Biology of Anaerobic Bacteria, edited by H . C . Dubourguier et al.
Elsevier Science Publishers B . V . , A m s t e r d a m , 1986 — Printed in T h e Netherlands
ANALYSIS OF FUNCTIONALLY RELATED GENE CROUPS IN METHANOGENIC BACTERIA J . S c h a l l e n b e r g1, M. Thomm2, K.O. S t e t t e r2, M. Moes1, M. T r u s s1, R. Allmansberger1, M. Bokranz1, E. Muth1 and A. K l e i n1
1Molecular Genetics, Dept. of B i o l o g y , P h i l i p p s U n i v e r s i t y , P.O. Box 1929, D-3550 Marburg (FRG)
2
I n s t i t u t e of Biochemistry, Genetics and M i c r o b i o l o g y , U n i v e r s i t y of Regens- burg, U n i v e r s i t ä t s s t r . 3 1 , 0-8400 Regensburg (FRG)
SUMMARY
S t r u c t u r a l genes of DNA-dependent RNA Polymerase and methyl reductase sub- u n i t s were cloned out of Methanobacterium thermoautotrophicum or Methanococcus y o l t a e . Their arrangements were determined. Both gene groups most l i k e l y form t r a n s c r i p t i o n u n i t s . S t r i k i n g peptide sequence homologies were detected between an RNA Polymerase subunit from the methanogen with corresponding ones from
£ L c o t* a n d y e a s t . Very high conservation of the Polypeptide sequences of two
corresponding reductase subunits was found among the two methanogens. Two small open reading frames of so f a r unknown f u n c t i o n were detected in between two reductase genes in Mc^ v o l t a e .
INTRODUCTION
Genetic studies on the molecular l e v e l can c o n t r i b u t e in various ways to the understanding of the biology of a group of p h y s i o l o g i c a l l y or p h y l o g e n e t i c a l l y r e l a t e d organisms.
The a n a l y s i s of the arrangement of genes involved in a metabolic pathway allows p r e d i c t i o n s about a p o s s i b l e r e g u l a t i o n of t h e i r e x p r e s s i o n . It might also lead to the d e t e c t i o n of genes which d e f i n e p r e v i o u s l y unknown components of the pathway. The a n a l y s i s of the gene s t r u c t u r e s leads to the deduction of primary structures of the gene products and permits the p r e d i c t i o n of secondary structures of the p r o t e i n s , w i t h i n c e r t a i n l i m i t s . F i n a l l y , the a n a l y s i s of r e - gions f l a n k i n g s t r u c t u r a l genes allows the d e t e c t i o n of S i g n a l s used f o r gene e x p r e s s i o n . Taken together these approaches help define the genetic basis f o r the p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the organism.
With b a c t e r i a l i k e the methanogens which almost always e x i s t as components of multiorganism c o n s o r t i a the understanding of gene s t r u c t u r e and expression mechanisms also gives Clues to the p o s s i b i l i t i e s and l i m i t s of the exchange of genetic Information among members of such b a c t e r i a l ecosystems. Our i n t e r - est in methanogens has two a s p e c t s . Methanogens are terminal l i n k s in anaero- bic degradative food chains and t h e r e f o r e play an important e c o l o g i c a l as well as biotechnological r o l e . In a d d i t i o n , methanogens are a major group of a r - chaebacteria, a group of procaryotes d i s t i n c t from both eucaryotes and eubac-
t e r i a . These organisms d i s p l a y c h a r a c t e r i s t i c features of the gene expression apparatus ( r e f s . 1 - 4 ) .
Two p r i n c i p a l approaches towards the e l u c i d a t i o n of the genetics of an o r - ganism are a v a i l a b l e . C l a s s i c a l genetics use mutants and e x p l o i t recombination and complementation s t u d i e s . Due to the lack of e f f i c i e n t gene t r a n s f e r Sys- tems t h i s approach has not been a p p l i c a b l e to methanogens even though mutants of various species are being accumulated ( r e f s . 5 - 1 0 ) . The a l t e r n a t i v e approach i s known as reverse g e n e t i c s . It S t a r t s from the gene products and employs gene technology in i d e n t i f y i n g the corresponding genes as w i l l be d e t a i l e d be-
low (compare F i g . 1).
