Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium tepidum By Natural Transformation

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation NIELS-ULRIK FRIGAARD* AND DONALD A. BRYANT Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania Journal Article Review Heather Jordan BMMB 507 April 8, 2003

What is Chlorobium tepidum ?

Moderately thermophilic green sulfur bacterium

Temperature: 47 o C

Temperature: 47 o C

Anaerobic, obligate autotrophs

Temperature: 47 o C

Live in sulfide-rich aquatic environments

Photo-oxidize reduced sulfur compounds (i.e., sulfide & sulfur)

Found in sediments, muds, microbial mats and anoxic & sulfide-rich waters

Temperature: 47 o C

Live in sulfide-rich aquatic environments

Photo-oxidize reduced sulfur compounds (i.e., sulfide & sulfur)

Found in sediments, muds, microbial mats and anoxic & sulfide-rich waters

Distinct phylogeny (not closely related to other bacteria)

Current Research Interests

Lithotrophic oxidation of sulfur compounds

CO 2 fixation

Photosynthetic electron transport

Energy transfer and organization of the chlorosomes

Biosynthesis and function of chlorophylls

Nitrogen fixation

Genome Highlights

1 circular DNA molecule

2,154,946 bp

G+C content 49.1%

2,284 ORFs

50% have been assigned a known function

Six plasmids (pAQ1-pAQ6)

4.6, 10.0,15.9, 31.0, 38.6 and 115.6 kb respectively

pAQ1 has been sequenced

Transformation of C. tepidum

Methods for natural transformation allow for targeted gene inactivation by homologous recombination

More than 30 mutants have been created with specifically inactivated genes

More than 30 mutants have been created with specifically inactivated genes

Revealed information about processes pertinent to biosynthetic pathways of carotenoids and bacteriochlorophylls to chlorosome proteins

Antibiotic resistance used as marker

Spectinomycin, streptomycin, Ampicillin & chloramphenicol

Can use natural transformation, chemical transformation & electroporation.

Most genes targeted for inactivation were chlorosomal proteins

Most genes targeted for inactivation were chlorosomal proteins

The only genes targeted for inactivation encode:

CsmC & CsmA (chlorosomal proteins)

Reaction center cytochrome c551 Prc

rbcL (Rubisco subunit)

Only CsmC & rbcL fully segregated

Nitrogen Fixation

Major Nutrient

Accounts for 11% of dry weight of bacterial cells

Nitrate reduction & assimilation is a high energy process

Up to 30 % of the electrons generated by photosynthetic H 2 O oxidation are consumed during the reduction of nitrate to ammonia

Cyanobacterial cells preferentially use reduced nitrogen sources

Ammonia & Urea

Nitrogen Fixation

Nitrogen is needed for the synthesis of amino acids & nucleotides

Organic Route: Breakdown of proteins

Inorganic Route: Nitrate Reduction

N fixation is an energetically costly process

Nitrogen fixation related ( nif ) genes are expressed under anaerobic conditions

nifD gene : encodes a subunit of nitrogenase

Study Objective

To form a foundation for the systematic targeted inactivation of genes in C. tepidum (the genome for which had been recently sequenced).

nifD gene used to formulate the general model

Inactivation of nifD expressed phenotypically (inability to grow diazetrophically)

Markers used include Spectinomycin-Streptomycin, Gentamicin & Erythromycin

Maps

NifHDK operon



Maps

NifHDK operon

Streptomycin-Spectinomycin Resistance Cassette



Maps

NifHDK operon

Gentamicin Resistance Cassette



Maps

NifHDK operon

Gentamicin Resistance Cassette

Erythromycin-Chloramphenicol Resistance Cassette



Creating a nifD knockout

Making pTN1CX

nifD knock-out construct for C. tepidum

Restriction Sites :

AhdI (6553)

HindIII (4018, 1639, 896)

ScaI (3139, 1570, 1560, 289)

Sty I (3378, 1390, 1377 339, 69)

SspI (2747, 2701, 1105)

Antibiotic Sensitivity

Temperature:

Lower than optimum (48 o C): 40 o C

Antibiotic resistance markers originate from mesophiles

Concentrations That Inhibit Growth:

Gentamicin, 100 μ g ml -1

Erythromycin, 2 μ g ml -1

Chloramphenicol, 30 μ g ml -1

Tetracycline, 1 μ g ml -1

Streptomycin & Spectinomycin, 300 μ g ml -1 & 150 μ g ml -1 (combined)

Kanamycin (100 μ g ml -1 +)

Ampicillin (100 μ g ml -1 +)

aadA Cassette:

Confers resistance to Streptomycin & Spectinomycin

Antibiotics Not Tested:

Amoxicillin, nalidixic acid, vancomycin, mitomycin C and colistin.

