1

Free Energies Calculations Basic skills:

1. Calculation of the change in free energy for a reaction from the free energies of formation of the reactants and products.

2. Interconversion of the change in free energy for a reaction and the equilibrium constant.

3. Changes in free energy for oxidation and reduction reactions. 4. Changes in free energy for chemiosmotic reactions. 5. Changes in free energy for photochemical reactions. 6. Some simple kinetics.

1. Calculation of the change in free energy for a reaction from the free

energies of formation of the reactants and products.

ΔG0 = 3ΔGf0(products) - 3ΔGf

0(reactants)

Free energy of formation (ΔGf0) is the free-energy change that occurs when

one mole of a compound in its standard state is formed from its elements in their standard states.

Example: D-glucose (C6H12O6) + 6O2 Y 6CO2 + 6H2O

Compound ΔGf

0 (kJ/mol) at 25 C Glucose -917 (aq) H2O -237 CO2 -386 (aq) O2 0 (g)

ΔG0 = 3ΔGf

0(products) - 3ΔGf0(reactants)

ΔG0 = 6(-237 kJ/mol) + 6(-386 kJ/mol) - (-917 kJ/mol) - 6(0 kJ/mol) ΔG0 = -1422 kJ/mol - 2316 kJ/mol + 917 kJ/mol = -2821 kJ/mol ΔG0 = -2870 kJ/mol (textbook value)

2

2. Interconversion of the change in free energy for a reaction and the equilibrium constant

For a reaction: aA + bB Y cC + dD (where H2O is neglected as a reactant)

ΔG = ΔG0 + RT ln CcDd where R = 8.3 J mol-1 K-1 and T = oKelvin AaBb

if ΔG = 0, ΔG0 = -RT ln CcDd = -2.3 RT log CcDd AaBb AaBb

by definition, when ΔG = 0, CcDd is the Keq or the equilibrium constant AaBb

for convenience, ΔG0 = -5.69 kJ/mol x log Keq at 25 C

Example: For a reaction: SO4-2 + H+ + 4H2 Y HS- + 4H2O,

where ΔG0' = -151.9 kJ/rxn; what would the concentration of HS- be at environmental concentrations of the other reactants?

[SO4

-2] = 10 mM [H+] = 10-7 M at pH 7, however this term is not used in the calculation [H2] = 10-5 atm, notice that this is the gaseous concentration in units of

atmospheres [H2O] = 56 M, however this term is also not used in the calculation

Since ΔG = 0, ΔG0 = -5.69 kJ/mol x log Keq and CcDd is Keq

AaBb

Now write: SO4-2 + H+ + 4H2 Y HS- + 4H2O

as SO4-2 + 4H2 Y HS-

Then: ΔG0 = -5.69 log [HS-]/([SO4

-2][H2]4) = -5.69log [HS-] + 5.69log([SO4

-2][H2]4) Rearrange to: -5.69log [HS-] = ΔG0 - 5.69log([SO4

-2][H2]4) log [HS-] = ΔG0/(-5.69) + log([SO4

-2][H2]4) = (-151.9 kJ/rxn)/(-5.69) + log(10-2 M) (10-5 atm)4 = 26.7 + log (10-22) = 4.7

[HS-] = 5 x 104 M So what!

3

3. Changes in free energy for oxidation and reduction reactions. Oxidation-reduction reactions are a special class of chemical reactions:

E = midpoint potential Eo = midpoint potential under standard conditions (pH = 0, 1 M

concentrations of the reduced and oxidized species, 273 K) Eo= = midpoint potential under standard conditions at pH = 7

E = Eo + 2.3RT log10([ox]/[red]) = Eo + (0.059 V.eq)(1/n.eq) log([ox]/[red])

n ö at 25 C

ΔG = -n öΔE where n = number of electrons transferred ö= Faraday=s constant or 96.5 kJ/V.mol.eq ΔE = change in midpoint potential

Examples: 1. What is the change in free energy for the reduction of fumerate by NADH?

Succinate/fumarate Eo= = +0.033 V NADH + H+/NAD+ Eo= = -0.320 V

ΔE = +0.033 V - (-0.320 V) = +0.353 V

ΔG = -n öΔE = -(2 eq)(96.5 kJ/V.mol eq)( 0.353 V) = -68 kJ/mol 2. What is the magnitude of ΔE for a couple involving 2e- transfers needed to drive ATP synthesis?

ΔG = -n öΔE = -2 öΔE rearranges to ΔE = -ΔG/2 ö ΔG = 48 kJ/mol for ATP biosynthesis under physiological conditions ΔE = - ΔG/2 ö = - (48 kJ/mol)/(2 eq) (96.5 kJ/V.mol.eq) = - 0.249 V

4

4. Changes in free energy for chemiosmotic reactions Concentration gradients across a membrane and membrane electrical potentials can also be expressed in terms of changes in free energy In the chemiosmotic hypothesis: Δp = Δψ - ZΔpH where

Δp = protonmotive force or the electrochemical potential difference of protons across a membrane, has units of V

Δψ = the electric potential difference across the membrane ΔpH = the difference in pH Z = 2.3RT/ö and converts the ΔpH to V

can be rewritten as Δp = Δψ - 0.059(ΔpH) for Δp in V at 25 C

Then: ΔG = -n öΔp where n = number of charges on the ion transferred

Δp = -ΔG/n ö Δp is composed of two components: the potential due to the concentration

difference acrossed the membrane and the electric potential due to the charge difference caused by the concentration difference

for the concentration difference, ΔG = 2.3 RT log([Xout]/[Xin]

= (5.7 kJ/mol)(log([Xout]/[Xin]) for the electrical potential, assume an ion of charge m is transported down a

potential of Δψ, then ΔG = -m öΔψ Combining the concentration and electrical components yields:

ΔG = -m öΔψ + 2.3 RT log([Xout]/[Xin] or ΔG = - öΔψ + 2.3 RT ΔpH for protons

5

5. Changes in free energy and photochemical reactions.

Properties of light: V = λf where V = velocity (in vacumn =3 x 1010 cm sec-1)

