Vol.

168,

No.

April

30,

1990

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

809-817

Pages

IDENTIFICATION,

SEQUENCE DETERMINATION,

AND EXPRESSION

FROM DESULFOVIBRIO Larry Department Received

COMMUNICATIONS

RESEARCH

R. Helms,

Grigorios

of Biochemistry,

March

27,

D. Krey,

The Ohio

OF THE FLAVODOXIN

GENE

SALEXIGENS

State

and Richard

P. Swenson

University,

Columbus,

OH 43210

1990

Restriction fragments of genomic DNA from Desulfovibrio salexigens (ATCC 14822) containing the structural gene coding for the flavodoxin protein were identified using the entire coding region of the gene for the Desulfovibrio vulgaris (Hildenborough) flavodoxin as a probe (Krey, G.D., Vanin, E.F., and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). A 1.4-kb PstI-Hind111 fragment was ultimately identified which contains an open readingfram=ding for a polypeptide of 146 amino acid residues that was highly homologous to the D. vulgaris flavodoxin, sharing a sequence identity of 55%. When compared to the X-ray crystal structure of the D. vulgaris protein, the homologous regions were largely confined to those portions of the protein which are in the immediate vicinity of the flavin mononucleotide cofactor binding site. Tryptophan-60 and tyrosine-98, which reside on either side of the isoalloxazine ring of the cofactor, are conserved, as are the sequences of the polypeptide loop that interacts with the phosphate moiety of the flavin. Acidic residues forming the interface of model electron-transfer complexes with certain cytochrome 5 proteins are retained. The flavodoxin holoprotein is overexpressed in E. coli from the cloned gene using its endogenous promoter. 01990 Academic Press, Inc. SW:

Flavodoxins

have

species

including

In each

case,

within

this

sequence this

structure,

however,

the

(3,

examination significant. redox

of the

structure from amino

the

FMN binding

site,

of a closely localize properties

over

which

are

regions

might

be important

bound

of flavodoxin

cofactor

primary

of the

structures

the

are of the

uncertainties by visual

However, sequences

should

to be functionally in the

regulation

transfer

structural

of

processes. gene

for

the

0006-291x/90 809

of

remarkably

residues

protein likely

and in electron

sequence

its

portions

themselves.

regions

the nucleotide

all

acid

family

of the

However,

of proteins

increasing amino

related these

are

family

homologous

For example, we reported

greatly

structures

(1).

The tertiary

extends

important

crystal

and D. vulgaris.

sources

of this

variation

Desulfovibrio

has had

(2).

of other

sequences

of functionally flavodoxin

D. vulgaris

elucidated

acid

of the

and characterized

from

a variety

Sequence

4).

members

D. desulfuricans,

isolated

flavodoxin

crystal

.including

more readily

Recently,

the

identification

inspection

has been

and those

different

in the

the

only

in several

D. salexigens,

protein

and X-ray

flavodoxin

quite

identified

D. gigas, the

group

homologous;

been

$1.50

CoDright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

168, No. 2, 1990

flavodoxin probe,

from

D. vulgaris

we report

Desulfovibrio

BIOCHEMICAL

here

(5).

the

cloning

salexigens

which

AND BIOPHYSICAL

Using

the

coding

and sequence codes

for

the

RESEARCH COMMUNICATIONS

region

from

of a fragment flavodoxin

from

this

gene

of genomic this

as a DNA from

organism.

