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Chapter 44 : Genomics and Proteomics of Foodborne Microorganisms
Category: Applied and Industrial Microbiology; Food Microbiology
This chapter outlines the basic concepts underlying genomics, proteomics, and microarray technologies of foodborne microorganisms. The heart of all genomics research lies in DNA sequencing. DNA sequencing method utilizes normal DNA replication with a template strand, a primer, DNA polymerase, and a mix of deoxynucleotide triphosphates (dNTPs). DNA microarray analysis is a relatively new technology that allows investigators to take a genome-wide approach to biological systems. While the details of the Gad system were described primarily by experiments with Escherichia coli, genomics and bioinformatics have enabled researchers to identify and study the effects of these genes in other microorganisms. Foods and their microenvironments could potentially be better designed and formulated to minimize the expression of undesirable pathogenic traits (e.g., acid tolerance, virulence, and toxin formation) or to optimize the expression of beneficial properties in desirable microorganisms (e.g., cryoprotection, acidification rates, and adherence to intestinal tissues). The nature of food microbiology has changed dramatically from its historical emphasis on microbial phenotypic properties and behavior to a new perspective dominated by genomic and comparative genomic information. The food microbiologists of the future will become increasingly reliant on genomics and the other omics technologies in their efforts to understand and control microorganisms associated with foods.
The dNTP (A) has a 3′ hydroxyl present on the deoxyribose that the ddNTP (B) does not. This stops DNA strand growth because DNA polymerase no longer has a way to connect the bases in the growing strand.
KEGG pathway map and predicted enzymes for folate metabolism in Lactococcus lactis. Pathway intermediates and reaction products are shown with EC numbers for enzymes which catalyze these reactions. Shaded boxes indicate catalytic activities encoded in the L. lactis genome.
Plasmid insertion into a gene through homologous recombination for inactivation of gene function. The phenotype of the mutant can then be analyzed to investigate the function of the gene.
A replacement or deletion mutant can be created by first cloning two noncontiguous portions of a gene into an integration vector. The vector integrates into a targeted gene within one region of homology (black or light gray regions). Excision of the plasmid from the integrant structure can occur in a manner that either resolves the original gene (wild type) or leaves the deleted version. Points of resolution at steps A, B, C, or D result in various combinations, as illustrated.
Proteomic methods. (A) 2D-PAGE/MS. In the first dimension, proteins are separated based on isolectric point (pI) by using isoelectric focusing (IEF). The proteins migrate along a pH gradient until they reach their pI, at which point they carry no net charge and stop migrating. In the second dimension, proteins are further separated according to molecular weight using sodium dodecyl sulfate-PAGE (SDS-PAGE). Gels are stained to identify protein bands. Individual spots (circles) are excised from the gel, trypsin digested, and sequenced using MS/MS. (B) Protein expression profiling. By overlaying images of 2D-PAGE gels, comparisons can be made between the proteomes of different organisms or differences in protein expression of a single organism in different conditions. Downregulated (dotted circles) and upregulated (solid circles) proteins can be visualized. Adapted from reference 53 .
GadA and GadB catalyze the exchange of the α-carboxyl of glutamate for a proton in the environment, leading to the creation of a molecule of carbon dioxide and one molecule of GABA. GadC is an antiporter that expels GABA from the cell and imports fresh glutamate.
The EvgA/S circuit of acid resistance regulation is dependent on a histidine protein kinase (EvgS) which senses an environmental change, causing it to activate its corresponding response regulator (EvgA), which is then able to act as a transcriptional regulator. The regulation follows a pathway to produce GadE, which ultimately induces the transcription of GadA and GadBC (see Fig. 44.6 ).
Bile salt hydrolases catalyze the hydrolysis of the peptide bond between the amino acid and the cholesterol-derived backbone of the bile salt.
Microorganisms of foodborne significance whose genomes have been sequenced