We have concentrated our e f f o r t s on the e l u c i d a t i o n of the s t r u c t u r e and f u n c t i o n of genes encoding two c o n s t i t u t i v e m u l t i s u b u n i t enzymes, namely DNA- dependent RNA Polymerase and methyl reductase of methanogenic b a c t e r i a . We hope to gain i n s i g h t into the s t r u c t u r e s of the gene C l u s t e r s , the t r a n s c r i p t i o n u n i t s and the nature of expression S i g n a l s . In a d d i t i o n , we compare the primary s t r u c t u r e s of the proteins with homologous p r o t e i n s of other organisms. In t h i s way, we expect to get Information about s t r u c t u r e - f u n c t i o n - r e l a t i o n s h i p s and about phylogenetic pathways leading to the present State of the genetic C o n s t i - t u t i o n of these two groups of f u n c t i o n a l l y r e l a t e d genes.
RESULTS
RNA Polymerase genes of methanogenic b a c t e r i a
( i ) I d e n t i f i c a t i o n of s t r u c t u r a l genes of Methanobacterium thermoautotrophi- cum. DNA-dependent RNA Polymerase of Mb^ thermoautotrophicum c o n s i s t s of at l e a s t 8 subunits (ref. 11). Employing a n t i s e r a r a i s e d against the i s o l a t e d sub- u n i t s of RNA Polymerase of Mb. thermoautotrophicum ( s t r a i n Winter) recombinant Plasmids could be i d e n t i f i e d w h i c h . c a r r i e d at l e a s t fragments of the s t r u c t u r a l genes of the four largest RNA Polymerase subunits A, B\ B " and C. These P l a s - mids are l i s t e d in Table 1. The methods used are o u t l i n e d in F i g . 1. F i g . 2
TABLE 1
pEx3l Plasmids ( r e f . 12) c a r r y i n g Mb. thermoautotrophicum ( s t r a i n Winter) RNA Polymerase gene fragments
Expressed subunit antigenic determinants Insert s i z e
A s t e r i s k s i n d i c a t e that the a n t i g e n i c determinants are c a r r i e d on f u s i o n p r o t e i n s . The cloning procedures have been described in r e f . 13.
A A*/C B 7 B ' B1 '*
B' 1
1.6 kb 2.6 kb 1.0 kb 0.2 kb 0.2 kb
Clontng of small DNA-fragments from tne metnanogen in E. coli vector yietding E. c o l l - « e - tnanogen gene fusions
Expression of E. coli-metnano- gen hybrid Peptides in E. coli clones using an E. coli Pro- moter
Protein purificatlon
Production of antlseru«
aqainst purifled Pro- tein
lamunological identtfl-
• catlon of clones pro- - ducing antigenic de- terminants of the pro- tein
Clonlng of nultigene DNA fragments fron the metnanogen in bacterio- phageX vectors
Expression of gene products fron
* Ts, Promoter
Isolation of Plasmids carrying relevant gene fragment fused to E. coli gene
Identification of phages carrying relevant genes oy DHA/OHA-hvoridt- zatton
F i g . 1. Flow diagram of methods employed i n t h i s study t o i d e n t i f y s t r u c t u r a l genes S t a r t i n g from the known gene products.
E E
1 1 E E
1 I .
1Kb
F i g . 2 . EcoRI r e s t r i c t i o n map of the Mb. thermoautotrophicum ( s t r a i n Winter) DNA segment c a r r y i n g the RNA Polymerase subunit genes A, B ' , B '1 and C. The bars above show i n s e r t s of X g t 1 0 v e c t o r s , the boxes below i n d i c a t e the l o c a - t i o n of the genes as determined by DNA/DNA-hybridization ( r e f . 1 4 ) .
shows the r e l a t i v e p o s i t i o n s of the four genes, taken from DNA/DNA-hybridization data ( r e f . 14) of plasmid against DNA from recombinant"Xphage taken from a genomic l i b r a r y of Mb. thermoautotrophicum generated i n the X i n s e r t i o n vector X g t 1 0 ( r e f . 15). The 4 kb EcoRI fragment comprising the 3 ' end of the A gene was subcloned i n the expression v e c t o r X g t H ( r e f . 16) which allowed t o e x - press the complete C gene product ( F i g . 3) encoded on the same fragment and thus to confirm both the neighborhood of the A and C genes and t h e i r common d i r e c t i o n of t r a n s c r i p t i o n .
