SymbiosisSymbiosis

Heather JordanHeather Jordan

BMMB 597DBMMB 597D

Biochemistry, Microbiology & Molecular BiologyBiochemistry, Microbiology & Molecular Biology

The Pennsylvania State UniversityThe Pennsylvania State University

University Park, PA 16802University Park, PA 16802

http://starklab.slu.edu/Bio2000/clover.jpg

What is symbiosis?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

What is symbiosis?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

Aeromonas veroniiAeromonas veronii

• HostHost: : Hirudo Hirudo medicinalismedicinalis (medicinal (medicinal leech)leech)

• TypeType: Digestive-tract: Digestive-tract

• LocationLocation: Extracellular: Extracellular

• FunctionFunction: ?: ?

http://yoda.ucc.uconn.edu/~mcbstaff/graf/Sym.htmlhttp://yoda.ucc.uconn.edu/~mcbstaff/graf/Sym.html

Vibrio fischeriVibrio fischeri

• HostHost: : Euprymna scolopesEuprymna scolopes (bobtailed squid) (bobtailed squid)

• TypeType: Light Organ : Light Organ

• LocationLocation: Extracellular: Extracellular

• FunctionFunction: Light production: Light production http://yoda.ucc.uconn.edu/http://yoda.ucc.uconn.edu/~mcbstaff/graf/Sym.html~mcbstaff/graf/Sym.html

BuchneraBuchnera

• HostHost: : AphidopideaAphidopidea (aphids) (aphids)

• TypeType: Nutritional: Nutritional

• LocationLocation: Intracellular: Intracellular

• FunctionFunction: Amino Acid Synthesis: Amino Acid Synthesis http://http://yoda.ucc.uconn.edu/yoda.ucc.uconn.edu/~mcbstaff/graf/Sym.html~mcbstaff/graf/Sym.html

ParasitismParasitism• Primarily injure host in 2 Primarily injure host in 2

waysways::– Consuming its tissueConsuming its tissue

• HookwormsHookworms• PlasmodiumPlasmodium

– Liberating toxinsLiberating toxins• Tetanus bacilliTetanus bacilli: secretes : secretes

toxin that interferes with toxin that interferes with synaptic transmissionsynaptic transmission

• Diptheria bacilliDiptheria bacilli: secretes : secretes toxin that inhibits protein toxin that inhibits protein synthesis by ribosomessynthesis by ribosomes

• Include viruses, bacteria, Include viruses, bacteria, fungi, protozoans, fungi, protozoans, nematodes, insects & nematodes, insects & arachnidsarachnids http://ftp.decsy.ru/nanoworld2002/v075/DATA/http://ftp.decsy.ru/nanoworld2002/v075/DATA/

mpeg/virus.mpgmpeg/virus.mpg

Symbiosis in the Microbial WorldSymbiosis in the Microbial World

• Most plants & animals live Most plants & animals live symbiotically with microbessymbiotically with microbes

• Favorite hiding places for bacterial Favorite hiding places for bacterial symbionts:symbionts:– Skin (Skin (Staphylococcus epidermidisStaphylococcus epidermidis))

– Digestive Tract (Digestive Tract (E. coliE. coli))

– Plant Roots (Plant Roots (RhizobiumRhizobium))

• Opportunistic PathogensOpportunistic Pathogens – Pseudomonas aeruginosaPseudomonas aeruginosa

http://www.steve.gb.com/http://www.steve.gb.com/vegetable_empire/the_dark_side.htmlvegetable_empire/the_dark_side.html

RhizobiumRhizobium, Root Nodules & , Root Nodules & Nitrogen FixationNitrogen Fixation

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

• ModelModel: Between soil microbes (: Between soil microbes (RhizobiumRhizobium) & legumes) & legumes– Legumes agriculturally valuable (soybeans are a good Legumes agriculturally valuable (soybeans are a good

source of protein!)source of protein!)

http://www.science.siu.edu/parasitic-plants/images/nodules.JPEG

http://www.agron.iastate.edu/plantscience/Beneficial_microbes.htm

The Perfect The Perfect Relationship!Relationship!

