Sequence of the gat operon for galactitol utilization from a wild-type strain EC3132 of Escherichia coli

BB

Bioehi~ic~a

ELSEVIER

et Biophysica A~ta Biochimica et Biophysica Acta 1262 (1995) 69-72

Short Sequence-Paper

Sequence of the gat operon for galactitol utilization from a wild-type strain EC3132 of Escherichia coli Barbara Nobelmann, Joseph W. Lengeler * Universitiit Osnabriick, Fachbereich Biologie / Chemie, D-49069 OsnabriJck, Germany Received 31 October 1994; revised 21 February 1995; accepted 27 February 1995

Abstract

The sequence of the gat operon for galactitol (Gat) utilization from a wild-type isolate of Escherichia coli, strain EC3132, is presented. The operon comprises 7 open reading frames (ORFs) called gatYZABCDR. The genes are transcribed from a promoter located upstream of gatY. Genes gatABC encode the substrate-specific domains IIA, IIB and IIC of a galactitol-specific Enzyme II (Eli cat) of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS); gatD encodes an NAD-dependent Gat l-phosphate dehydrogenase; and gatY an enzyme which hydrolyses tagatose 1,6-bisphosphate; gene gatZ is required in a cell to show a Gat + phenotype, but its physiological function has not yet been identified; gatR encodes a repressor tbr the gat operon. All genes are highly similar to the gat genes from E. coli K-12; in this organism they map at 46.70 min of the gene map, equivalent to about 2180-2186 kbp. Keywords: Galactitol; PTS; gat operon; DNA sequence; (E. coli); (Strain EC3132)

In Escherichia coli, the hexitol galactitol (Gat) is transported and phosphorylated through the PEP-dependent galactitol:phosphotransferase system (PTS) [1-3]. During PTS-dependent transport and phosphorylation of a carbohydrate, a phosphoryl-group is transferred by autophosphorylation from PEP to the first of the general PTS components, the Enzyme I (EI, gene ptsI). The phosphate is transferred from phospho-EI through a Histidine-protein or HPr (gene ptsH) to the various substrate-specific Enzymes II (EII) (for reviews see [4,5]). It is accepted first by domain IIA, then by domain IIB. It is transferred in a final step through the membrane-bound and substrate-specific transporter domain IIC to the substrate which thus is phosphorylated during the process. The different domains of an EII may be fused into a single protein, or they can be separated into two or three distinct proteins. Galactitol 1-phosphate (GatlP) is generated during galactitol transport through EII cat, and converted by an NAD-dependent GatlP-dehydrogenase (gene gatD) into tagatose 6-phosphate (Tag6P) [1,3,6]. Tag6P is phospho-

The sequence data reported in this paper have been submitted to the EMBL/GenBank DFDJB databases under the accession number X79837. * Corresponding author. Fax: + 4 9 541 9692870. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

SSD10167-4781(95)00053-4

rylated through the enzyme phosphofructokinase I (gene pfkA) to tagatose 1,6-bisphosphate, and hydrolysed by the enzyme ketose-bisphosphate-aldolase (gene kba) into dihydroxyacetone phosphate and glyceraldehyde phosphate. These two genes are not encoded in the gat operon [3]. The gat operon is expressed constitutively in all Gat ÷ strains of E. coli K-12, due to a gatR49 mutation present in the original Lederberg strain [3]. In W3110, a close relative of the Lederberg strain, an IS3E insertion was detected in the gene gatR (K.E. Rudd, personal communication), thus explaining the constitutive expression of the gat operon. In this paper we present the complete sequence of a 6074 bp DNA fragment cloned on pBNL6 (Figs. 1 and 2), which contains seven gat genes from a wild-type isolate, strain EC3132, of E. coli [7]. Similar to E. coli K-12, EC3132 also expresses the gat genes in a constitutive way. Sequencing was done by the dideoxy-chain termination method [8] using the T7 sequencing kit (Pharmacia Freiburg, Germany) and [35S]dATP. The sequence was determined completely from both DNA strands except for the region between bp 1-400 in which short stretches were sequenced only from one DNA strand. In Fig. 1, the restriction map of the sequenced part of the fragment cloned on pBNL6 is given together with a schematic