(1 1 ) Arrangement of Polymerase genes in other methanogenic b a c t e r i a . We wanted t o determine the gene order of the i d e n t i f i e d s t r u c t u r a l genes f o r the A, B ' , B " and C subunits of the RNA Polymerase i n other methanogenic b a c t e r i a . Experiments performed towards t h i s goal showed that DNA sequence homologies among d i f f e r e n t members of the group only allowed h y b r i d i z a t i o n between Methano-
1 2 3
F i g . 3 . I d e n t i f i c a t i o n of vector encoded C subunit Polypeptide by an immunoblot technique (compare r e f . 1 3 ) . Lanes, 1: Extract from Mb. thermoautotrophicum
( s t r a i n W i n t e r ) ; 2 : E x t r a c t from E. c o l i c o n t a i n i n g an expression plasmid c a r r y - ing a 2.6 kb genomic DNA fragment
of~W7
thermoautotrophicum; 3 : P r o t e i n o b - tained f r o m ' X g t n c a r r y i n g a 4 . 0 kb W7 thermoautotrophicum DNA fragment which overlaps the one contained i n the p l a s m i d .B E BE E Mb.th«rmo- J 1 U -
B* 8 "
autotrophicum (MARBURG) E B B E E 8 Mb.formi- J i i i u ocum
E Cl ECl E D ECI E J i ir i i L _ J u Mc. volta«
F i g . 4 . Gene order of RNA Polymerase subunit s t r u c t u r a l genes i n methanogenic b a c t e r i a . Genome fragments are shown with relevant r e s t r i c t i o n S i t e s and t h e areas i n d i c a t e d to which probes of i d e n t i f i e d Mb. thermoautotrophicum gene fragments (open bars) of t h e i n d i c a t e d genes h y 5 r i d i z e .
E = EcoRI, B = BamHI, Cl = C l a l r e s t r i c t i o n S i t e s
bacterium and Methanococcus s p e c i e s . Mb^ thermoautotrophicum probes d i d not h y b r i d i z e with DNA of Methanosarcina b a r k e r i . The h y b r i d i z a t i o n between
Mb. thermoautotrophicum and Mc. v o l t a e i s e s p e c i a l l y remarkable because of the d i f f e r e n c e i n the DNA GC contents of these two members of d i f f e r e n t Orders o f methanogenic b a c t e r i a ( r e f s . 17, 1 8 ) . As can be seen from F i g . 4 the gene order of the four Polymerase genes mapped i s i d e n t i c a l i n a l l four organisms in which
i t couid be i n v e s t i g a t e d .
( i i i ) P o s s i b l e r e l a t i o n s h i p between RNA Polymerase genes among methanogenic and s u i f u r dependent a r c h a e b a c t e r i a . Methanogenic b a c t e r i a are a subgroup of a r c h a e b a c t e r i a , which d i f f e r from both eubacteria and eucaryotes with respect to various features of the gene expression apparatus i n c l u d i n g t h e i r RNA p o l y - merases ( r e f . 19).
