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 proteinMutant-Analysis, Genotype-Phenotype correlation, crosses, gene transfer

molecular genetics: Isolate und Analyse DNA, cloning, in vitro Mutagenesis

“Reverse genetics”:Protein Genephenotypeconstruction of mutants; analyze in vivo function („phenotype“)

prokaryotic eukaryoticmolecular 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

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- -

- - - -

Forces:-Effective charge of the molecule-Electrical field strength-cancelled by Friction (stokes law)-gel matrix = sieve Small DNA molecules migrate faster

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+

Agarose gel electrophoresis

Stokes law: F = 6πrην

F = frictional force

r = particle radius

η = fluid viscosity

v = particles speed

Working with DNAC. Denaturation/Hybridization

Tm = 81.5 + 16.6(log10[Na+]) + 0.41(fraction G+C) -600/N

„probe“

„Southern“ hybridization

DNA topologyDefinition: knot-like arrangements that segments of DNA may assume.

positive supercoil: twisted in same direction as right handed helixnegative supercoil: DNA twisted opposite to hight handed helix

DNA topology: requires constraints

„Relaxed“ DNA supercoiled DNA constraint

Proteins

circular DNA

DNA topoisomerasesTopoisomerase I

nicks one strand of DNA double helixrelaxes 1 negative supercoil per nickimportance: in front of replication fork

Topoisomerase IIcuts both strands of DNA double helixrelaxes or introduces 2 supercoils per cutimportance: DNA gyrase from E. coli (replication, decatenation)

supercoiled plasmids

non-denaturing agarose gel-electrophoresisstaining of DNA: ethidium bromide

plasmid (neg. supercoiled)

denatured supercoiled(non-digestible)

plasmid (relaxed)

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+

chromosomal DNA(contamination)

-R +R -R +R

plasmid multimers

DNA topology (Bacteria, Archaea)

chinolones (i.e. nalidixic acid)fluorochinolones (i.e. ciprofloxacin)novobiocin

Gyrase inhibitors = antibiotics:

Ciprobay

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 plasmidsshotgun

physical map of R. sphaeroides

Genome sequencing

1977 Bacteriophage FX174 5 kb1982 Phage l 48 kb1990 Vaccinia 192 kb1991 Cytomegalovirus 229 kb1992 Marchantia polymorpha Mitochondrium 187 kb1992 Marchantia polymorpha Chloroplast 121 kb1993 Variola (Pocken) 186 kb1995 Haemophilus influenzae Rd 1830 kb1995 Mycoplasma genitalium 580 kb1996 Saccharomyces cerevisiae 12’500 kb1996 Mycoplasma pneumoniae 816 kb1996 Methanococcus jannaschii 1665 kb1996 Synechocystis PCC6803 3573 kb1997 Escherichia coli 4639 kb1997 Bacillus subtilis 4200 kb1997 Helicobacter pylori 1668 kb

Chromosome organization

Prokaryotes: few introns, little repetitive DNA (Alu etc)Sequence database: http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi

(141 on 8.4.02)

(15 on 8.4.02)

(41 finished + unfinished on 8.4.02)

Science. 1997 277(5331):1453-74.

rrn operons

tRNA genes

REP sequences

G/C composition of +/- strand

phage proteins

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 genomesmutations

Rearrangements, deletions, horizontal gene transfer(Lawrence (1997) Trends Microbiol. 5: 355-359)

Original chromosome

4800 kb 100 Million years

Final chromosome

4800 kb

3000 kb new DNA

620 kb „old“ DNAdeleted

2380 kb of new DNAlost right away

620 kb new

DNA integratedE. coli(old)

E. coli(new)

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 mutagenesisdeletion 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!!!

Minimal number of protein genes: 470 (Mycoplasma genitalium)

Genome size: 4.6 Mbp 1.8 Mbp 0.6 Mbp

RUSSELL F. DOOLITTLE Nature 416, 697 - 700 (2002)

Genes „lost“ in the smallest genomes

bacteria living inside host cells

a „minimal metabolism“

Microbiol Mol Biol Rev. 2004 Sep;68(3):518-37

genome: prediction of metabolic pathwaysHelicobacter pylori 26695circular genome1,667,867 bpNature 388, 1997, S. 545

Nature 388, 1997, S. 545

H. pylori: prediction of metabolic capacity

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

in total: 9 aa biosynthesis pathways missing7 aa biosynthesis pathways incomplete

T. whipplei cultivated in fibroblast cell line (HEL)

axenic medium: DMEM (aa), 10% FCS,1% glut., non-essential aa

The Lancet (2003), 362, pp. 447-449

Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus3.7 Mbp50% GC3584 Orfsmany: DNases, proteases, RNases, glycanases, lipases

A Predator Unmasked: Life Cycle of Bdellovibrio bacteriovorus from a Genomic PerspectiveSnjezana 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 feedback-loops (plasmid/host factors)

- very abundant in nature:300 identified in E. coli isolates

Plasmids

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.

paramters of interest: example: cloning vector

size small

copy number high (10->100)

selection marker antibiotic resistance

host range narrow (safety)

fertility (conjugation?) non conjugative

ori of replication (incompatibility group) i.e. ColE1

additional genes i.e. lacZ- (insert screening)

Plasmid encoded phenotypes

Examples of naturally ocurring plasmids

F-Plasmid Fertility plasmid for conjugative transfer of genes

R-Plasmids Antibiotics resistancee.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

LactococcusplasmidpK214

29871 bp

5000

10000

15000

20000

25000

inv soj 03orf59

orf60 orf61

repB

orf63

oriT

nel

IS904

mef214

phnBp35

IS904#IS215binL

rob

IS214-III

str

repDnick site

mob

cat

rep#

IS214-II

orf7 (Tn916)

orf9 (Tn916)

27

tetS

IS214-I

ChloramphenicolR

tetracyclinR

streptomycinR

replication:repB + iterons

macrolideefflux pump

ChloramphenicolR

tetracyclinR

streptomycinR

replication:repB + iterons

macrolideefflux pump

Overview:

2 ways to acquire resistance:mutation of a target geneacqusition of novel genes (source antibiotic producers)

genetic basis of spread of antibiotics resistancefactors involved in spread of resistance:

medicineagriculture

Antibiotic resistance in pathogenic bacteria

Where does antibiotic resistance come from?

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.

pBR322

ancestor of most general cloning vectors:

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.)

stx1

stx2

E. coli K12 vs. O157 Sakai

LEE-locus

stx2

stx2

Phage „cargo“ genes

Evolution of bacterial genomesMutations

Rearrangements, deletions, horizontal gene transfer(Lawrence (1997) Trends Microbiol. 5: 355-359)

Original chromosome

4800 kb 100 Million years

Final chromosome

4800 kb

naked DNAplasmidsphages

620 kb „old“ DNA(includes plasmids, phages)

includes:plasmidsphages

620 kb new

DNA integratedE. coli(old)

E. coli(new)