B. Nobelmann, J. W. Lengeler / Biochimica el Biophysica Acta 1262 (1995) 69-72

70

presentation of the seven identified open reading frames (ORFs). In Fig. 2, the complete DNA sequence is shown together with the deduced amino acid sequences and with the putative regulatory elements. Responsible for galactitol transport and phosphorylation is a galactitol-specific EII Gat as shown before [2,3]. Transformation with pBNL6 of E. coli C or Klebsiella pneumoniae KAY2026, strains which lack all gat genes according to Southern hybridization experiments ([9,10] and our unpublished results), gave fully positive Gat + colonies with normal transport and GatlP dehydrogenase activities (data not shown). Similar results were obtained after transforming JWLI93, a GatD- mutant [2]. Upon transformation with a truncated pBNL5, JWL193, but not the other two strains, was transformed to a Gat + phenotype. According to these data, pBNL5 must carry and express gatD. Three complete (gatB, C,D) and two incomplete (gatA' and gatR') ORFs are encoded on pBNL5, a subclone which carries the 3' EcoRI fragment of pBNL6. Gene gatD corresponds to a protein of 346 residues (M r 37422). The protein contains a putative NAD-binding site with good similarity to the consensus sequence [11] which is located within a hydrophobic region (residues 155-200) of the otherwise hydrophilic protein. The protein shows similarity to other polyalcohol dehydrogenases, e.g., the glucitol dehydrogenase from Bacillus subtilis and members of the eukaryotic alcohol dehydrogenase family [12]. It could thus correspond to the GatlP dehydrogenase encoded on pBNL5. The product of gatC corresponds to a strongly hydrophobic protein of 427 residues ( M r 45 570). Because it is the only hydrophobic protein encoded on pBNL5 or pBNL6, it should represent the membrane-bound translocator IIC Gat. Sequence comparison with other members of the different EII families revealed 24% identical amino acids between residues 220-365 of the putative IIC G~t and IIC Fru (residues 306-451) from E. coli, corroborating this hypothesis. Genes gatA and gatB encode two soluble

SphI

EcoRV

BarnHl Sphl

Y~nI Smal

EcoRV

NruI NOel

ii

i

Pstl

Clal

~ ,

~

g~v

I

j

INdoI

iPvull

i H i n c l I Nrul ; I~el BamH[ ~HinclIXmnIEcoRI Sphl HincIII Smal i PvulI Stul i Nrul i~

c:::::>~ gatA 9atB

9atZ

I

I

i

P~ull i: Hindlll Xhol : HindlII l EcoRl

>,

3:, c::>

9ate

I

~atD

I

r

9~tR'

I

I ~74

bp=

Fig. 1. Restriction map of the gat operon from E. coli, strain EC3132. Seven putative open reading frames and the direction of their transcription are indicated by arrows. The 6074 bp DNA fragmentsymbolisedby the black box complementsall Gat- strains of E. coli and K. pneumoniae to a Gat+ phenotype. Part of the gatR gene was deleted during cloning of the genes from the chromosomeof EC3132.

