September 9, 1997                             MAST 634               Lecture II: Hydrothermal Vents 

Important Points of Last Lecture (9/4/97)  

Molecular tools for identifying organisms 
ribosomal RNA 
Three Kingdoms: Eukarya, Bacteria, and Archaea 
Review 
          Ribosome structure 
          Function of mRNA, tRNA, & rRNA in protein synthesis 
          Base composition and pairings in DNA and RNA 
                A-T, G-C, A-U, G-C 

Form of primary production: chemoautotrophs 

Can think of all life as redox reactions 

                                                      e^- donor         e^- acceptor 
       Animals (heterotrophs):    C₆H₁₂O₆ + 6O₂ = 6CO₂ + 6H₂O 
       Chemo lithotrophs      :        H₂A + O₂ = A + H₂O 

A bit more complicated than that 
          energy ATP 
          reducing power NAD(P)H (=NADPH or NADH) 
                    Necessary for reducing compounds 
                            E.g. CO₂ ---->  ----> C₆H₁₂O₆ 

                                                  C source     e^- source     Energy  
            Heterotrophs                   organics         --->          ---> 
            Photoautotrophs                 CO₂          H₂O           light 
           Chemoautotrophs                CO₂              inorganic compounds 
           Chemolithotroph 
           Photoorganotrophs              CO₂        organics        light 

What are chemoautotrophs? 
          bacteria 
          archaea 
very specialized; cannot use organic compounds 
first organisms on earth? 

         Sulfide-oxidizing bacteria 

First note: H₂S = HS^- + H⁺ = S^2- + 2H⁺ 
        pka        7.0             13.0 

 

So, at pH 8.0 (pH of Seawater) mostly HS^- 

                                                                (delta)  G°´  
                           HS^- + 20₂ = SO₄^2- + H⁺           -794 
            S^o + 1½O₂ + H₂O = SO₄^2- + 2H⁺          -585 

                                               Oxidation State  
                                       HS^-                    -2 
                                       S^o                  0 
                                       SO₄             +5 

Some important examples 
          Nitrification 
                NH₄⁺ ----> NO₂^- ---->NO^-₃ 

             Ammonium     nitrite          nitrate 
oxidation      -3             +3              +5 
state 

     Important in 
          soils: 
         ocean: 
 

 

     Common traits: redox reaction 

          1. O₂ is e^- acceptor 
          2. Inorganic compound e^- donor 
 
     Compound must be "reduced", i.e. have e^- 

              Possible           Not possible  
                  HS^-                  SO₄^2- 
                 NH₄⁺               NO₃^- 
                 Fe²⁺                 Fe³⁺ 

How to calculate energy yield?      p 434-441 
                                                      Chap. 3 

Gibbs free energy 
     (delta)  G   =  (delta)  H         -T(delta)S 
                                  |                     | 
                            Enthapy        Entropy; change in order 

(delta) G > O; change in heat endergonic; not spontaneous 
(delta) G = O; forward and backward reaction balance equilibrium 
(delta) G < O; exergonic; spontaneous reaction 

Redox reaction 
    see handout 

Example:     NO₂^- oxidation with O₂ 

Divide into half-reactions 
          ⁺³NO₂^- ----> ⁺⁵NO₃^- + 2e^- 
         ½ O₂ + 2e^- ----> H₂O 

Look for reduction potential in table 

                                                               E^o´  (V)  
           ½O₂ + 2H⁺ + 2e = H₂O              + 0.815 
     NO₃^- + 2H⁺ + 2e = NO₂^- + H₂O      + 0.42 

Notes 
     Relative to 2H⁺ + 2e^- = H₂ 
     E^o´   : std biochemical state 
     E^o´   : std chemical state 
     e^- on left; reduction 

sum of half reactions:    (delta) E^o´   = E^o´   _{e acceptor}  - E^o´   _{e donor} 

In this example 
          acceptor: O₂ 
          donor : NO₂ 
    (delta) E^o´   = 0.815 - (+0.42) 
             = +0.39 V 

How to relate to (delta)   G? 
              (delta) G = -n F E derivation in book 
                         N = number of electrons 
                         F = Faraday's const. 

