Symbiosis Presentation

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Published on June 1, 2008

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Symbiosis Heather Jordan BMMB 597D Biochemistry, Microbiology & Molecular Biology The Pennsylvania State University University Park, PA 16802 http://starklab.slu.edu/Bio2000/clover.jpg

What is symbiosis? Interactions between species usually involve food Competition Predation Defense (against being eaten) http://www.exatec.unisinos.br/~osorio/mmidia/trab-gb/pac-man/pacman.mpg

Interactions between species usually involve food

Competition

Predation

Defense (against being eaten)

What is symbiosis? Relationships in which at least 1 member benefits. Defined by relationship of other member: Parasitism : Injured (Sea Lamprey on Fish) Commensalism : Unaffected (Barnacles on Whales) Mutualism : Benefit (Lichens) Typically the larger organism is the ‘host’ and the smaller is the ‘symbiont ’ http://www.whaleswim.com/en/information/video.html

Relationships in which at least 1 member benefits. Defined by relationship of other member:

Parasitism : Injured (Sea Lamprey on Fish)

Commensalism : Unaffected (Barnacles on Whales)

Mutualism : Benefit (Lichens)

Typically the larger organism is the ‘host’ and the smaller is the ‘symbiont ’

Aeromonas veronii Host : Hirudo medicinalis (medicinal leech) Type : Digestive-tract Location : Extracellular Function : ? http://yoda.ucc.uconn.edu/~mcbstaff/graf/Sym.html

Host : Hirudo medicinalis (medicinal leech)

Type : Digestive-tract

Location : Extracellular

Function : ?

Vibrio fischeri Host : Euprymna scolopes (bobtailed squid) Type : Light Organ Location : Extracellular Function : Light production http://yoda.ucc.uconn.edu/~mcbstaff/graf/Sym.html

Host : Euprymna scolopes (bobtailed squid)

Type : Light Organ

Location : Extracellular

Function : Light production

Buchnera Host : Aphidopidea (aphids) Type : Nutritional Location : Intracellular Function : Amino Acid Synthesis http://yoda.ucc.uconn.edu/~mcbstaff/graf/Sym.html

Host : Aphidopidea (aphids)

Type : Nutritional

Location : Intracellular

Function : Amino Acid Synthesis

Parasitism Primarily injure host in 2 ways : Consuming its tissue Hookworms Plasmodium Liberating toxins Tetanus bacilli : secretes toxin that interferes with synaptic transmission Diptheria bacilli : secretes toxin that inhibits protein synthesis by ribosomes Include viruses, bacteria, fungi, protozoans, nematodes, insects & arachnids http://ftp.decsy.ru/nanoworld2002/v075/DATA/mpeg/virus.mpg

Primarily injure host in 2 ways :

Consuming its tissue

Hookworms

Plasmodium

Liberating toxins

Tetanus bacilli : secretes toxin that interferes with synaptic transmission

Diptheria bacilli : secretes toxin that inhibits protein synthesis by ribosomes

Include viruses, bacteria, fungi, protozoans, nematodes, insects & arachnids

Symbiosis in the Microbial World Most plants & animals live symbiotically with microbes Favorite hiding places for bacterial symbionts: Skin ( Staphylococcus epidermidis ) Digestive Tract ( E. coli ) Plant Roots ( Rhizobium ) Opportunistic Pathogens Pseudomonas aeruginosa http://www.steve.gb.com/vegetable_empire/the_dark_side.html

Most plants & animals live symbiotically with microbes

Favorite hiding places for bacterial symbionts:

Skin ( Staphylococcus epidermidis )

Digestive Tract ( E. coli )

Plant Roots ( Rhizobium )

Opportunistic Pathogens

Pseudomonas aeruginosa

Rhizobium , Root Nodules & Nitrogen Fixation Virtually all roots have symbiotic associations with soil microbes (i.e., 90% land plants with soil fungi) Model : Between soil microbes ( Rhizobium ) & legumes Legumes agriculturally valuable (soybeans are a good source of protein!) http://www.science.siu.edu/parasitic-plants/images/nodules.JPEG http://www.agron.iastate.edu/plantscience/Beneficial_microbes.htm

Virtually all roots have symbiotic associations with soil microbes (i.e., 90% land plants with soil fungi)

Model : Between soil microbes ( Rhizobium ) & legumes

Legumes agriculturally valuable (soybeans are a good source of protein!)