proteins with 150 (M r 16911) and 94 residues (M, 10 188), respectively. They are needed for galactitol transport and phosphorylation as shown by in vitro phosphorylation assays and thus seem to correspond to IIAGat and IIB aat. We have not been able as yet to identify which protein corresponds to which domain. Together with IIC cat they would constitute an EII Gat of normal size (671 amino acids) [5]. The most distal of the cloned genes, gatR, is incomplete. The cloned part corresponds to the 68 amino-terminal residues of a DNA-binding protein with a characteristic helix-turn-helix motif. The sequence shows similarity to the homologous protein GatR from E. coli K-12, strain W3110 (98% identical residues; K.E. Rudd, personal communication), and to GutR (33% identical residues) [13], the repressor of the gut operon for glucitol degradation [14]. It is unknown whether an IS3E insertion also inactivated gatR in strain EC3132, thus causing its constitutive expression. This seems, however, very likely because most strains of E. coli contain an IS3E copy at about 46.6 min of the gene map. The function of two additional genes, gatY and gatZ, is not yet known exactly. Their inactivation through a partial deletion which removed the 3'-end of gatY and the 5'-end of gatZ, or through insertion of an w-fragment into gatZ eliminates the Gat + phenotype. Complementation with both genes in trans restored the Gat + phenotype of the deletion mutant but not of the insertion mutant. Because the w-fragment carries transcription and translation stop signals in all three reading frames, this result indicates that the promoter for these gat genes must be located in front of gatY. GatY (286 residues, M r 31055) and GatZ (387 residues, Mr 42 319) however, may be two enzymes involved in galactitol metabolism. The sequence of GatY has similarity to other ketose bisphosphate aldolases, and pBNL6 encodes such an activity. The aldolase encoded by gene kba [3] may thus be a general enzyme while GatY could correspond to the real tagatose 1,6-bisphosphate aldolase. GatZ, finally, does not resemble other proteins, in particular not GutM, the second regulatory gene of the gut operon [13]. Based on these results, we conclude in summary that all 7 genes form an operon whose equivalent in E. coli K-12 is transcribed in a counter-clockwise way. The exact location of the physiological promoter (probably upstream of gatY), and the physiological role of GatZ have yet to be determined. The gat operon is present in some strains of E. coli and absent from others [3]; it shows a mutual exclusion with the atl-rtl genes for D-arabinitol and ribitol degradation [9,10]; it maps at 46.7 rain of the gene map (our unpublished results), equivalent to 2180-2186 kbp of the physical map, in the immediate neighbourhood of an IS3E element (K.E. Rudd, personal communication); hence, it may be a recent acquisition of E. coli from its 'collective chromosome' [15]. The G + C content of the genes varies from 52.6% (gatY), to 49.6% (gatZ), 42.6% (gatA),

B. Nobelmann, J. W. Lengeler / Biochimica et Biophysica Acta 1262 (1995) 69-72

XS CaU C~

~C~ C~

C~G ~CS G~

TGA ~C

~G

~

~ZC T~A rrc rT~ C~C ~T~ ~G

T=A TTA AO3 C~

ACG CCT TAT ACG ~C

CAT ~

~0 CTS ACC ~C~ GC~ ~S C~

L~C ~

AGC A~

A~

~

~5 TCC GTA cr~ cc~ TT~ TG~ aC~ ~C

C ~ T T C A ~ G a ~ A T CO;, T G ~ o r s ~ e ~ G C ~

ATC TC~ ATA GC~ ~

ACC CCG TAT GTC ~A

~

~C

~'~A C T A T ~

C~T ~A

~T

2SS 270 ~5 ,~.~C ACO C~C ACT ? r n CT.~ CC~ e r r CCC TAT CC.~ C-AG GCC ~TT ? r r

TT¢ TGA TTa ATT TTC O~a~ ~T~ TCO T r r

~TT TCA r T ~ T~T TTT O ~

;~T¢ ¢ 4 ~ ~ ' r

T'm ~

~

~0 GC~

TC~ ?r.~ r c r

~e~e

l~)Ar~

~

20SS 207o A~GTTTATG~G~CATT~ACC~TTAC~ACC~GA~G~TAT~ M V Y E A H S T D Y Q T R 211S ~COATCACr~CAATA~ R D H F A I

L

K

217S A T A ~ G ~ ~ G ~ Z F A L A Q

ATC

C~O ~ r O C r ~ ~ C Q M L N

c~c H

CT¢ S~ a E

~T S

A.~C GCA GaG C~C 0 0 ¢ G~T TAT ~CC~ s r r N A O R G G Y A V

ACS AT~ C~ T ~ ¢

Ore V

~ V

GT~ S~ V ~

~C

30~ CTG TeA

C~

ATA

~C~ Ar~ GC~ ~A~ ~ A M A K ~

CC~ GC~ a r c A.~T ATT p A F ~ x

A C e OCT G C C A A ¢ ¢ ' ~ 7 C ~ T G C ~ C O S GTC T A A N L H A ~ V

3d%A " ~ K F

GAC ~T D D

~T H

~C L

~AC CAT C~C AC~ D H H T

A T C G C T SAG ~.~C C T T C O T T C T G G C G T G C G C T C A G T C A T G A T T G A C G C C I A ~ N L R S G V R S v M I D A