• ~90% of legumes become ~90% of legumes become nodulatednodulated

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

• RhizobiaRhizobia fix nitrogen gas for fix nitrogen gas for the “breadwinner” (plant)the “breadwinner” (plant)

– N is most limiting element in N is most limiting element in terrestrial ecosystemsterrestrial ecosystems

http://www.dpi.qld.gov.au/fieldcrops/3256.html

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 N2 NH3 requires 16 ATP & enzymes (nitrogenase) to break N-bonds

• N2 + 3H2 energy > 2NH3

http://www.donkeyshow.org/movies/

Nitrogen Cycle Reactions• Nitrogen Fixation:

– Rhizobium– 1st step in synthesis of nitrogenous

compounds

• Ammonification:– Break down proteins in dead organisms– Release ammonium ions– Ammonifying bacteria

• Nitrification:– Nitrosomonas & Nitrobacter– 2-step process

• OxidizedNitratesNitrites (used by plants)

• Denitrification:– Denitrifying bacteria

– Nitrates reducedN2 (completing cycle)

http://www.uoguelph.ca/http://www.uoguelph.ca/%7Emgoss/seven/nodules.jpg%7Emgoss/seven/nodules.jpg

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

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

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

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

Rhizobium etli possesses 2 regulatory cascades Rhizobium etli possesses 2 regulatory cascades containing nitrogen-fixing genes. What are they?containing nitrogen-fixing genes. What are they?

• Cascade # 1:Cascade # 1:– Controls Expression of:Controls Expression of:

• The nifHDK operons (both copies)The nifHDK operons (both copies)• 33rdrd nifHnifH gene (not linked to other gene (not linked to other nifnif genes) genes)• Production of melanin (black pigment)Production of melanin (black pigment)

– Dependent on transcriptional regulator Dependent on transcriptional regulator nifAnifA

• Cascade # 2:Cascade # 2:– Dependent on fixL genes (signal trigger unknown)Dependent on fixL genes (signal trigger unknown)

– Controls Expression of:Controls Expression of: rpoN1rpoN1 • Controls growth on CControls growth on C44-dicarboxylic acids & on some N-sources.-dicarboxylic acids & on some N-sources.

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

rpoNrpoN is found in a wide variety of microorganisms. is found in a wide variety of microorganisms. Describe the examples given in the paper.Describe the examples given in the paper.

• Caulobacter crescentusCaulobacter crescentus::– Transcription is 10-fold induced during swarmerTranscription is 10-fold induced during swarmerstalk transitionstalk transition

• Rhodobacter capsulatusRhodobacter capsulatus::– Transcription is under control of 2 promotersTranscription is under control of 2 promoters

• Upstream of Upstream of NifUNifU22 & expressed in presence of nitrogen & expressed in presence of nitrogen

• Autoactivated by RpoN & NifAAutoactivated by RpoN & NifA

• Rhodobacter sphaeroidesRhodobacter sphaeroides::– Genetic evidence supports the presence of a 2Genetic evidence supports the presence of a 2ndnd copy copy

• Bradyrhizobium japonicumBradyrhizobium japonicum::– The only bacterium in which 2 genes are regulated are separately The only bacterium in which 2 genes are regulated are separately

regulated (the 1regulated (the 1stst is activated by FixLJ-FixK2 cascade during is activated by FixLJ-FixK2 cascade during microaerobiosis & 2microaerobiosis & 2ndnd is negatively autoregulated) is negatively autoregulated)

• Azohrizobium caulinodansAzohrizobium caulinodans::– Genetic evidence also supports the presence of a 2Genetic evidence also supports the presence of a 2ndnd copy copy

What do the authors of this paper hypothesize?What do the authors of this paper hypothesize?

• Given that:Given that:– rpoN2 is transcribed by rpoN2 is transcribed by

RpoN1 & NifA under RpoN1 & NifA under microaerobic conditionsmicroaerobic conditions

– Expression of Expression of rpoN1rpoN1 & & rpoN2rpoN2 are mutually are mutually exclusive (exclusive (rpoN1rpoN1=free-=free-living & living & rpoN2rpoN2=symbiotic)=symbiotic)

• Then it would seem thatThen it would seem that::– rpoN2rpoN2 could be regulated could be regulated

by a symbiosis-specific by a symbiosis-specific signalsignal

http://www.science.uwaterloo.ca/http://www.science.uwaterloo.ca/~tcharles/rhizobium.html~tcharles/rhizobium.html

What originally led the authors to suspect that there may be a What originally led the authors to suspect that there may be a 22ndnd rpoN rpoN gene?gene?