λ= wavelenght in cm (100 nm = 10-6 cm) f = frequency in sec-1

light energy E = hf = hc/λ where h= Planck=s constant = 6.6 x 10-34 j.sec

1 einstein = 1 mole of quanta of light = 6.02 x 1023 quanta

E in kJ/einstein = 1.2 x 105/ λ in nm

hence, the energy in 680 nm light is 176 kJ/einstein 870 nm light is 138 kJ/einstein

6. Some simple kinetics

Imagine a simple reversible reaction 1:

A º B , k+1 is the rate constant of the forward reaction k-1 is the rate constant of the reverse reaction Keq = k+1/k-1 = e-ΔG/RT

Imagine a complex series of reversible reactions:

1 2 3 A º B º C º D

ΔG1-3 = ΔG1 + ΔG2 + ΔG3

K1-3 = K1K2K3 = k+1k+2k+3/k-1k-2k-3

where Kx is the equilibrium constant for reaction x

k+x is the forward rate constant, vf = k+x[S] k-x is the reverse rate constant, vr = k-x[P]

Nomenclature of prokaryotes

Prokaryotes are named according to the binomial ,rrr.- of naming plants and animals. A11species are named by a binary combination consisting of the genus mme and the specificepithet.

All species are based upon a type strain. A type strain is made up of livingcultures (when possible) of an organism descended from a strain designated as thenomenclatural type when the species name is first proposed. The strain should havebeen maintained in pure culture and its characteristics should closely agree with those inthe original description.

The type strain does not have to be and frequently is not typical of all the strainsof the species. It serves as a reference point if the taxonomy is later revised.

When the type strain is lost, a neotype strain may be proposed. The neotypestrain should have properties similar to those of the original description.

All higher taxa have a type chosen from the lower taxa. For instance, a genus hasa type species and an order has a type genus. In this way, all the taxonomic ranks areultimately connected to a biological specimen or type strain.

However, the species is the only taxonomic rank which is defined by a consensusof workers in many different fields.

Prokaryotic taxonomy is governed by the International Committee on Systematic Prokaryotes(ICSP; formerly the International Committee on Systematic Bacteriology,ICSB), which is acommittee of the International Union of Microbiological Societies (IUMS). The IUMS itselfis a loose federation of national societies, like the American Society for Microbiolory or ASM.The International Journal of Systematic and Evolutionary Microbiology (IJSEM, formerlythe International Joumal of Systematic Bacteriology or IJSB) is the official publication of theICSP.

Prokaryotic nomenclature is governed by the International Code of Nomenclature ofBacteria (1990 Revision), also called the Bacteriological Code.

Only taxa that are validly published have any official standing in the BacteriologicalCode. Taxa described before 1980 were validly published in the Approved Lists ofBacterial Names. For taxa described since then, the original proposal must either bepublished in the IJSB or, if published elsewhere, appear on the Validation List of IJSB.The effective publication date is the date of validation.

Currently, all valid names of prokaryotes are listed at: Jean Euzeby's website "List ofProkaryotic names with Standing in Nomenclature" at: http://uurrv.bacterio.cict.frl

Definition of a prokaryotic species

What is diversity and how is it measured? One can imagine many types of diversity:morphological, functional, evolutionary, etc. Each type of diversity might be of interest fordifferent reasons. Each type of diversity can be used as the basis for the species definition.

Morphological diversity: early editions of Bergey's Manual classified organismsaccording to shape because this characteristic could be easily measuredFunctional diversity: in the study of pathogenesis, one is interested in the functionaldiversity-- the abilify to cause different types of diseases in different hosts. (Plantpathologists frequently name species of bacteria according to the plant they infect)Similarly, microbial ecologists frequently discuss groups of prokaryotes according totheir role in geochemical cycling, ie. nitrifiers, methylotrophs.Evolutionary diversity: considers the ancestry of the organisms. Implies that organismswhose last common ancestor is more ancient and more diverse. Unlike other types,evolutionary diversity can lead to a "natural" system of classification.

Systematics: the scientific study of the kinds and diversity of organisms and of any and allrelationships among them; the classification of organisms in an ordered system designed toindicate natural relationships.

Classification: the ordering of organisms into groups on the basis of their relationships: genetic,phenetic, or phylogenetic

Taxonomy: the theory and practice of classi$ing organisms.

Identification: the process of placing an individual into a given group

Taxon (Taxa): a taxonomic group of any rank that is sufficiently distinct to be worthy of beingassigned to a specific category

Advantages of a natural classification: it is explanatory, it is predictive, it is conceptual (ie., itfollows from and adds to our concept of what is a prokaryote).

Phylogeny: the evolutionary history of an organism. The phylogeny of prokaryotes has beendifficult to study until the recent advent of molecular techniques. Currently, it is the basis forprokaryotic systematics.

The library metaphor. Imagine that each prokaryote is a book in a library and your goal is tolearn the contents of every book. If the library is organized arbitrarily, then you have to readvery book to learn the contents of the library. If the library is organized in a natural system, youonly have to read all of some books and only parts of the other books. The remaining contents ofthe library can then be infened from the location of the books. The downside is that you alsohave to know the organizational principles.

Numerical Taxonomy. Based upon the principles of Adanson, an 18th century botanist. Aphenetic analysis in which taxa are defined on the basis of overall similarity according to allpossible tests of all possible strains where the results.of each test are weighld equally.

number of tests: <40 not statistically significant, 100-200 usually optimalnumber of strains: upper limit usually about 300characteristics for classification: care must be taken to choose characters representative of

many properties of the cell, ie. morphology, biochemistry, cell wall chemistry,nutrition, etc.

unit character: a taxonomic character of two or more states which can not be subdividedlogically, test are designed to delineate the unit characters

Characters commonl used in numerical taxonom ofClass of character example

morphology

physiological

biochemical

chemical composition

cultural

nutritional

drug sensitivities

selological

genetic

cell shape

presence offlagella

presence ofspores

anaerobic growth, reduce nihate

oxidase or catalase activity

acid production from galactose

presence of lysine in cell wall

colony appearance

ability to grow on acetate as sole carbon source

penicillin resistance

agglutination with antiserum

mol%o G+C of DNA

sensitivity to a phage

Because of the large number of tests, characters must be coded for computer analysis. Typicallyeach test is scored as: 0, 1, or NC (where NC is no comparison). the coding straiegy issomewhat arbitrary and can be used to weigh certain tests and outcomes.