The Perfect Relationship! ~90% of legumes become nodulated Rhizobium gets a nice place to live (plant provides nutrients & energy to the bacteria) Rhizobia fix nitrogen gas for the “breadwinner” (plant) N is most limiting element in terrestrial ecosystems http://www.dpi.qld.gov.au/fieldcrops/3256.html

~90% of legumes become nodulated

Rhizobium gets a nice place to live (plant provides nutrients & energy to the bacteria)

Rhizobia fix nitrogen gas for the “breadwinner” (plant)

N is most limiting element in terrestrial ecosystems

Nitrogen Fixation Atmospheric : Lightning (only a small amount) Industrial : Haber process (used to make fertilizers) Biological : N-fixing bacteria fix 60% of nitrogen gas Reduction of N 2  NH 3 requires 16 ATP & enzymes (nitrogenase) to break N-bonds N 2 + 3H 2 energy > 2NH 3 http://www.donkeyshow.org/movies/

Atmospheric : Lightning (only a small amount)

Industrial : Haber process (used to make fertilizers)

Biological : N-fixing bacteria fix 60% of nitrogen gas

Reduction of N 2  NH 3 requires 16 ATP & enzymes (nitrogenase) to break N-bonds

N 2 + 3H 2 energy > 2NH 3

Nitrogen Cycle Reactions Nitrogen Fixation : Rhizobium 1 st step in synthesis of nitrogenous compounds Ammonification : Break down proteins in dead organisms Release ammonium ions Ammonifying bacteria Nitrification : Nitrosomonas & Nitrobacter 2-step process Oxidized  Nitrates  Nitrites (used by plants) Denitrification : Denitrifying bacteria Nitrates reduced  N 2 (completing cycle) http://www.uoguelph.ca/%7Emgoss/seven/nodules.jpg

Nitrogen Fixation :

Rhizobium

1 st step in synthesis of nitrogenous compounds

Ammonification :

Break down proteins in dead organisms

Release ammonium ions

Ammonifying bacteria

Nitrification :

Nitrosomonas & Nitrobacter

2-step process

Oxidized  Nitrates  Nitrites (used by plants)

Denitrification :

Denitrifying bacteria

Nitrates reduced  N 2 (completing cycle)

What are Rhizobium ? In the soil, free-living & motile Cannot fix nitrogen & have a different shape than Rhizobium found in root nodules Free-Living : Regular, straight rods Symbiont : Irregular cells (bacteroids); usually club or Y-shaped. Feed on remains of dead organisms 6 genera : Rhizobium, Bradyrhizobium, Azorhizobium, Photorhizobium, Siorhizobium & Mesorhizobium

In the soil, free-living & motile

Cannot fix nitrogen & have a different shape than Rhizobium found in root nodules

Free-Living : Regular, straight rods

Symbiont : Irregular cells (bacteroids); usually club or Y-shaped.

Feed on remains of dead organisms

6 genera : Rhizobium, Bradyrhizobium, Azorhizobium, Photorhizobium, Siorhizobium & Mesorhizobium

How does Rhizobium form root nodules? Roots of legumes produce flavenoids Different flavenoids attract different Rhizobia Rhizobia produce Nod-factors (sensed by plant)  synthesis of acyl homoserine lactones (signal)  AHL-based quorum sensing Root hair curls & Rhizobium invades cell wall invaginations which form tubes called infection threads Thread grows through root hair cells & penetrates other nearby cells by branching  network of tubes  nodule Differentiation into bacteroids (carry out N-fixation) http://www.cfr.washington.edu/classes.esc.210/Lecture%20Notes/Lecture%2023/lec23.htm

Roots of legumes produce flavenoids

Different flavenoids attract different Rhizobia

Rhizobia produce Nod-factors (sensed by plant)  synthesis of acyl homoserine lactones (signal)  AHL-based quorum sensing

Root hair curls & Rhizobium invades cell wall invaginations which form tubes called infection threads