MATERIAL!ZANDllBTBODS: Desulfovibrio salexigens cultures obtained from American Type Culture Collection (ATCC 14822) were revived and grown in modified Baar's medium under anaerobic conditions (6). E. coli strains XLl-Blue (Stratagene) and AGl were grown in LB medium. Genomic DNA was purified as described previously (5). A 450-bp Hind111 fragment (designated DVF450H3) containing the entire coding region ofhe D. vulgaris flavodoxin (DVF) gene (5) was radiolabeled by a modification of the method of Feinberg and Vogelstein (7) and used as a probe for flavodoxin structural genes. DNA restriction fragments that putatively contain the D. salexigens flavodoxin (DSF) gene were identified by hybridization of Southern blots of ClaI-digested D. salexigens genomic DNA with the radiolabeled DVF450H3Tobe as follows. The -ClaI restriction fragments were separated by agarose gel electrophoresis and transferred to GeneScreen Plus membranes (DuPont) by the Southern blot procedure. Membrane filters were prehybridized by established methods and hybridized overnight at 55'C with the radiolabeled probe (6.2 x 106 dpm). Filters were washed twice, each for 20 min at 55OC in 3x SSC/O.5% SDS, dried, and subjected to autoradiography. A genomic mini-library was constructed using the Bluescript vector (Stratagene) as follows. Genomic DNA was digested to completion with the restriction enzyme -ClaI and fragments size-selected by centrifugation though a linear NaCl gradients as described previously (5). Appropriately sized fragments were ligated into the ClaI site in the Bluescript vector using conditions described by Grundstrcet al. (8). E . coli XLl-Blue cells were transformed by the procedure described by Hanahan (9) and transformation mixtures plated onto GeneScreen Plus membranes which had been placed on LB media supplemented with ampicillin (75 vg/ml) and tetracycline (15 l.rg/ml). Colony hybridizations were as follows. All plates were replica plated onto GeneScreen Plus membranes and allowed to grow to l-2mm in diameter (ca. 8 hrs at 37OC). The filters were first laid for 7 min on Whatman 3MM filter paper saturated with 0.5M NaOH and then washed with 1M Tris-HCl, pH 7.4 for 5 min and finally for 5 min with O.SM Tris-HCl, pH 7.4, containing 1.5M NaCl. The filters were then scrubbed with a gloved hand in 6x SSC/O.5% SDS at 25'C and allowed to air dry. Filters were prehybridized at 65'C with sonicated and denatured Bluescript vector (5 ug/ml) to minimize nonspecific interactions of the probe with this Hybridization with radiolabeled DVF450H3 proceeded overnight at cloning vector. 65OC. The filters were washed twice in lx SSC/O.5% SDS at 65OC, each for 20 min. Positive colonies were visualized by autoradiography and rescreened by hybridization at 65OC of the DVF450H3 probe to Southern blots of C&I-digested recombinant plasmid preparations as described above. Restriction fragments were subcloned into Bluescript for restriction mapping and sequence determination. Nucleotide sequences were determined using the dideoxy nucleotide chain termination method of Sanger et al. (10). All other recombinant DNA procedures were taken from Maniatis et al. (11) unless otherwise noted. DNA and protein sequences were analyzed using software from DNAstar, Inc. The amino acid similarity (PAM) matrix described by Lipman and Pearson (12) was used in the protein sequence comparisons. Purification of the DSF protein expressed in E. coli and SDS-polyacrylamide gel electrophoresis were carried out essentially as described by Krey et al. (5). RESDL'J3ANDDISCUSSION Isolation nucleotide study,

of the sequence

the

entire

gene

for coding

the

- We have

previously

flavodoxin

region

of the

from

cloned Desulfovibrio

D. vulgaris 810

and determined

flavodoxin

vulgaris (DVF)

the (5). gene

In this (as the

BIOCHEMICAL

Vol. 168, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

kb

3.6

Figure 1. Autoradiogram of the Southern blot of Desulfovibrio salexigens genomic DNA digested with the restriction endonucleases -1 (Lane 1) and NcoI was to a radiolabeled 450bp HindI11 fragment containing (Jane 2). Hybridization the entire coding region of the flavodoxin gene from Desulfovibrio vulaaris.