Good general reference: Dunn G and Everitt BS (1982) An introduction to mathematicaltaxonomy. Cambridge Univ. Press.

Numerical taxonomy leads to one concept of a species [Liston, Wiebe, and Colwell (1963) J.Bacteriol. 85 : 1061-10701 :

Hypothetical Median Organism: described by the collection of characters typical of

most of the strains of a species, all of the strains will have a similarity coefficient of > 75

% to the hypothetical median organism

The median organism describes the typical strain not the type strain.

l regus -1. ( 'a lcula led tntd ian organisn forP sc ud ot t t o t tas uer u 17 i r t oso

2 2!

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I t o o %

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r lotol occu,rlnc! rnqroup ol 3f, ! lrqn3

rrc. 3. Frequencg oJ5paired characler occTtri'encc(rnininrunr nol in e-l uded) .

Short rodsRound or s l igh t lv t r -

pered errdsSingle and pl irsi l Iot i le, polur f lugel laCl ranr-negati veGra l ' , t rans lucent\ lediurn colonr' (2 to 5

ntm)Entire edgeDiffusible green, f f t to-

rescent pigmentPe l l i c le , n rodemte .

even turbiditr 'L i ro r l ' t .n a t , J , L

.A.gur not digerted

Celn t in l ique f iedL i tn r i rs n r i l k uc id and

pep tonizedNHr fronr pept.oneN O 3 - N O 1 + g a sUrease prodr rcedCatal ase-posi t i veGab1' 's c1'tochlonre ox-

iduse -pos i t i veI iovac's oxidase-posi-

t iveCi t rc te -pos i t i veH trgh and Leifson-.

aerobie (gluco-"e-posi t ive I

I)enici l l inr '-e producedPteri din - i nsensi t i ve

'o'lII

q 8 . 1r ln i[ 5 i9 lo 4 iE I! , l. i

t l ,

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I r , / r \ - It0 rr | 20 tO r€^r 40 30 r^r. 60

,,"lli-';,',ff J!il",',:!,,,:::8";:,ii,i,i:{JIi!,#!Limitations of numerical taxonomy:1. For routine classification, the process is very time consuming2. Not truly objective due to difficulty in determining unit characteristics and in sampling arepresentative population3. Sensitive to error in test design in a very complex fashion

Advantages of numerical taxonomy1 . Knowledge of the properties of an organism is useful for other purposes besides classification.2. Many of the techniques are accessible to most microbiologists3. Provides a description of the organism

Ar.

Report of the Ad IIoc Committee on Reconciliation of Approaches to Bacterial Systematics(Inter. J. System. Bacteriol. 37 U9871463-464) established three criteria for definition of aprokaryotic species:

Members of the same species:1. would possess >70% DNA hybridization2. with <5 oC change in the melting temperafure (ATm) of DNA:DNA hybrids3. have similar phenotypic properties

Thermal stability (AT.) is measured as the difference in temperafure required to melthalf of the hybrid duplex versus half of the homologous duplexes. The thermal stabilityof the DNA:DNA duplexes is correlated to DNA sequence similarity not the extent ofhybridization. AT* increases about I oC for every 1.7 % base pair mismatch (Caccone eta1., J. Mol. Evol.27:212-216\

'Tig. l. Graph of data in Table

l. Numbers corrcspond to left-most column in Table l. Barsindicate I standard crror.Regression line is drawn usingall points without constraint ofpassing througi the origin. SeeTable 2 for alternative analyses.

. 5 6 7

Z b 9 m i s m a t c h

Genospecies: species defined on the basis of DNA sequence similarity or other geneticcharacteristics, such as criteria 1 and 2 above.

A second species concept: if two organisms are simiiar, the DNA sequence of theirchromosomes should be similar. DNA-DNA hybridization attempts to measure theextent of this similarity by measuring the ability of denatured DNA from one organism toreanneal to DNA from another organisms (or form duplexes). The extent of duplexformation (hybridization) is an indirect measure of the extent of DNA sequencesimilarity.

Deceptively simple! %hybndization and ATm depend cntically on the experimental conditionsand protocol used, ie. different methods may yieid different results! See Brenner et al. (1969) J.Bacteriol. 98: 637-350.

E.coli - A.oerooener 6O'c E.coli - E.coli

;q. COl l - E . col i 75oc

20

o l n

q

" t o

E.coli -S.lyphimurium 60nc

- , , - , . . , IL C o i l - H m [ 0 0 t t t s i

. Frc. l. Thermal elution profiles of reassociatecl ,tt,tterspecies etrterohacterial DNA duplexes. (A) pro- 'lilr.s obrainetl .[rom 75 C rectctiotrs. (B) proJiles ob-t,tti.tt('d J'ro,n 60 C retrctions. A 0.1-pg ilnount of 32p-luheletl E. coli DNA Jragnettts *,us iucubcttet! with it.tJtproxinately 500 ps of unlabeletl DNA I'rugnenrs ,tront the indicuted org.utisn$ irt I ml ol 0.t2 t pB Irrtr lll Iv at 60 or 75 C. Tlrc samples vere loudetl Ittttlo. ltytlro.v.vaputite cctlumns uul elutetl uul assul,ed Iut ttescribed in fularerials and tllerhods.

i

E.coli-A.ocrogcnca 75'c ! 6o"cI

.4M PB

Surprisingly, things seem to work! Note the correlation between DNA:DNA hybridization andthe phenetic similarity.