Since a l l organisms contain RNA polymerases t h i s enzyme lends i t s e l f f o r studies on the e v o l u t i o n of gene and enzyme s t r u c t u r e . It has been found t h a t w i t h i n the archaebacteria the RNA Polymerase s t r u c t u r e s d i f f e r . One i n t e r e s t i n g d i f f e r e n c e occurs among the methanogenic b a c t e r i a and the s u i f u r dependent genus Sulfolobus as schematically shown i n the i n s e r t of F i g . 5b. The l a r g e s t
subunit of the Sulfolobus enzyme has a n t i g e n i c determinants which appear d i s - t r i b u t e d on two subunits of the methanogen enzymes, namely B' and B1' . As shown above, the genes f o r these two subunits are located next t o each other on the chromosome. It appeared i n t e r e s t i n g t o look f o r the s t r u c t u r e of the i n t e r g e n i c region between the B* and B ' ' genes i n Methanobacterium i n order to get a c l u e concerning a p o s s i b l e phylogenetic pathway leading from a Polymerase subunit B type found i n s u i f u r dependent archaebacteria t o the methanogen B 7 B " subunit arrangement. Sequence determination i n between a sequence coding f o r a B' a n t i - genic determinant and a B " coding sequence i n Mb^ thermoautotrophicum (both c h a r a c t e r i z e d by a n t i s e r a a f t e r expression on Plasmids) showed two open reading frames which o v e r l a p . The sequence ATGA includes both the stop codon TGA t e r - minating the B' open reading frame and an AT6 codon most l i k e i y c o n s t i t u t i n g the s t a r t of the B " gene ( F i g . 5 a ) . It i s c l e a r from t h i s sequence that the d e l e t i o n of one base at the overlap would lead t o the f u s i o n of the two genes or e i s e these two genes could have o r i g i n a t e d from one gene by a one base p a i r
I n s e r t i o n ( e . g . the A or T of the AT6 codon).
QI, Methanobacterium
B' ' ß. Bo
-GGGTACACTCTCCATATGAGGAGGGTTGTATCA- —
B
Sulfolobus
F i g . 5 . a) Overlapping open reading frames at t h e presumptive border of the B' and BM Polymerase subunit genes of Nfc. thermoautotrophicum ( s t r a i n W i n t e r ) , b) Schematic representation of the gene arrangements i n Sulfolobus and Methano- bacterium (redrawn from r e f . 19). Sequence determination was performed using the chemical cleavage method ( r e f . 2 0 ) .
(iv) S t r u c t u r a l homologies o f RNA poiymerases o f Mb. thermoautotrophicum, E s c h e r i c h i a c o l i and Saccharomyces c e r e v i s i a e . As mentioned above the archae- b a c t e r i a l RNA poiymerases d i f f e r from both the e u b a c t e r i a l and e u c a r y o t i c e n - zymes. Yet, a common ancestor i s very l i k e l y and immunologicai studies have indeed i n d i c a t e d s t r u c t u r a l r e l a t i o n s h i p s among poiymerases o f a l l three groups ( r e f . 19). We have t h e r e f o r e compared a v a i l a b l e sequences o f E. c o l i , yeast and Mb. thermoautotrophicum Polymerase genes and found an example o f s t r i k i n g Pep- t i d e sequence homology (as derived from the corresponding DNA sequences). This
i s shown i n F i g . 6 and points t o a h i g h l y conserved domain o f the large RNA Polymerase subunits of the three organisms, ß1, RP021 and A o f E. c o l i , S . c e r e - v i s i a e and Mb. thermoautotrophicum, r e s p e c t i v e l y ( r e f . 2 1 ) .
SC -Met Gly Gly Arg Glu Gly Leu He ASD Ihr Aia Völ Lys Ihr Ala Glu Ihr Gly Tyr
< • ) • • • < • ) < • ) • • (•) • • MT -Met Gly Gly Arg Glu Gly Leu tel Aso Ihr Ala Ile Arg Ihr Ala Gin Ser Gly Tyr
* (•) • • * • • « • « * » • EC -Hls Gly Ala Arg Lys Gly Leu Ala Asp Thr Ala Leu Lys Ihr Ala Asn Ser Gly Tyr SC üe Gin Arg Arg Leu val Lys Ala Leu Glu Asp Ile Met Völ-
C) • MT Met Gin Arg Arg Leu tel Asn Ala Leu Gin Asp Leu Ihr Völ-
» • • • * • * • EC Leu Thr Arg Arg Leu Va\ Asp Val Ala Gin Asp Leu vai Völ-
F i g . 6 . Sequence homologies o f RNA Polymerase subunits. The data f o r E. c o l i
a n d S« c e r e v i s i a e were taken from r e f . 2 1 . The methyl reductase gene group
In c o n t r a s t t o RNA Polymerase methyl CoM reductase component C (methyl r e - ductase) i s an enzyme found only i n methanogenic b a c t e r i a . I t i s the c e n t r a l enzyme i n the energy metabolism, c a t a l y z i n g the terminal Step o f carbon r e - duction t o methane ( r e f . 4 ) . This r e a c t i o n i s g e n e r a l l y performed a t the e x - pense of a methyl group i n i t i a l l y bound t o an unique coenzyme (coenzyme M).