450 bp HindIII

fragment,

identification

and isolation

DVF450H3 3.8

probe

was found

and 7.2-kb

ively,

clones,

cloned eight

a plasmid

with

colonies

--PstI-ClaI

the

fragment

responsible located

found

insert

flavodoxin

fragments

(3.0

vector. hybridizing

hybridization

at the

extreme 811

of molecular

to the

3 '-end

of the

sizes

The of

DNA, respect-

in E. coli

XLl-Blue

-ClaI

DVF450H3

was further

gene.

genomic

to 5.0-kb) to the

in the

(DSF)

Of approximately

to hybridize

also for

as a probe

or NcoI-digested was prepared

size-selected

Bluescript

were

a 3.8-kb

The region

of -ClaIA mini-library

1. with

into

to two

blots

in Figure

was used

D. salexigens

to hybridize

by transformation

fragments

DVF450H3)

of the

in Southern

as shown

cells

designated

restriction 1600 recombinant probe

and contain

probe. confined insert

to a l.O-kb

of one of these

Vol.

168,

No.

clones.

Analysis

this

clone

BIOCHEMICAL

2, 1990

of

AND

the nucleotide

was missing

at least

BIOPHYSICAL

sequence 12% of the

of this 3'-end

(based

on homology

to the DVF sequence).

orientation

of the

gene was from

the -PstI site other restriction

with

larger WI-GRI

and various more

fragment

Southern The

=I

fragments

likely

bind

1200 recombinant

the

probe

hybridized

to the

l.O-kb

nucleotide

sequence

Nucleotide 1.4-kb

in Figure this

--PstI-ClaI analysis.

2.

The nucleotide for

derived

from

this

to the

of 148 amino

DNA sequence

homology significantly

D. vulgaris

site"

homologous

very

"-10

site"

A/T-rich

the

share

the to 3'

on

to the

sequence

isolated

that of the

consensus

of the

optimal

insert

DSF sequence

in Figure with

residues.

also from

shown

for

5'-flanking

beginning

acid

a protein will

follow.

at nucleotide

for frame

corresponding

sequence is very

of codon

3, is highly

open-reading the

sequences

-109

initiation

of 278 nucleotides

of this

promoter

Examination

the

in Figure

portion

shown

The amino

comparison

observed

of the

3.

D. vulgaris,

alignment

for

map and sequencing

entire

acid

a is

gene

revealed

of E . coli (13). to the

similar

-87

is

homologous

The region bounded by these two sites length for the separation of these two

Pst1 -i.j' -__

ClaI I

+RI

@+x11 -__ 3'

LOObp Figure 2. Partial restriction map and kb PstI-WI11 restriction fragment. region for the Desulfovibrio salexigens regions sequenced on each DNA strand.

to

which

was chosen

the

at nucleotide

promoters.

1.4-kb

clones

observed

sequence

A detailed

-TAAATT-

the

were

nucleotide

The G + C content

beginning

and is

with

frame,

than

colonies

beginning

146 amino

a sequence

Examination

of E. coli

six

reported

frame

residues.

lower

-TTCACATwhile

is

protein

acid

of 63%.

(62%).

highly

The sequence

of

flavodoxin

47%, a value

is

for 5'

A l.l-kb

identified

restriction

contained

open-reading

The DSF and DVF genes

to the

the

gene.

were

One of these

of the

sequence

a polypeptide

consisting

"-35

fragment

- The partial

an open-reading

homologous

regions

that

structural

a plasmid

determination fragment

revealed

ATG and coding

from

entire

clones,

probe.

analysis

in the

%I-Hind111

sequence

sequence

and to contain

sequence

involved

of the

region

Knowing

genomic DNA was -ClaI site, endonucleases to generate

--PstI-Hind111 double-digests using

Of approximately

strategy

coding

that

of these the WI-ClaIfragment as a probe. PstI-Hind111 fragment was chosen for cloning. A mini-library of -fragments was prepared and screened as described --PstI-Hind111

size-selected strongly

the

suggested

to the

and a 1.4-kb

blots

1.4-kb

above.

to contain

COMMUNICATIONS

fragment

of the

DSF protein digested

RESEARCH

nucleotide The darkened flavodoxin.

