Complete Microbial Genome Sequences Research Articles

The Long and Short of Genome Sequencing: Using a Hybrid Sequencing Strategy to Sequence Oral Microbial Genomes.

Since our chapter on genome sequencing using the GS-FLX pyrosequencer in the First Edition of this book, significant advances have been made in next-generation DNA sequencing (NGS) technology. Not only has the GS-FLX become extinct, but the more recent introduction and establishment of the so-called third-generation DNA sequencers by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) has revolutionized genomics yet again by generating ultra-long (>100,000 basepair) sequence reads concomitant with an incredible reduction in cost per sequenced basepair. Unfortunately, the ultra-high sequence yields of third-generation sequencers are compromised by their inherent sequencing error rates, prompting an alternative sequencing strategy, i.e., a hybrid sequencing strategy, which combines PacBio/ONT primary datasets with complementary datasets generated by mainstream short-read NGS platforms, e.g., Illumina or Ion Torrent. Although the concept of a hybrid sequencing strategy is not new, existing yields and accuracy of ultra-long and short-read sequencing technologies makes such a strategy achievable, resulting in complete genome sequences in one hit. In this chapter, we describe our updated laboratory and bioinformatic protocols that will allow the average research group to obtain complete oral microbial genome sequences assembled from a combination of DNA sequence data generated by NGS and third-generation platforms.

Tracing Lifestyle Adaptation in Prokaryotic Genomes

Lifestyle adaptation of microbes due to changes in their ecological niches or acquisition of new environments is a major driving force for genetic changes in their respective genomes. Moving into more specialized niches often results in the acquisition of new gene sets via horizontal gene transfer to utilize previously unavailable metabolites, while genetic ballast is shed by gene loss and/or gene inactivation. In some cases, larger genome rearrangements can be observed, such as the incorporation of whole genetic islands, providing a range of new phenotypic capabilities. Until recently these changes could not be comprehensively followed and identified due to the lack of complete microbial genome sequences. The advent of high-throughput DNA sequencing has dramatically changed the scientific landscape and today microbial genomes have become increasingly abundant. Currently, more than 2,900 genomes are published and more than 11,000 genome projects are listed in the Genomes Online Database‡. Although this wealth of information provides many new opportunities to assess microbial functionality, it also creates a new array of challenges when a comparison between multiple microbial genomes is required. Here, functional genome distribution (FGD) is introduced, analyzing the diversity between microbes based on their predicted ORFeome. FGD is therefore a comparative genomics approach, emphasizing the assessments of gene complements. To further facilitate the comparison between two or more genomes, degrees of amino-acid similarities between ORFeomes can be visualized in the Artemis comparison tool, graphically depicting small and large scale genome rearrangements, insertion and deletion events, and levels of similarity between individual open reading frames. FGD provides a new tool for comparative microbial genomics and the interpretation of differences in the genetic makeup of bacteria.

Commentary: Call for a Quality Standard for Sequence-Based Assays in Clinical Microbiology: Necessity for Quality Assessment of Sequences Used in Microbial Identification and Typing

Subsequent to the early days of radioisotope sequencing, DNA sequencing technologies have evolved rapidly, giving rise to techniques that are more sensitive and rapid and provide longer sequence reads. These incremental improvements in technology and in the automation of modern sequencing platforms have resulted in applications of DNA sequencing across virtually all biological science disciplines. In microbiology, this has led to a significant increase in the number of available complete microbial and viral genome sequences and, consequently, to increasing use of DNA sequence analysis in clinical microbiology laboratories, where the results may be influential on consequent patient management. The evolution of Sangerbased technologies is now being complemented by the revolution of second- and third-generation sequencing technologies that, potentially, could provide whole-microbial-genome analysis at the clinical bench and have a greater, more direct impact on patient and outbreak management. While the technologies have continued to evolve, common standards for the description of DNA sequence data quality still do not exist. Further, utilization of DNA sequence data requires comparison with other similar sequence data held in private or public databases. Similarly, there is no process for the accreditation of these databases and the analyses that they provide to ensure that they are fit for purpose. We strongly urge that action is required to address this. CURRENT USES OF SEQUENCING IN CLINICAL MICROBIOLOGY DNA sequencing is being introduced into microbial diagnosis particularly for the identification of esoteric organisms where traditional methods fail or where the delay to definitive identification by traditional means is significant to patient treatment. The most common locus used for bacterial identification is the 16S rRNA gene (6). In general, the complete sequence data rather than individual bases are important when making comparisons to existing data to make the identification. Other loci, in particular rpoB and gyrB (19, 31), are often used for genera whose species are more difficult to resolve with the 16S locus. Because the identification may be used in clinical management to alter treatment regimens, the quality of the data used to make this identification is crucial. Sequence-based typing methodologies, typified by multilocus sequencing typing (MLST), are often used for typing in epidemiological studies and population biology. The results do not usually have a clinical impact in the short term but may be of great importance in medico-legal cases when identifying source of infection, and therefore data quality is essential. Crucially, with these techniques, a single-base change alters the type; therefore, the quality at individual bases is as relevant as

