HSP phylogeny?

[Peter Gogarten]

In article <PWLEPP-151294102114 at schmidt2.mph.msu.edu>, PWLEPP at rrn.mph.msu.edu (Paul Lepp) says:
>
>A few months ago there was a discussion here about phylogenies based on
>heat shock proteins and a number of other proteins.  From what I remember
>it seemed that the argument was made that eukaryotes could have arisen from
>the fusion of a gram pos. and an archea.  Could someone be so kind as to
>pass along a few cites on the HSPs and the gram pos./archea  connection. 
>Much appreciated.
>
>Paul Lepp


These are the references concerning the actual analysis and data 
of the HSP70 homologues:
R. S. Gupta, K. Aitken, M. Falah & B. Singh (1994) Cloning of 
  Giardia lamblia heat shock protein HSP70 homologs: 
  implications regarding origin of eukaryotic cells and of 
  endoplasmic reticulum [see comments] Proc Natl Acad Sci U S A 
  91: 2895-2899  (1994) [94211768]
Gupta, R.S., Golding, G.B. (1993) Evolution of HSP70 gene and its 
 implications regarding relationships between archaebacteria, 
 eubacteria and eukaryotes. J. Mol. Evol. 37:573-582
 Gupta, R.S. and Singh, B. (1992) Cloning of the HSP70 gene from 
 Halobacterium marismortui: relatedness of archaebacterial 
 HSP70 to its eubacterial homologs and a model of the 
 evolution of the HSP70 gene. J. Bacteriol. 174, 4594-4605.

 As the HSP70 homologues suggest relationships very distinct from 
ATPases (and 16S rRNA) I had a closer look at the data.  The 
sequences show very convincingly a close association between 
HSP70 homologues from gram positive bacteria and from 
archaebacteria or Archaea (so far only sequences from two 
Euryarcheota).  The obtained tree are similar to the 
glutaminsynthetase data (see citations below).

 Concerning the close relationship between gram negative bacteria 
and eukaryotes I think the authors and others completely mis- (or 
over-)interpret the data.  The trees they Gupta et al. calculated 
are all unrooted.  If one uses midpoint rooting (i.e., one 
assumes a molecular clock) the root is placed between the 
eukaryotes on one side and all the prokaryotes on the other side 
(This was done by Sharon Shtang in here dissertation at the Univ. 
of Toronto).  The same result is obtained if one uses an 
'outgroup': 
The front half of the HSP70 homologues is homologous to the MreB 
proteins of E.coli and Bacillus (i.e. a gram positive and a gram 
negative). We (Elena Hilario and I) did some so far unpublished 
analyses including these proteins in a phylogenetic analyses of 
the HSP70 homologues.  Using parsimony, distance matrix or 
maximum likelihood analysis, the MreB proteins always group 
between Eukaryotes on one side and all the prokaryotes on the 
other.  As far as I can see there is no indication in the HSP70 
data what so ever indicating a close association between gram 
negative bacteria and eukaryotes.  The insertion that according 
to Gupta et al unites gram negative eubacteria and eukaryotes 
appears in our analysis as an insertion that unites gram 
positives and archaebacteria.  A problem with the use of the mreB 
proteins as an outgroup is that so far these mreB proteins have 
only been found in eubacteria.  Therefore, it might be that the 
duplication that gave rise to HSP70 and mreB proteins occurred 
only after the last common ancestor.  However, the duplication 
certainly preceded the separation of gram positives from purple 
bacteria; therefore, the grouping of the archaebacterial HSP70s 
with the gram positives is incompatible with the molecular 
phylogenies that find a clear separation between archae- and 
eubacteria.  This position of the "root" is found, if we use a 
computer generated alignment without further selection by hand, 
but it is also obtained when we used only those regions of the 
mreB proteins that were highly conserved and that could be 
aligned without difficulties.  

            |-------- mreB E.coli
 |----------|
 |          |-------- mreB Bacilli 
 |           
 |         |--------- eukaryotic HSP70 hom. 
 |         |         
 |---------|
           |     |--- gram negative bacteria     
           |-----|
                 |   |-- gram positive bacteria
                 |---|
                     |-- archaebacteria



Citations concerning glutaminsynthetases:

Kumada, Y., Benson, D.R., Hillemann, D., Hosted, T.J., Rochford, 
 D.A., Thompson, C.J., Wohlleben, W., Tateno, Y. (1993) 
 Evolution of the glutamine synthase gene, one of the oldest 
 existing and functioning genes. Proc. Natl. Acad. Sci. USA 
 90: 3009-3013

Tiboni, O., Cammarano, P., Sanangelantoni, A.M. (1993) Cloning 
 and sequencing of the gene encoding glutamine synthase I from 
 the archaeum Pyrococcus woesei: Anomalous phylogenies 
 inferred from analysis of archaeal and bacterial glutamine 
 synthase I sequences. J. Bacteriol. 175: 2961-2969

Brown, J.R., Masuchi, Y., Robb, F.T., Doolittle, W.F. (1994) 
 Evolutionary Relationships of Bacterial and Archaeal 
 Glutamine Synthetase Genes. J. Mol. Evol. 38, 566-576

 Some other molecular markers that suggest (or rather are compatible 
with) a close association between gram positives and 
archaebacteria (with +/- good resolution): 

Carbamylphosphate synthetase
Lazcano, Puente, Gogarten, unpublished.

Glutamate dehydrogenases,
Benachenhou-Lafha et al., 1993; Hilario and Gogarten, 1993.

F-ATPase subunit encoding DNA isolated from Methanosarcina 
Sumi et al., 1992; Hilario and Gogarten, 1993. 


Benachenhou-Lafha, N., Forterre, P. and Labedan, B. (1993) 
 Evolution of glutamate dehydrogenase genes: evidence for two 
 paralogous protein families and unusual branching patterns of 
 the archaebacteria in the universal tree of life. J. Mol. 
 Evol. 36, 335-346.

Hilario, E., Gogarten, J.P. (1993) Horizontal Transfer of ATPase 
 Genes -The Tree of Life becomes a Net of Life.  BioSystems 
 31, 111-119

Sumi, M., Sato, M.H., Denda, K., Date, T., Yoshida, M. (1992)  A 
 DNA fragment homologous to F1-ATPase beta subunit was 
 amplified from genomic DNA of Methanosarcina barkeri. 
 Indication of an archaebacterial F-type ATPase. FEBS. Lett. 
 314: 207-210


********************************************
  J. Peter Gogarten
  University of Connecticut
  Dept. Molecular and Cell Biology
  75 North Eagleville Rd.
  Storrs, CT 06269-3044

  Phone: USA 203 486 4061
  FAX:   USA 203 486 1784
  e mail: Gogarten at UConnVM.uconn.edu 
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