I

.Frc. 1. Congruenie between DNA pairing andphenetic similarity (modifiedfrom relerence nZ9\. Cir-cles show the per cent DNA pairing and rhe pir rrutphenetic simihrity of comparisons between sirains oflhe genera Pseudomonas, Xanthomonas, Agrobacterium,

'

Rhizobium, Vibrio, and Chromobacteriui. fhe DNedata are f'ront De Ley et al. (132), Heberteirt et al. (206\ .and Colwell (99), whereas the phenetic data are l'roiLysenko (294), Heberlein et .al. (206), and Cilwell(99), shown as closed circles, or are.estimatecl from in-

formation of Dye (146, open circles). Although tltesestudies .are not strictty comparable, they appear Jrominternal evidence to be reasonably so. Arrotus show thestandard errors attributable to e.rperimenta! or statisti-cal data (based on details given by the authois). It canbe seen that the scatter is of the same order oi'magni_tude as tteice tlte standard errors (approximatinI n95/6 confdence limits). The highest circle repreientsan'anonnlous reaction (greater pairing vith o ili6rrentstrain than vith the strain that was.llrc source ol tlrcDNA),

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Do prokaryotic species exist in nature? Three points of view:

1. Species are real entities that are discovered orrevealed by classification.

2. Nature produces individuals and species have no actual existence. Species have beeninvented so that we can refer to great numbers of individuals collectively.

3. Species are phenotypically similar groups that represent adaptive peaks. Thus, speciesare formed by att interaction between phylogeny and ecology.

Clonal model of population structure

Populations of bacterial species are basically clonal. Even though the number of individuals maybe on the order of 1020, these individuals are recently descendents of a much smaller (102-104)grollp of ancestors.

population: a group of related individuals in a specified area.clone: a group of genetically identical or nearly identical cells that owe their similarity toa recent descent from a common ancestor in the absence of chromosomal recombination(or genetic exchange)

Evidence for a low rate of recombination:

1. In chemostats, the rates of phage-mediated and conjugative plasmid-mediaterecombination are very low, about equal to the rate of spontaneous mutations (10-8 per cellgeneration)

2. In natural populations, therecombination. If recombination wasrandom.

variations in allozymes is consistent with very low rates offrequent, the distribution of allozyme (see below) would be

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How general are these results?

Index of Association : Io : Vo/Vu-l

Vo : observed variance between electrophoretic types (ET)

V, : expected variance between electrophoretic types

In populations of prokaryotes reproducing by binary fission, Iodepends on the relative rates of I) divergence of hneages throughfixation of new mutations arfi2) transfer of genes betweenlineages by recombination

Io: 0 is evidence for a high relative rate of recombination

D : mean genetic difference per locus between strains

Smith, J.M., N.H. Smith, M. O'Rourke, and B.G. Spratt.1993.How clonal are bacteria? Proc. Natl. Acad. Sci. USA 90:4384-4388.

Frc. 1. Rcprcscntations of population structurcs. (z{ and A)Populations consisting of isolates that are scparated into two majoibranchcs. Tbc population stnrctur€ in r{ is clonal at ell lcvcls, suchthat thc dendrogram is an cvolutionary trcc: no rccombinationoccurs, cither bctween isolatcs ia thc same, or diffcrent, braochcs ofthc tr€e. Ln 8, recombination docs not occur bctwccn isolatcs fromthc two major branches, but frcqucnt recombination occurs bctwecnisolates within cach major branch. Thc structurc within thescbranchcs is thus nct.likc rathcr than trce-likc, as rcprcscnted by thccxpandcd scction of onc of thc oajor branchcs. (C) An cpidcmicstructurc in which thcrc is frequcnt rccombination within all mcm-bcrs of the population, such that thc structurc is a net rathcr than aFec. Howevcr, occasionally a highly successful individual arises andincrcases rapidly in &cquency to producc an cpidcmic clonc.

Tablc 1. Mcasures of association between loci in bactcria

All isolates ETs only

BacteriaNo. ofisolatcs Rcf.IetIe'

No. ofETs

No. ofloci

N, gonorrhoeaeN. meningitidisHae mo p hil us i ntl ue nzae

"Division 1""Division 2"

SalmonellaS. panamaS. pararyphi BS. typhimuriumS. pararyphi CS. choleraesuis

Rhizobium melilotiDivision ADivision B

LegionellaL. pneumophilal. "species 1"

Bordetella bronchisepticaPseudomonas syringae

P, syringae tomato.P. syringae syringae

227688

22092LL7

921495

9911E340100L74232208

71

17014324

30423L76

t933156*42L4

1061314t79

1150341562509

2L1046

915t7L7L7242424242424L4l47471

1)

2215262626

0.309 0.04 r 0.09 0.4140.507 1.% I 0.05 0.5470.226 5.40 = 0.03 0.5340.1E6 4.42 ! 0.03 0.4520.361 2.30 t 0.14 0.4540.355 3.11 = 0.04 0.3650.038 -I5A=n.161 o.B80.074 2.68 t 0.13 j 0.1680.035 1.03 + 0.10t 0.1170.054 4j66 i o.ii 0.1430.036 2.57 + 0.L4 0.17t0.238 6.34 ! 0.09 0.4950.101 0.47 t 0.10 0.2330.219 0.24 * 0.29 0.2490.359 _ _ _4,53 t-0.1-L _-0.420O.Uz li49 + 0.12 0.3120.107 7.45 ! 0.29 0.1820.@7 1.29 r 0.08 0.24E0.47 1E.35 = 0.29 0.6860.076 3.39 t 0.33 0.1t00.480 t.42 + 0.57 0.480

-0.16 t 0.170.21 + 0.0t1.25 + 0.180.90 t 0.211.20 t 0.37l.3E + 0.140.28 r 0.39 -\

0.36 r 0.37 ,41.22 t 0.33 ll

J-97 _=-Pr6'I1.36 t 0.42-tr4.74 ! 0.I9

-0.24 + 0.240.01 + 0.35l^E_ r_qJg_0.69 + 0.201.93 r 0.470.99 r 0.30

12.11 = 0.,t41.68 t 0.671.42 + 0.57

t9

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28

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ET, electrophoretic type; D, mean genetic distance per locus between strains.'Standard errors are for the null hypothesis of independence between loci,

l"t.O'R., unpublished data.rFifty-six clusters identified by Musser et al, (27).

