Module 1-1 Bacterial Genetics

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Module 1-1Bacterial Genetics

• Organization of bacterial chromosome Prokaryotic
  DNA replicate, transcription translation

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Topics

• Bacterial chromosome, structure organization
• Prokaryotic DNA replication, transcription,
  translation
• Prokaryotic regulation of gene expression
• Mutations and Selection
• Extra-chromosomal elements.
• - Bacteriophages
• - Plasmid DNA

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Prokaryote

• the genome of prokaryotes is not in a separate
  compartment, haploid. Single chromosome it is
  located in the cytoplasm (although sometimes
  confined to a particular region called a
  nucleoid). Prokaryotes contain no
  membrane-bound organelles their only membrane is
  the membrane that separates the cell form the
  outside world. Nearly all prokaryotes are
  unicellular.

Eukaryotes are defined as having their genetic
material enclosed in a membrane-bound nucleus,
separate from the cytoplasm. In addition,
eukaryotes have other membrane-bound organelles
such as mitochondria, lysosomes, and endoplasmic
reticulum. almost all multicellular organisms
are eukaryotes.
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Prokaryote cond..

• Prokaryotes are haploid, and they contain a
  single circular chromosome. In addition,
  prokaryotes often contain small circular DNA
  molecules called plasmids, that confer useful
  properties such as drug resistance. Only
  circular DNA molecules in prokaryotes can
  replicate.

Eukaryotes are often diploid, and eukaryotes have
linear chromosomes, usually more than 1.
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Prokaryote cond..

• In prokaryotes, translation is coupled to
  transcription translation of the new RNA
  molecule starts before transcription is finished.
  

In eukaryotes, transcription of genes in RNA
occurs in the nucleus, and translation of that
RNA into protein occurs in the cytoplasm. The
two processes are separated from each other.
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Bacteria

• Bacteria review
• one-celled organisms
• prokaryotes
• reproduce by mitosis
• binary fission
• rapid growth
• generation every 20 minutes
• 108 (100 million) colony overnight!
• dominant form of life on Earth
• incredibly diverse

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Bacterial genome

• Single circular chromosome
• haploid
• naked DNA
• no histone proteins
• 4 million base pairs
• 4300 genes
• 1/1000 DNA in eukaryote

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No nucleus!

• No nuclear membrane
• chromosome in cytoplasm
• transcription translation are coupled together
• no processing of mRNA
• no introns
• but Central Dogma still applies
• use same genetic code

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Bacterial Chromosome

• Molecules of double-stranded DNA
• Usually circular
• Tend to be shorter
• Contains a few thousand unique genes
• Mostly structural genes
• Single origin of replication

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Bacterial Chromosome cond..

• The bacterial chromosome is found in region
  called the nucleoid (not membrane-bounded- so the
  DNA is in direct contact with the cytoplasm)

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Bacterial Chromosome cond..
Bacterial Genome is haploid, single chromosome

• The circularity of the bacterial chromosome was
  elegantly demonstrated by electron microscopy in
  both Gram negative bacteria (such as Escherichia
  coli) and Gram positive bacteria (such as
  Bacillus subtilis).
• Bacterial plasmids were also shown to be
  circular.
• Linear chromosomes found in Gram-positive
• Borrelia Streptomyces.

Not all bacteria have a single circular
chromosome some bacteria have multiple circular
chromosomes, and many bacteria have linear
chromosomes and linear plasmids.
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Bacterial Chromosome cond..