Optimization of Transformation

Transformation Frequency :

100 μl late exponential-phase culture

Incubated with 1 μg of Ahd I-digested pTN1G4

In 10 hours, frequency reached 2E-7 to 3E-7

Corresponds to 100 transformants per μg DNA

:. Most transformation events occurred at the beginning of the experiment and were stable.

• Gentamicin-resistant transformants ° Transformation Frequency

Liquid Suspensions & Transformation :

Same method as for plates

Incubated with 1 μg DNA

Then plated on selective plates

Highest transformation frequency 1 order of magnitude lower than transformation frequencies on agar plates

Why?

“ DNA may interact differently than in liquid suspension… allow for increased uptake of DNA by the cells.”

• Gentamicin-resistant transformants ° Transformation Frequency

Stationary vs. Late Log Phase Cells :

Cells are competent in both phases

Stationary cells gave ½ as many transformants as the late-exponential-phase cells

Linearized DNA

Increasing amounts of DNA yielded an increased transformation frequency

Increasing the DNA from 0.1-10 μg increased the frequency only 3-fold

Suggests that 10 μg of DNA is close to the saturation amount

Transformation frequency with linear plasmid was an order of magnitude higher than with circular plasmid.

Difference is probably due to DNA binding and uptake mechanisms of the cell.

Effect of Variation in Length of Homologous Flanking DNA

When a plasmid construct is made for gene inactivation by homologous recombination, it is typically advantageous to include a large region of the homologous DNA to increase the probability of homologous recombination.

Restriction endonuclease sites and toxic gene products may impose limits on the length of homologous DNA that can be cloned.

To determine the length of homologous flanking DNA on transformation, 3 constructs for inactivation were made.

{ { {

To determine the length of homologous flanking DNA on transformation, 3 constructs for inactivation were made.

Gentamicin resistance marker is inserted in the middle

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Plasmids were digested with :

Ahd I, which cuts only once

Eco RI, which cuts twice.

{ { {

Transformation frequencies with :

2.93 kb homologous DNA

Similar regardless of enzyme used for linearization

Transformation frequency was 1 order of magnitude lower when the plasmid was digested with Eco RI (as oppsed to Ahd I)

No transformation observed regardless of enzyme

Why?

Some bacteria partially degrade absorbed DNA via exonuclease activity.

Given this:

A homologous flanking region of about 1 kb should be used in transformation

Linearize with a plasmid that leaves dispensible flanking DNA at the ends of the fragment

Separate nontransforming DNA from transforming (produced by digest)

Nontransforming may compete with transforming DNA for uptake into the cells

Supported by side experiment in which 20 μg of sonicated chromosomal DNA from Synechococcus to a C. tepidum transformation mixture (containing 1 μg of linearlized DNA) decreased the transformation frequency an order of magnitude.

Various Selection Markers

3 constructs for nifD inactivation were made with different antibiotic resistance markers

Transformation was about the same when the Gentamicin and erythromycin-chloramphenicol resistance markers were used

Oddly, only resistant to erythromicin (even though both genes were present). Confirmed by southern hybridization.

Chloramphenicol marker didn’t function because:

Either the expressed protein is not functional in C. tepidum

Or the promoter is too weak in C. tepidum

Neither of these possibilities were investigated further

Transformation efficiency was 4 orders of magnitude higher with the aadA marker than the other two ( aaC1 & ermC )

The reason for this may lie in the genomic sequence

aadA marker contains a 59-bp “recombinational hot spot”

All 22 mutants created were first screened for antibiotic resistance and then for the expression of the desired phenotype.

Test of Transformants

Expected phenotype of transformants is the inability to reduce dinitrogen.

Results confirmed that mutants had lost nitrogen fixation ability and that mutations were fully segregated.

PCR analysis amplified a 0.41 kb fragment in WT and 1.46 kb fragment in the mutants.

PCR with primers specific for the aaC1 did not produce a product in WT but amplified a 0.75 kb product in the mutants.

  

Conclusions

Genes in C. tepidum can be insertionally inactivated by natural transformation & homologous recombination

The following markers were used successfully:

Gentamicin ( aacC1 )

Erythromycin ( ermC )

Streptomycin-Spectinomycin ( aadA )

This marker gave significantly higher transformation than the others

Guidelines for Routine Gene Inactivation by Natural Transformation

Use cells from at least 100 μl of a late-exponential liquid culture

Use linearized DNA (1-10μg) with sequences of at least 0.5 kb of flanking homologous DNA

Transforming cells should be spotted on agar surface & incubated for 10-20 hours at 40 o C

Shorten incubation time for higher temperatures

Transformation may be done by scraping cells off of a plate and incubating the tranforming mixture overnight on a nonselective plate.

The cells should then be re-streaked the following day .

References

Frigaard, N.U., and Bryant, D.A. (2001) Chromosomal Gene Inactivation in the Green Sulfur Baterium Chloroboum tepidum by Natural Transformation. App. & Env. Microbiol. 2538-2544.

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