实验基础

Understanding molecular mechanisms of protein secretion by bacteria requires the purification of secretion machinery components and the isolation of complexes between the secretion machinery and substrate proteins. Here, we describe methods for the purification of protei ...

The ultrastructure of bacteria is only accessible by electron microscopy. Our insights into the architecture of cells and cellular compartments such as the envelope and appendages is thus dependent on the progress of preparative and imaging techniques in electron microscopy. Here, I g ...

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 d ...

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 ...

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 cytop ...

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 ...

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. Monitor ...

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 prote ...

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 kn ...

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 ...

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 rea ...

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 q ...

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 ...

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 ...

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. Sin ...

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 i ...

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 ...

Investigation of secretion systems is often critical to understanding the virulence mechanisms of bacterial pathogens. With estimates as high as 30–40% of proteins secreted or localized to the cell envelope, information about the subcellular localization and organization of sec ...

In the clinical microbiology laboratory advances in nucleic acid detection, quantification, and sequence analysis have led to considerable improvements in the diagnosis, management, and monitoring of infectious diseases. Molecular diagnostic methods are routinely used to ...

PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency ...