Download Microbiology

Microbial genetics
Genetics
DNA, its manipulation and the consequences for the organism.
Aims to understand cellular functions and properties of organisms.
classical genetics:
Phenotype genotype  protein
Mutant-Analysis, Genotype-Phenotype correlation, crosses, gene transfer
molecular genetics: Isolate und Analyse DNA, cloning, in vitro Mutagenesis
“Reverse genetics”:
Protein Genephenotype
construction of mutants; analyze in vivo function („phenotype“)
prokaryotic  eukaryotic
molecular biology
central dogma of molecular biology:
Structure of bacterial DNA
- chemical structure: A, C, G, T (like eukaryotes, exception: unmethylated CpG)
-„free“ state (not enclosed by a membrane)
- nucleoid ≙ eukaryotic chromosome
„nucleoid“
Properties of DNA
- 1 bp = 0.34 nm (along helix axis)
- 1 helical turn = 10.4 bp
- 1 nucleotide = 330 Da, 1 bp = 660 Da
- 100 nt: fairly rigid
- chromosomal DNA: flexible
- resistant to alkaline treatment
UV absorption spectrum:
-1 OD260 double stranded DNA = 50 μg/ml
-1 OD260 single stranded DNA = 33 μg/ml
Agarose gel electrophoresis
-
---- -
+
Forces:
-Effective charge of the molecule
-Electrical field strength
-cancelled by Friction (stokes law)
-gel matrix = sieve
 Small DNA molecules migrate faster
Stokes law: F = 6πrην
F = frictional force
r = particle radius
η = fluid viscosity
v = particles speed
Working with DNA
C. Denaturation/Hybridization
„probe“
„Southern“ hybridization
Tm = 81.5 + 16.6(log10[Na+]) + 0.41(fraction G+C) -600/N
DNA topology
Definition: knot-like arrangements that segments of DNA may assume.
positive supercoil: twisted in same direction as right handed helix
negative supercoil: DNA twisted opposite to hight handed helix
DNA topology: requires constraints
„Relaxed“ DNA
supercoiled DNA
constraint
Proteins
circular DNA
DNA topoisomerases
Topoisomerase I
nicks one strand of DNA double helix
relaxes 1 negative supercoil per nick
importance: in front of replication fork
Topoisomerase II
cuts both strands of DNA double helix
relaxes or introduces 2 supercoils per cut
importance: DNA gyrase from E. coli (replication, decatenation)
supercoiled plasmids
-R +R -R +R
chromosomal DNA
(contamination)
plasmid multimers
plasmid (relaxed)
plasmid (neg. supercoiled)
denatured supercoiled
(non-digestible)
+
non-denaturing agarose gel-electrophoresis
staining of DNA: ethidium bromide
DNA topology (Bacteria, Archaea)
Ciprobay
Gyrase inhibitors = antibiotics:
chinolones (i.e. nalidixic acid)
fluorochinolones (i.e. ciprofloxacin)
novobiocin
Bacterial genome:
• Size: 5 x 105 – 107 bp
• Chromosome(s)
–
–
–
–
–
mostly haploid (generally one chromosome)
mostly circular
supercoiled
organized in nucleoid (1 histone like protein/100 bp)
contains essential genes (+ non-essential genes, mobile
elements)
• Plasmids (facultative)
• Phages (facultative)
Genomics = analyzing the genome
• Genetic mapping: markers (conjugation, transduction)
• Physical map: restriction map (size; #chromosomes,
linear/circular)
• high resolution map: clone overlapping DNA fragments
• sequencing:
ordered library of plasmids
shotgun
physical map of R. sphaeroides
Genome sequencing
1977
1982
1990
1991
1992
1992
1993
1995
1995
1996
1996
1996
1996
1997
1997
1997
Bacteriophage FX174
Phage l
Vaccinia
Cytomegalovirus
Marchantia polymorpha Mitochondrium
Marchantia polymorpha Chloroplast
Variola (Pocken)
Haemophilus influenzae Rd
Mycoplasma genitalium
Saccharomyces cerevisiae
Mycoplasma pneumoniae
Methanococcus jannaschii
Synechocystis PCC6803
Escherichia coli
Bacillus subtilis
Helicobacter pylori
5 kb
48 kb
192 kb
229 kb
187 kb
121 kb
186 kb
1830 kb
580 kb
12’500 kb
816 kb
1665 kb
3573 kb
4639 kb
4200 kb
1668 kb
Chromosome organization
(141 on 8.4.02)
(15 on 8.4.02)
(41 finished + unfinished on 8.4.02)
Prokaryotes: few introns, little repetitive DNA (Alu etc)
Sequence database: http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi
rrn operons
tRNA genes
REP sequences
G/C composition
of +/- strand
phage proteins
Science. 1997 277(5331):1453-74.
