1. Chapter 15
Microbial Genomics

2. 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

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

4. Dideoxyadenosine Triphosphate (ddATP)
Figure 15.1

5. 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

6. The Sanger Method
Figure 15.2

7. 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

8. Table 15.1

9. Whole-Genome Shotgun Sequencing
Figure 15.3

10. 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)

11. 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

12. Finding Potential Protein Coding Genes
Figure 15.4

13. Table 15.2

14. 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

15. 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

16. 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.

17. 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

18. 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.

19. 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

20. 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

21. 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

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

23. 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

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

25. 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