• Bacterial chromosomal DNA is usually a circular
  molecule that is a few million nucleotides in
  length
• Escherichia coli ? 4.6 million base pairs
• Haemophilus influenzae ? 1.8 million base pairs
• A typical bacterial chromosome contains a few
  thousand different genes
• Structural gene sequences (encoding proteins)
  account for the majority of bacterial DNA
• The nontranscribed DNA between adjacent genes are
  termed intergenic regions

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Chromosome of E. coli
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Chromosomal Map of Bacteria
Circular genetic map of E coli. Positions of
representative genes are indicated on inner
circle. Distances between genes are calibrated in
minutes, based on times required for transfer
during conjugation. Position of threonine (thr)
locus is arbitrarily designated as 0 minutes, and
other assignments are relative to thr.
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The Complete Sequence of Escherichia coli
Chromosome
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Key features of bacterial chromosomes
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Compaction

• Typical bacterial chromosome must be compacted
  about 1,000-fold
• Bacterial DNA is not wound around histone
  proteins to form nucleosomes
• Proteins important in forming loop domains
• Compacts DNA about 10-fold
• DNA supercoiling
• Topoisomerases twist the DNA and control degree
  of supercoiling

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Comparison of a gene in bacteria

• The length of a typical bacterial operon (usually
  about 3 genes), is about as long as the entire
  bacterial cell !

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The Operon Model
The position that a given gene occupies on a
chromosome
The operon model of prokaryotic gene regulation
was proposed by Fancois Jacob and Jacques Monod.
Groups of genes coding for related proteins are
arranged in units known as operons. An operon
consists of an operator, promoter, regulator, and
structural genes. The regulator gene codes for a
repressor protein that binds to the operator,
obstructing the promoter (thus, transcription) of
the structural genes. The regulator does not have
to be adjacent to other genes in the operon. If
the repressor protein is removed, transcription
may occur.
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The Operon Model
Operons are either inducible or repressible
according to the control mechanism. Seventy-five
different operons controlling 250 structural
genes have been identified for E. coli. Both
repression and induction are examples of negative
control since the repressor proteins turn off
transcription.
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The Operon Model
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Extra-chromosomal Elements

• DNA molecules that replicate as discrete genetic
  units in bacteria are called replicons.
• Extrachromosomal replicons
• - bacteriophages
• - plasmids (non-essential replicons)
• These determine resistance to antimicrobial
  agents or production of
• virulence factors.

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• Bacteriophage (from 'bacteria' and Greek f??e??
  phagein "to eat")
• is any one of a number of viruses that infect
  bacteria. The term is commonly used in its
  shortened form, phage.

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A plasmid is an extra-chromosomal DNA molecule
separate from the chromosomal DNA which is
capable of replicating independently of the
chromosomal DNA. In many cases, it is circular
and double-stranded. Plasmids usually occur
naturally in bacteria, but are sometimes found in
eukaryotic organisms.
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Bacteria genetics
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Bacteria have 4 important advantages for
"traditional types of genetic experiments
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Genetic material in Bacteria
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Thank U!!!
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Nucleic Acid
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Nucleic Acid (DNA RNA)

• Nucleic acids are polynucleotides, consist of
  repeating nucleotide units
• Each nucleotide contains one phosphate group, one
  sugar (pentose
• or deoxypentose) and one base (purine or
  pyrimidine).
• Phosphodiester bonds link the 3'-OH of one
  nucleotide sugar to the
• 5'-OH group of the adjacent nucleotide
  sugar.
• In DNA the sugar is D-2-deoxyribose in RNA the
  sugar is D-ribose.
• RNA has a hydroxyl group on the 2' carbon of
  the sugar.
• In DNA the purine bases are adenine (A) and
• guanine (G), and the pyrimidine bases are
  thymine
• (T) and cytosine (C).
• In RNA, uracil (U) replaces thymine.
• Chemically modified purine and pyrimidine bases
• are found in some bacteria and
  bacteriophages.

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Nucleic Acid Structure

• DNA is a double-stranded helix two strands are
  anti-parallel.
• Double helix is stabilized by H bonds between
  purine pyrimidine bases on the opposite
  strands. A pairs T by 2 H bonds G pairs C by 3
  H bonds.
• Two strands in DNA helix are complementary, ie.
  dsDNA contains equimolar amounts of purines (A
  G) and pyrimidines (T C), with A T and G C.
  