812

sequencing scheme for the 1.4 region represents the coding The arrows indicate the

Vol.

BIOCHEMICAL

166, No. 2, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AGACTAACAATAAAAATTTTGTTTTCTCACAAACAACATTTTGMC~GCATATATCGGTACGTC~~GGAGTGCATA -160 ACACCACTCAGGCGCGGCGTAlULCACTCAATTACCGATT~CTGACATTTTCACATTTTC~~TAT~TAT~TTG -80 -35 -10 ATATTGACTTTGMTTTCACTCTCACTACAllACTAACACMCGTTATC~CCGATTACAGA~TAT~~ACACT SD

-1

ATG TCC AAA TCA CTG ATC GTT TAC GGCTCT ACT ACC GGA AAT ACT GM ACA GCCGCCGAA 60 Met Ser Lys Ser Leu Ile Val Tyr Gly Ser Thr Thr Gly Asn Thr Glu Thr Ala Ala Glu 1 10 20 TAC GTG GCT GAA GCGTTT GM AAC AAA GAA ATT GAT GTG GAA CTT AAA AAT GTT ACT GAT 120 Tyr Vial Ala Glu Ala Phe Glu Asn Lys Glu Ile Asp Val Glu Leu Lys Asn Val Thr Asp 30 40 GTC AGT GTT GCCGAT CTC GGCAAC GGA TAC GAC ATC GTG CTA TTC GGCTGC TCT ACCTGG 180 Val Ser Val Ala Asp Leu Gly Asn Gly Tyr Asp Ile Val Leu Phe Gly Cys Ser Thr Trp 50 60 GGCGAA GAA GAA ATT GAA TTG CAGGAC GAC TTC ATC CCCCTC TAC GAT TCC CTC GAA AAC 240 Gly Glu Glu Glu Ile Glu Leu Gin Asp Asp Phe Ile Pro Leu Tyr Asp Ser Leu Glu Asn 70 80 GCA GAC CTG AAG GGCAAG AAA GTA TCT GTC TTC GGA TGC GGT GAC TCC GAT TAC ACT TAT 300 Ala Asp Leu Lys Gly Lys Lys Val Ser Val Phe Gly Cys Gly Asp Ser Asp Tyr Thr Tyr 90 100 TTT TGC GGT GCA GTA GAT GCCATC GAA GA4 AAA CTC GAA AAA ATG GGGGCCGTC GTC ATA 360 Phe Cys Gly Ala Val Asp Ala Ile Glu Glu Lys Leu Glu Lys Met Gly Ala Val Val Ile 110 120 GGT GAC:AGC CTC AAG ATC GAT GGCGAC CCGGAA CGCGAT GAGATT GTA AGC TGG GGT TCA 420 Gly Asp Ser Leu Lys Ile Asp Gly Asp Pro Glu Arg Asp Glu Ile Val Ser Trp Gly Ser 130 140 GGA ATA GCGGACAAA ATT TAA ATTAATACAGCTCAGCTATCCTTACGAAG Gly Ile Ala Asp Lys Ile *** Figure 3. Nucleotide sequence of the flavodoxin gene from Desulfovibrio salexigens. The sequence shown is the nontranscribed strand. The translated amino acid sequence for the flavodoxin protein is indicated below the open coding region of the gene. SD, potential Shine-Dalgarno sequence; "-35" and "-lo", putative promoter sites.

sites.

The sequence

nearly

identical

separated that

from

observed

to the the

located

-AGGAGG-

just

Shine-Dalgarno

initiation

site

codon

in other

bacteria

10

20

by five

(14).

upstream observed

of these

40

initiation

in E. coli.

nucleotides,

Because

30

to the

codon This

a separation strong

50

region similar

similarities,

70

60

D.s. MSKSLIWGSTTGNTETAAEYVAEAFENKEIDVELKNVTDV III,,,III,,I 8I I I I I, I, I :I; :I .:.: . :I: ::.:.I..::! :~:~:~~:~~~)~~::.,,I,,,,,,,: I:I:III,~III,III D.v. MPKALIVYGSTTGNTEYTAETIARELADAGYEVDSRDAASF II" * * 80

