Springer protocols feed by Microbiology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Current Landscape of Biomolecular Approaches for Assessing Biodegradation of Aromatic Hydrocarbons
The ability of bacteria to degrade hazardous pollutants is a valuable tool that can be employed for cleaning contaminated sites. As a result of the complex mixtures of organic compounds present in contaminated areas, the combined genetic information of more than one organism is necessary to enhance the degradation process. Aromatic compounds are believed to constitute approximately 25% of all biomass on earth. Community profiling and other molecular techniques, such as quantitative real-time PCR and fluorescence in situ hybridization, provide the phylogenetic context of the potential key genes associated with the degradati...
Source: Springer protocols feed by Microbiology - October 12, 2016 Category: Microbiology Source Type: news
Primers: Functional Genes for Nitrogen-Cycling Microbes in Oil Reservoirs
Microbial communities found in the subsurface are important in the biogeochemical cycling of nitrogen (N) both in the oxidative and reductive processes, and changes in their functional structure might affect the stability of a petroleum reservoir. In petroleum reservoirs, where in situ conditions are predominantly anoxic, denitrification involving the stepwise reduction of nitrate (NO3−) via nitrite (NO2−) and nitric oxide (NO) to nitrous oxide (N2O) or dinitrogen gas (N2) is a major process. Microorganisms may also decompose organic N to ammonium (NH4
+) by ammonification, which can subsequently...
Source: Springer protocols feed by Microbiology - October 12, 2016 Category: Microbiology Source Type: news
Primers: Bacterial Genes Encoding Enzymes for Aerobic Hydrocarbon Degradation
Alkanes are saturated hydrocarbons that are ubiquitous in the environment. Microbial degradation pathways evolved to activate and catabolise these compounds in order to gain energy and building blocks for cell growth. These pathways involve a number of hydroxylases, which primarily differ according to the nature of the hydrocarbon itself (e.g. aromatic or aliphatic). Given the widespread distribution of alkanes in the environment, a number of variants of such enzymes are present among microbes. Hence, primers designed to detect such environmental variants would require a database with a sufficiently large number of sequenc...
Source: Springer protocols feed by Microbiology - October 12, 2016 Category: Microbiology Source Type: news
Primers: Functional Genes and 16S rRNA Genes for Methanogens
To date, a great number of oligonucleotide probes/primers targeting phylogenetic markers of methanogenic archaea (methanogens), such as 16S rRNA and the gene for theα-subunit of methyl-coenzyme M reductase (mcrA), have been developed and used for the identification and quantification of individuals and groups of methanogens in environmental samples. These probes/primers were designed for different taxonomic levels of methanogens and have been used for studies in environmental microbiology as hybridization probes or PCR primers of qualitative and quantitative molecular techniques, such as high-throughput sequencing, q...
Source: Springer protocols feed by Microbiology - October 12, 2016 Category: Microbiology Source Type: news
Introduction to Genetic, Genomic and System Analyses of Pure Cultures
Although considered to be only a minute fraction of global biodiversity, the handling of the culturable microbiota is still essential for exploring the interface of the bacterial world with lipids, hydrocarbons and other chemicals. This endeavour requires a suite of wet and computational tools that are the subject of the present volume. The protocols detailed below go from generating large volumes of data with a suite of omics to methods for distilling such a data into information and this in turn into new and useful knowledge. To this end, in silico approaches have to go hand-in-hand with new strategies for genome editing...
Source: Springer protocols feed by Microbiology - December 31, 2015 Category: Microbiology Source Type: news
Introduction to Systems and Synthetic Biology in Hydrocarbon Microbiology: Applications
Contemporary Microbial Biotechnology is experiencing a rapid transition between being a mostly trial-and-error endeavour towards becoming a quantitative and predictable branch of contemporary research. A key ingredient of this shift involves the adoption of Systems and Synthetic Biology approaches for either revisiting typical themes (e.g. bioproduction of added-value molecules) or to develop altogether new ones (such as engineering of sensor/actuator devices). The first wave of goods reaching the biotechnological sector largely comes from metabolic engineering of microorganisms for biofuels, fine chemicals and high-added ...
