Genomic Analysis Of Nitrogen Fixation In Synechococcus Sp. PCC 7002 and Chlorobium tepidum

75 %
25 %
Information about Genomic Analysis Of Nitrogen Fixation In Synechococcus Sp. PCC 7002 and...

Published on June 1, 2008

Author: Water_Xta1

Source: slideshare.net

Description

See Title

Genomic Analysis of Nitrogen fixation in Synechococcus sp. PCC 7002 and Chlorobium tepidum Heather Jordan Department of Biochemistry and Molecular Biology The Pennsylvania State University University Park, Pennsylvania 16802 March 18, 2003

Heather Jordan

Department of Biochemistry and Molecular Biology

The Pennsylvania State University

University Park, Pennsylvania 16802

March 18, 2003

Bryant Lab Research Interests Physiology of growth at low temperature in cyanobacteria The role of multiple sigma factors in gene expression in cyanobacteria The structure, function & biogenesis of PI rxn center Alternative electron transport pathways in cyanobacteria Photosynthetic apparatus in green sulfur bacteria (chlorosome) Physiology & genetics of green sulfur bacteria C. tepidum & C. vibrioforme Bacteriochlorophyll biosynthesis in green sulfur bacterium C. tepidum

Physiology of growth at low temperature in cyanobacteria

The role of multiple sigma factors in gene expression in cyanobacteria

The structure, function & biogenesis of PI rxn center

Alternative electron transport pathways in cyanobacteria

Photosynthetic apparatus in green sulfur bacteria (chlorosome)

Physiology & genetics of green sulfur bacteria C. tepidum & C. vibrioforme

Bacteriochlorophyll biosynthesis in green sulfur bacterium C. tepidum

Bryant Lab Research Interests Chlorobium tepidum Synechococcus sp. PCC 7002

Chlorobium tepidum

Synechococcus sp. PCC 7002

What is cyanobacteria? 7500 species Greatest diversity in fresh water Pigmentation Chlorophyll Carotenoids Phycobillins Add biomass & nitrogen to barren areas Found in a wide variety of environments ranging from hot springs to glacier ice Recently found in the most barren area of Antarctica where no other life is found. May also be endolithic

7500 species

Greatest diversity in fresh water

Pigmentation

Chlorophyll

Carotenoids

Phycobillins

Add biomass & nitrogen to barren areas

Found in a wide variety of environments ranging from hot springs to glacier ice

Recently found in the most barren area of Antarctica where no other life is found.

May also be endolithic

What is cyanobacteria? Morphological Hierarchy: Single cells Colonies Filaments Branched, unbranched or clustered

Morphological Hierarchy:

Single cells

Colonies

Filaments

Branched, unbranched or clustered

Evolutionary Significance Important source for fixed nitrogen Cyanobacteria most likely origin of chloroplasts Endosymbiont Theory Supported through analysis of photosynthetic apparatus & C assimilation Components (LHC) of C. tepidum chlorosome resembles plant chloroplasts Mechanism of CO 2 fixation distinct from Calvin cycle found in high plants. Prominent role in global carbon cycling Constitute some of the oldest fossils (over 2.7 billion years old) Oldest oxygen-producing organism Profoundly changed the earth’s atmosphere.

Important source for fixed nitrogen

Cyanobacteria most likely origin of chloroplasts

Endosymbiont Theory

Supported through analysis of photosynthetic apparatus & C assimilation

Components (LHC) of C. tepidum chlorosome resembles plant chloroplasts

Mechanism of CO 2 fixation distinct from Calvin cycle found in high plants.

Prominent role in global carbon cycling

Constitute some of the oldest fossils (over 2.7 billion years old)

Oldest oxygen-producing organism

Profoundly changed the earth’s atmosphere.

Diversity of Cyanobacteria Differences probably due to adaptation to different environments Sequencing projects seek to establish Which genes are common to all Which genes are specific to ecological niches

Differences probably due to adaptation to different environments

Sequencing projects seek to establish

Which genes are common to all

Which genes are specific to ecological niches

The Ecological Significance of Cyanobacteria Can lead to erosion in limestone & coral reefs Grazing animals eat the cyanobacteria and subsequently reduce the amount of limestone present, Can lead to limestone deposits Some marine species precipitate CaCO 3

Can lead to erosion in limestone & coral reefs

Grazing animals eat the cyanobacteria and subsequently reduce the amount of limestone present,

Can lead to limestone

deposits

Some marine species

precipitate CaCO 3

Nitrogen Fixation Nitrogen is needed for the synthesis of amino acids & nucleotides Organic Route: Breakdown of proteins Inorganic Route: Nitrate Reduction Nitrate reduced to nitrite via nitrate reductase Nitrite reduced to ammonia via nitrite reductase Ammonia incorporated into amino acids N fixation is an energetically costly process