90

100

110

120

130

140

D.s. DSLENADLKGKKVSVFGCGDSDYTlFCGAMAIEEKLEKMGP--ERDEIVSWGSGIADKI III,II ,,IltII1I,,I, ..,I :: ;:;::;;;; II.II.I. IIII... .,r.r(.,. :: : 1, ,,,I... :l:ll: I11III.I.I11II,I11I,II~--I, D.v. DSLEETGAQGRKVACFGCGDSSYEYPCGAMAIEEKLKNLG~IVQDGLRIDGDP~DIVGW~~GAI * " Figure 4. Comparison of the flavodoxin protein sequences from D. vularis (D.v.) and D. salexinens (D.s.) using the PAM matrix of Lipmann and Pearson (ref 12). Identical (I), conservative (:), and semiconservative (.) homologies are shown. Underscored regions form portions of the FMN binding site in the D. vulRaris the FMN isoalloxazine ring; *, acidic flavodoxin. W, aromatic residues flanking residues possibly involved in complex formation with cytochrome c3 (ref 16). 813

is is to

Vol.

168,

No.

expression

of this

promoter

might

near

carboxyl bend

a-helix.

value the

for

homologous

regions immediate known

predominantly cofactor

DVF in this

lo-15

region.

striking

FMN is

the

which

lie

on the

(2).

These other

inner

Clostridum

properties

side

The portions acid

residues

immediate

adjacent

to the

residue form

This

of the

is

in Clostridium of the

protein,

in that MP, which

apparently

very

that

the points rotates

61-64

is

identical

to

bonding group

as

DSF (2). ring

of

system,

respectively

theme

is

is

repeated

by no means

in many flavodoxins involving

the

suggests

on the

these

form

that

chemical

aromatic

nature

nonidentical,

a loop

which

flavin.

Glycine-61 of the

upon

aromatic

but

carbonyl

away from

814

a loop

of the

the of

(15). flank

amide

forms

and tyrosine-98

mutagenesis

homologous

edge of the

of

isoalloxazine

studies

dependent

sequence

of hydrogen

general

conservation

which

binding

phosphate

the

but

our

protein

group

in the

ring

This (4)),

positions

Residues

C(4)-N(S)-C(6)

noteworthy

flavin

in DSF.

strongly

which

of the

for

in DVF by in vitro FMN are

conserved

types

with

composition

cofactor

typtophan-60

Indeed,

polypeptide

vicinity.

of the

Their

at these

of the

conserved

residues

MP (3)). role.

portions

site

nidulans

in DSF and DVF are highly

in their DSF.

bound

chains

are

Anacystis

residues

of the

acid

face

conserved

functional

of these

amino

are

DVF, the

atoms

binding

aromatic

and outer (e.g.,

an important

redox

of the

flavodoxins

substitution the

of the

of the

similar

and the

(1).

The DSF sequence

that

chain

acid

phosphate

of

for

data

terminus

final

consistent

amino

published

amino

and oxygen

feature

the

terminal (3).

then, side

presence

(e.g.,

likely, residues

daltons,

a highly

their

to the

ribityl

two residues

universal implies

is

these

of the

The most

near

bonds

the

identity

weight

mononucleotide

share

eliminate

to be conservatively

with

in D. vulgaris)

It

between

as portions

the

acids

would precedes

found

to those

structures

and hydrogen

interactions

in

flavodoxin

residue

a sequence

Also,

flavin

to reveal

share

structure

confined of the

"deletion"

15,814

of

was observed

after

immediately

is

consistent crystal

largely

vicinity

(residues

frame

to that

aligned

alignment

The molecular

protein.

is

X-ray

hydroxyamino

surrounds

well

were

this which

respectively.

purified

DSF was compared

was introduced

two proteins

reading

sequence

to the

are

which

D. salexiRens

better

26% and 8% were

open

the

gene

When compared

All

this

for

deletion

protein

the

homologous,

from

site.