If recombination is common with prokaryotic species, can it be observed by sequencecomparisons? Compare gene tree versus organismal-tree.

Gene tree: represents the phylogenetic relationships of an individual gene in a group of

strains

Organismal tree: represents the phylogenetic relationships of the whole organisms

Dykhuizen, D.E., and L. Green. Recombination in Escherichia coli and the definition ofbiological species. J. Bacteriol. 173: 7257 -7268.

FlG. 1. Effect of rccoobination on tbe apparcnt phylogcneticrelationship of thrce strains. fm gene trei-ofi.ili *iff put strainsB and C together, whereas thc g"* t .. fo, an! oif,", gcnc *iU pta.rtrains A and-B r,ogethcr. Thi-slifc;n;;;ft;; lrees is causcdby thc transfer.of gene X from an aDcestor 6f etrain C into aoanccstor of strain B.

tld(rM202l)

g,a(nmrse)

ona(Rrrt_Dg)

@(rP)

snd (RMztzI)

l!g(RM221H)

sd(RM20rc)

et dfti+tJ9A)

sq(rT2)

s,l(RntgtF)

4(aamr)

4(nz)

!e(RM20lc)

g2(nure)

E(RM2l7r)

r'p ( nlt zo B)

rrp (rma5e)

3q(nmzzrx)

r'p (ruzoz t)

!p( tr2)

phoA ( RM 708)

etpA(PUJ9A)

dsa(rMrs€)

dsa(RM2tzT)

Q!9A(RM2?rH)

&a(nuretr)

elbA(Kt2)

plsA(RM20tC)

*9A(rM202r)

0.il0 0.099 0.0tt oo/, 0.066 0.055 0.0.. 0033 0.022 o.oil o.oooGmeric Drrsrr (D)

0.ilo 0066 00.rGrnrtc Dirtorr (D)

0.0t3 00il o.oto 0.@6 0.@t 0.6rD;rracr (D)

omt 0.00t o.m0.@9 0.&aGortrc

5'ph"s7hq hanle1nl =

dcl/,ayaasc

FU = d[kal/oe 7loV6</os'-lg , fo1rb7to" J,>nti,{iu lahds

Multilocus sequence typing:

Sequences short portions of many genes froni large numbers of strains of a species todetermine phylogenetic relationships. Typicaliy uses 5-10 housekeeping genes. FromMaiden, M.C.J. et al. 1998. Proc. Natl. Acad. Sci. USA 95: 3140-3145. Multilocussequence typing: A portable approach to the identification of clones within populations ofp atho genic microorganisms.

1.0Linkage Oistance

0.8 0.6 0.4 0.? 0.0

Table 1. Gene frasments used in MLSTanalvsis

Subgrouns

VII

VIvAfiI

IV-1/IV-2

UIA/III

A4cluster

ET.37complex

STs 18-20sT-21

r T <

complex

sT.36

lineage

FragmentGene size, bp

Anomalies'

Alleles Variable sitss MLEE MLST

abcZl $3adk 465aroE 490

sdh 501mtg 497pdhC 480pgm 450piA 432pip 417ppk 579serC 451

1 )

10l d

16L O

1 1

36191 1

t )

3816628o l

8077

zd

77o /

3 (2) 4 (3)0 0

) 1

1 t

a a

J J' l t / 1 1 \ 1 < / 1 / \rJ \ r ' . ,

7 71 ^t >

1 1 r / ? \ t r / 1 < \. - \ , / . 1 \ . J )

'The numbers of alleles h groups of at least four strains in thereference set of 107 meningococcal isolates that are inconsistent withstrain relationships previously determined by MLEE or within theclustering obtained by MLST at a genetic distance of <0.35 in Fig.1. The numbers in parentheses are the numbers of anomalous alleleswhen replicate anomalies within a given grouping are counted onlyonce.

lThe six genes used to generate Fig. 2 are shown in boldface. Thesegenes provided data that were consistent both between MLEE andMLST as well as within MLST groups. Tlte mtg gene fragment wasexcluded from the set of six because of its close physical linkage toaroE

Dendrogram of genetic relationships among 107strains based on 6 gene fragments. Linkagedistance is indicated by a scale at the top, and theMLEE or ST assigmlents of lineages are indicatedby shaded rectangles. The asterisk indicates ST-21(serogroup A strain B534)

ffiFJFI

ffitillHIffiFiltitilHIE1lg

fii!

Hf;fli$IJ

METHANAOGENESIS

What are the methanogenic prokaryotes?

Strictly anaerobic archaea that obtain most or all of their energy for growth from theprocess of methane biosynthesis. Many are also autotrophic and also obtain all theircarbon for biosynthesis from COr.

Mini-methane producers: many anaerobes produce small amounts of methane as thebyproduct of their metabolism (usually methylcobamides are an intermediate in animportant pathway). However, methanogenesis is not coupled to energy biosynthesis andthese bacteria are NOT considered methanogens.

What is so special about the methanogenic archaea?

Methanogens are archaea and possess many of the unusual features of these prokaryotes,including isoprenoid-based lipids, diverse cell wall structures lacking murein, eukaryoticlike RNA polymerase and DNA replication

Methanogens are the only cultivated archaea that are not limited to extremehabitats such as high temperature or high salinity. However, some methanogensare hyperthermophiles or extremely halophilic.

Methanogens are a very diverse group within the archaea and includes specieswith very different growth preferences, morphologies, and other cellularproperties. All methanogens are related to each other to the exclusion of otherprokaryotes, indicating that this is a very ancient, physiological group.

