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

Chromosomal Gene Inactivation in the Green Sulfur BacteriumChromosomal Gene Inactivation in the Green Sulfur BacteriumChlorobium tepidum Chlorobium tepidum by Natural Transformationby Natural Transformation

NIELS-ULRIK FRIGAARD* AND DONALD A. BRYANTNIELS-ULRIK FRIGAARD* AND DONALD A. BRYANTDepartment of Biochemistry and Molecular Biology, The Pennsylvania State Department of Biochemistry and Molecular Biology, The Pennsylvania State

University, University Park, PennsylvaniaUniversity, University Park, Pennsylvania

Journal Article ReviewJournal Article ReviewHeather JordanHeather Jordan

BMMB 507BMMB 507

April 8, 2003April 8, 2003

What is What is Chlorobium tepidumChlorobium tepidum?? Moderately thermophilic green Moderately thermophilic green

sulfur bacteriumsulfur bacterium– Temperature:Temperature: 47 47ooCC



Anaerobic, obligate autotrophsAnaerobic, obligate autotrophs




Live in sulfide-rich aquatic Live in sulfide-rich aquatic environmentsenvironments– Photo-oxidize reduced sulfur Photo-oxidize reduced sulfur

compounds compounds (i.e., sulfide & (i.e., sulfide & sulfur) sulfur)

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




Live in sulfide-rich aquatic Live in sulfide-rich aquatic environmentsenvironments– Photo-oxidize reduced sulfur Photo-oxidize reduced sulfur

compounds compounds (i.e., sulfide & (i.e., sulfide & sulfur) sulfur)

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

Distinct phylogeny Distinct phylogeny (not closely (not closely related to other bacteria)related to other bacteria)

Current Research InterestsCurrent Research Interests Lithotrophic oxidation of Lithotrophic oxidation of

sulfur compoundssulfur compounds

COCO22 fixation fixation

Photosynthetic electron Photosynthetic electron transporttransport

Energy transfer and Energy transfer and organization of the organization of the chlorosomeschlorosomes

Biosynthesis and function of Biosynthesis and function of chlorophyllschlorophylls

Nitrogen fixationNitrogen fixation

Genome HighlightsGenome Highlights 1 circular DNA molecule1 circular DNA molecule

2,154,946 bp2,154,946 bp

G+C content 49.1%G+C content 49.1%

2,284 ORFs2,284 ORFs– 50% have been assigned a 50% have been assigned a

known functionknown function

Six plasmids (pAQ1-pAQ6)Six plasmids (pAQ1-pAQ6)– 4.6, 10.0,15.9, 31.0, 4.6, 10.0,15.9, 31.0,

38.6 and 115.6 kb 38.6 and 115.6 kb respectivelyrespectively

pAQ1 has been sequencedpAQ1 has been sequenced

Transformation of Transformation of C. tepidumC. tepidum

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



More than 30 mutants have been created with specifically inactivated genesMore than 30 mutants have been created with specifically inactivated genes



More than 30 mutants have been created with specifically inactivated genesMore than 30 mutants have been created with specifically inactivated genes

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

Transformation of Transformation of C. tepidumC. tepidum Antibiotic resistance used as markerAntibiotic resistance used as marker

– Spectinomycin, streptomycin, Ampicillin Spectinomycin, streptomycin, Ampicillin & chloramphenicol& chloramphenicol



Can use natural transformation, Can use natural transformation, chemical transformation & chemical transformation & electroporation.electroporation.




