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

0 %
100 %
Information about Chromosomal Gene Inactivation In The Green Sulfur Bacterium Chlorobium...

Published on June 1, 2008

Author: Water_Xta1

Source: slideshare.net

Description

see title

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

Moderately thermophilic green sulfur bacterium

Temperature: 47 o C

What is Chlorobium tepidum ? Moderately thermophilic green sulfur bacterium Temperature: 47 o C Anaerobic, obligate autotrophs

Moderately thermophilic green sulfur bacterium

Temperature: 47 o C

Anaerobic, obligate autotrophs

What is Chlorobium tepidum ? Moderately thermophilic green sulfur bacterium Temperature: 47 o C Anaerobic, obligate autotrophs 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

Moderately thermophilic green sulfur bacterium

Temperature: 47 o C

Anaerobic, obligate autotrophs

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

What is Chlorobium tepidum ? Moderately thermophilic green sulfur bacterium Temperature: 47 o C Anaerobic, obligate autotrophs 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)

Moderately thermophilic green sulfur bacterium

Temperature: 47 o C

Anaerobic, obligate autotrophs

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

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

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

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

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

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

More than 30 mutants have been created with specifically inactivated genes

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

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

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

Transformation of C. tepidum Antibiotic resistance used as marker Spectinomycin, streptomycin, Ampicillin & chloramphenicol

Antibiotic resistance used as marker

Spectinomycin, streptomycin, Ampicillin & chloramphenicol

Transformation of C. tepidum Antibiotic resistance used as marker Spectinomycin, streptomycin, Ampicillin & chloramphenicol Can use natural transformation, chemical transformation & electroporation.

Antibiotic resistance used as marker

Spectinomycin, streptomycin, Ampicillin & chloramphenicol

Can use natural transformation, chemical transformation & electroporation.

Transformation of C. tepidum Antibiotic resistance used as marker Spectinomycin, streptomycin, Ampicillin & chloramphenicol Can use natural transformation, chemical transformation & electroporation. Most genes targeted for inactivation were chlorosomal 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

Transformation of C. tepidum Antibiotic resistance used as marker Spectinomycin, streptomycin, Ampicillin & chloramphenicol Can use natural transformation, chemical transformation & electroporation. 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

Antibiotic resistance used as marker

Spectinomycin, streptomycin, Ampicillin & chloramphenicol

Can use natural transformation, chemical transformation & electroporation.

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

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

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

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 

NifHDK operon

Maps NifHDK operon Streptomycin-Spectinomycin Resistance Cassette 

NifHDK operon

Streptomycin-Spectinomycin Resistance Cassette

Maps NifHDK operon Streptomycin-Spectinomycin Resistance Cassette Gentamicin Resistance Cassette 

NifHDK operon

Streptomycin-Spectinomycin Resistance Cassette

Gentamicin Resistance Cassette

Maps NifHDK operon Streptomycin-Spectinomycin Resistance Cassette Gentamicin Resistance Cassette Erythromycin-Chloramphenicol Resistance Cassette 

NifHDK operon

Streptomycin-Spectinomycin Resistance Cassette

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)

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.

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

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.

Optimization of Transformation 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

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.”

Optimization of Transformation 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.

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.

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.

Effect of Variation in Length of Homologous Flanking DNA 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.

Effect of Variation in Length of Homologous Flanking DNA To determine the length of homologous flanking DNA on transformation, 3 constructs for inactivation were made. Gentamicin resistance marker is inserted in the middle { { {

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

Gentamicin resistance marker is inserted in the middle

Effect of Variation in Length of Homologous Flanking DNA Plasmids were digested with : Ahd I, which cuts only once Eco RI, which cuts twice. { { {

Plasmids were digested with :

Ahd I, which cuts only once

Eco RI, which cuts twice.

Effect of Variation in Length of Homologous Flanking DNA Transformation frequencies with : 2.93 kb homologous DNA Similar regardless of enzyme used for linearization 1.08 kb homologous DNA Transformation frequency was 1 order of magnitude lower when the plasmid was digested with Eco RI (as oppsed to Ahd I) 0.29 kb homologous DNA No transformation observed regardless of enzyme Why? Some bacteria partially degrade absorbed DNA via exonuclease activity.

Transformation frequencies with :

2.93 kb homologous DNA

Similar regardless of enzyme used for linearization

1.08 kb homologous DNA

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

0.29 kb homologous DNA

No transformation observed regardless of enzyme

Why?