and e l e c t r o n s derived from hydrogen with the help o f a hydrogenase as Schema- t i c a l l y shown i n F i g . 7 . The enzyme has been p u r i f i e d from d i f f e r e n t methano- gens and shown t o contain three subunits, named o<, ß and ^ ( r e f . 2 2 ) . I t i s a very abundant p r o t e i n c o n s t i t u t i n g roughly 5% o f the t o t a l c e l l u l a r p r o t e i n in Mc_^ v o l t a e .
( i ) Gene arrahgement and Expression. We have p r e v i o u s l y described t h a t the genes encoding the three known subunits o f methyl reductase are arranged i n the order ß , ^ and o< i n Mc. v o l t a e ( r e f . 2 3 ) . They are located i n c l o s e p r o x i m i t y to each o t h e r . This same gene order o f the three genes has a l s o been found i n Mb. thermoautotrophicum (Marburg) (Bäumner and Bokranz, unpublished r e s u l t s ) . The genes probably c o n s t i t u t e a t r a n s c r i p t i o n u n i t ( r e f . 13 and unpublished
F i g . 7. Schematic r e p r e s e n t a t i o n of the terminal Steps of C l - r e d u c t i o n in methanogenic b a c t e r i a . HY - hydrogenase, RE = methyl reductase, CoM = c o - enzyme M ( t h i o e t h a n e s u l f o n a t e ) .
observations).The high amount of the enzyme requires very e f f i c i e n t expression of the genes which occur only once in the genome of the c e l l . This e f f i c i e n t expression w i l l most l i k e l y e n t a i l coordination of t h a t expression which at f i r s t approximation could be accomplished by c o t r a n s c r i p t i o n on a p o l y c i s t r o - n i c messenger molecule.
Düring sequence a n a l y s i s we have r e c e n t l y detected two small open reading frames intervening between the ß and ^ reductase genes ( F i g . 8 ) .
F i g . 8 . Arrangement of methyl reductase genes and two interspersed open reading frames of unknown f u n c t i o n in Mc. v o l t a e .
Their f u n c t i o n i s unknown and i t w i l l be i n t e r e s t i n g to see whether s i m i l a r genes are found in the same P o s i t i o n i n other methanogens. In a common t r a n - s c r i p t i o n u n i t with the three s t r u c t u r a l genes of the reductase the small open reading frames would be t r a n s c r i b e d at the same r a t e as the reductase genes.
It has been found p r e v i o u s l y that s t r u c t u r a l genes i n methanogens are p r e - ceded by sequences which are complementary to the 16S rRNA 3* end ( r e f . 2 4 ) .
They are assumed to c o n s t i t u t e ribosome binding S i t e s ( r e f s . 25, 2 6 ) , i . e .
t r a n s l a t i o n S i g n a l s . Such Signals i n f r o n t of the two reductase genes ^ a n d o <
and the two open reading frames i n between the reductase ß and ^ genes are shown together with the complementary 3* terminal sequence of the 16S rRNA i n F i g . 9 .
3 ' C C U C C A C U A G - 16S RNA
A G G T G A T C t t A T G y -Gen G A G G T G A a C c t a t A T G o c -Gen
A G G T a A a a a a A T G ORF-1 G t a G a G c T g t t A T G ORF-2
F i g . 9 . P u t a t i v e ribosome binding S i t e s i n f r o n t of methyl reductase genes and and ORF 1 and 2 . The shown RNA sequence i s t h e 3* terminus of rnethano- coccal 16S rRNA ( r e f . 2 4 ) .