Nitrogen is needed for the synthesis of amino acids & nucleotides

Organic Route: Breakdown of proteins

Inorganic Route: Nitrate Reduction

Nitrate reduced to nitrite via nitrate reductase

Nitrite reduced to ammonia via nitrite reductase

Ammonia incorporated into amino acids

N fixation is an energetically costly process

Nitrogen Fixation Often have symbiotic relationships with other organisms Lichens, Hornworts, Azolla (fern), Cyads, Gunnera (flowering plant) Heterocysts are sites for N fixation Provide an anaerobic environment Oxygen inhibits nitrogenase Internal membranes are thylakoids that have lost chlorophyll (sites for nitrogenase) Connected to vegetative cells via microplasmodesmata (pore in end wall)

Often have symbiotic relationships with other organisms

Lichens, Hornworts, Azolla (fern), Cyads, Gunnera (flowering plant)

Heterocysts are sites for N fixation

Provide an anaerobic environment

Oxygen inhibits nitrogenase

Internal membranes are thylakoids that have lost chlorophyll (sites for nitrogenase)

Connected to vegetative cells via microplasmodesmata (pore in end wall)

Nitrogen Fixation In non-heterocystous cyanobacteria (i.e., Synechococcus ), N is fixed aerobically Cyanobacteria unique in that they can perform both N fixation & oxygenic photosynthesis N-fixation occurs when PSII is not oxidizing H 2 O to O 2 .

In non-heterocystous cyanobacteria (i.e., Synechococcus ), N is fixed aerobically

Cyanobacteria unique in that they can perform both N fixation & oxygenic photosynthesis

N-fixation occurs when PSII is not oxidizing H 2 O to O 2 .

Nitrogen Fixation Genes NAR1 ( Synechococcus ) nifD ( C. tepidum ) Nitrogen fixation related ( nif ) genes are expressed under anaerobic conditions Nitrate Assimilation Related ( NAR ) genes NAR1 gene encodes a chloroplast membrane protein involved in nitrite transport Nitrite reduced to ammonia

NAR1 ( Synechococcus )

nifD ( C. tepidum )

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

Nitrate Assimilation Related ( NAR ) genes

NAR1 gene encodes a chloroplast membrane protein involved in nitrite transport

Nitrite reduced to ammonia

Project 1: Genomic Analysis of Nitrogen Fixation in Synechococcus sp. PCC 7002

Optimal Growth Conditions for Synechoccus Temperature: 38 o C Likes brackish water Obligate requirement for B12 Facultative photoheterotroph Tolerant of high light intensities (up to 5000  E m -2 s -1 )

Temperature: 38 o C

Likes brackish water

Obligate requirement for B12

Facultative photoheterotroph

Tolerant of high light intensities (up to 5000  E m -2 s -1 )

Other Characteristics of Synechococcus Also known as Agmenellum quadrupiplicatum strain PR-6 Isolated by Chase Van Baalen in 1961 from a marine mud sample in Puerto Rico Naturally transformable Among fastest-growing cyanobacteria (doubling time under opt. conditions = 3.5 hours) Cells 1.5-2.5  m in size. Usually occur as single cells but sometimes in clusters Gram negative

Also known as Agmenellum quadrupiplicatum strain PR-6

Isolated by Chase Van Baalen in 1961 from a marine mud sample in Puerto Rico

Naturally transformable

Among fastest-growing cyanobacteria (doubling time under opt. conditions = 3.5 hours)

Cells 1.5-2.5  m in size.

Usually occur as single cells but sometimes in clusters

Gram negative

Other Characteristics of Synechococcus Foundation of the marine food web Primary producers on a global scale One of the most numerous genomes on Earth Obligately marine 1/3 of open ocean isolates possess a type of swimming motility not seen in any other type of microbe (propel at speeds of up to 25 mm/sec) Motility in response to small gradients of nitrogenous compounds

Foundation of the marine food web

Primary producers on a global scale

One of the most numerous genomes on Earth

Obligately marine

1/3 of open ocean isolates possess a type of swimming motility not seen in any other type of microbe (propel at speeds of up to 25 mm/sec)

Motility in response to small gradients of nitrogenous compounds

Hypothesis: Knocking out NAR1 should result in physiologically interesting phenotypes   Do this by inserting a Sp r cassette

Knocking out NAR1 should result in physiologically interesting phenotypes

Do this by inserting a Sp r cassette

Checking for segregation & orientation Segregation: Is the insert present? YES! Orientation: Is the insert going in the proper direction? Will find out tomorrow. 

Segregation: Is the insert present?

YES!

Orientation: Is the insert going in the proper direction?

Will find out tomorrow.

Project 2: Genomic Analysis of Nitrogen Fixation in Chlorobium tepidum

Optimal Growth Conditions for C. tepidum Found in sediments, muds, microbial mats and anoxic & sulfide-rich waters Anaerobic, obligate autotrophs Photo-oxidize reduced sulfur compounds (i.e., sulfide & sulfur) Temperature: 47 o C

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

Anaerobic, obligate autotrophs

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

Temperature: 47 o C

Other Characteristics 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 Can use natural transformation, chemical transformation & electroporation. Most genes targeted for inactivation were chlorosomal proteins Inactivation of nifD expressed phenotypically (inability to grow diazetrophically) Markers used to date include Spectinomycin-Streptomycin, Gentamicin & Erythromycin

Antibiotic resistance used as marker

Spectinomycin, streptomycin, Ampicillin & chloramphenicol

Can use natural transformation, chemical transformation & electroporation.