Some bacteria partially degrade absorbed DNA via exonuclease activity.

Effect of Variation in Length of Homologous Flanking DNA 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.

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.

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.

Various Selection Markers 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

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

Various Selection Markers 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.

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.   

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

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 .

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. http:// geoweb . princeton . edu /research/ biocomplexity /index.html http://www. bact . wisc . edu / microtextbook /Metabolism/ NitrogenAssim .html http://www. bigelow .org/ cytometry /Image_gallery/SYN.html http://www. biologie . uni - hamburg .de/b-online/library/ webb /BOT311/ Cyanobacteria / Cyano .html http://www. bmb . psu . edu / deptpage /faculty/ bryant / bryant .html http://www. bmb . psu . edu /faculty/ bryant /lab/index. htm http://www. bom . hik .se/~ njasv / disp .html http://www. cbs . dtu . dk /services/ GenomeAtlas /Bacteria/ Chlorobium / tepidum /TLS/ Ctepidum . htm http://www. dsmz .de/strains/no012025. htm http://www. er .doe. gov /production/ ober / gc / omp .html http://www. jgi .doe. gov /JGI_microbial/html/ synechococcus / synech _content.html http://www. ornl . gov / TechResources /Human_Genome/ publicat /99santa/158.html Sakamoto, T., Inoue-Sakamoto, K. and Bryant, D.A. (1999) A Novel Nitrate/Nitrite Permease in the Marine Cyanobacterium Synechococcus sp. Strain PCC 7002. Journal of Bacteriology. 7363-7372.

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.

http:// geoweb . princeton . edu /research/ biocomplexity /index.html

http://www. bact . wisc . edu / microtextbook /Metabolism/ NitrogenAssim .html

http://www. bigelow .org/ cytometry /Image_gallery/SYN.html

http://www. biologie . uni - hamburg .de/b-online/library/ webb /BOT311/ Cyanobacteria / Cyano .html

http://www. bmb . psu . edu / deptpage /faculty/ bryant / bryant .html

http://www. bmb . psu . edu /faculty/ bryant /lab/index. htm

http://www. bom . hik .se/~ njasv / disp .html

http://www. cbs . dtu . dk /services/ GenomeAtlas /Bacteria/ Chlorobium / tepidum /TLS/ Ctepidum . htm

http://www. dsmz .de/strains/no012025. htm

http://www. er .doe. gov /production/ ober / gc / omp .html

http://www. jgi .doe. gov /JGI_microbial/html/ synechococcus / synech _content.html

http://www. ornl . gov / TechResources /Human_Genome/ publicat /99santa/158.html

Sakamoto, T., Inoue-Sakamoto, K. and Bryant, D.A. (1999) A Novel Nitrate/Nitrite Permease in the Marine Cyanobacterium Synechococcus sp. Strain PCC 7002. Journal of Bacteriology. 7363-7372.

??Questions??

Add a comment

Related presentations

Related pages

Chromosomal Gene Inactivation in the Green Sulfur ...

... the Green Sulfur Bacterium Chlorobium tepidum by ... inactivation of genes in C. tepidum, ... gene inactivation by natural transformation ...
Read more

Chromosomal Gene Inactivation in the Green Sulfur ...

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum ... chromosomal DNA from ... gene inactivation by natural transformation ...
Read more

Chromosomal Gene Inactivation in the Green Sulfur ...

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum ... chromosomal genes of Chlorobium tepidum ... natural transformation ...
Read more

Chromosomal Gene Inactivation in the Green Sulfur ...

... green sulfur bacterium Chlorobium tepidum by ... chromosomal genes of Chlorobium tepidum by ... gene inactivation by natural transformation ...
Read more

Chromosomal Gene Inactivation in the Green Sulfur ...

routine gene inactivation by natural transformation: (i) ... green sulfur bacterium Chlorobium ... mophilic green sulfur bacterium Chlorobium tepidum ...
Read more

Chromosomal Gene Inactivation in the Green Sulfur ...

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum b.
Read more

Gene inactivation in the cyanobacterium Synechococcus sp ...

... green sulfur bacterium Chlorobium tepidum ... Inactivation of a chromosomal gene is ... bacterium Chlorobium tepidum by natural transformation ...
Read more

Chromosomal Gene Inactivation in the Green Sulfur ...

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation.
Read more

Gene Inactivation in the Cyanobacterium Synechococcus sp ...

the green sulfur bacterium Chlorobium tepidum ... Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation ...
Read more