Methanogens are necessary for many anaerobic fermentations and prevent theaccumulation of toxic organic acids. By reducing the H, and formate concentrations, theyallow other anaerobes to obtain more energy during the fermentation of organic polymersto the substrates for methanogenesis (interspecies Hr-transfer). Of special interest, theyare intracellular and extracellular symbionts of a variety of free-living anaerobic protists.

Methanogens catalyze a major link in the C-cycle, probably l-2 % of the carbon fixed inphotosynthesis each year passes through the methanogenic pathways

CHo is a significant trace gas in the earth's atmosphere that has been increasing atabout I o/o per year for the last 200 years

CHo is a greenhouse gas and may contribute 10-20 % of the total increase inatmospheric warming where CO, contributes 50 % of the total increase.

Much of the CHo produced by methanogens never enters the atmosphere but isconsumed by methane-oxidizing bacteria, called methanotrophs

Methanogens

-anaerobic archaea that obtain all or most of their energy for growth from the process ofmethane synthesis from a limited number of substrates including: H, + COr, formate, acetate,methylamines, methylsulfides, methanol, and a few alcohols (isopropanol). All methanogensknown are obligate methane-producers.

f Melhanosarcina barkn' f l l

s M r l r t a r r , : . . r r c I \ . ' l h r r r n ^ n t , , l A l 2 lMethanosrcina lrisia j ll

Melhancecoides methylulens {41Methanohalophilus mahii [5]

Methanohalophilus oregonansis [6]Melhanolobus lindarius [7]

Methanohalobium evesligatum [8]Melhanohalophilus zhilanae l9l

Methanosaeta lhermoacetophila l l 0l- Melhanosaela concil i i 1111l-ueif,'a n & uri5u s-." ;--' g ,i t r zJ

Methantrulleus thermophiticus [1 3]Melhanoculleus bourg€nsis [1 4]

Melhan€enium tatiff is [1 5]Methanospirillum hungatei J1 6l

Melhancorpusculum paryum [1 7]Methanogenium organophilum l t 8l

Methanomicrobium mobile [1 9]- Melhanoplanus l imicola I20l- Halolerax volcanii 12lI

Halobaclerium halobium [22]Halocacus morrhuae [231

Halobaclerium marismortui t24lI nermoprasma acidophitum {251

Sanla Barbara Channel bacterioF)tankton DNA clone SBAR16 [26]Woods Hole bacterioptankton DNA ctone WHARN lATj

- Archaeoolobusfuloidusl2Sl- Melhanolhermus leryidus l29l

Methanobaclerium thermoautotrophicum [30]Methanobrevibactef arboriphilus [31 ]

Melhanosphaera stadtmanii {321Melhanobaclerium tormicicum [33]

Methanoccus thermolithotf ophicus [37]'Melhanococcus asolicus" l38l -Methanococcus maripaludrs [39] {

Melhanoc@cus vannie||i l40l- Melhanmoccus voltae l41l

' rierrioc-mi.rsie'Ter]42- - -'lr,rErnEnfr yiis-ran?l-ei T45l

- -' -''

Table 3.1 Methanogenic reactions

Reactants

4 H r + H C O 3 - + H *4 H C O r - + H * + H 2 O

4CO + 5Hr0

2CHsCHrOH

4CH3OH

4(CHr)3-NHt + gHrOD

2(CH3)r-S + 3H2O'

cH3oH + H'

Products Hlmol CH

cH"coo- + H,o cH4 + HCOr-

cH. + 3HrOCH. + 3HCO3-

cH. + 3HCO3- +3H*

2CHjCOo- + H* +

3CH. + HCO3- +H2O + Ht

9CH. +3HCO3- +4NH.' + 3H*

3CHo + HCO3- +2H2S + H*

cHl + H2O

Most methanogensMany hydrogenotrophic

methanogens- l!)6 Metlanobacterium and

Metlnnosarcina-l 16 Some hydrogenotophic

Metlanosarcina and

- 105 Metlunosarciru and othermethylotrophicmethanogens

-76 Metlanosarcita and other Imethylotrophicmethanogens

-49 Somemethylotrophicmethanogens

- I 13 Metlnnosplnera stadt-zanii, methylotrophic

- 135- 145 Ar- cJtrci+7

dcet;clc'-s,f11

m"A1' ot4o'Jovv

methanogens

'Other short chain alcohols are utilized including isopropanol.oOther methylated amines utilized include dimethylamine and methylamine.Tr,lethyl mercaptan (methane thiol) is also used.oAGo' values from Thauer et al.. 1977.

AC'

In anaerobic environments, CO, is the most abundant electron acceptor. However,methanogenesis seems to be the anaerobic respiration of last resort, and other processesdominate when their electron acceptorc are present.

Table 3.2 H, and acetate utilization by Fe" reducing bacteria, sulfate reducing

Reactants Products

LC"',Hlrxn

4H, * 8Fer*4 H 2 + S O n 2 - + H *4 H r + H C O r - + H *4 H r + 2 H C O r - + H *C H 3 C o o - + 8 F e r ' + 4 H r ocHscoo- + so.2-

8H* + 8Fe2*HS- + 4HzOcH4 + 3HrOcH3coo- + 4HrO2 H c o r - + 8 F e z ' + 9 H '2HCOr- + HS-

-914-152- 135- 105-809-47- 3 1cH.coo- + H,o cH4 + HCOr-

cx")l<

o

oAG"' values from (Thauer et al., 1917).

Figure 3.1. Effect of H, partial pressure on the free energy of methanogenesis, sulfate

reduction, or acetogenesis using H, . The equations for the reactions are in Table 3.2. The

dots show typical H, thresholds for the various microbial groups shown in Table 3'5. The

concentrations for the products and reactants other than H, are: HCOr- and SO.2-,20

mM; CH.,0.5 atm; HS- = I mM; acetate, l0 mM'

Habitat H,(Pa)'

Lake Mendota sediments

Knaak Lake sediments

Rice paddY

Sewage sludge

Rumen fluid (basal level)

Rumen fluid (Post-

4 .8J . I

2 ; l27.