Environmental Microbiology: Current Technology and Water Applications

“Environmental Microbiology” is the study of the composition and physiology of microbial communities in the environment. Microbial life is amazingly diverse and microorganisms literally cover the planet. One of the major prerequisites for a healthy life is an adequate supply of safe drinking water. Waterborne diseases are still a major cause of death in many parts of the world, particularly in young children, the elderly, or those with compromised immune systems. As the epidemiology of waterborne diseases is changing, there is a growing global public health concern about new and reemerging infectious diseases that are occurring through a complex interaction of social, economic, evolutionary, and ecological factors. An important challenge is therefore the rapid, specific and sensitive detection of waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. However, newer enzymatic, immunological and genetic methods are being developed to replace and/or support classical approaches to microbial detection. Moreover, innovations in nanotechnology and nanosciences are having a significant impact in biodiagnostics, where a number of nanoparticle-based assays and nanodevices have been introduced for biomolecular detection. Molecular techniques based on genomics, proteomics and transcriptomics are rapidly growing as complete microbial genome sequences are becoming available, and advances are made in sequencing technology, analytical biochemistry, microfluidics and data analysis. While the clinical and food industries are increasingly adapting these techniques, there appear to be major challenges in detecting health-related microbes in source and treated drinking waters. This is due in part to the low density of pathogens in water, necessitating significant processing of large volume samples. From the vast panorama of available molecular techniques, some are finding a place in the water industry: Quantitative PCR, protein detection and immunological approaches, loop-mediated isothermal amplification (LAMP), microarrays. This book gives a detailed insight related to all these aspects. The book is divided into 12 chapters. In the first chapter, the authors has discussed in detail about various molecular methods for the detection of microbes in source and drinking water. In second chapter, various advancements in methods used earlier have been given for microbial concentration by reducing the volume of water samples. Third chapter deals with the detection of water borne pathogens by using antibodies and gene-based recognition chemistries as biosensors. In fourth chapter, details regarding screening of multiple waterborne pathogens are given based on low cost technology of loop-mediated isothermal amplification (LAMP), a relatively new DNA amplification technique. In fifth chapter, the authors have stressed the use of microarray technique, whereby, the detection of multiple genetic sequences from different viruses, bacteria, protozoa, and other emerging pathogens is possible. This is advantageous and eliminates all the hurdles faced with earlier techniques. In sixth chapter, the authors have highlighted in detail the general approaches for the growth of overlooked obligate intracellular microorganisms using amoebae as a host. Alternative approach for the isolation of new/novel potentially pathogenic amoeba-resisting microorganisms (ARM) is also described. Chapter seven describes the combination of Raman spectroscopy with Fluorescence in situ Hybridization (FISH) and Stable Isotope Probing (SIP) to gather information about heterogeneity within populations and the role of spatial organization within the environment. In chapter eight, details regarding the detection of viability of organisms and the functional gene expression by using various molecular techniques are given. In chapter nine, the authors have stressed the use of GS-FLX pyrosequencer, a next generation sequencing approach for characterization of microbial community structures in recreational waters and primary sources of faecal pollution. In chapter 10, the author has emphasized the monitoring of faecal pollution by microbial indicators rather than by direct pathogen detection with its advantages. Details regarding use of molecular methods to track faecal sources of pollution in environmental waters are also given. Chapter 11 gives an insight of the various microarrays available for environmental analysis with special reference to the use of Geochips, a powerful tool for rapid, sensitive and specific examination of microbial communities in a high-throughput manner. In chapter 12, the authors have discussed the use of microbes as microbial bioreporter, for its use in detecting chemicals or bacterial pathogens. Their possible use as a biosensor for rapid and versatile water quality monitoring tool is also discussed. This book this focuses on the current and future technologies as well as its applications related to environment with special reference to water microbiology. It will be of immense help to water and environmental microbiologists as well as regulators and is recommended for all life science professionals and scientists.

