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.Electrophysiological Characterization of Bacterial Pore-Forming Proteins in Planar Lipid Bilayers
Together with patch-clamp, the planar lipid bilayer technique is one of the electrophysiological approaches used to study the biophysical properties of bacterial pore-forming proteins. Electrophysiological studies have provided important insight into the mechanistic details underlying the function of this class of proteins. Although there are different apparatus designs and variations to the process of obtaining channel recordings, the general architecture of a planar lipid bilayer setup involves two compartments filled with an ionic solution and separated by a septum with a micro-aperture, where a phospholipid bilayer is ...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Patch Clamp Electrophysiology for the Study of Bacterial Ion Channels in Giant Spheroplasts of E. coli
Ion channel studies have been focused on ion channels from animal and human cells over many years. Based on the knowledge acquired, predominantly over the last 20 years, a large diversity of ion channels exists in cellular membranes of prokaryotes as well. Paradoxically, most of what is known about the structure of eukaryotic ion channels is based on the structure of bacterial channels. This is largely due to the suitability of bacterial cells for functional and structural studies of biological macromolecules in a laboratory environment. Development of the “giant spheroplast” preparation from E. coli cells was ...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Isolation of Bacteria Envelope Proteins
Proteomic analysis on cell envelope proteins from Gram-negative bacteria requires specific isolation techniques. We found that conventional extraction methods such as osmotic shock cause extracts to be heavily contaminated with soluble cytoplasmic proteins. These cytoplasmic protein contaminants constitute the major signal in proteomic analysis and can overwhelm the signals coming from genuine envelope components. After extensive testing of various protocols for the preparation of envelope contents, we found that a modified version of the method of Oliver and Beckwith consistently produces the cleanest extract of periplasm...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Using Reporter Genes and the Escherichia coli ASKA Overexpression Library in Screens for Regulators of the Gram Negative Envelope Stress Response
We describe methods for screening the E. coliASKA overexpression library for clones that lead to altered expression of reporter genes. First, a promoter of interest is cloned upstream of either the lacZor luxCDABEgenes to yield reporter genes in which transcription is proportional to the levels of β-galactosidase or luminescence produced by strains carrying the reporter. The ASKA library is then condensed into two 96-well plates resulting in mixed preparations of 12 plasmids in each well. The plasmids in each well are transformed into the reporter strain and transformants are screened for either altered β-galacto...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Protein Disulfide Bond Formation in the Periplasm: Determination of the In Vivo Redox State of Cysteine Residues
Many proteins secreted to the bacterial cell envelope contain cysteine residues that are involved in disulfide bonds. These disulfides either play a structural role, increasing protein stability, or reversibly form in the catalytic site of periplasmic oxidoreductases. Monitoring the in vivo redox state of cysteine residues, i.e., determining whether those cysteines are oxidized to a disulfide bond or not, is therefore required to fully characterize the function and structural properties of numerous periplasmic proteins. Here, we describe a reliable and rapid method based on trapping reduced cysteine residues with 4′-...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
A Bird’s Eye View of the Bacterial Landscape
Bacteria interact with the environment through their cell surface. Activities as diverse as attaching to a catheter, crawling on a surface, swimming through a pond, or being preyed on by a bacteriophage depend on the composition and structure of the cell surface. The cell surface must also protect bacteria from harmful chemicals present in the environment while allowing the intake of nutrients and excretion of toxic molecules. Bacteria have evolved four main types of bacterial cell surfaces to accomplish these functions: those of the typical gram-negative and gram-positive bacteria, and those of the Actinobacteria and Moll...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Subfractionation and Analysis of the Cell Envelope (Lipo)polysaccharides of Mycobacterium tuberculosis
The cell envelope ofMycobacterium tuberculosis, the causative agent of tuberculosis in humans, is the source of carbohydrates of exceptional structure which play essential roles in the physiology of the bacterium and in its interactions with the host during infection. Much of what is known about their biosynthesis was derived from the phenotypic analysis of knockout or conditional knockout mutants of mycobacteria generated by random or specific insertional mutagenesis. Here, we describe the current techniques used to subfractionateM. tuberculosiscells and investigate major quantitative and qualitative changes in their cell...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Extraction of Cell Wall-Bound Teichoic Acids and Surface Proteins from Listeria monocytogenes