1872,000

362828

203I,400

15,000

( l )( l )(2)( l )(3)(3)

' (Pascals, I atm = l0l. l x 105 Pa''References: l, Conrad et al', 1986; 2, Conrad et al'' 198?; 3' Smolenski and Robinson'

1988.

H2 Partial Pressure (atm)

Threshold: the lowest concentration of a substrate which can be metabolized or degraded byan organism. Set by the thermodynamics of the reaction. When the concentration is verylow, the degradation no longer provides sufficient energy to support growth.

Table 3.5 Thresholds for hydroggnotrophic anaerobes

AGo' H. Threshold -Organism Electron Accepting Rxn (UlmolHr) (Pa) (nM)

Acetobacterium woodii CO, ---r acetateMethanospirillum hungatei COt + Q11oMethanobrevibacter smithii CO, -> Q11nDesulfuvibrio desulfuricans SOo' -> l{r5Acetobacterium woodii caffeate + hydrocaffeate -85.0 0.3 2.3Wolinella succinogenes fumarate + succinate -86.0 0.002 0.015tvolinel/a succinogenes NOr- --+ NH." -149. 0.002 0.015

oData from Cord-Ruwisch et al. (1988).

- 10

Methanothrixthreshold

Methathreshold

-26.1 52. 390.-33.9 3.0 23.-33 .9 10 . 75 .-38 .9 0 .9 6 .8

-?-Y

CI

-5010-6 1O-7 10-6 1O-5 1O-4 1O_3 f i_z 1O-1 100

Acetate (M)Figure 3.2. Effect ofacetate concentration on the free energy available from methanogen-esis from acetate (Table 3.1) by mesophilic acetotrophic methanogens. It is assumed thatthe thresholds for acetate are I mM for Methanosarcina (Fukazaki et al. , 1990) and 5 g,Mfor Methanothrrx (Jenen et al., 1990), and that the HCor- concentration is l0 mM andthe CHo partial pressure is 0.5 atm.

Table 3.6 Apparent iK. values and minimum thresholds for acetate catabolism byacetotroohic anaerobic cultures.

Organism Apparent K^ Reference" Threshold Reference'

M e t hano s arc ina bar ke ri F waroM e t ha no sa rc i na barke ri 227Methanothrix sp.M et lanothrix soe hnge ni i Opfi konMethanothrix sp. CALS-lM.ethanothrix soehngenii GP IMethanothrix soehngenii MT-lTAM organismAcetate-oxidizing syntrophic cultureD e s ulfobac te r po s t ga t e i

0.069 3 -0.8 4 0.005 5

I 0.620 23 t . 2 3

7 -8 0.075 ;

>0.2 mM 9t -

3. t4.5

6 0 .012 60 . 8 6 7

' -> 0 . 1

0.490.8 mM

0.23

Figure 22.7 Pathways of methanogenesis. (a) Methylcoenryme M (methyl-S-CoM) is a central intermediate inmethanogenesis from C02, acetate, and methanol or meth-ylamina. For some of the reactions, either Fa2e or H2 is theelctron donor. In cass'designated by "2e," the irnmediateelctron donor is not known. Shaded reactions indicate p-

tential points of coupling with the proton motive force.WR = methanofuran, tlaMPT lletrahydromethano-pterin, HS-HTP = 7-mercaptoheptanoylthreoninephos-phate. (b) The aceticlastic reaction for bios,mthesis ofmethyl-HaMPT from aretatr,. Fdo,. and Fd,x6 are theoxidized and reducd forms of fenedoxin, respectively.

Table 3.E Estimates of CHo rpleased into the atmosfhere, based on estimates of Tyler(1991).

CH, Emission (l1tz glyr)

BiogenicLivestockTermitesRice paddy fieldsNatural wetlandsLandfillsOceansTundra

NonbiogenicCoal miningNarural gas flaring and ventinglndustrial and pipeline lossesBiomass bumingMethane hydratesVolcanoesAutomobiles

TotalTotal biogenicTokl anthropogenic

Methanogens catalyze a major linkin the C cycle. Thus, l-2 % of thecarbon fixed in photosynthesis eachyear passes through methanogenicpathways.

CHo is a greenhouse gas and hasbeen increasing at about | % peryear for the last 200 years. Thecrurent concentration is about 2ppm. Increases of CH4 in theearth's atmosphere may contributeabout 20 % of the total globalwanning (if that occurs) while CO2contributes about 50 %.

8Gr0025-1501rr20

t2G|005-70l-20l-5

rL35lG-301545lo-40240.50.5

355-870302-71520t441

I

I

I

I

I

I

I

I

I

I

Methanogenesis requires a large number of special coenzymes. Many of these coenzymesare virtually unique to these organisms. However, some of them (coenzyme Foro andmethanopterin) were flust discovered in methanogens but subsequently .ho*tt to be widelydistributed. DON'T PANIC!!

a - - - - + Formylmethanofunn

5.CH'NH&{-\-6 i

x

"6 b to

Tetrahvdromethan op terin(HTMPT)

.., \rH-R il iH l l'\ryT\r'""'i

l l l l r,,./\.,,/tr*Acn,

;n r r \ N i i t l

5-form,vherahldromerhanopterin 5,lGmethenyltetrahldromethanopterin 5,lGmethylenetetmhydromethanopterin $meth1'ltetrahl'dromethanopterin(HCO - Hr\.1PT)

l 0

Coenzvme Foon Coen4me M

-O3FCHTCHt:SL

Methl'l coen4me M

-O3S-CHeCH;j19Hi

7-mercaptohepunovlth reoninephosphate (HSHTP)

