Bacteria structure, enzymic analysis

IMSs have been employed as detectors for well-established methods for the determination of bacteria and enzyme-linked immunosorbent assays (ELISA). In ELISA methods, primary antibodies attach to epitopes on the bacterial wall. Each antibody has a structure containing numerous epitopes that can be associated with a secondary antibody. The secondary antibody also has a region with enzymatic activity. This enzymatic region is able to react with a substrate to cleave a product that either is colored and can be determined by a spectrophotometer or is volatile and can be determined by headspace analysis. In the method developed by Snyder et al. and quantitatively explored by Smith et al.," the final product exhibited a distinctive negative product ion peak, as observed with a mobility spectrometer. This was accomplished with the widely deployed military-grade CAM, which was used without modification and suggested that it could serve as a potential bacteria analyzer, provided reagent kits and an inlet adaptor were also distributed. 

After a desired clone is obtained and mapped with restriction enzymes, further analysis usually depends on the deteonination of its nucleotide sequence. The nucleotide sequence of a new gene often provides clues to its function and the structure of the gene product. Additionally, the DNA sequence of a gene provides a guidepost for further manipulation of the sequence, for example, lea ding to the production of a recombinant protein in bacteria. 

Due to phase variation, there are fluctuations in expression levels of certain enzymes in bacteria, therefore, not all colonies or cells make the same structure of lipid A species. A micro-extraction method for extraction of lipid A from a single colony has been developed (Zhou et al., 2009). This method uses microwave-assisted enzymatic digestion and sodium acetate hydrolysis, suitable to analyze lipid A from both cell samples and an individual colony. Because the clean up of SDS is very time-consuming, and the contaminated SDS would seriously interfere with the analysis by mass spectrometry, the proteinase K, instead of SDS, is used to disrupt the cells. Using this method, the entire process for lipid A preparation only takes about 2 h with a detection limit to 1 (xg. 

Among the outer membrane enzymes, OmpT is a special protease that has been implicated in the pathogenicity of bacteria. It is monomeric with the active center pointing to the outside (Vandeputte-Rutten et al., 2001). Another enzyme, the phospholipase A OmpLA, produces holes in the outer membrane when it is activated. The activation process has not yet been clarified, but it is known to require a dimerization of OmpLA in the membrane. The activation by dimer formation has been verified by a crystal structure analysis of an OmpLA dimer that was produced by a reaction with an inhibitor (Snijder et al., 1999). It showed that each active center contained a catalytic triad Ser-His-Asn on one subunit and an ox-anion hole formed by an amide together with a hydrated Ca2+ ion on the other. The active centers are well placed for deacylating lipopolysaccha-rides of the external leaflet of the outer bacterial membrane. OmpLA functions in the secretion of colicins and virulence factors. 

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