分子微生物生态学手册

分子微生物生态学手册内容摘要：

crosslinking interbridges of the peptidoglycan layers of the cell walls of bacteria. The applicability of Proteinase K for disrupting bacterial cell walls is enhanced by its insensitivity to specific chelating agents, allowing it to be utilised in bination with EDTA and lysozyme. However, the peptide interbridges of the cell walls of different species, formed by different binations of ponent amino acids, with inherently different susceptibilities to cleavage, may be more or less resistant to Proteinase K lysis. While lysozyme and proteinase K are, probably, the enzymes most monly used for the disruption of bacterial cells, additional bacterial celldisrupting enzymes also have been reported with broad or narrow specificities. Other muramidases, mutanolysin and lysostaphin react, analogous to lysozyme, at the peptide linkages in the cell walls, although the species which are susceptible to these enzymes differ from those which are affected by lysozyme [2, 20, 26]. Subtilisins are extracellular proteases, produced by Bacillus spp., exhibiting a broad specificity in hydrolysing most peptide and ester bonds [24]. They are not inactivated by chelating agents, which makes them applicable in bination with EDTA. The application of achromopeptidase has been limited to the disruption of Grampositive cells, principally staphylococci [9], although applications with other bacteria have been reported. Cell disruption by detergent treatments Detergents provide effective, yet relatively gentle, means for disrupting cells, binding strongly to proteins and causing irreversible denaturation. Further, conditions which cause dissociation of protein (., high pH, low and high ionic strength, etc.) tend to enhance, as well, the solubilisation efficiencies of detergents [7]. Detergents are particularly effective for disrupting bacteria when their cell walls have been damaged (., through the actions of metal chelating agents, lysozyme and Proteinase K) prior to their addition to the cell suspension. Sodium dodecyl sulfate (SDS) is an anionic detergent which reacts, at low concentrations, at protein hydrophobic sites, binding cellular proteins and lipoproteins, forming SDSpolypeptide micellar plexes, and effectively denaturing them and promoting the dissociation of nucleic acids [17]. Further, SDS inhibits nucleases and does not interact with the hydrophilic nucleic acids. Some proteins form SDS plexes only after they have been heated or treated with reagents (., mercaptoethanol) to cleave intraprotein disulfide bonds. Nlauroylsarcosine (Sarcosyl), empirically, may be more effective at denaturing cellular polysaccharide material and can be used, instead of SDS, for the disruption of bacterial cells (., Azotobacter, Beijerinckia, Klebsiella, etc.) which produce copious amounts of capsule. Cetyltrimethyl ammonium bromide (CTAB), a cationic detergent, has been used extensively in the preparation of nucleic acids from fungi and plants, when large amounts of polysaccharide materials tend to interfere with the extraction. However, CTAB also has been proven useful for DNA extractions from bacterial cells by denaturing and precipitating the cell wall lipopolysaccharides and proteins [12]. In the presence of monovalent cation (., Na+) concentrations above M, DNA will remain soluble. Nonpolar detergents, including the Triton X series, Tween series, Nonidet P40, etc., are generally “milder” solubilising agents than the polar detergents and they seem to have a much more limited ability to initiate the disruption of bacterial cells. Cell disruption by “physical” methods Bacteria whose cell walls are not susceptible to enzymatic and detergent treatments may be disrupted using “harsher” (., also on the DNA) methods which may be described, arbitrarily, as “physical” or “mechanical” [ 10,11,14,19]. Such methods generate DNA which is often sheared and usually not of the relatively uniform, large, molecular weight that can be attained using enzymatic and detergent disruption. Thus, such methods may not be appropriate for preparing DNA for specific analytical techniques. However, in instances wherein it has not been critical that the DNA be of uniform high molecular weight, methods employing a French pressure cell or a sonicator have been used with success. The use of glass particles with the (mini)bead beater is particularly effective for disrupting most bacteria and is the method of choice for the preparation of DNA from bacterial cells in problematic matrices (., soils) [23]. Additionally, a method for the production of high molecular weight DNA from Grampositive and acidfast bacteria using a microwave oven has been described [1]. However, the efficacies of such methods, all of which require additional, specialised, equipment, have been limited, in most cases, in the range of bacteria for which a given method can be applied. A further application which has been shown to be effective, particularly in bination with other steps, for disrupting extremely recalcitrant bacteria is the freeze (in liquid nitrogen) and fast thaw (at 95– 98176。 C) technique. This method is often used in procedures for extracting nucleic acids directly from environmental samples, such as soil and sediment [22]. Such a treatment enhances bacterial cell disruption (., particularly species producing protective capsular slime and those involved in the formation of biofilms) by inducing phase changes in cell membranes through successive, rapid, extremes in temperature which render cells more susceptible to enzymatic and detergent lysis. Nucleic acid extractions The isolation of DNA from cells (., selectively eliminating other cellular ponents except the DNA) is the most straightforward of the three general steps. The methods of choice for extractions, traditionally, have involved the app。