Exploring Microbial Genome Sequences to Identify Protein Families on the Grid

The analysis of microbial genome sequences can identify protein families that provide potential drug targets for new antibiotics. With the rapid accumulation of newly sequenced genomes, this analysis has become a computationally intensive and data-intensive problem. This paper describes the development of a Web-service-enabled, component-based, architecture to support the large-scale comparative analysis of complete microbial genome sequences and the subsequent identification of orthologues and protein families (Microbase). The system is coordinated through the use of Web-service-based notifications and integrates distributed computing resources together with genomic databases to realize all-against-all comparisons for a large volume of genome sequences and to present the data in a computationally amenable format through a Web service interface. We demonstrate the use of the system in searching for orthologues and candidate protein families, which ultimately could lead to the identification of potential therapeutic targets.

A phylogenomic analysis of the Actinomycetales mce operons

BackgroundThe genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins.ResultsThe M. tuberculosis mce loci each include two yrbE and six mce genes, which have homology to ABC transporter permeases and substrate-binding proteins, respectively. Operons with an identical structure were identified in all Mycobacterium species examined, as well as in five other Actinomycetales genera. Some of the Actinomycetales mce operons include an mkl gene, which encodes an ATPase resembling those of ABC uptake transporters. The phylogenetic profile of Mkl orthologs exactly matched that of the Mce and YrbE proteins. Through topology and motif analyses of YrbE homologs, we identified a region within the penultimate cytoplasmic loop that may serve as the site of interaction with the putative cognate Mkl ATPase. Homologs of the exported proteins encoded adjacent to the M. tuberculosis mce operons were detected in a conserved chromosomal location downstream of the majority of Actinomycetales operons. Operons containing linked mkl, yrbE and mce genes, resembling the classic organization of an ABC importer, were found to be common in Gram-negative bacteria and appear to be associated with changes in properties of the cell surface.ConclusionEvidence presented suggests that the mce operons of Actinomycetales species and related operons in Gram-negative bacteria encode a subfamily of ABC uptake transporters with a possible role in remodeling the cell envelope.

Reconstruction of ancient genome and gene order from complete microbial genome sequences

Microbial genome sequences provide us with the fossil records for inferring their origination and evolution. Assuming that current microbial genomes are the evolutionary results of ancient genomes or fragments and the neighboring genes in ancient genomes are more likely neighbors in current genomes, in this paper we proposed a paleontological algorithm and assembled the orthologous gene groups from 66 complete and current microbial genome sequences into a pseudo-ancient genome, which consists of continuous fragments of various sizes. We performed bootstrap resampling and correlation analyses and the results showed that the assembled ancient genome and fragments are statistically significant and the genes of the same fragment are inherently related and likely derived from common ancestors. This method provides a new computational tool for studying microbial genome structure and evolution.