E. coli K12 chromosome
4.7 x 106 bp, circular
4288 genes; 38% unknown
7 rRNA operons
86 tRNA genes
2192 transcriptional units, 73% monocistronic; 6% >4 genes
start codons: 83% ATG; 14% GTG; 3% TTG; 1 x ATT; 1 x CTG
405 genes with start/stop overlap
largest orf = 2383 aa protein
average orf = 317 aa
314 rep elements
E. coli chromosome
bacterial genomics
genome comparison:
conserved ORF (minimal genome??)
mechanism of genome expansion/contraction
evolution of bacteria (commensal  pathogen)
genome analysis:
metabolic functions
prediction of nutrient requirements
study characteristics
Evolution of bacterial genomes
mutations
Rearrangements, deletions, horizontal gene transfer
(Lawrence (1997) Trends Microbiol. 5: 355-359)
620 kb „old“ DNA
deleted
620 kb new
E. coli
(old)
Original
chromosome
4800 kb
DNA integrated
3000 kb
new DNA
E. coli
(new)
2380 kb of new DNA
lost right away
100 Million years
Final
chromosome
4800 kb
Minimal gene set
- metabolites can be imported; proteins not  relies on own gene set
Minimal gene set =
„least # of genes required for life in extremely rich media“
experimental:
transposon mutagenesis
deletion of each single orf
computational:
small genomes of parasites/endosymbionts
(Mycoplasma, Buchnera)
conserved genes between small genomes (… 150-300 ?)
….there may not be a singular exact answer!!!
Genome size:
4.6 Mbp
1.8 Mbp
0.6 Mbp
Minimal number of protein genes: 470 (Mycoplasma genitalium)
Genes „lost“ in the smallest genomes
bacteria living inside host cells
RUSSELL F. DOOLITTLE Nature 416, 697 - 700 (2002)
a „minimal metabolism“
Microbiol Mol Biol Rev. 2004 Sep;68(3):518-37
genome: prediction of metabolic pathways
Helicobacter pylori 26695
circular genome
1,667,867 bp
Nature 388, 1997, S. 545
H. pylori: prediction of metabolic capacity
Nature 388, 1997, S. 545
Analysis of „non-cultivatable“ bacteria
<< 5 % of all bacteria have been cultured
bacterial consortia in nature:
soil
gut
water
genomics approach:
DNA isolation + sequence analysis (Cosmids, shotgun)
sequence assembly
prediction of metabolic capacity
 study composition of consortium
 design appropriate culture media
Genome based design of Media for
Tropheryma whipplei
T. whipplei cultivated in fibroblast cell line (HEL)
The Lancet (2003), 362, pp. 447-449
in total: 9 aa biosynthesis pathways missing
7 aa biosynthesis pathways incomplete
axenic medium: DMEM (aa), 10% FCS,
1% glut., non-essential aa
Bdellovibrio bacteriovorus
Bdellovibrio bacteriovorus
3.7 Mbp
50% GC
3584 Orfs
many: DNases, proteases, RNases, glycanases, lipases
A Predator Unmasked: Life Cycle of Bdellovibrio
bacteriovorus from a Genomic Perspective
Snjezana Rendulic et al., Science Jan 30 2004: 689-692.
Bdellovibrio bacteriovorus: hydrolytic enzymes
Bacterial genome:
• Size: 5 x 105 – 107 bp
• Chromosome(s)
–
–
–
–
–
mostly haploid (generally one chromosome)
mostly circular
supercoiled
organized in nucleoid (1 histone like protein/100 bp)
contains essential genes (+ non-essential genes, mobile
elements)
• Plasmids (facultative)
• Phages (facultative)
Plasmids
no „house keeping“/essential genes
- size: 1 – 1000 kb (< 5% of chrom.)