Thread grows through root hair cells & penetrates other nearby cells by branching  network of tubes  nodule

Differentiation into bacteroids (carry out N-fixation)

2 Basic Types of Root Nodules Determinate : Short, predestined lifespan (days-weeks) New nodules form & old nodules sloughed off (older branches) as root grows Soybean nodules Indeterminate : Longer lifespan (many months) New root cells become infected by old cells Plants with apical meristem (alfalfa) http://www.biologie.uni-hamburg.de/b-online/library/webb/BOT311/Roots/RootSymbioses.htm

Determinate :

Short, predestined lifespan (days-weeks)

New nodules form & old nodules sloughed off (older branches) as root grows

Soybean nodules

Indeterminate :

Longer lifespan (many months)

New root cells become infected by old cells

Plants with apical meristem (alfalfa)

http://redescolar.ilce.edu.mx/redescolar/Revista/06/articulos/06.html

Rhizobium etli possesses 2 regulatory cascades containing nitrogen-fixing genes. What are they? Cascade # 1: Controls Expression of: The nifHDK operons (both copies) 3 rd nifH gene (not linked to other nif genes) Production of melanin (black pigment) Dependent on transcriptional regulator nifA Cascade # 2: Dependent on fixL genes (signal trigger unknown) Controls Expression of: rpoN1 Controls growth on C 4 -dicarboxylic acids & on some N-sources. Inactivation represses expression of nifH (microaerobic) & melanin production (free-living)

Cascade # 1:

Controls Expression of:

The nifHDK operons (both copies)

3 rd nifH gene (not linked to other nif genes)

Production of melanin (black pigment)

Dependent on transcriptional regulator nifA

Cascade # 2:

Dependent on fixL genes (signal trigger unknown)

Controls Expression of: rpoN1

Controls growth on C 4 -dicarboxylic acids & on some N-sources.

Inactivation represses expression of nifH (microaerobic) & melanin production (free-living)

rpoN is found in a wide variety of microorganisms. Describe the examples given in the paper. Caulobacter crescentus : Transcription is 10-fold induced during swarmer  stalk transition Rhodobacter capsulatus : Transcription is under control of 2 promoters Upstream of NifU 2 & expressed in presence of nitrogen Autoactivated by RpoN & NifA Rhodobacter sphaeroides : Genetic evidence supports the presence of a 2 nd copy Bradyrhizobium japonicum : The only bacterium in which 2 genes are regulated are separately regulated (the 1 st is activated by FixLJ-FixK2 cascade during microaerobiosis & 2 nd is negatively autoregulated) Azohrizobium caulinodans : Genetic evidence also supports the presence of a 2 nd copy

Caulobacter crescentus :

Transcription is 10-fold induced during swarmer  stalk transition

Rhodobacter capsulatus :

Transcription is under control of 2 promoters

Upstream of NifU 2 & expressed in presence of nitrogen

Autoactivated by RpoN & NifA

Rhodobacter sphaeroides :

Genetic evidence supports the presence of a 2 nd copy

Bradyrhizobium japonicum :

The only bacterium in which 2 genes are regulated are separately regulated (the 1 st is activated by FixLJ-FixK2 cascade during microaerobiosis & 2 nd is negatively autoregulated)

Azohrizobium caulinodans :

Genetic evidence also supports the presence of a 2 nd copy

What do the authors of this paper hypothesize? Given that: rpoN2 is transcribed by RpoN1 & NifA under microaerobic conditions Expression of rpoN1 & rpoN2 are mutually exclusive ( rpoN1 =free-living & rpoN2 =symbiotic) Then it would seem that : rpoN2 could be regulated by a symbiosis-specific signal http://www.science.uwaterloo.ca/~tcharles/rhizobium.html

Given that:

rpoN2 is transcribed by RpoN1 & NifA under microaerobic conditions

Expression of rpoN1 & rpoN2 are mutually exclusive ( rpoN1 =free-living & rpoN2 =symbiotic)

Then it would seem that :

rpoN2 could be regulated by a symbiosis-specific signal

What originally led the authors to suspect that there may be a 2 nd rpoN gene? rpoN1 null mutants had phenotypic defects under free-living conditions Symbiotic N 2 -fixation was not affected Sequenced, designed primers (conserved regions) & cloned it out. Map of region containing rpoN2 gene (Fig. 1)

rpoN1 null mutants had phenotypic defects under free-living conditions

Symbiotic N 2 -fixation was not affected

Sequenced, designed primers (conserved regions) & cloned it out.

