages from metagenomic samples can become a reality. CopyrightDissertation (MSc)--University of Pretoria, 2009.Biochemistryunrestricte