September 9, 1997                                MAST 634                          Lecture 2: 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 

Source of energy for hydrothermal vents 
      reduced inorganic compounds, mainly HS- (hydrogen sulfide) 

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 

 

                                                                  (delta)G^o'  

          HS^- + 20₂ = SO₄^2- + H⁺                         -794 
          S^o + 1O₂ + H₂O = SO₄^2- + 2H⁺      -585 

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

Some important examples 
        Nitrification 
             NH₄⁺ --->NO₂^- ---> N₃O^- 

                   Ammonium           nitrite          nitrate 
oxidation            -3                   +3               +5 
  state 
     Important in 
         soils: 
         ocean: 

 

      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 deltaS 
                  |            | 
             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^- 
1/2 O₂ + 2e^- ----> H₂O 

Look for reduction potential in table 

                                                                           E^o (V)  
              1/2 O₂ + 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 delta E derivation in book 
      N = number of electrons 
      F = Faraday's const. 

Note: if delta E > O, then delta G <O, i.e. reaction 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       -2823 k J mol^-1 
               
See page 43 

NADH + 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. 

 

          Delta G = 2.3 RT[pH_In^- pH_out] + ZF delta psi 
                                                 | 
                                 Membrane potential 

Mitochondria 

      delta pH = 0.75 higher outside 
      delta psi = 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 
      original 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

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.