• The mole fraction of G C in DNA varies
  widely among different bacteria.
• DNA is supercoiled and tightly packaged.
• The extent of sequence homology between DNAs from
  different microorganisms determines how closely
  related they are (eg. 16sRNA sequence)
• RNA exists as a single-stranded molecule forms
  hairpin loops (secondary structure) due to
  intra-molecular base-pairing.

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• DNA Replication in Bacteria
• The DNA replicates semiconservatively
• - Each strand in dsDNA serves as a
• template for synthesis of a new
• complementary strand.
• - Result daughter dsDNA molecule -
• contains one old polynucleotide strand
• and one newly synthesized strand.
• Replication of chromosomal DNA in
• bacteria starts at a specific chromosomal
• site called the origin of replication and
• proceeds bi-directionally until the process is
  
• completed.

Autoradiograph of intact replicating chromosome
of E coli. Bacteria were radioactively labeled
with tritiated thymidine
X
Y
.
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• DNA Replication in Bacteria
• DNA replication is initiated whenever cells
  divide, so in rapidly
• growing bacteria a new round of chromosomal
  replication begins
• before an earlier round is completed.
• The origin regions specifically and transiently
  associate with
• the cell membrane after initiation of DNA
  replication. Membrane
• attachment directs separation of daughter
  chromosomes.
• Time required for replication of the entire
  chromosome is
• about 40 minutes (500 1000 nucleotides /
  sec)
• Replicated chromosomes are partitioned into
  each of the
• daughter cells.

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Central Dogma of Molecular Biology How does the
sequence of a strand of DNA correspond to the
amino acid sequence of a protein?  

• DNA codes for RNA production.
• RNA codes for protein production.
• Protein does not code protein, RNA
• or DNA production.
• The end.
• Or in the words of Francis Crick Once
  information has passed into
• protein, it cannot get out again!

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• Revision of the "Central Dogma"
• CAN go back from RNA to DNA (reverse
  transcriptase)
• RNA can also make copies of itself (RNA
  polymerase)
• Still NOT possible from Proteins back to RNA or
  DNA
• Not known mechanisms for proteins making copies
  of themselves.

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• Gene Expression
• Expression of genetic determinants in bacteria
  involves the
• unidirectional flow of information from DNA to
  RNA to
• protein.
• Two processes involved are transcription and
  translation.

 
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Transcription Translation Prokaryotic vs
Eukaryotic cells In a prokaryotic cell, which
does not contain a nucleus, this process happens
at the same time. In Eukaryotic cells, occur at
different cell compartments.
Prokaryotic cell
Eukaryotic cell
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• Transcription
• The DNA-directed synthesis of RNA is called
  transcription.
• Transcription produces RNA molecules that are
  complimentary
• copies of one strand of DNA.
• Only one of the dsDNA strands can serve as
  template for
• synthesis of a specific mRNA molecule.
• mRNAs transmit information from DNA, and each
  mRNA in
• bacteria function as a template for synthesis
  of one or more
• specific proteins.

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• Translation
• The process by which the nucleotide sequence of
  an mRNA molecule
• determines the primary amino acid sequence of
  a protein.
• Ribosomes are complexes of ribosomal RNAs
  (rRNAs) and several
• ribosomal proteins.
• Ribosomes with the aid of transfer RNAs
  (tRNAs), amino-acyl tRNA
• synthesases, initiation factors and elongation
  factors are all involved in
• translation of each mRNA into corresponding
  polypeptide (protein).

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• Translation
• Initiated at an AUG codon for methionine.
• Codons are translated sequentially in mRNA from
  5' to 3'.
• The corresponding polypeptide chain / protein is
  assembled
• from the amino terminus to carboxy terminus.
• The sequence of amino acids in the polypeptide
  is, therefore,
• co-linear with the sequence of nucleotides in
  the mRNA and the
• corresponding gene.