What phenotypic defects were observed when rpoN1 and rpoN2 were inactivated? rpoN1: Phenotypic defects during free-living growth WT levels of symbiotic N-fixation Reduced expression during microaerobiosis & symbiosis rpoN2: No phenotypic defects while free-living 90% less N-fixation

rpoN1:

Phenotypic defects during free-living growth

WT levels of symbiotic N-fixation

Reduced expression during microaerobiosis & symbiosis

rpoN2:

No phenotypic defects while free-living

90% less N-fixation

Why do the authors think that rpoN2 may have been acquired via lateral gene transfer (as opposed to gene duplication)? G+C content between rpoN genes differ ( rpoN1 =64% & rpoN2 =56%) Amino acid sequences divergent RpoN1 : S. meliloti (87%), Rhizobium sp. Strain NGR234 (65%) http://www.ambassadors.net/archives/issue8/symposium_select.htm

G+C content between rpoN genes differ ( rpoN1 =64% & rpoN2 =56%)

Amino acid sequences divergent

RpoN1 : S. meliloti (87%), Rhizobium sp. Strain NGR234 (65%)

Why was media containing ammonium, nitrate and alanine used (testing under aerobic & microaerobic conditions)? What happened when glutamine and mannitol were used in the media? NH 4 + , NO 3 - & Ala reduce aerobic growth of rpoN1 mutant rpoN2 mutants grew like wild-type (aerobic & microaerobically) rpoN1 & double mutants grew alike (produced copious amounts of exopolysaccharides; indicative of low intracellular fixed N

NH 4 + , NO 3 - & Ala reduce aerobic growth of rpoN1 mutant

rpoN2 mutants grew like wild-type (aerobic & microaerobically)

rpoN1 & double mutants grew alike (produced copious amounts of exopolysaccharides; indicative of low intracellular fixed N

How did the mutations affect nodulation in P. vulgaris ? How did they impact the ability to fix nitrogen? Are both of these genes necessary for nitrogen fixation? Shape, size & dry weight unchanged More nodules on nifA (smaller)

Shape, size & dry weight unchanged

More nodules on nifA (smaller)

How did the mutations affect nodulation in P. vulgaris ? How did they impact the ability to fix nitrogen? Are both of these genes necessary for nitrogen fixation? Over 90% less in rpoN2 mutant strain FAJ1169 None in nifA & double mutant ( rpoN1/rpoN2 )

Over 90% less in rpoN2 mutant strain FAJ1169

None in nifA & double mutant ( rpoN1/rpoN2 )

How did the mutations affect nodulation in P. vulgaris ? How did they impact the ability to fix nitrogen? Are both of these genes necessary for nitrogen fixation? rpoN2 is essential but rpoN1 is not.

rpoN2 is essential but rpoN1 is not.

How did the authors know that the absence of transcription of the rpoN2 gene were not responsible for the observed phenotypes of FAJ1173? When they attempted to determine the function of orf180 , they inserted a Ω –Km cassette & grew the mutant on various N sources Did this interposon create a polar mutation? Tested by complementation with a plasmid containing orf180 and an inactivated rpoN2 gene. Since there was no complementation, it would seem that orf180 & rpoN2 are transcribed by an upstream promoter. :. Transcription was not responsible for the phenotype.

When they attempted to determine the function of orf180 , they inserted a Ω –Km cassette & grew the mutant on various N sources

Did this interposon create a polar mutation?

Tested by complementation with a plasmid containing orf180 and an inactivated rpoN2 gene.

Since there was no complementation, it would seem that orf180 & rpoN2 are transcribed by an upstream promoter.

:. Transcription was not responsible for the phenotype.