7~S OCT ~ T G~

~CG CAT ~

T~T CXC C~r CC~ Ca~ ~C~ ~ Y H H P L A I

CGC T T Z C A T ~ T C ~ C C ~ T C a ~ O v ~ v

?SO ATT T~ ~G

~

~T

GTC ~

~.~ ~

G C ~ GAG C r G G G ~ C ~ A t L G Q

?~S GAG GTG G ~

GAT TTT ~C

C T T C ~ C OGC C A ~ ~ L ~ ~ Q S

?SO CAT

CAT CAT GT~ D ~ v

~5S s?~ 88~ soo CA;, ~T¢ aAT GAA GC¢ GAT GCr T r T TAT ~C¢ AAC ¢¢C ~CT ¢~G GCG C~T ~ , ~ TTT ~CC GA~ Q V N E A D A F Y T N P A Q A R E F A E

~C~ ACC ~

9~S ~ r ' r OAT "rcc ~ c

~0 ~ c ~ ~T¢ OCC x r ¢

~C

~

CAT ~C H ~

C~ U

~S G

~

AAT

T A C C T G A C C OCG C A C CCC Z A A GCG A C C C A T CCC CGG GAT T A T TTG C A S T O 9 G C T ~ a r A ~ ~ S A T ~ P ~ O Y L 0 S A ~

TCC S

AT~ TCC ~

SC~ ~T~ A ~

~ ¢ O T C A C,S S ~ A ~ S U G

T~ S

ACT AAG OAT ATT ~ T ~ D ~ 0

~TC ~AC eTT ~CA ACG OAe CTG ~

COC ~ A r ~ ~

~T~ V

CGC A C e T C C C A T T T ~

~T~ V

~C S

~.~ ~

ACA ~

~T

a

~¢

Ar~

A~

Stirt ~A Z

CAr H

ATe I

~SC G

~ T A T ~ C T C A GTC TGT r C r Z C S V C S

G C A T T T O A T C ~ C ~ A C A G C A C ~ CGC ~ A r ~ R ~ S r ~ •

~

Trr G

GCA rAT

CeC ~

T~¢ C

C ~ G C,~C a ~ S S C ~ T ~ Z ~ a ~ " St*p attr

AC~ "era ATT ~C¢

CGS CAT ~

C C ~ TTG GTT A T C G A ~ ~ C S ~ ~ V I ~ ~

CT~ AT~ ~ L I E

w

C== CaA V ~

TTT ,:~ ~ ~

C~T GAS ~T ~ S ~

O

E

N

A

e

A

r'rC

~T

A~

ATT eAT

O

GTT C~T ~C¢ ~¢

O~T C¢C A~A CC~ T~A GCA ,:C~ G~ ~ ~ P L ~ ~ ~

~Cr

C~T

Git~

SC¢ aT A ,

Sea A

a¢: T

T A AA¢ ¢~ C N e

145~ 1470 1485 O C ~ G T T A T ~ C C 7 , ~ a ~ T G ~ C A C C C G C ~ GAC T T T CGC G A A T T T ~ T T G ~ T ~ ~ T P ~ ~ F a S Z V