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• The Genetic code
• The "universal" genetic code employed by most
  organisms is a triplet code and it determines how
  the nucleotides in mRNA specify the amino acids
  in the polypeptide.

• 61 of 64 possible trinucleotides (codons) encode
  specific amino acids.
• 3 remaining codons (UAG, UAA or UGA) code for
  termination of translation (nonsense codons do
  not specify any amino acids)
• Exceptions
• UGA as a tryptophan codon in some species of
  Mycoplasma and in mitochondrial DNA.
• Few codon differences in mitochondrial DNAs from
  yeasts, Drosophila, and mammals.

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Gene expression occurs in 2 steps Transcription
of the information encoded in DNA into a molecule
of RNA Translation of the information encoded in
mRNA into a defined sequence of amino acids in a
protein.
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Tutorial

• The sequence of one strand of DNA is
• 5 GGGTAAGCTTATCCCGTA 3
• 3 CCCATTCGAATAGGGCAT 5
• The sequence of the complementary strand from 5
  to 3 is
• A) CCCATTCGAATAGGGCAT
• B) TACGGGATAAGCTTACCC
• C) GGGTAAGCTTATCCCGTA
• D) ATGCCCTATTCGAATGGG

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• The following is the sense strand of the DNA
  sequence. Give the amino acid sequence of the
  protein generated
• after translation.
• 5 ATGGGGTACTACCATCCCAATCATCCCAATAGGTACCCC 3
• TRANSCRIPTION
• 5 AUGGGGUACUACCAUCCCAAUCAUCCCAAUAGGUACCCC 3
• TRANSLATION
• Met Gly Tyr Tyr His Pro Asn
  His Pro Asn Arg Tyr Pro
• 5AUG GGG UAC UAC CAU CCC AAU CAU
  CCC AAU AGG UAC CCC 3

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References

• Charlebois, R. 1999. Organization of the
  Prokaryotic Genome. ASM Press, Washington, D.C.
• Casjens, S. 1998. The diverse and dynamic
  structure of bacterial genomes. Ann. Rev. Genet.
  32 339-377.
• Casjens, S. 1999. Evolution of the linear DNA
  replicons of the Borrelia spirochetes. Curr.
  Opin. Microbiol. 2 529-534.
• Chen, C. 1996. http//www.ym.edu.tw/ig/cwc/end_tro
  ubles/End_Troubles.html
• Jumas-Bilak et al. 1998. Unconventional genomic
  organization in the alpha subgroup of the
  Proteobacteria. J. Bacteriol. 180 2749-2755.
• Kobryn K, Chaconas G. 2001. The circle is broken
  telomere resolution in linear replicons. Curr
  Opin Microbiol. 4(5) 558-564.
• Suwanto, A., and S. Kaplan. 1989. Physical and
  genetic mapping of the Rhodobacter sphaeroides
  2.4.1 genome presence of two unique circular
  chromosomes. J. Bacteriol. 171 5850-5859.
• Suwanto, A and S. Kaplan. 1992. Chromosome
  transfer in Rhodobacter sphaeroides Hfr
  formation and genetic evidence for two unique
  circular chromosomes. J. Bacteriol. 174
  1135-1145.
• Trucksis et al. 1998. The Vibrio cholerae genome
  contains two unique circular chromosomes. Proc.
  Natl. Acad. Sci. USA 95 14464-14469.
• Volff, J.-N., and J. Altenbuchner. 2000. A new
  beginning with new ends linearisation of
  circular chromosomes during bacterial evolution.
  FEMS Microbiol. Lett. 186 143-150.
• Yang CC, Huang CH, Li CY, Tsay YG, Lee SC, Chen
  CW. 2002. The terminal proteins of linear
  Streptomyces chromosomes and plasmids a novel
  class of replication priming proteins. Mol
  Microbiol. 43(2) 297-305.