Metabolism and Genomics: Adventures Derived From Complete Genome Sequencing

The genomic era has provided us with hundreds of complete microbial genome sequences (current estimates as of July 12, 2005, are 266 microbial genomes completed and an additional 730 in progress; see http://www.GenomesOnline.org ) (Mongodin et al. 2005a). Collectively, the sequencing of individual genomes and whole communities has enabled the realization of a level of genetic diversity and complexity that was previously unappreciated (Venter et al. 2004; Mongodin et al. 2005b). This is particularly evident when the results of these endeavors are related to the study of physiological processes and metabolic capabilities of both the individual species and community members from a range of environments. Often, species are found to harbor the genetic material for metabolic pathways that had not been identified or tested in the laboratory setting, and it has become increasingly evident that we are some distance away from understanding the tremendous biological, physiological and metabolic diversity and potential that clearly exists in the microbial world. The chemical process of sequencing allows for the determination of the primary structure of a region of DNA (the main information carrier in a cell). The result of this process is a determination of the exact order of the four-nucleotide building blocks (adenine, cytosine, guanine and thymidine abbreviated A, C, G, T, respectively) that make up the DNA region in question. Completing the entire genome sequence of an organism thus provides a comprehensive representation of the entire sequence of the organism under study and its genome structure including the presence of chromosomes and in the case of prokaryotes, the presence of plasmids. Upon completing the sequence of a microbial genome, a thorough analysis of the genetic data should follow (detailed in Fig. 1⇓). This process typically begins with the identification of all open reading frames (ORFs). A variety of ORF finding …

Seeing is believing: the impact of structural genomics on antimicrobial drug discovery.

Over the past decade, the availability of complete microbial genome sequences has led to changes in the strategies that are used to search for novel anti-infectives. However, despite the identification of many new potential drug targets, novel antimicrobial agents have been slow to emerge from these efforts. In part, this reflects the long discovery and development times that are needed to bring new drugs to market and the bottlenecks at the stages of identifying good lead compounds and optimizing these leads into drug candidates. Structural genomics will hopefully provide opportunities to overcome these bottlenecks and populate the antimicrobial pipeline.

Genomics-based identification of targets in pathogenic bacteria for potential therapeutic and diagnostic use

The availability of numerous complete microbial genome sequences has profoundly altered our understanding of a number of fundamental biological processes. For example the enzymes involved in aminoacyl-tRNA (AA-tRNA) synthesis, the key process responsible for the accuracy of protein synthesis, have been found to be highly species-specific. In particular, a number of pathogens contain certain pathways of AA-tRNA synthesis that are unrelated to those found in their mammalian hosts. Since AA-tRNA synthesis is indispensable for cell viability, the discovery of pathogen-specific pathways and enzymes presents novel therapeutic and diagnostic targets. Here we will review recent advances in the elucidation of AA-tRNA synthesis pathways and discuss the possible pharmaceutical exploitation of these discoveries. In particular, the integration of genomic and biochemical approaches to identify novel targets for the treatment of Chlamydial infections and the diagnosis and treatment of Lyme disease will be presented.

Testing ancient RNA–protein interactions

The past decade in molecular biology has seen remarkable advances in the study of the origin and early evolution of life. The mathematical tools for analyzing DNA and protein sequences, coupled with the availability of complete microbial genome sequences, provide insight almost as far back as the age of the nucleic acids themselves. Experimental evolution in the laboratory and especially in vitro evolution of RNA provide insight into a hypothetical world where RNA, or a close relative, may have debuted as a primary functional and informational molecule. The ability to isolate new functional RNAs from random sequences now ultimately makes the world of possible primitive chemical interactions accessible even when the molecules or reactions are no longer present in modern species. Thus we can at last form direct experimental tests of specific models for the origin of RNA-protein associations, such as those that influenced the genetic code. This marks a turning point for probing the origin and early history of life at the molecular level.

Findings emerging from complete microbial genome sequences

Sixteen microorganisms, including one eukaryote, four archaeons, and 11 eubacteria, have been completely sequenced and published. More than 50 genomes are scheduled to be completed by the year 2000. This explosive growth of information is forcing change in many scientific disciplines (e.g. bioinformatics and molecular genetics), spawning new fields, and even changing the way scientific information is used and shared. Novel, global genome sequence comparisons seem slow to appear but the infrastructure for these projects is being built, and we expect exciting developments in the near future.

Microbial pathogenesis: genomics and beyond.

The growing number of complete microbial genome sequences provides a powerful tool for studying the biology of microorganisms. In combination with assays for function, genomic-based approaches can facilitate efficient and directed research strategies to elucidate mechanisms of bacterial pathogenicity. As genomic information accrues, the challenge remains to construct a picture of the biology that accurately reflects how individual genes collaborate to create the complex world of microbial specialization.