• rpoN1rpoN1 null mutants had phenotypic defects under free-living null mutants had phenotypic defects under free-living conditionsconditions

• Symbiotic NSymbiotic N22-fixation was not affected-fixation was not affected

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

Map of region containing rpoN2 gene (Fig. 1)

What phenotypic defects were observed when What phenotypic defects were observed when rpoN1rpoN1 and and rpoN2rpoN2 were inactivated? were inactivated?

• rpoN1:rpoN1:– Phenotypic defects during free-living growthPhenotypic defects during free-living growth

– WT levels of symbiotic N-fixationWT levels of symbiotic N-fixation

– Reduced expression during microaerobiosis & symbiosisReduced expression during microaerobiosis & symbiosis

• rpoN2:rpoN2:– No phenotypic defects while free-livingNo phenotypic defects while free-living

– 90% less N-fixation90% less N-fixation

Why do the authors think that Why do the authors think that rpoN2rpoN2 may have been may have been acquired via lateral gene transfer (as opposedacquired via lateral gene transfer (as opposed

to gene duplication)?to gene duplication)?

• G+C content G+C content between between rpoNrpoN genes differ genes differ ((rpoN1rpoN1=64% & =64% & rpoN2rpoN2=56%)=56%)

• Amino acid Amino acid sequences sequences divergent divergent – RpoN1RpoN1: : S. melilotiS. meliloti

(87%), (87%), RhizobiumRhizobium sp. Strain NGR234 sp. Strain NGR234 (65%)(65%)

http://www.ambassadors.net/archives/issue8/symposium_select.htm

Why was media containing ammonium, nitrate and alanine used (testing Why was media containing ammonium, nitrate and alanine used (testing under aerobic & microaerobic conditions)? What happened when under aerobic & microaerobic conditions)? What happened when

glutamine and glutamine and mannitol were used in the media?mannitol were used in the media?

• NHNH44++, NO, NO33

-- & Ala reduce aerobic growth of & Ala reduce aerobic growth of rpoN1rpoN1 mutant mutant

• rpoN2rpoN2 mutants grew like wild-type (aerobic & microaerobically) mutants grew like wild-type (aerobic & microaerobically)• rpoN1rpoN1 & double mutants grew alike (produced copious amounts of & double mutants grew alike (produced copious amounts of

exopolysaccharides; indicative of low intracellular fixed Nexopolysaccharides; indicative of low intracellular fixed N

How did the mutations affect nodulation in How did the mutations affect nodulation in P. vulgarisP. vulgaris? ? How did How did they impact the ability to fix nitrogen? Are both of these they impact the ability to fix nitrogen? Are both of these

genes necessary for nitrogen fixation?genes necessary for nitrogen fixation?

• Shape, size & dry weight unchangedShape, size & dry weight unchanged• More nodules on More nodules on nifAnifA (smaller) (smaller)

How did the mutations affect nodulation in How did the mutations affect nodulation in P. vulgarisP. vulgaris?? How did How did they impact the ability to fix nitrogen? they impact the ability to fix nitrogen? Are both of these Are both of these

genes necessary for nitrogen fixation?genes necessary for nitrogen fixation?

• Over 90% less in Over 90% less in rpoN2rpoN2 mutant strain FAJ1169 mutant strain FAJ1169• None in None in nifAnifA & double mutant ( & double mutant (rpoN1/rpoN2rpoN1/rpoN2))

How did the mutations affect nodulation in How did the mutations affect nodulation in P. vulgarisP. vulgaris?? How did How did they impact the ability to fix nitrogen?they impact the ability to fix nitrogen? Are both of these Are both of these

genes necessary for nitrogen fixation?genes necessary for nitrogen fixation?

• rpoN2rpoN2 is essential but is essential but rpoN1 rpoN1 is not.is not.

How did the authors know that the absence of transcription How did the authors know that the absence of transcription of the of the rpoN2rpoN2 gene were not responsible for the gene were not responsible for the

observed phenotypes of FAJ1173?observed phenotypes of FAJ1173?