Gram-positive bacteria contain a cell wall consisting of a thick peptidoglycan layer decorated with surface proteins and polysaccharide-based polymers. The latter include the wall teichoic acids (WTAs), which are anionic glycopolymers covalently linked to the peptidoglycan matrix. They are constituted by a long backbone containing sugars of various sizes (trioses to hexoses) which can be reduced (polyols as in Listeria) or oxidized (uronic acids) and can undergo a variety of species- or often strain-specific modifications and substitutions. These confer unique biochemical properties to WTAs and any defect in the modificati...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
In Vitro Peptidoglycan Synthesis Assay with Lipid II Substrate
Bacterial cell wall peptidoglycan is synthesized from lipid II precursor by two reactions. Glycosyltransferases polymerize the glycan chains and transpeptidases form the peptide cross-links. The bifunctional class A penicillin-binding proteins catalyze both of these reactions. Here, we describe an in vitro peptidoglycan synthesis assay utilizing radiolabeled lipid II substrate to monitor simultaneously peptidoglycan glycosyltransferase and transpeptidase activities. The products of the reaction are separated by high-pressure liquid chromatography and quantified by flow-through scintillation counting. (Source: Springer prot...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Quantitative and Qualitative Preparations of Bacterial Outer Membrane Vesicles
Gram-negative bacterial outer membrane vesicle production and function have been studied using a variety of quantitative and qualitative methods. These types of analyses can be hampered by the use of impure vesicle preparations. Here we describe a set of techniques that are useful for the quantitative analysis of vesicle production and for preparative yields of highly purified vesicles for studies of vesicle function or composition. Procedures and advice are also included for the purification of vesicles from encapsulated and low-yield strains. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
The Outer Membrane of Gram-Negative Bacteria: Lipid A Isolation and Characterization
The isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) are important methodologies utilized to gain understanding of the Gram-negative cell envelope. Here, we describe protocols often employed by our laboratory for small- and large-scale isolation of lipid A from bacterial cells. Additionally, we describe various methodologies including isolation of radiolabeled lipid A, thin layer chromatography, and various mass spectrometry methods. Tandem mass spectrometry is an integral tool for the structural characterization of lipid A molecules, and both coventional collision induced dissociation (CID)...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Assembly of Bacterial Outer Membrane Proteins
Various methods that are routinely used to study the subcellular localization of membrane proteins in wild-type Gram-negative bacteria fall short in genetic studies addressing the biogenesis of outer membrane proteins (OMPs). Here, we describe three biochemical methods that can be used in such studies to evaluate the proper assembly of OMPs into the outer membrane. The methods are based on (1) the differential electrophoretic mobility of folded and nonnative OMPs, (2) the intrinsically high protease resistance of folded OMPs, and (3) the observation that integral membrane proteins are not extracted from the membrane in sol...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Production and Crystallization of Bacterial Type V Secretion Proteins
X-ray crystallography has become the most powerful approach to determine the three dimensional structures of proteins. The major bottleneck issues in protein crystallography are the availability of high-quality protein samples and the production of diffracting crystals. Since the type V secretion pathway involves unusually large substrate proteins (passenger domains or TpsA) and membrane proteins (β-barrel domains or TpsB), crystallography of type V secretion proteins deals with additional challenges in protein production and crystallization efforts. This chapter presents essential procedures used to generate successf...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Isolation of Bacterial Type IV Machine Subassemblies
The bacterial type IV secretion systems (T4SSs) deliver DNA and protein substrates to bacterial and eukaryotic target cells generally by a mechanism requiring direct contact between donor and target cells. Recent advances in defining the architectures of T4SSs have been made through isolation of machine subassemblies for further biochemical and ultrastructural analysis. Here, we describe a protocol for isolation and characterization of VirB protein complexes from the paradigmatic VirB/VirD4 T4SS of Agrobacterium tumefaciens. This protocol can be adapted for isolation of T4SS subassemblies from other gram-negative bacteria ...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Pore Formation by T3SS Translocators: Liposome Leakage Assay
Gram-negative bacteria utilize a dedicated membrane-embedded apparatus, the type III secretion system (T3SS), to inject proteins into host cells. The passage of the proteins across the target membrane is accomplished by a proteinaceous pore—the translocon—formed within the host-cell cytoplasmic membrane. Translocators bound to their chaperones can be expressed in Escherichia coli and subsequently dissociated from the chaperone by guanidine treatment. The pore formation properties of the translocators can then be studied by an in-vitro liposome leakage assay. Sulforhodamine-B is encapsulated within lipid vesicle...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
