Chapter 15 Microbial Genomics

Genomics – genetic information Genome – all the genes in an organism study of molecular organization of genomes, their information content, and gene products they encode divided into three areas structural genomics physical nature of genomes – DNA sequence functional genomics – protein encoded how genome functions comparative genomics compares genomes of different organisms – determine differences and similarities

Determining DNA Sequences Sanger Method most commonly used method referred to as the chain-termination DNA sequencing method uses dideoxynucleoside triphosphates (ddNTP)

Dideoxyadenosine Triphosphate (ddATP) Figure 15.1

Sanger method mix single strands of DNA with primer, DNA polymerase I, 4 deoxynucleotides (one of which is radiolabeled), small amount of one ddNTP DNA synthesis occurs; random insertion of ddNTP generates DNA fragments of different lengths four reactions carried out; each with different ddNTP fragments in each reaction mixture separated electrophoretically gel autoradiographed and sequence read

The Sanger Method Figure 15.2

Whole-Genome Shotgun Sequencing developed in 1995 by J. Craig Venter and Hamilton Smith four stage process library construction generates clones of portions of genome random sequencing determines sequences of clones fragment alignment and gap closure editing

Table 15.1

Whole-Genome Shotgun Sequencing Figure 15.3

Bioinformatics analysis of genome data using computers (data base) Combines biology, math, computer science, statistics generates data on genome content, structure, and arrangement also provides data on protein structure and function uses annotation to determine location of genes on newly sequenced genome further examination carried out using in silico analysis (computer analysis)

Genome Annotation process that locates genes in the genome map identifies each open reading frame (potential gene) in genome a reading frame > 100 codons that is not interrupted by a stop codon there is an apparent ribosomal binding site at the 5’ end and terminator sequences at the 3’ end uses databases to assign tentative function of gene

Finding Potential Protein Coding Genes Figure 15.4

Table 15.2

Functional Genomics determination of how genome works (metabolic pathways, transport systems, regualatory and signal transduction mechanism) from alignment (base by base comparison of two or more gene sequences) of gene sequences paralogs – genes arose from gene duplication common ancestral gene – single organism orthologs – genes very similar and are predicted to have same function – different organisms also involves analysis of translated amino acid sequence of presumed genes provides understanding of protein structure and function motif, a short pattern of amino acids, may represent a functional unit within the protein,such as the active site

DNA Microarray Analysis (Gene Chips) can determine which genes are expressed at a specific time arrays are solid supports to which DNA is attached each DNA spot represents a single gene or ORF

Gene Microarrays (gene chips) when the genes are expressed? spotted arrays prepared by robotic application of DNA known DNA attached to glass (genes in an organism) Repressible or inducible genes Environment often influences the expression of certain genes Upregulated, down regulated, rate of expression is expressed at a constant rate.

Analysis of Gene Expression using Microarrays based on hybridization between gene and the targets (the nucleic acids to be analyzed) targets are labeled with fluorescent dyes and then incubated with the gene chip unbound target is washed off the chip is scanned with laser beams to detect fluorescence which indicates that hybridization has occurred

A Microarray System for Monitoring Gene Expression acidic Neutral green neutral Figure 15.9 Helicobacter pylori stomach Acidic neutral - peptic ulcer Certain genes Repressed, induced In an acidic env.

Comparative Genomics provides information about genome structure and evolution as well as on lateral gene transfer (LGT) - horizontal, a major evolutionary force in short-term microbial evolution and long-term speciation is an important tool in understanding how microbes adapt to different ecological niches and in the development of new therapeutic agents such as vaccines

Proteomics the study of the proteome, the entire collection of proteins in an organism produces provides information about genome function not available from mRNA studies information determines what is actually happening in cell is referred to as functional proteomics proteome often analyzed by two-dimensional gel electrophoresis

Two-Dimensional Gel Electrophoresis Based Based on the ph gradient content of AA in proteins isoelectric focusing Each protein shows up as a spot At specific location on the gel Figure 15.11 Up-regulation Down regulation Genes under different conditions

Genomic Analysis of Pathogenic Microbes has provided information about virulence and evolution as well as potential targets for therapy or vaccines

More Findings… comparison of S. aureus and S. epidermidis genomes used to track evolution of antibiotic resistance and virulence origin of ~1,700 shared genes is difficult to determine most strain-specific genes thought to have been introduced by prophages, transposons, insertion sequences and plasmid mediate gene transfer both strains thought to have acquired ability to synthesize a capsule, a major virulence factor, from Bacillus anthracis Artificial heart valves, medical implants

More Findings… Bacillus anthracis (cause of anthrax) genomic analysis suggests it was derived from an insect-infecting ancestor

More Findings… Mycobacterium tuberculosis >250 genes are devoted to lipid metabolism much of its energy may be obtained from degradation of host lipids two families of proteins with unknown function represent ~ 10% of the genome