Most genes targeted for inactivation Most genes targeted for inactivation were chlorosomal proteinswere chlorosomal proteins




Most genes targeted for inactivation Most genes targeted for inactivation were chlorosomal proteinswere chlorosomal proteins

The only genes targeted for The only genes targeted for inactivation encode:inactivation encode:– CsmC & CsmA CsmC & CsmA (chlorosomal proteins)(chlorosomal proteins)– Reaction center cytochrome c551 PrcReaction center cytochrome c551 Prc– rbcL rbcL (Rubisco subunit)(Rubisco subunit)

Only CsmC & rbcL fully segregatedOnly CsmC & rbcL fully segregated

Nitrogen FixationNitrogen Fixation• Major NutrientMajor Nutrient

• Accounts for 11% of dry weight of Accounts for 11% of dry weight of bacterial cellsbacterial cells

• Nitrate reduction & assimilation Nitrate reduction & assimilation is a high energy processis a high energy process

• Up to 30 % of the electrons Up to 30 % of the electrons generated by photosynthetic Hgenerated by photosynthetic H22O O oxidation are consumed during the oxidation are consumed during the reduction of nitrate to ammoniareduction of nitrate to ammonia

• Cyanobacterial cells Cyanobacterial cells preferentially use reduced preferentially use reduced nitrogen sourcesnitrogen sources

• Ammonia & UreaAmmonia & Urea

Nitrogen FixationNitrogen Fixation

Nitrogen is needed for the synthesis of Nitrogen is needed for the synthesis of amino acids & nucleotidesamino acids & nucleotides

• Organic Route:Organic Route: Breakdown of proteins Breakdown of proteins

• Inorganic Route:Inorganic Route: Nitrate Reduction Nitrate Reduction

• N fixation is an energetically costly N fixation is an energetically costly processprocess

Nitrogen fixation related (Nitrogen fixation related (nifnif) genes are ) genes are expressed under anaerobic conditionsexpressed under anaerobic conditions

nifD genenifD gene: encodes a subunit of : encodes a subunit of nitrogenasenitrogenase

Study ObjectiveStudy ObjectiveTo form a foundation for the systematic targeted To form a foundation for the systematic targeted

inactivation of genes in inactivation of genes in C. tepidumC. tepidum (the genome for (the genome for which had been recently sequenced).which had been recently sequenced).

– nifD gene used to formulate the general modelnifD gene used to formulate the general model

– Inactivation of nifD expressed phenotypically Inactivation of nifD expressed phenotypically (inability to (inability to grow diazetrophically)grow diazetrophically)

Markers used include Spectinomycin-Streptomycin, Gentamicin & Markers used include Spectinomycin-Streptomycin, Gentamicin & ErythromycinErythromycin

MapsMaps NifHDK operonNifHDK operon


Streptomycin-Streptomycin-Spectinomycin Spectinomycin Resistance Resistance CassetteCassette



Gentamicin Gentamicin Resistance Resistance CassetteCassette



Gentamicin Gentamicin Resistance Resistance CassetteCassette

Erythromycin-Erythromycin-Chloramphenicol Chloramphenicol Resistance Resistance CassetteCassette

Creating a nifD knockoutCreating a nifD knockout

Making pTN1CXMaking pTN1CX– nifD knock-out construct for nifD knock-out construct for

C. tepidumC. tepidum

Restriction SitesRestriction Sites::– AhdI (6553)AhdI (6553)– HindIII (4018, 1639, 896)HindIII (4018, 1639, 896)– ScaI (3139, 1570, 1560, 289)ScaI (3139, 1570, 1560, 289)– Sty I (3378, 1390, 1377 339, Sty I (3378, 1390, 1377 339,

69)69)– SspI (2747, 2701, 1105)SspI (2747, 2701, 1105)

Antibiotic SensitivityAntibiotic Sensitivity Temperature:Temperature:

– Lower than optimum (48Lower than optimum (48ooC): 40C): 40ooCC– Antibiotic resistance markers Antibiotic resistance markers

originate from mesophilesoriginate from mesophiles

Concentrations That Concentrations That Inhibit Growth:Inhibit Growth:– Gentamicin, 100 Gentamicin, 100 μμg mlg ml-1-1

– Erythromycin, 2 Erythromycin, 2 μμg mlg ml-1-1

– Chloramphenicol, 30 Chloramphenicol, 30 μμg mlg ml-1-1

– Tetracycline, 1 Tetracycline, 1 μμg mlg ml-1-1

– Streptomycin & Spectinomycin, Streptomycin & Spectinomycin, 300 300 μμg mlg ml-1-1 & 150 & 150 μμg mlg ml-1-1 (combined)(combined)

