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Microbiology Spectrum Apr 2014Transcription factors (TFs) play a central role in regulating gene expression in all bacteria. Yet until recently, studies of TF binding were limited to a small number... (Review)
Review
Transcription factors (TFs) play a central role in regulating gene expression in all bacteria. Yet until recently, studies of TF binding were limited to a small number of factors at a few genomic locations. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) provides the ability to map binding sites globally for TFs, and the scalability of the technology enables the ability to map binding sites for every DNA binding protein in a prokaryotic organism. We have developed a protocol for ChIP-Seq tailored for use with mycobacteria and an analysis pipeline for processing the resulting data. The protocol and pipeline have been used to map over 100 TFs from Mycobacterium tuberculosis, as well as numerous TFs from related mycobacteria and other bacteria. The resulting data provide evidence that the long-accepted spatial relationship between TF binding site, promoter motif, and the corresponding regulated gene may be too simple a paradigm, failing to adequately capture the variety of TF binding sites found in prokaryotes. In this article we describe the protocol and analysis pipeline, the validation of these methods, and the results of applying these methods to M. tuberculosis.
Topics: Binding Sites; Chromatin Immunoprecipitation; Computational Biology; DNA; Genetics, Microbial; Molecular Biology; Mycobacterium tuberculosis; Protein Binding; Sequence Analysis, DNA; Transcription Factors
PubMed: 26105820
DOI: 10.1128/microbiolspec.MGM2-0035-2013 -
Frontiers in Cellular and Infection... 2012
Topics: Evolution, Molecular; Gene Transfer, Horizontal; Genetic Variation; Genetics, Microbial; Selection, Genetic
PubMed: 23091803
DOI: 10.3389/fcimb.2012.00127 -
Molecular Microbiology Jun 1999Microbial genome sequencing is driven by the need to understand and control pathogens and to exploit extremophiles and their enzymes in bioremediation and industry. It...
Microbial genome sequencing is driven by the need to understand and control pathogens and to exploit extremophiles and their enzymes in bioremediation and industry. It is hard for the traditional bacteriologist to grasp the scale and pace of the venture. Around two dozen microbial genomes have now been completed and, within a decade, genomes from every significant species of bacterial pathogen of humans, animals and plants will have been sequenced. Indeed, we will often have more than one sequence from a species or genus--for example, we already have sequences from two strains of Helicobacter pylori, from two strains of Mycobacterium tuberculosis and from three species of Pyrococcus. However, genome sequencing risks becoming expensive molecular stamp-collecting without the tools to mine the data and fuel hypothesis-driven laboratory-based research. Bioinformatics, twinned with the new experimental approaches forming functional genomics', provides some of the needed tools. Nonetheless, there will be an increasing need for us to explore the detailed implications of genomic findings. Microbial genome sequencing thus represents not a threat, but an exciting opportunity for molecular microbiologists.
Topics: Computational Biology; Databases, Factual; Genetics, Microbial; Genome; Humans; Internet; Sequence Analysis, DNA
PubMed: 10490383
DOI: 10.1046/j.1365-2958.1999.01427.x -
Research in Microbiology Jun 2014
Topics: Gene Expression Regulation, Bacterial; Genetics, Microbial; History, 20th Century; Molecular Biology; Operon
PubMed: 24853964
DOI: 10.1016/j.resmic.2014.05.012 -
Research in Microbiology Jun 2014
Topics: Gene Expression Regulation, Bacterial; Genetics, Microbial; History, 20th Century; Molecular Biology; Operon
PubMed: 24859143
DOI: 10.1016/j.resmic.2014.05.017 -
Microbiology Spectrum Aug 2014Genetic strategies have yet to come into their own as tools for antibiotic development. While holding a lot of initial promise, they have only recently started to bear... (Review)
Review
Genetic strategies have yet to come into their own as tools for antibiotic development. While holding a lot of initial promise, they have only recently started to bear fruit in the quest for new drug targets. An ever-increasing body of knowledge is showing that genetics can lead to significant improvements in the success and efficiency of drug discovery. Techniques such as high-frequency transposon mutagenesis and expression modulation have matured and have been applied successfully not only to the identification and characterization of new targets, but also to their validation as tractable weaknesses of bacteria. Past experience shows that choosing targets must not rely on gene essentiality alone, but rather needs to incorporate knowledge of the system as a whole. The ability to manipulate genes and their expression is key to ensuring that we understand the entire set of processes that are affected by drug treatment. Focusing on exacerbating these perturbations, together with the identification of new targets to which resistance has not yet occurred--both enabled by genetic approaches--may point us toward the successful development of new combination therapies engineered based on underlying biology.
Topics: Anti-Bacterial Agents; Bacteria; Drug Discovery; Genetics, Microbial; Molecular Biology
PubMed: 26104208
DOI: 10.1128/microbiolspec.MGM2-0030-2013 -
FEMS Yeast Research Sep 2015In recent years, there has been a noticeable rise in fungal infections related to non-albicans Candida species, including Candida glabrata which has both intrinsic... (Review)
Review
In recent years, there has been a noticeable rise in fungal infections related to non-albicans Candida species, including Candida glabrata which has both intrinsic resistance to and commonly acquired resistance to azole antifungals. Phylogenetically, C. glabrata is more closely related to the mostly non-pathogenic model organism Saccharomyces cerevisiae than to other Candida species. Despite C. glabrata's designation as a pathogen by Wickham in 1957, relatively little is known about its mechanism of virulence. Over the past few years, technology to analyse the molecular basis of infection has developed rapidly, and here we briefly review the major advances in tools and technologies available to explore and investigate the virulence of C. glabrata that have occurred over the past decade.