~ C C C,~T ~ A P ~

c

S~3 v

CAA ACC ATC ~ ~ T ~ ~

~ C C T T C T C ~ CA(; G C S ~ A

GT~ ~TT ~CC ~AT r~r V I A ~ C

ATT A~r t I

CTC ~C L C

A

E

M

CTT ~AT ~¢~

ACG ~ T T ~ C G G ~ T V A E

C~ ~

~C C

~C~ ~

CTO L

~T¢ V

~AT S

1501 TTT ACG ArT Z T I

Ca¢ D

C~T ~ ~ L

~ O

=~

v

E

V

K

C~ T ~ C ~

~a

T C ~ ATG

GCT G C T G T ~ ~ A V

L

CTT TGC T T T C C T L ¢ F

GCG GAA AGT GTG GCG ACA GAT TGC CAG CGT GAG CAA CTG AGC TAT GTC ATT GGC Ace GAA A ~ S V A T ~ C ~ ~ E Q ~ S Y v ~ G T

GTT C~

GTT ¢CG GG¢ GGT GAC GCC ~C

W

~ L

E

2101 ~AGTC V

21~0 2~4S 2161 ~ C T C G G T C C C O ~ A A C C ~ S ~ A ~ S ~ Q C O V G P A L ? F A L R E A

223S C T ~ G ~ G T A A T T ~ O ~ G ~ A ~ L A V I E E V M r

~ D

2~S0 ~ C ~ C ~ P 0 Y

E

22SS ~ T A C ~ ~ W K K Y r

229S 2~I0 A C G ~ T ~ ~C~TTCA~ACTGGATATT~TTAC~CC~T~GATCGTA~TTAT T G F N D S L L D I R Y S L S D

22el ~TATTAT~T R

2nS R

2221

2341 I

R

Y

SCC ATT GAS T=A GTA CAC ATT ACC COG GTT m~

2r70 23~ 2~ ~ T ~ C G T C G ~ A ~ A T G A ~ G ~ T ~ G ~ C

~

A~ ~

~ATC~C~CATGATT~GT~GTA~TCCT~ACAATTTGAA~CATT~GTC Z I L W ~ " S~o~ Oa~Z 247S 2490 CGG~ ~ATCAGCAATACCGCAT~TTGA~ATG~AT~A~TAT~TG~TTG~C

~SOS

2S~

G C C T A T C G C T A C ~ C T G T G ~ G ~ T A A ~ C ~ T A T A ~ A ~ C C ~ T G ~ C G T A G C ~ T . ~ V R S 2S~ S

~

V

2~0 D

R

S

E

V

2~4~

2~S

G

I

~

~ T G T G G T T ~ T G A T A ~ T ~ C ~ G G ~ T T A A ~ G C C ~ G ~ ~TTCC~ O V V ~ D T W ~ A L Z ~ E ~ E F P

L

T

H

I

G

N

E

ACC~r~nTO~TGaO~CAC~CCAW~C~T~C~CAT~TOa~nC~AWC~TOCT T ~ I M L E Q H A I A I p H C E A I

M

H

L

A

A

~75 2790 2~0S ~ G T C G T ~ G C C A T T T A T C ~ T T A A G G C ~ A ~ T ~ G ~ T ~ C A ~ G ~ G A T K S S A I Y L L R P T N K V H F Q Q A D

GATGAT~CGACGTGGCGGTATCGTTGG~A~OC~TTAA~G~ D D N D V A V S L V I A

~5 9~0 nCT c a r C~T ~ ' m T~'r ~CC ~ ¢

975 990 1305 1020 G C A C C G G T G C'fT G A T T T T T C T CGA C ' ~ G A G ~ A C A T T CGC CAG TCG G T G A A C T T A C C G C T G A P V L D F S R L E N I ~ Q W V N h P L

GTG ~S V U

Y

ATG TAC ~ ' m ~TA TC~

SS5 S~O 5~5 60O A T C A T C GCC GG& A C G C C T G G C K C A T T T A C T C A T G C T G G T A C A C ~ A A T C T O TTO G C G C T G i i A O T P G T F T H A O T E N L L A L