- double stranded DNA
- supercoiled
- 1 to >100 copies / cell
- autonomous replication („replicon“)
-replication controlled by feedbackloops (plasmid/host factors)
- very abundant in nature:
300 identified in E. coli isolates
Plasmids
paramters of interest:
size
copy number
selection marker
host range
fertility (conjugation?)
ori of replication (incompatibility group)
additional genes
example: cloning vector
small
high (10->100)
antibiotic resistance
narrow (safety)
non conjugative
i.e. ColE1
i.e. lacZ-a (insert screening)
Nomenclature for recombinant Plasmids:
pXY000
XY: shorthand for name of researcher
000: continuous numbering
zB. pBR322 is the 322th plasmid
made by Bolivar and Rodriguez.
Plasmid encoded phenotypes
Examples of naturally ocurring plasmids
F-Plasmid
Fertility plasmid for
conjugative transfer of genes
R-Plasmids
Antibiotics resistance
e.g. amp, kan, tet, cam
ColE1
Production von colicin, a
bacteriocin against E. coli
Ti
Tumor initiation in plants
pSym
symbiotc plasmid for nodulation
and N2-fixation
Tol
degradation of toluene
E. coli
F-plasmid
99,159 bp
replication + segregation
tetS

IS214-I inv soj 03
orf59
orf60
tetracyclinR
orf61
replication:
repB +
iterons
27
orf9 (Tn916)
repB
orf7 (Tn916)
25000
orf63
5000
Lactococcus
rep#
plasmid
cat  ChloramphenicolR pK214
29871 bp 10000
20000
mob
IS214-II
oriT
nel
IS904
nick site
15000
repD
str 
mef214
streptomycinR
IS214-III
rob
binL
IS215
IS904#
phnB
p35
macrolide
efflux pump
tetracyclinR 
replication:
repB +
iterons

ChloramphenicolR

streptomycinR
macrolide
efflux pump
Where does antibiotic resistance come from?
Overview:
2 ways to acquire resistance:
mutation of a target gene
acqusition of novel genes (source antibiotic producers)
genetic basis of spread of antibiotics resistance
factors involved in spread of resistance:
medicine
agriculture
Antibiotic resistance in pathogenic bacteria
plasmids = tools in modern molecular biology
vector
property_______________________
Cloning:
obtain/analyze a specific piece of DNA
Expression:
regulated promotor
Shuttle:
promotors/ori for diverse organisms
Mobilizable:
transferable by conjugation
broad host range:
works in diverse bacteria
suicide:
site directed mutagenesis
cosmids:
plasmid/phage lambda etc.
ancestor of most general cloning vectors:
pBR322
pGEX-3X
Bacterial genome:
• Size: 5 x 105 – 107 bp
• Chromosome(s)
–
–
–
–
–
mostly haploid (generally one chromosome)
mostly circular
supercoiled
organized in nucleoid (1 histone like protein/100 bp)
contains essential genes (+ non-essential genes, mobile
elements)
• Plasmids (facultative)
• Phages (facultative)
Bacteriophages
=
bacterial viruses
Bacteriophages: plaque formation
Virulent phages
Temperate
phages
Phage lambda
genome
E. coli O157 Sakai
LEE-locus
(Type III secr.)
stx2
stx1
Q
Shiga toxin 1A S
SpLE6
SpLE5
yj
S
S
cI
cro
cII
O
P
int
xis
exo
gam
bet
kil
cIII
ssb
sieB
E. coli K12 vs. O157
Sakai
N
stx2
Sp18
SpLE4
LEE-locus
C.
SpLE3
Sp17
C.
Sp15
SpLE2
Sp14
Sp13
Sp12
Sp11
C.
Sp10
Sp9
Sp8
Sp6 Sp7
SpLE1
Sp5
Sp4
Sp3
Sp2
Sp1
KpLE2
CP4-57
CP4-U
KpLE1
CP4-44
Qin
Rac
e14
DLP
CP4-6
K-12
stx2
O157 Sakai
Sp16
Phage „cargo“ genes
Evolution of bacterial genomes
Mutations
Rearrangements, deletions, horizontal gene transfer
(Lawrence (1997) Trends Microbiol. 5: 355-359)
620 kb „old“ DNA
(includes plasmids, phages)
620 kb new
E. coli
(old)
Original
chromosome
4800 kb
DNA integrated
naked DNA
plasmids
phages
includes:
plasmids
phages
100 Million years
E. coli
(new)
Final
chromosome
4800 kb