• When they attempted to determine the function of When they attempted to determine the function of orf180orf180, they , they inserted a inserted a ΩΩ–Km cassette & grew the mutant on various N –Km cassette & grew the mutant on various N sourcessources

• Did this interposon create a polar mutation?Did this interposon create a polar mutation?• Tested by complementation with a plasmid containing Tested by complementation with a plasmid containing orf180 orf180

and an inactivated and an inactivated rpoN2rpoN2 gene. gene.• Since there was no complementation, it would seem that Since there was no complementation, it would seem that orf180 orf180

& & rpoN2rpoN2 are transcribed by an upstream promoter. are transcribed by an upstream promoter.

:. :. Transcription was not responsible for the phenotype.Transcription was not responsible for the phenotype.

Why did the authors test for the production of melanin? What does Why did the authors test for the production of melanin? What does rpoNrpoN have to do with the production of melanin? Is have to do with the production of melanin? Is rpoN1rpoN1

necessary for melanin synthesis? necessary for melanin synthesis? rpoN2rpoN2??

• Production of melanin previously shown to be dependent of Production of melanin previously shown to be dependent of NifA & RpoN1NifA & RpoN1

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

• rpoN1rpoN1 is required but is required but rpoN2rpoN2 is not (melanin synthesis) is not (melanin synthesis)

The authors stated that The authors stated that rpoN1rpoN1 and and rpoN2rpoN2 are active under are active under different physiological conditions. Are these different physiological conditions. Are these

observations explained by the differential expression observations explained by the differential expression of both genes?of both genes?

• rpoN1rpoN1 (independent of N-conc. & autoregulating) essential during free-living (independent of N-conc. & autoregulating) essential during free-living growthgrowth

• rpoN2rpoN2 required for symbiosis required for symbiosis:. :. rpoN1rpoN1 expressed in aerobic environment & expressed in aerobic environment & rpoN2rpoN2 is the one being actively is the one being actively

transcribed in bacteroids.transcribed in bacteroids.

What was concluded about the expression patterns What was concluded about the expression patterns of of rpoN2rpoN2 with relation to with relation to orf180orf180??

• Expression patterns are identical (coordinate Expression patterns are identical (coordinate expression of both genes)expression of both genes)

According to the authors, no active RpoN2 protein is formed under free-According to the authors, no active RpoN2 protein is formed under free-living aerobic or microaerobic conditions in the living aerobic or microaerobic conditions in the rpoN1rpoN1 mutant. mutant. How did they know this? How did they know this? How could RpoN2 protein facilitate How could RpoN2 protein facilitate

turning off the turning off the rpoN1rpoN1 gene during symbiosis? 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 NO3

- while both are active in microaerobic environments

According to the authors, no active RpoN2 protein is formed under free-According to the authors, no active RpoN2 protein is formed under free-living aerobic or microaerobic conditions in the living aerobic or microaerobic conditions in the rpoN1rpoN1 mutant. mutant. How did they know this?How did they know this? How could RpoN2 protein facilitate How could RpoN2 protein facilitate

turning off the turning off the rpoN1rpoN1 gene during symbiosis? gene during symbiosis?

• Free-Living MicroaerobicFree-Living Microaerobic: Expression of : Expression of rpoN1rpoN1 ↓↓ & Transcription of & Transcription of rpoN2rpoN2 activated activated

• Activation depends on presence of RpoN1 & NifA (this is why there Activation depends on presence of RpoN1 & NifA (this is why there was no growth by was no growth by rpoN1rpoN1 mutant on Ala) mutant on Ala)

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

• :. No active RpoN2 formed under free-living aerobic conditions :. No active RpoN2 formed under free-living aerobic conditions (microaerobic in (microaerobic in rpoN1rpoN1); both are functional during free-living growth ); both are functional during free-living growth on NOon NO33

-- while both are active in microaerobic environments while both are active in microaerobic environments

• RpoN2 is strongly expressedRpoN2 is strongly expressed

• rpoN2 mutant still produces WT levels of melaninrpoN2 mutant still produces WT levels of melanin

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

• During symbiosis, unlike During symbiosis, unlike rpoN1rpoN1, , rpoN2rpoN2 is highly activated (requires is highly activated (requires NifA, except while free-living)NifA, except while free-living)

Therefore…Therefore…

• Symbiotic expression of Symbiotic expression of rpoN2rpoN2 involves a involves a symbiosis-specific mechanism!symbiosis-specific mechanism!

The EndThe End