GTC ~C V S

A

2Z90 ~20S ~ A ~ G A G ~ G ~ ~ A ~ G C C C ~ G ~ T ~ C ~ C ~ T ~ C I E Q E L I A P E R a s G ¢

2JSS T A C ~ C ~ T ~ G T C G G A ~ Ae_~ ~ T K

20as T

CL~ C~T

~C

CC~ CAT TT~ ~

71

2~9S

~ L

I

V

E

N

~0

P

Q

~x

~ T C C G C ~ G Q

2~5

294~

29SS 2~70 2S~S ~001 ~ A T C A C T ~ C C T G ~ A C C ~ G T T ~ T ~ C T T C ~ n ~ A T O ~ O ~ T T C A

~0~S 30~0 a~S ~0~S ~0a~ : A C G ~ T ~ A T C C C T C ~ T ~ C ~ T ~ C T A ~ T A ~ ~ ~ C ~ G A ~ A ~ G T C O E " ~ K R K I ~ V st~G.e~ st.~at~ ~07S 30S0 n0s ~2~ G~C~AGGCGCGG~GCGACCTCTA~ATGG~GCGG~ ~ A ~ ~ G ~ T ~ T A C G G A V A T S T M A A E E I K E L C

~ G A G T C A T ~ T A ~ C C T G ~ G ~ T T A A T C C ~ T G T ~ G ~ T G ~ A T A ~ A C C T A T Q S H N I P V E L ~ Q C R V N E I E T ¥

ATGGATGGCGTG~TT~ATATGCACCA~GCCA~GTGGAT~A~TTTT~CGATA~ M D G V H L I C T T A R V D R S

F

G

D

I

C C ~ A G T T ~ C ~ C A ~ C C T T ~ T T T ~ G T G T C ~ T A T C ~ L V ~ G M P F V S G V G I E A

L

Q

N

K

R

Y

I

L ~ G * M F S E V M Stop Gat~ Start Gate 3375 ]390 C T C ~ C C ~ A C G G ~ A T G ~ C C A A ~ T C A T C A ~ A ~ T ~ T ~ L G ~ T V M L P I V I I I F S K T

I

3405 I

L

L

D

3421 ~ A T A T T A ~ C A ~ G M

~ G ~ A ~ C G A T T G C ~ T ~ G ~ T C ~ C A T A T C ~ A ~ C ~ T G T T ~ C A ~ C

C~GTGA~GSC~AATG~GATTCCA~GGTCmGCGG~

~ G ~ A ~ O ~ G ~ T

TTCGAC~G~T~GCATGTGGTCGATGTC~CT~CCG~CTCTT~C~ATGACC~

3~1S 3630 G~TCGC~ATTG~C~GTGG~ATTC~ATTGCGA~GGTT~CG~GCGATGCTA A S Q ~ A L V ~ I A I L V ~

3661

3g~5 V

A

~

L

C~ACCCOTATGACG~GTGGTA~TGTTGATATCTGG~TATC~CATATGACCTTC

ACCGGCGCA~CTGCAT~GG~ACC~TT~TCGATGATAGGGATGG~GGTGTGGTA T G A L L H L A T G S W M I G M

A

G

V

V

ATT~CGCGGCGTTTG~TAT~GCTC~CGAC~T~GCC~CGATACCCGA~T~C

1875 1890 190S 1920 CAT GCC GCC AAT ACT TTA OnT ACG CAT CAA AAG GCC TTT ATT GCC CGT GGG CTG GCA GAG D A A N T ~ ~ T H O K A F I A R G L A E

CCG ~A A L

ACa CCC ~ T ~ V

~T ~

~CC ~TT G~ ~ ~ V

ATT ATC CAT TAT CAG :CG 7~ : I H y O p O

OI"O ¢ ~ V ~

:CA C~¢ ~r~ e~ P e V ~

G~.~ G C A C~J3 C C G ~ ~ A Q P L

Tar GAT CAC ~C r = ~ S

~T

~ T G C T ~ T ~ A ~ C T A T ~ T C G A G ~ T C C C ~ C G T T ~ C C G T A T T ~ T ~ C O C C

G C G C A A T G G A T A G~.~ A A C A C C C G A A o w ~ ~ N T R

Fig. 2. DNA sequence and deduced amino acid sequence of the gat genes gatYZABCDI~. The postulated 'helix-turn-helix' motif and the putative NAD-binding sites are underlined, putative ribosome binding sites (RBS) for each gene are indicated, alternative start and RBS sites of gatY marked with brackets. The promoter(s) located in front of gatY, of gatZ, or of gatY and of gatZ has (have) not been identified thus far.