Note: if E > O, then (delta) G <O, i.e. rx goes! 
     so, to decide if X and Y are donor or accept, see which has larger E^o´   (potential) 

0.39 V * 96,494 J V^-1 mol^-1 *2 = 75270 J mol^-1 
                                                 = 75.3 kJ mol^-1 

Is this a lot?    No, small compared to oxidative phosphorylation 
                   C₆H₁₂O₆ + 6O₂ = 6CO₂ + 6H₂O -2823K J mol^-1 

See page 43  

NADH + ½ O₂ = NAD⁺ + H₂O 
                             (delta) G^o = -218 kJ mol^-1 

Again, energy obtained from oxidation of NO₂^- small to that requred to synthesize NADH 
Need more NO₂^- than expected for NADH synthesis, because also need to synthesize ATP 

Reduction of 1 NAD requres 5 NO₂^- 

How does microbe get reducing power and energy? 

Reverse e^- flow 
     Details not important, but main features are 

1. Oxidation of NO₂^- creates 
     - Proton gradient 
     - charge gradient 
2. Series of cytochromes mediates e^- flow like in respiration. 
 
 

 

 
Proton-motive force 

(delta) G = 2.3 RT[pH_In^- pH_out] + ZF (delta and psi symbols missing) 
                                                                           | 
                                                             Membrane potential 

Mitochondria 

          (delta)pH = 0.75 higher outside 
        (delta & ps)= 0.168 V 

~ 210,00 V cm^-1 over 80A 

o.:(delta)G = -21.5 kJ mol^-1 

Bottom Line 
     not much energy in oxidizing inorganic compounds 
     see table on pagae 284 in Gottschalk (on reserve) 

Importance of symbiotic chemoautotrophic Bacteria at Hydrothermal vents 

Vent communities supported by chemoautotrophy 
     - novel; usually phototrophy 
     - more novel: symbiosis 

First Example 
     Colleen Cavanaugh ~ 1980 

Phylum Vestimentifera 
     Riftia pachyptila 

Evidence of symbiosis 
     bacteria-like particles 
     presence of lipo polysaccharide (LPS) 
          -endotoxin 
          -unique to G - bacteria 

enzyme unique to autotrophy 
               Rubisco: CO₂-fixing enzyme 
              other enzymes 
              APS - reductase 
              ATP - sulfurylase 

But impossible to culture symbiont 

     How to study 
     ori ginal study on tube worm 
     Dan Distel (Norm Pace's lab) (1988) 

               Isolate RNA 

                      |         Reverse transcriptase 
                      |           (Originally from NA virus) 
                      |                     David Baltimore, Nobel Prize ~ 1975 
                      |             Using primers specific to 16S RNA 

              16S rDNA 
 
                       | 

              Sequence 

Results 
     each animal species had a different symbiont 

Note limitations 
     1. Difficult to work with RNA 
     2. Reverse transcriptase linear increase in DNA; used with other primers 

Distel and Cavanaugh (1994) 

How do different types of symbiontic bacteria compare? 

                 DNA 

                     |        PCR with "universal" primers for 16S 

             16S rDNA 

                     |        Clone into "TA vector" 

Separate clones, each with separate 16S gene 
 
What is cloning? 

Putting "foreign" DNA into a self reproducing vector which in turn goes into host (usually bacteria) 

See p 901 of book for overview of cloning 

One plasmid + insert ----> one bacterium ----> one colony (many bacteria) on 

Cloning PCR products 
     example where insert is "made" by PCR 
     other cases where insert comes directly from organisms 

What does PCR product look like?       Theory    "blunt" ends                                    0.1 kb - 10 k kb 

In fact: 

 

                       Design vector with T 

 

                                    Need to "cement" vector-insert with ligase 

What did they find? 
     1. Chemoauto trophic symbionts very different from methanotrophs, although both proteobacteria 
     2. Some similarity between symbionts and known methanotrophs -- evidence that symbiont is a                   methanotroph.