COMMUNICATIONS

can be directly

structure,

of the

sequence,

from

reported

in the

RESEARCH

endogenous

A somewhat

a double

adjustment,

remaining

derived

derived

two sequences

DVF crystal

surface

this

semiconservatively the

if

to the

on the

Of the

protein

the

sequence

identity.

terminus

With

55%.

4, the

of sequence

By analogy

130. a short

BIOPHYSICAL

using

- The protein

in Figure

regions the

in E. coli

protein

comparisons

As shown

several

AND

be anticipated.

Sequence DVF.

BIOCHEMICAL

2, 1990

the

reduction

is

positioned

is

conserved

homologous

flavin of the

in

amino especially

the

in

glycine oxidized

flavodoxin

to the

Vol.

semiquinone the

to establish

flavin

(3).

for

This

more negative

surface

and the

transposition flanking

of the binding

in

several

isoalloxazine site

acidic.

for

at this

location

observed

so-called

90's

heteroatoms hydrogen ring

bonding (2).

similar are

asymmetric

in DSF,

is

with

the

similar

proteins.

, with

distribution

radical

creating

residues

conserved.

It

in DVF that

are

with

the

in

the

FMN

cofactor.

slightly residues

net more noted

gel electrophoretic analysis of Pip.ure 5. Sodium dodecyl sulfate-polyacrylamide the expression of recombinant D. salexiaens flavodoxin in transformed E. coli. Electrophoresis was performed on 16% (w/v) polyacrylamide eels as described previously (ref 5). -Lane 1, extract of unt;a&formed cell;; Lane 2, cells transformed with the Bluescript plasmid; Lane 3, cells transformed with the plasmid with the 1.4-kb &'&I-Hind111 insert containing the entire coding region and 5'-flanking sequence for the D. salexiEens flavodoxin; Lane 4, purified The faint trailing band is an apparent artifact D. salexisens flavodoxin (Note: as it also appears after re-electrophoresis of the excised lower band). 815

is

subnucleus

The calculated

of charged

two

of the

acid

identity

DSF being

a

by the

pyrimidine

of sequence

of interactions acidic

flavin

exception

identically

of

partially

provided the

95 and loo-102

degree

types quite

of the

that With

least

and hydroxyamino

interactions

The high

DSF and DVF at pH 7 are general

loop

of residues

form

than

acidic

atom on N(5)

to be at residues

in DVF.

homologous

as a group, the

neutral

potential

in DSF implies

Also,

blue

glutamate

the

backbone

thought

by three

residues

the hydrogen

is

followed

serine

tyrosine-98,

Flavodoxins, charges

of the

of conservatively

peptide

involved

is

bond with

interaction

stabilization

glycine

aspartate

This

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a new hydrogen

semiquinone.

responsible

the

BIOCHEMICAL

168, No. 2, 1990

in

Vol.

168, No. 2, 1990

DVF is

also

surface the ion

residues

postulated

The sequence

results

are

surface

that

within

homologous

in these

Gene expression the

5'-flanking

sequences possibility using band

in cells

regions cells This

transformed

with

extractable perhaps,

wildtype E. coli

to have DSF. gene using

from the

even within

expressed

only

A-:

and

These

the

entire proteins

where

from

homology

no detectable region vectors

of the

the 5 '-end

was supported

protein cells

5, Lanes

or

1 and 2).

flavodoxin

an exogenous

(Lane

4).