Most genes targeted for inactivation were chlorosomal proteins

Inactivation of nifD expressed phenotypically (inability to grow diazetrophically)

Markers used to date include Spectinomycin-Streptomycin, Gentamicin & Erythromycin

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 pAQ1 has been sequenced Six plasmids (pAQ1-pAQ6) 4.6, 10.0,15.9, 31.0, 38.6 and 115.6 kb respectively

1 circular DNA molecule

2,154,946 bp

G+C content 49.1%

2,284 ORFs

50% have been assigned a known function

pAQ1 has been sequenced

Six plasmids (pAQ1-pAQ6)

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

nifD Details nifD gene encodes a subunit of nitrogenase Located in the middle of the nifHDK operon 

nifD gene encodes a subunit of nitrogenase

Located in the middle of the nifHDK operon

Creating a nifD knockout Making pTN1CX nifD knock-out construct for C. tepidum 6,553 bp nifD (~1100  2550) 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

6,553 bp

nifD (~1100  2550)

Restriction Sites:

AhdI (6553)

HindIII (4018, 1639, 896)

ScaI (3139, 1570, 1560, 289)

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

SspI (2747, 2701, 1105)

Hypothesis: Since targeted inactivation of nifD using antibiotics as markers has worked successfully in the past, use of a slightly altered cassette containing the same markers should work as well.

Since targeted inactivation of nifD using antibiotics as markers has worked successfully in the past, use of a slightly altered cassette containing the same markers should work as well.

nifD Project Outline —————— I am here. —————— Previous End

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. Herdman, M., Janvier, M. Waterbury, J.B., Ripka, R., Stanier, R.Y., and Mandel, M. (1979a) Deoxyribonucleic acid base composition of cyanobacteria. J. Gen. Microbiol. 111, 63-75. 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 Roberts, T.M. and Koths, K.E. (1976) The blue-green alga Agmenellum quadriplicatum contains covalently closed DNA circles. Cell 9, 551-557. Van Baalen, C. (1962) Studies on marine blue-green algae. Bot. Mar. 4, 129-139.

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.

Herdman, M., Janvier, M. Waterbury, J.B., Ripka, R., Stanier, R.Y., and Mandel, M. (1979a) Deoxyribonucleic acid base composition of cyanobacteria. J. Gen. Microbiol. 111, 63-75.

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

Roberts, T.M. and Koths, K.E. (1976) The blue-green alga Agmenellum quadriplicatum contains covalently closed DNA circles. Cell 9, 551-557.

Van Baalen, C. (1962) Studies on marine blue-green algae. Bot. Mar. 4, 129-139.

Acknowledgements Joel Graham Niels-Ulrik Frigaard

Joel Graham

?? Questions??

Add a comment

Related presentations

Related pages

Synechococcus sp. Strain PCC 7002 Transcriptome ...

Synechococcus sp. Strain PCC 7002 Transcriptome: Acclimation to Temperature, ... Synechococcus sp. PCC 7002 ... Chlorobium tepidum ...
Read more

CyanOmics: an integrated database of omics for the model ...

... an integrated database of omics for the model cyanobacterium Synechococcus sp. PCC 7002. ... both the carbon and nitrogen ... one genomic dataset, 29 ...
Read more

One-step plasmid construction for generation of knock-out ...

Construction of PCR primers. Primers for inactivation of Synechococcus sp. PCC 7002 genes were designed based on the annotated genome (GenBank accession ...
Read more

Synechococcus - Wikipedia, the free encyclopedia

Synechococcus; Synechococcus PCC 7002 cells in ... urea as a sole nitrogen source. Marine Synechococcus are traditionally not ... Prochlorococcus sp. ...
Read more

PLOS ONE: Anchoring a Plant Cytochrome P450 via PsaM to ...

... (primer pair e and f, subpanel C) in the transformed Synechococcus sp. PCC 7002 genomic ... bacterium Chlorobium tepidum using ... fixation, biofuels ...
Read more

Effects of Modified Phycobilin Biosynthesis in the ...

... Colonies of Synechococcus sp. strain PCC 7002 transformed ... Functional genomic analysis of the HY2 ... bacterium Chlorobium tepidum using in ...
Read more

The Biosynthetic Pathway for Myxol-2′ Fucoside ...

... (Myxoxanthophyll) in the Cyanobacterium Synechococcus sp ... Synechococcus sp. strain PCC 7002 ... bacterium Chlorobium tepidum using ...
Read more

Frontiers | Synechococcus sp. Strain PCC 7002 ...

When grown at high salinity, Synechococcus sp. PCC 7002 had ... 7002 transcriptome: acclimation to temperature, ... Chlorobium tepidum using ...
Read more

CiteSeerX — Effects of Modified Phycobilin Biosynthesis ...

... {Effects of Modified Phycobilin Biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002 ... Chlorobium tepidum ... genomic analysis ...
Read more