72

B. Nobelmann, J.W. Lengeler / Biochimica et Biophysica Acta 1262 (1995) 69-72

~

97S CACGAT~TTCACGC~ L , D : Q R

3990 40US ~GGTC~TTr~CGAGCCTGTCACCGTG~a~A~ F G p F ~ E ~ V T V G

K

40~S

40S0

409S

4110

A ~ GCG GCA GTT A ~ T A A V M L

M 4001

CAC C ~ ,

GCC G~A A V

4:41 CCC ATC ATG OAr GGT TTA D G L

412S

~ O TTG ATG CCA Cca GTG ATT ~ L M P R V I K p I

M

4tSS 4,~0 4185 ACCCCTATC~Cr~GCAGGCC~TAGr~TTT~eAaGCG~Gr=¢GGCGGCCAGGAG~C T p l A E Q A R S R L Q A K F G 421S Cr~ATTGGCCTGO~TCCCGCATTA~ ~ I O ~ D V A L L

V

406S

CCC CTG ATT ATe GG¢ A r c ~ ¢ G¢~ G~T TAC CAT CT¢ ~ a T CrAiG G L I I G I L A G Y D V K G V L Q I

K

42O0

G

Q

E

F

4n0 4~4S ~GGG~CATA~CGGTGGrAT~SCAAGC~G ~ H T ~ V V S A S L

4~6~

aTVTTVATCCCG~CACCATVrrAA~T~Cr~r~T~VOWGC~T~rCAGGTG~CC~ Z F I P L T I L I A V C V p G N Q V

L

p

F

R

G

G

D

L

A

T

I

G

F

F

V

43.9% (gatB), 51.4% (gatC), 48.3% (gatD) and 48.5% (gatR'). Except for the putative hydrophilic domains of the EII Cat, all other genes show the typical G + C content of E. coli (from 48% to 52%) [16]. The hypothesis of a recent horizontal transfer of the gat genes, at least from an organism with a clearly different G + C content, is not supported by these results.

4021

r

A

M

A

V

A

V

H

References

~TCTGTTCCGCACCTTAATCTCGGGTGTCATCATTA~AGCATCACCC~ATCG~

T

Q

T

I

~

L

H

T

~

L

A

A

N

A

G

A

L

K

A

[1] [2] [3] [4]

G

GOTATGG~G~TCAATGGATACAGGG~TTCTCC~TTACC~GTTACTGATTCA~T

TTCTCCCCGC~ATA~CCCGG~TCA~ATT~TCGGCGC~TTTATCTGACCGGTATTT

[5] TCATGAC~G~CGTA~AG~CG~GTTTTA~A~C~GA=A~GC~CTCGCAG~T

~TTTTTACC~A~GGGA~T~TCCCCC~AA~ATC~GTTTTTATG~ ~

GTG ~ T

GAT ACT GAT ~ T

ATC G ~

CGC GTT GCA G ~ A G C

GT= ATT C ~

S~

K

[6] [7]

TCAG' V

S

ATT ~

CAT

[8] ~g~TGAGGTGCGGGTA~ATTGCCAGCT~GGATTATGT~TTCCGATTTACCCAGA

ATTT~

~

[9] [10]

~T~TGCACATTATTATUCAATAACGTTAGGCCATG~TTTAGCGOCTAT

[11]

ATTGATGCTGTG~ATCC~TGTTGATGATTTACATC~GGCG~TGCGG~=CCTOTOTG

C~TTATTACCC~T~ACT~TCCAGAGTGTTTG~G~TTATTCCCAGTGCGCA P L L P C F T C P ~ C L Y O F Y

S

Q

C

[12]

A

~TATGATT~ATTGGCT~CGGCOTGATGGTGGA~TGCTG~TATATTGTCGTT~G

[13] [14]

