微生物学技术

The development of analytical methods enabling the accurate identification and enumeration of bacterial species colonizing the oral cavity has led to the identification of a small number of bacterial pathogens that are major factors in the etiology of periodontal disease. Further, t ...

Surface plasmon resonance (SPR) is a powerful tool for kinetic analyses of protein–protein interactions. Here we describe the application of this method to study interactions of membrane proteins involved in multidrug efflux in Escherichia coli.These so-called multidrug efflux ...

Confocal laser scanning microscopy (CLSM) is a useful research tool to explore the exopolysaccharides (EPS) in bacterial biofilms. Here, we describe the analysis of different biofilms of Myxococcus xanthusformed in a modified chamber slide system with CLSM. In conjunction with seve ...

Polysialic acid capsules are the major virulence factors in Escherichia coliK1, K92, and groups B and C meningococci. The sialic acid monomers (2-keto-3-deoxy-5-acetamido-7,8,9-d-glycero-d-galacto-nonulosonic acids) comprising these homopolymeric polysaccharide ch ...

A large number of bacterial species move smoothly on solid surfaces in the absence of extracellular �organelles. In the deltaproteobacterium Myxococcus xanthus, this surface motion, termed gliding motility, involves a novel macromolecular machinery Agl-Glt. During the motili ...

Pili or fimbriae are recognized as essential virulence determinants assembled on the bacterial surface. Gram-positive bacteria produce covalently linked pilus structures that are distinct from gram-negative counterparts. In this chapter, we describe three commonly used te ...

Curli are proteinaceous fibrous structures produced on the surface of many gram-negative bacteria. As a major constituent of the extracellular matrix, curli mediate interactions between the bacteria and its environment, and as such, curli play a critical role in biofilm formation. Cur ...

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 ...