– Kanamycin (100 Kanamycin (100 μμg mlg ml-1-1 +) +)– Ampicillin (100 Ampicillin (100 μμg mlg ml-1-1 +) +)

• aadA Cassette:aadA Cassette:–Confers resistance to Streptomycin & Confers resistance to Streptomycin & SpectinomycinSpectinomycin

–Antibiotics Not Tested:Antibiotics Not Tested:– Amoxicillin, nalidixic acid, vancomycin, Amoxicillin, nalidixic acid, vancomycin, mitomycin C and colistin. mitomycin C and colistin.

Optimization of TransformationOptimization of Transformation Transformation FrequencyTransformation Frequency::

– 100 100 μl late exponential-μl late exponential-phase culturephase culture

– Incubated with 1 μg of Incubated with 1 μg of AhdAhdI-I-digested pTN1G4digested pTN1G4

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

Corresponds to 100 Corresponds to 100 transformants per transformants per μg DNAμg DNA

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

• Gentamicin-resistant transformantsGentamicin-resistant transformants

° Transformation FrequencyTransformation Frequency

Optimization of TransformationOptimization of Transformation Liquid Suspensions & Liquid Suspensions &

TransformationTransformation::– Same method as for platesSame method as for plates– Incubated with 1 Incubated with 1 μg DNAμg DNA– Then plated on selective Then plated on selective

platesplates– Highest transformation Highest transformation

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

– Why?Why? ““DNA may interact DNA may interact

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

• Gentamicin-resistant transformantsGentamicin-resistant transformants

° Transformation FrequencyTransformation Frequency

Optimization of TransformationOptimization of Transformation Stationary vs. Late Log Phase Stationary vs. Late Log Phase

CellsCells:: Cells are competent in both Cells are competent in both

phasesphases Stationary cells gave ½ as Stationary cells gave ½ as

many transformants as the many transformants as the late-exponential-phase cellslate-exponential-phase cells

Linearized DNALinearized DNA Increasing amounts of DNA Increasing amounts of DNA

yielded an increased yielded an increased transformation frequencytransformation frequency

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

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

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

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

Effect of Variation in Length of Homologous Flanking DNAEffect of Variation in Length of Homologous Flanking DNA

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

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


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

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

Gentamicin resistance marker is inserted in Gentamicin resistance marker is inserted in the middlethe middle

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Plasmids were digested withPlasmids were digested with::– AhdAhdI, which cuts only onceI, which cuts only once– EcoEcoRI, which cuts twice.RI, which cuts twice.

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Transformation frequencies withTransformation frequencies with::– 2.93 kb homologous DNA2.93 kb homologous DNA

Similar regardless of enzyme used for linearizationSimilar regardless of enzyme used for linearization

– 1.08 kb homologous DNA1.08 kb homologous DNA Transformation frequency was 1 order of magnitude lower when the plasmid Transformation frequency was 1 order of magnitude lower when the plasmid

was digested with was digested with EcoEcoRI (as oppsed to RI (as oppsed to AhdAhdI)I)

– 0.29 kb homologous DNA0.29 kb homologous DNA No transformation observed regardless of enzymeNo transformation observed regardless of enzyme

Why?Why?– Some bacteria partially degrade absorbed DNA via exonuclease Some bacteria partially degrade absorbed DNA via exonuclease

activity.activity.