It can be seen that the complementarity i s much weaker with the Signals i n f r o n t of the small open reading frames than with those i n f r o n t of the r e d u c - tase genes which might i n d i c a t e d i f f e r e n t i a l expression on the t r a n s l a t i o n a l
l e v e l .
( i i ) Homology of the methyl reductase oC genes of Mc. voltae and Mb. thermo- autotrophicum (Marburg). It i s c l e a r that the methyl reductases i n methanogens are homologous enzymes. S t i l l , , comparison of t h e i r s t r u c t u r e s in d i f f e r e n t me- thanogens may y i e l d i n s i g h t i n t o the f u n c t i o n a l Organization of the p r o t e i n s . It i s conceivable that d i s t a n t l y r e l a t e d methanogens have h i g h l y conserved s e - quences only in f u n c t i o n a l l y important domains l i k e c o f a c t o r and Substrate binding S i t e s and the r e a c t i v e c e n t e r .
Nothing i s known so f a r about the s t r u c t u r e - f u n c t i o n - r e l a t i o n s h i p of t h e reductase. We have s t a r t e d comparing the primary s t r u c t u r e s of the enzymes by DNA sequence determination of t h e i r s t r u c t u r a l genes i n Mc. voltae (an AT r i e h mesophilic methanogen) and Mb. thermoautotrophicum which has a higher 6C c o n - tent and i s t h e r m o p h i l i c . Very high conservation has been found in t h e f i r s t p a i r of genes looked a t , theoC genes. The homology i s roughly 71% on both the protein and the DNA l e v e l s . Base exchanges i n the wobble p o s i t i o n of t h e codons allow adaptation t o the GC contents of the DNA without changing the r e s u l t i n g Polypeptide sequence.
F i g . 10 demonstrates t h i s e f f e c t i n an extremely conserved r e g i o n : even c o n -
s e r v a t i v e amino acid exchanges such as serine to threonine at the corresponding P o s i t i o n f o l l o w that r u l e .
MT -GAC CCT GTC AGG GTG TCA CTT GAC GTT GTG GCA ACC GGT GCA ATG CTC TAC GAC CAg ATC
»** *«» »• * •** * » • « « » * •*« •» *»• • •««. »«« «« # # # MV -GAC CCT GTT GAG CAA TCA TTA GAG GTA GTT GCA ACT GGT GCT GCT TTA TAC GAC CAA ATC MT -Asp Pro toi Arg toi Ser Leu Asp toi toi Ala Thr Gly Ala Met Leu Tyr Asp Gin Ile
• • • • • < • ) # • • • •
MV - A S P Pro toi Glu Gin Ser Leu Glu toi toi Ala Thr Gly Ala Ala Leu Tyr Asp Gin Ile
MT TGG CTA GGA TCA TAC ATG TCA GGT GGT GTC GGA TTC ACA CAG TAC GCC ACA GCA GCA TAC MV TGG CTT GGT TCA TAC ATG TCT GGT GGT GTA GGA HC ACA CAA TAT GCT ACA GCA TCA TAC MT Trp Leu Gly Ser Tyr Met Ser Gly Gly Völ Gly Rie Thr Gin Tyr Ala Thr Ala Ala Tyr MV Trp Leu Gly Ser Tyr Met Ser Gly Gly toi Gly Rie Thr Gin Tyr Ala Thr Ala Ser Tyr
HT ACA GAC AAC ATA CTT GAC GAC TTC ACC TAC TTC GGT AAG GAG TAC GTG GAA GAC AA6 TAC- MV ACA GAT GAC ATC TTA GAT GAC TTC TCA TAC TAC GGA TAC GAA TAC GTA GAG AAA AAA TAC- MT Thr Asp Asn Ile Leu Asp Asp Rie "Dir Tyr Rie Gly Lys Glu Tyr \öl Glu Asp Lys Tyr-
* * (#) * (#)
MV Thr ASP ASP Ile Leu Asp Asp Rre Ser Tyr Tyr Gly Tyr Glu Tyr toi Glu Lys Lys Tyr-
F i g . 10. Sequence homologies of a DNA and corresponding Polypeptide sectiorr of the methyl reductase o( genes from Mc. voltae (MV) and Mb. thermoautotrophicum
(Marburg) (MT).