Topics: Animals; Candida glabrata; Candidiasis; Disease Models, Animal; Genetics, Microbial; Humans; Molecular Biology; Virulence; Virulence Factors
PubMed: 26205243
DOI: 10.1093/femsyr/fov066 -
Croatian Medical Journal Aug 2005The field of microbial forensics was formalized because of the need for attribution in events where a bioweapon has been used. Microbial forensics has its origins in... (Review)
Review
The field of microbial forensics was formalized because of the need for attribution in events where a bioweapon has been used. Microbial forensics has its origins in traditional forensics, microbiology, and epidemiology. Microbial forensics can be defined as a scientific discipline dedicated to analyzing evidence for attribution purposes from a bioterrorism act, biocrime, hoax, or inadvertent microorganism/toxin release. This is a very challenging task, since there are myriad microorganisms that can pose a threat, and analytical methods need to be used reliably. The Scientific Working Group on Microbial Genetics and Forensics (SWGMGF) has addressed some quality assurance and control issues, and particularly validation criteria (focusing on preliminary validation) due to the dynamic nature of evolving investigations. Unique identification of a microorganism may never be possible. Yet, qualitative and/or quantitative assessments of the evidence can be made. One approach to provide direction on gaps in the microbial forensics effort is to perform an end-to-end retrospective analysis of past cases. As an example, the case of a gastroenterologist who was accused of second degree attempted murder of his paramour using HIV as the weapon was reviewed. The scientific evaluation involves epidemiology, molecular biology, phylogenetics, and legal deliberations.
Topics: Bioterrorism; Crime; Forensic Medicine; Genetics, Microbial; HIV; HIV Seropositivity; Humans; United States
PubMed: 16100753
DOI: No ID Found -
Microbiology Spectrum Feb 2014For bacterial model organisms like Escherichia coli and Bacillus subtilis, genetic tools to experimentally manipulate the activity of individual genes have existed for... (Review)
Review
For bacterial model organisms like Escherichia coli and Bacillus subtilis, genetic tools to experimentally manipulate the activity of individual genes have existed for decades. But for genetically less tractable yet medically important bacteria such as Mycobacterium tuberculosis, such tools have rarely been available. More recently, several groups developed genetic switches that function efficiently in M. tuberculosis and other mycobacteria. Together these systems utilize six transcription factors, eight regulated promoters, and three regulatory principles. In this chapter we describe their design features, review their main applications, and discuss the advantages and disadvantages of regulating transcription, translation, or protein stability for controlling gene activities in bacteria. Genetic elements that enable specific and quantitative control over the activity of individual genes are irreplaceable components of the modern genetic toolbox. They facilitate not only the purification of proteins for biochemical, structural, or immunological studies but can also be applied to improve our understanding of in vivo gene functions. Until recently, only one such tool was available for use in mycobacteria, and its applicability in slowly growing mycobacteria was limited. But during the last decade at least a dozen new systems have been developed. In this chapter we review the design, components, and regulatory mechanisms of the different systems and discuss their main applications.
Topics: Gene Expression Regulation, Bacterial; Genetic Vectors; Genetics, Microbial; Molecular Biology; Mycobacterium
PubMed: 26082123
DOI: 10.1128/microbiolspec.MGM2-0018-2013 -
American Journal of Physiology.... Jul 2009Microbes have been widely used in experimental evolutionary studies because they possess a variety of valuable traits that facilitate large-scale experimentation. Many... (Review)
Review
Microbes have been widely used in experimental evolutionary studies because they possess a variety of valuable traits that facilitate large-scale experimentation. Many replicated populations can be cultured in the laboratory simultaneously along with appropriate controls. Short generation times and large population sizes make microbes ideal experimental subjects, ensuring that many spontaneous mutations occur every generation and that adaptive variants can spread rapidly through a population. Another highly useful experimental feature is the ability to preserve and store ancestral and evolutionarily derived clones. These can be revived in parallel to allow the direct measurement of the competitive fitness of a descendant compared with its ancestor. The extent of adaptation can thereby be measured quantitatively and compared statistically by direct competition among derived groups and with the ancestor. Thus, fitness and adaptation need not be matters of qualitative speculation, but are quantitatively measurable variables in these systems. Replication allows the quantification of heterogeneity in responses to imposed selection and thereby statistical distinction between changes that are systematic responses to the selective regimen and those that are specific to individual populations.
Topics: Adaptation, Physiological; Biomedical Research; Cryopreservation; Environment; Evolution, Molecular; Gene Expression Regulation; Genetics, Microbial; Genotype; Hydrogen-Ion Concentration; Microbiological Techniques; Mutation; Phenotype; Population Density; Reproducibility of Results; Reproduction; Selection, Genetic; Time Factors
PubMed: 19403860
DOI: 10.1152/ajpregu.90562.2008