Source: Springer protocols feed by Microbiology - December 31, 2015 Category: Microbiology Source Type: news
Restriction & ndash;Modification Systems as a Barrier for Genetic Manipulation of Staphylococcus aureus
Genetic manipulation is a powerful approach to study fundamental aspects of bacterial physiology, metabolism, and pathogenesis. Most Staphylococcus aureus strains are remarkably difficult to genetically manipulate as they possess strong host defense mechanisms that protect bacteria from cellular invasion by foreign DNA. In S. aureus these bacterial & ldquo;immunity & rdquo; mechanisms against invading genomes are mainly associated with restriction & ndash;modification systems. To date, prokaryotic restriction & ndash;modification systems are classified into four different types (Type I & ndash;IV), all of which have been f...
Source: Springer protocols feed by Microbiology - November 23, 2015 Category: Microbiology Source Type: news
Understanding Staphylococcal Nomenclature
Bacteria are often grouped by a variety of properties, including biochemical activity, appearance, and more recently, nucleic acid sequence differences. In the case of human pathogens, significant work goes into “typing” strains to understand relatedness. This is especially true when trying to understand the epidemiology of these organisms. In attempts to group Staphylococci, a variety of methods and nomenclatures have been employed, which can often serve as a point of confusion to those entering the field. Therefore, the intent of this chapter is to give a brief overview of some common methods and associated n...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Rapid Amplification of cDNA Ends for RNA Transcript Sequencing in Staphylococcus
Rapid amplification of cDNA ends (RACE) is a technique that was developed to swiftly and efficiently amplify full-length RNA molecules in which the terminal ends have not been characterized. Current usage of this procedure has been more focused on sequencing and characterizing RNA 5′ and 3′ untranslated regions. Herein is described an adapted RACE protocol to amplify bacterial RNA transcripts. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Batch Transduction of Transposon Mutant Libraries for Rapid Phenotype Screening in Staphylococcus
In the gram-positive pathogen Staphylococcus aureus, transposon mutagenesis is a useful method of screening large numbers of mutants for a given phenotype. However, constructing a transposon mutant library can take several months of work and validation in the laboratory. In this chapter, we describe a method for batch transduction of existing transposon mutations into new genetic backgrounds. Transduction in S. aureus is accomplished quickly and easily in most commonly used laboratory strains. The method described herein utilizes transduction to facilitate the rapid creation of new libraries and quick screening in strains ...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Electrophoretic Mobility Shift Assays
Experimental demonstration of regulatory protein interactions with the sequences upstream of potential target genes is an important element in gene expression studies. These experiments termed electrophoretic mobility shift assays (EMSAs) provide valuable insight into the mechanism of action of transcription factors. EMSAs combined with downstream applications such as transcriptional analysis help uncover precisely how regulatory proteins control target gene expression. This chapter comprises a guideline for expression and purification of recombinant transcription factor proteins followed by a detailed protocol for EMSAs. ...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Conjugative Transfer in Staphylococcus aureus
The acquisition of plasmids has led to a significant increase in antimicrobial resistance within the staphylococci. In order to study these plasmids effectively, one must be able move the plasmid DNA into genetically clean backgrounds. While the smaller staphylococcal class I (1–5 kb) and class II (10–30 kb) plasmids are readily transferred using bacteriophage transduction or electroporation, these methods are inefficient at moving the larger class III (30–60 kb) plasmids. This review describes methods to transfer class III plasmids via conjugative mobilization. (Source: Springer protocols ...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
De Novo Assembly of Plasmids Using Yeast Recombinational Cloning
Molecular cloning is a cornerstone of modern biology laboratories. However, traditional cloning can be time-consuming and problematic. We outline herein a method that utilizes the endogenous gap repair system of yeast cells to clone and assemble DNA constructs. This system is simple, cheap, and requires minimal reagents. It can be used for the assembly of both simple (single DNA fragments) and complex (multiple DNA fragments) constructs into plasmids. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression
Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a “two-step” qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specif...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
RNA-Sequencing of Staphylococcus aureus Messenger RNA
RNA-sequencing (RNA-seq) is a technique that employs next-generation DNA-sequencing technology to simultaneously sequence all of the RNA transcripts in a cell. It can provide valuable insights into transcript and operon structure, and is rapidly replacing expression microarrays as the technique of choice for determining global gene expression profiles in bacteria. Herein we outline the procedures involved in performing RNA-seq with samples of RNA from Staphylococcus aureus. We draw particular attention to key aspects of sample preparation, such as RNA integrity and removal of ribosomal RNA, and provide details of critical ...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