Given this:Given this:– A homologous flanking region of about 1 kb should be used in A homologous flanking region of about 1 kb should be used in

transformationtransformation– Linearize with a plasmid that leaves dispensible flanking DNA at the Linearize with a plasmid that leaves dispensible flanking DNA at the

ends of the fragmentends of the fragment– Separate nontransforming DNA from transforming (produced by Separate nontransforming DNA from transforming (produced by

digest)digest) Nontransforming may compete with transforming DNA for uptake into the Nontransforming may compete with transforming DNA for uptake into the

cellscells Supported by side experiment in which 20 Supported by side experiment in which 20 μg of sonicated chromosomal DNA μg of sonicated chromosomal DNA

from from SynechococcusSynechococcus to a to a C. tepidumC. tepidum transformation mixture (containing 1 μg transformation mixture (containing 1 μg of linearlized DNA) decreased the transformation frequency an order of of linearlized DNA) decreased the transformation frequency an order of magnitude.magnitude.

Various Selection MarkersVarious Selection Markers

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

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

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


Chloramphenicol marker didn’t function because:Chloramphenicol marker didn’t function because:– Either the expressed protein is not functional in Either the expressed protein is not functional in C. tepidumC. tepidum – Or the promoter is too weak in Or the promoter is too weak in C. tepidumC. tepidum

Neither of these possibilities were investigated furtherNeither of these possibilities were investigated further


Transformation efficiency was 4 orders of magnitude higher with Transformation efficiency was 4 orders of magnitude higher with the the aadAaadA marker than the other two ( marker than the other two (aaC1aaC1 & & ermCermC))

The reason for this may lie in the genomic sequenceThe reason for this may lie in the genomic sequence– aadA marker contains a 59-bp “recombinational hot spot” aadA marker contains a 59-bp “recombinational hot spot”

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

Test of TransformantsTest of Transformants Expected phenotype of Expected phenotype of

transformants is the inability to transformants is the inability to reduce dinitrogen.reduce dinitrogen.

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

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

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

ConclusionsConclusions Genes in Genes in C. tepidumC. tepidum can be can be

insertionally inactivated by natural insertionally inactivated by natural transformation & homologous transformation & homologous recombinationrecombination

The following markers were used The following markers were used successfully:successfully:– Gentamicin (Gentamicin (aacC1aacC1))– Erythromycin (Erythromycin (ermCermC))– Streptomycin-Spectinomycin (Streptomycin-Spectinomycin (aadAaadA))

This marker gave significantly higher This marker gave significantly higher transformation than the otherstransformation than the others

Guidelines for Routine Gene Inactivation by Natural TransformationGuidelines for Routine Gene Inactivation by Natural Transformation

Use cells from at least 100 Use cells from at least 100 μl of a late-exponential liquid cultureμl of a late-exponential liquid culture Use linearized DNA (1-10μg) with sequences of at least 0.5 kb of flanking Use linearized DNA (1-10μg) with sequences of at least 0.5 kb of flanking

homologous DNAhomologous DNA Transforming cells should be spotted on agar surface & incubated for 10-20 Transforming cells should be spotted on agar surface & incubated for 10-20

hours at 40hours at 40ooCC– Shorten incubation time for higher temperaturesShorten incubation time for higher temperatures

Transformation may be done by scraping cells off of a plate and incubating Transformation may be done by scraping cells off of a plate and incubating the tranforming mixture overnight on a nonselective plate.the tranforming mixture overnight on a nonselective plate.– The cells should then be re-streaked the following dayThe cells should then be re-streaked the following day..

ReferencesReferences Frigaard, N.U., and Bryant, D.A. (2001) Chromosomal Gene Inactivation in the Green Frigaard, N.U., and Bryant, D.A. (2001) Chromosomal Gene Inactivation in the Green

Sulfur Baterium Chloroboum tepidum by Natural Transformation. App. & Env. Sulfur Baterium Chloroboum tepidum by Natural Transformation. App. & Env. Microbiol. 2538-2544.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 Sakamoto, T., Inoue-Sakamoto, K. and Bryant, D.A. (1999) A Novel Nitrate/Nitrite

Permease in the Marine Cyanobacterium Synechococcus sp. Strain PCC 7002. Permease in the Marine Cyanobacterium Synechococcus sp. Strain PCC 7002. Journal of Bacteriology. 7363-7372.Journal of Bacteriology. 7363-7372.

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