HcH, ot l

o c H - o - P - o H

lnEcurcurcurcHrcgrcurlNHJH AIcooH

- - -Methanofuran (MFR)

cooH o cooH o cooH ot - i l l i l i i l A

HOOCCH,CHICHCHCH,CH,CNHCHCH,CH,CNHCHCH,CH,CNHCH:CH: \,[

OCHZ

cooH

C-l acceptor

[5 , l0 - (=cH-) H lMm] (cH" = HIMPT) ( C H l - H ' - M P T )

g", {"--1'f--f"t'1il |'\*ictt,

N

OFTOF|cHt t l ? f' '? cooH ' o cooH

t i l l

cooH

cH?cHcHcHCHz- O -P- O- CH- C-NH- CH- CH?CHCNH- CHt l lI oH cH,

Y +';

Coenzyme Fo,

HOOC

".,J H H NHrNOC

*t-\

N N

@, I * MF+H2

$+ tt'o

CH=H4MPT

.t*roro",-$* to'o

CH2 =11oYPr1

FqzoHzFqzo

cH3cooH

H4MPT [CO

cH3oHff

HS-CoM H2O

FIG. 3. Pathways of methanogenesis. Reactions involved in energy conservation are boxed. Tbe reduction of methyl-CoM (reactions 6 and 7) is common to allmethanogenic substrates. During methane formation from H2 plus CO2. reactions 1 to 5 proceed in the direction of C02 reduction. Tbe methyl groups of methanoland acetate enter the central pathwal at the level of HoMPT. During methanogenesis ftom metbanol, one-fourth of the methanol is oxidized to -Or b,t the reversalof reactions I to 5; lhe six reducing equivalenc gained are used to reduce 3 mol of methanol to methane. During methanogenesis fiom acetate, the carboryl group isoxidized to CO2 and the eleclrons gained are used to reduce the methyl group to acetale. Fo-, oxidized form of coenzyme For: Fo2eH2. reduccd form of Foro: HS-dM,CoM (2-mereptoethanesulfonate): HS-CoB, CoB (7-mercaptoheptanoylthreonine phosphate); CoM-S-S-CoB, heterodisulfide ot-HS-CoU and HS-CoB.Enzvmes: .t.formyl'MF dehydrogenase; 2, formyl-MF:HoMPT formyltransferase and methenyl-HoMPT cyclohydrolase; 3, F12a-dependenr merhylene-HoMPT dehydrogenase: 4,Fo26-dependent methl'lene-HoMPT reductase; J, methyl-H4Mt'T:CoM-methyltransferase; 6, methyl-CoM reductase;'7, beterodisuifide reduct"se syjtemldifferenrelectron donor s)stems are indicated).

FrzoH:

2H+Frzo'2 H *

2H+

2H+

-S-S-CoB + 2 H+

CoM-S-S-CoB + 2 H+

HS-CoM+ HS-CoBHS-CoM+ HS-CoB

FIG'4.Tentat iveschemeofelectronf lowandProtont lanslocat iondur ingf,o"ioOi*fna. reduction with H, as electron donor' This. re-action sequence ls

i"ti '"ir.ir'.""tenesis from Hrlco.. Thit scheme is valid for methylotrophic

;;ih.;;;;;;;i-. for hydrogenotr6phic metha-noBens do not contain cvlo-

.rtt"rli?"Jtrt. ii.s.n..lr.irtt"nophenazine (MP) has nor been verified The

heterodisulfide ,iductar. is not indicited to be a Proton pump. but this cannol

b" ;l;J out a priori. This scheme is based on the experimentally derived

tai.ftl".t"i' of ':

,o c H* translocared/methyl group reduced P' periplasm:

Ct"t, +optaimi. membrane; C, cytoplasm For explanations' see the text

P CM c

FIG. 5. Tentative scheme of electron flow and proton translocation duringheterodisulfide reduction with ForoHt as electron donor. This reaction sequenceis pan of methanogenesis from methanol, methylamines, and formate. Thisecheme is valid for methylotrophic methanogens onll' (see the legend to Fig. 4).F.a. coenz,vme Faa: MP, methanophenazine: P, periplasml CM. qtoplasmicmembrane; C, rytoplasm. For explanations, see the text.

-+--HS-coM' l'- H4MPT

curfs-cov

HS-CoB H2;Fa2eH2:[COj; forrny]-MCoM-S-S-CoB -t+ 2 H+; F42g; CO2l CO2 + MF

hydrogenase

odisulfiisulfi

FIG. 6. Tentative sche6e of elecron 6or and proton rranslocation coupled to heterodisulfide reduction with CO as electron donor. This reaction sequence is part

of methalogenesis from acetate. The presence of methanophenazine lurfin acetate-grown cells has not been veri6ed. Fd, fenedoxin; P, periplasm; cM, cytoplmmic

oembrane; C, cyroplasm. For explanations, see the text.

2H+

co

reductaseCM

[CO]+Fd+H2o

2H+

CoM-S-S-CoB + 2 H+

HS-CoM+ HS-CoB

z n + z e

CO2+ FdH2

FdHr- fcrredoxin: methanoohen azineoxidoreductase

2 H +

2 H + + 2 e

IH

FIG. 7. Strucrure and rcactivity of Eetbanophenazine, a membrane-integralclectron and hydrogen carricr of methanogens.'

cH3 - H4MPT

AGo'= = -15 kJ/mol

H4MPT

HS-CoM

AGo'= = -15 U/mol

CHr-S-CoM

FIG 8. Tentative scheme of the reaction mechanism of the Na*-translocating methyl-HoMPT:CoM-metbyltransferase. The enz,vme is a multisubunit enzvrreconsisting of eight nonidentical subunits in unknown sloichiometry. The reaction can bi ai"iAea into two partial reactions, methylirion and aemettrytarion oi ancnzlrne-bound corrinoid cofactor. The demethylation reaction is apparently coupled to Na* transpon. C6(I)

"nO Co(III) denoti diferent valence itates of the

enzyme -bound corrinoid cofacror. HS-CoM, CoM (2-mercaptoethanesulfonate); P, periplasm; CM, cytoplasmic mcmbraneiC, $optasm. for explanations, see the ten.

4o* MP'"o