It of the

in part

for

E. coli

reported

obtained

extracts

for for

the

the

was observed

should

D. vulgaris

but,

copy number

that

those

promoter

from

expression

(5).

occur

by SDS-poly-

4-6% of total

with with

might

the

and 5'-flanking

and the high

sharply

raised

(Figure

was isolated

consistent

promoter

research

gene

of

consensus

of untransformed

the purified

level

regions

of an intense

the

insert

protein

contrast

expression

of Health

high

properties

modification

presence

the

and the

extracts

in extracts with

This

D. vulgaris strong

the

between

in E. coli

of E. coli

lacking

promoter

results

This Institutes

the

endogenous

after

of the

and -129;

similarities

to be at approximately

in E. coli. molecular

These

containing

comigrates

estimated

with

These

Four

donor/acceptor

sequence

flavodoxin

absent

a remarkably

plasmids

-95, that

form

cytochrome.

in DSF.

homology

gene

reveal

is

vector

band

were

consistent

and found wildtype

protein

cloned

plasmids

3) that the

proteins,

Bluescript

National

with

5, Lane

a strong

Examination did

transformed

levels

residues

model.

(Asp-69,

with

was noted.

of the

electrophoresis

additional

that

of

cytochrome

of the

suggesting

D. salexigens

promoter.

(Figure

Expression

that

expression

gel

with

formation

above,

in E. coli

endogenous

acrylamide

of the

promoters that

its

these

as a glutamate

regions

several

flavodoxins. - As described

sequence

for

and certain

surface

conserved

that

and stabilization

models,

on the

appears

in complex

suggested

flavodoxin

consistent

identically Asp-62,

may be involved

is maintained

are

residue,

have formation

In these

residues

here

are

the

(16).

basic

involved located

studies in the

between

transfer

presented

The fifth

Glu-66).

modeling

forms

corresponding

residues

residues

that

electron

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

may be important

complex

during

pairs

acidic

Molecular

conserved.

acidic

proteins

BIOCHEMICAL

be noted,

in

however,

gene was efficiently coding

region

by a grant

(5). from

the

(GM36490).

REFERENCl?S 1. 2. 3. 4.

Moura, I., Moura, J.J.G., Bruschi, M., and LeGall, J. (1980) Biochim. Biophys. Acta, 591, 1-8. Watenpaugh, K.D., Sieker, L.C., and Jensen, L.H. (1973) Proc. Natl. Acad Sci. USA 70, 3857-3860. Mayhew, S.G. and Ludwig, M.L. (1975) in The Enzymes (Boyer, P.D., ed.), 3rd edn., Vol 12, pp 57-118. Smith, W.W., Pattridge, K.A., Ludwig, K.L., Petsko, G.A., Tsernoglou, D., Tanaka, N., Yasunobu, K.T. (1983) J. Mol. Biol. 165, 737-755. 816

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5. 6. 7. 8.

9. 10. 11. 12. 13. 14.

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Krey, G.D., Vanin, E.F., and Swenson, R.P (1988) 3. Biol. Chem263, 1543615443. Postgate, J.R. (1984) The Sulfate-Reducing Bacteria, 2nd ed., p.32, Cambridge University Press, Cambridge, England. Feinberg, A.P., and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. Grundstrom, T., Zenke, W.M., Wintzerith, M., Matthes, H.W.D., Staub, A., and Chambon, P. (1985) Nucleic Acids Res. 13, 3305-3316. Hanahan, D. (1983) J. Mol. Biol. 166, 557. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Prod. Nat11. Acad. Sci. U.S.A. 74, 5463-5467. and Sambrook, J. (1982) Molecular Cloning: A Maniatis, T., Fritsch, E.F., Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Lipman, D.J., and Pearson, W.R. (1985) Science 227, 1435-1441. Hawley, D.K., and McClure, W.R. (1983) Nucleic Acids Res. 11, 2237-2255. Storm0 G.D., Schneider, T.D., and Gold, L. (1982) Nucleic Acids Res. 10, 2971-2975.

15. Swenson, R.P., Eren, M., and Krey, G.D. (1988) J. Cell Biol 107, 621a. 16. Stewart, D.E., LeGall, .I., Moura, I., Moura, J.J.G., Peck, Jr., H.D., Xavier, A.V., Weiner, P.K., and Wampler, J.E. (1988) Biochemistry 27, 24442450.

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