It w i l l be i n t e r e s t i n g to see whether or not the high conservation of the Polypeptide sequence of t h e o < s u b u n i t i s exceptional compared to the other sub>
u n i t s thus p o i n t i n g to a c e n t r a l f u n c t i o n a l r o l e of t h a t subunit.
DISCUSSION
Our studies on the RNA Polymerase of Mb. thermoautotrophicum strengthen the hypothesis that RNA polymerases are p h y l o g e n e t i c a l l y derived from one p r e - cursor in a l l organisms. This i s i n f e r r e d from the sequence homology between the large subunits of a e u b a c t e r i a l , eucaryotic and a r c h a e b a c t e r i a l gene p r o - ducts ( f i g . 6 ) .
The arrangements of homologous b a c t e r i a l RNA Polymerase genes d i f f e r . We have found a conserved gene order and close p r o x i m i t i e s of the s t r u c t u r a l genes
f o r the largest RNA Polymerase subunits in the i n v e s t i g a t e d methanogenic b a c t e - r i a . These genes are l i k e l y to form a t r a n s c r i p t i o n u n i t i n Mb. thermoauto- trophicum since the d i r e c t i o n of t r a n s c r i p t i o n i s i d e n t i c a l f o r a l l genes (our unpublished r e s u l t s ) . Due to the s e r o l o g i c a l r e l a t i o n s h i p between the homolog- ous subunits t h i s arrangement can be compared to the corresponding gene order
i n E- c° l i (ref. 2 7 ) . In t h i s organism only two subunits are expressed from a t r a n s c r i p t i o n u n i t , namely the genes rpoB and rpoC. They code f o r the subunits ß and ß' corresponding to the B'1 and A subunits in the methanogens, r e s p e c - t i v e l y . The rpoD gene coding f o r the er subunit in E. c o l i and"corresponding to the C gene in the methanogens i s not c o t r a n s c r i b e d with the rpoB and rpoC genes but located d i s t a n t from these genes. This may have f u n c t i o n a l reasons.
since Q" i s a s p e c i f i c i t y f a c t o r and can be repl^ced by other subunits de- pending on the p h y s i o l o g i c a l State of the c e l l .
The s u r p r i s i n g f i n d i n g of two small open reading frames in the presumptive methyl reductase t r a n s c r i p t i o n u n i t i n Mc. voltae i s of s p e c i a l i n t e r e s t to u s .
The reductase has very low a c t i v i t y in the p u r i f i e d form which i s composed of the three mentioned subunits w , ß and ^ together w i t h the necessary n i c k e l containing c o f a c t o r . The d e t e c t i o n of f u r t h e r components might help d e f i n e a reason for the f u n c t i o n a l d e f i c i e n c y of the enzyme i n v i t r o . It must be s t r e s s e d , however, t h a t the Organization of genes in a common t r a n s c r i p t i o n u n i t need not mean that t h e i r products are parts of a p r o t e i n complex.
The comparison of the two methyl reductase oC genes from Mb. thermoauto- trophicum and Mc. voltae has e l i c i t e d strong homology of the p r o t e i n s . Most of the base exchanges in the s t r u c t u r a l genes are c o n s i s t e n t with adjustment t o the d i f f e r e n t o v e r a l l GC Contents of the DNA in the two organisms. The c o n c e r - vation of the Polypeptide sequences in these organisms might r e f l e c t c o n - s t r a i n t s due to e s s e n t i a l f u n c t i o n a l domains. S p e c i f i c conclusions must, how- ever, await the e l u c i d a t i o n and comparison of a l l enzyme s u b u n i t s .
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