Why did the authors test for the production of melanin? What does rpoN have to do with the production of melanin? Is rpoN1 necessary for melanin synthesis? rpoN2 ? Production of melanin previously shown to be dependent of NifA & RpoN1 No production in nifA or double mutant ( rpoN genes can substitute for each other) rpoN1 is required but rpoN2 is not (melanin synthesis)

Production of melanin previously shown to be dependent of NifA & RpoN1

No production in nifA or double mutant ( rpoN genes can substitute for each other)

rpoN1 is required but rpoN2 is not (melanin synthesis)

The authors stated that rpoN1 and rpoN2 are active under different physiological conditions. Are these observations explained by the differential expression of both genes? rpoN1 (independent of N-conc. & autoregulating) essential during free-living growth rpoN2 required for symbiosis :. rpoN1 expressed in aerobic environment & rpoN2 is the one being actively transcribed in bacteroids.

rpoN1 (independent of N-conc. & autoregulating) essential during free-living growth

rpoN2 required for symbiosis

:. rpoN1 expressed in aerobic environment & rpoN2 is the one being actively transcribed in bacteroids.

What was concluded about the expression patterns of rpoN2 with relation to orf180 ? Expression patterns are identical (coordinate expression of both genes)

Expression patterns are identical (coordinate expression of both genes)

According to the authors, no active RpoN2 protein is formed under free-living aerobic or microaerobic conditions in the rpoN1 mutant. How did they know this? How could RpoN2 protein facilitate turning off the rpoN1 gene during symbiosis? Free-Living Microaerobic : Expression of rpoN1 ↓ & Transcription of rpoN2 activated Activation depends on presence of RpoN1 & NifA (this is why there was no growth by rpoN1 mutant on Ala) No production of melanin/Expression of nifH-gusA fusion plasmid :. No active RpoN2 formed under free-living aerobic conditions (microaerobic in rpoN1 ); both are functional during free-living growth on NO 3 - while both are active in microaerobic environments

Free-Living Microaerobic : Expression of rpoN1 ↓ & Transcription of rpoN2 activated

Activation depends on presence of RpoN1 & NifA (this is why there was no growth by rpoN1 mutant on Ala)

No production of melanin/Expression of nifH-gusA fusion plasmid

:. No active RpoN2 formed under free-living aerobic conditions (microaerobic in rpoN1 ); both are functional during free-living growth on NO 3 - while both are active in microaerobic environments

According to the authors, no active RpoN2 protein is formed under free-living aerobic or microaerobic conditions in the rpoN1 mutant. How did they know this? How could RpoN2 protein facilitate turning off the rpoN1 gene during symbiosis? Free-Living Microaerobic : Expression of rpoN1 ↓ & Transcription of rpoN2 activated Activation depends on presence of RpoN1 & NifA (this is why there was no growth by rpoN1 mutant on Ala) No production of melanin/Expression of nifH-gusA fusion plasmid :. No active RpoN2 formed under free-living aerobic conditions (microaerobic in rpoN1 ); both are functional during free-living growth on NO 3 - while both are active in microaerobic environments RpoN2 is strongly expressed rpoN2 mutant still produces WT levels of melanin Since rpoN2-gusA & orf180-gusA fusions are activated, rpoN1 can’t be repressed During symbiosis, unlike rpoN1 , rpoN2 is highly activated (requires NifA, except while free-living)

Free-Living Microaerobic : Expression of rpoN1 ↓ & Transcription of rpoN2 activated

Activation depends on presence of RpoN1 & NifA (this is why there was no growth by rpoN1 mutant on Ala)

No production of melanin/Expression of nifH-gusA fusion plasmid

:. No active RpoN2 formed under free-living aerobic conditions (microaerobic in rpoN1 ); both are functional during free-living growth on NO 3 - while both are active in microaerobic environments

RpoN2 is strongly expressed

rpoN2 mutant still produces WT levels of melanin

Since rpoN2-gusA & orf180-gusA fusions are activated, rpoN1 can’t be repressed

During symbiosis, unlike rpoN1 , rpoN2 is highly activated (requires NifA, except while free-living)

Therefore… Symbiotic expression of rpoN2 involves a symbiosis-specific mechanism!

Symbiotic expression of rpoN2 involves a symbiosis-specific mechanism!

The End

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