R

[15] S2~S 5eSl 52~ A GT T AG~ A TAT ~ T ~G ~ GIG A A[ C C G ATT~CCTG~G TG C T n A TL T C AL G~CA G~GT x C GQ CG~ C

GCA ~

~29~ AGT G ~ ACG G ~ 8 ~ T A Z

531~ ATC GAC ATT ~ c n I S S Z

s~s~

~TGCGATG

G

A

V

5326

TCA ~ K

~

~ L

s~z

S2~2 n

A

[16]

2~312

GCA ~ GCA ~ A L A K S

TCT TTC F

s~B~

S40~

~ACAT~CAGCCTTG~ATGAGCGCGC~C~A~CAG~CGTTTTA

M

Q

T

CGCGACGTG~U~

~G

A

F

N

~TCAG

S

L

E

M

S

A

p

M

Q

G

V

L

~ATC~CGAGACGG~TOTCCCGC~ACCGTCG~

GCG GTA GAG ATT GCC GGG CCT CAT GCC CAG CTG GCG C ~

GAT CTG CAT TTA ACA T ~ D L H L T S T

Q

ACA A ~ TTT GGC ~ T F G K I L

GT0 GGC ACG ~ G

ATA TTA CGT ~ ~ R K E L T V

CAT ~ G

CTG ACG G T T ATC I

G~CAGCTGGATG~CTACTCCAGCC~T~C~GGQCAG~GTGGG~A~G~C~

TTG~GACAQ~CGT~GTTAAGC~GG~C:A~AATCG~CAC~TGGAAGC~G~

AGC TTC ACC CAG GTG GT~ CGT GAC ATC G ~

CGT ~ T

GCT ATG CCG ~ C

~

G~

TTG CTC

ATTCCCTGA~CCGCGGGCCAGCGTGATGCT~CCCGGTATTGTGC~CAGATCA~ : p .

CACC~T~GTCCCCCTTCG~TACA~AGCCAC~TTGAATAT~TAAAT~ACOA~

TCA~TCGA~CGAA~G~T~GATCATCC~AGTG~TG~C~GAACCGTG~TGTT

CAGGAT~GGCGG~GTA~TG~GCCT~G~G~ACAATC~TGCCGAT~CGC~

m

D

L

A

e

V

F

A

A

S

E

A

T

I

R

A

D

F

,

~

F

t~CO~ ~ ~GGCG~G~ACGCGC~TCATSGCGGTG~G~ ~ATAATOTCC~T L E Q K G V V T R F R G G A A K I M S G se~4

Fig. 2 (continued).

Lengeler, J.W. (1975) J. Bacteriol. 124, 26-28. Lengeler, J.W. (1975) J. Bacteriol. 124, 39-47. Lengeler, J.W. (1977) Mol. Gen. Genet. 152, 83-91. Meadow, N.D., Fox, D.K. and Roseman, S. (1990) Annu. Rev. Biochem. 59, 497-542. Postma, P.W., Lengeler, J.W. and Jacobson, G.R. (1993) Microbiol. Rev. 57, 543-594. Wolff, J.B. and Kaplan, N.O. (1956) J. Bacteriol. 71,557-564. Bockmann, J., Heuel, H. and Lengeler, J.W. (1992) Mol. Gen. Genet. 235, 22-32. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. Link, C.D. and Reiner, A.M. (1983) Mol. Gen. Genet. 189, 337-339. Woodward, M.J. and Charles, H.P. (1983) J. Gen. Microbiol. 129, 75-84. Wierenga, P.K., Terpsta, P. and Hol, W.G.J. (1986) J. Mol. Biol. 187, 101-107. Ng, K., Ye, R., Wu, X. and Wong, S. (1992) J. Biol. Chem. 267, 24989-24994. Yamada, M. and Saier, M.H. Jr. (1988) J. Mol. Biol. 203, 569-583. Lengeler, J.W. and Steinberger, H. (1978) Mol. Gen. Genet. 164, 163-169. Sprenger, G.A. and Lengeler, J.W. (1987) Mol. Gen. Genet. 209, 352-359. Brenner, D.J. (1984) in Bergey's manual of systematic bacteriology, Wol. 1 (Holt, J.G., ed.), pp. 408-516, Williams and Wilkins, Baltimore.