Molec. gen. Genet. 152, 83-91 (1977) © by Springer-Verlag 1977

Analysis of Mutations Affecting the Dissimilation of Galactitol (Dulcitol) in Escherichia coli K 12 J. Lengeler Institut fiir Biochemie, Genetik und Mikrobiologie, Lehrstuhl BiologieIX, Universit/it Regensburg, Universit~itsstral3e 34, D-8400 Regensburg, Bundesrepublik Deutschland

Summary. Three mutations clustered at 45.5 min of the genetic map of E. coli K12 have been shown previously (Lengeler, 1975a) to affect specifically galactitol transport via an enzyme II-complex aat (gatA) of the PEP dependent phosphotransferase system and a soluble, N A D dependent dehydrogenase (gatD). In the present report data are given further supporting the existence of a gat operon, made up by a control gene gatC and the structural genes gatA and gatD. The enzyme II-complex aat is shown to catalyze the formation of galactitol-l-P and the dehydrogenase to catalyze the reversible conversion of galactitol-l-P and D-tagatose-6-P. Loss of a phosphofructokinase activity controlled by the gene pfkA prevents growth on galactitol and concomitantly the formation of Dtagatose-l,6-P2, while the suppressing mutation pfkB1 restores a phosphofrucokinase activity and growth on galactitol. As shown further the erratic growth behaviour of E. coli K12, B and C on galactitol is apparently due to a temperature sensitive ketose-bis-phosphate aldolase inactive at temperatures > 35 ° C. This enzyme reacts with D-tagatose-l,6-P2 and to a lesser extent with D-fructose-l,6-P2 and thus is able to suppress fda mutations. It is controlled by a new gene locus kba located within 1 rain of the marker argG, remoted from the gat operon and the genefda. Galactitol dissimilation in E. coli K12 thus seems to be via galactitol-l-P - D-tagatose-6-P - D-tagatose-l,6-P2 to dihydroxyacetone-P + glyceraldehyde-P, controlled by the genetic loci gatC A D, pfkA, pflcB-1 and kba respectively.

Introduction

In contrast to other Enterobacteriaceae (Gutnick et al., 1969 ; Veron et Le minor, 1975) wild type strains

of E. coli B and K12 as a general rule are unable to grow at 37 ° C on galactitol, one of the naturally occuring four hexitols. Positive derivatives can be isolated and have been described before (Monod, 1942; Wolff and Kaplan, 1956 b; Lengeler, 1975 a). Galactitol has been shown (Lengeler, 1975b) to be taken up and phosphorylated by an enzyme IIcomplex ~at of the PEP dependent phosphotransferase system (Roseman, 1969). Furthermore some evidence has been given which suggests the involvement of a dehydrogenase converting galactitol-l-P into D-tagatose-6-P in the galactitol dissimilation in E. coli B (Wolff and Kaplan, 1956b) and K12 (Lengeler, 1975a). In E. coli 1(12 mutations in three genes, clustered at 45.5 rain of the genetic map, were shown to affect specifically galactitol dissimilation (Lengeler, 1975a). Phosphorylation of D-tagatose-6-P by ATP dependent kinases and cleavage of the resulting D-tagatose-l,6-Pz by aldolases is known to occur in yeast and mammalian cells (references in Stribling and Perham, 1973) and has been shown recently to be part of the lactose-galactose metabolism in Staphylococcus aureus (Bisset and Anderson, 1973; 1974). In the present report genetical and biochemical data are presented which proof the three phosphates mentioned to be intermediates in galactitol dissimilation and identify the enzymes and genes involved. Based on unpublished data this sequence has been suggested before (Lengeler, 1975b). While preparing this manuscript, data have been published proposing a similar pathway for galactitol dissimilation in Klebsiella pneumoniae (Markwell et al., 1976).

Materials and Methods

Chemicals. PIPES (Piperazine-N,N'-bis(2-ethanesulfonicacid) and D-tagatose were obtained from Sigma Chemie GmbH, Mtinchen. Hexitol- and hexose-phosphates as well as the auxiliary enzymes

84

J. L e n g e l e r : M u t a t i o n s o f G a l a c t i t o l D i s s i m i l a t i o n

were o b t a i n e d f r o m C . F . B o e h r i n g e r u. S 6 h n e , M a n n h e i m . G a l a c t i tol-l-P was prepared from D-galactose-6-P by borohydride reduct i o n a t p H 10 a c c o r d i n g to W o l f f a n d K a p l a n ( 1 9 5 6 b ) . 14C-galactitol w a s f r o m T h e R a d i o c h e m i c a l C e n t r e , A m e r s h a m .

to 1 x l01° b a c t e r i a / m l . T h e s e w e r e i n c u b a t e d w i t h 14C-galactitol a t 50 g M a n d 5.5 m C i / m M o l . A f t e r 1, 5, 10 a n d 20 m i n the cells w e r e c e n t r i f u g e d , r e s u s p e n d e d in 1.0 ml w a t e r a n d k e p t f o r 20 m i n a t 100 ° C. T h e p r e c i p i t a t e finally w a s c e n t r i f u g e d a n d the s u p e r n a tant used for chromatography.

Extraction of Labelled Carbohydrate-Phosphates from Cells. In ord e r to m i n i m i z e the a m o u n t o f i n o r g a n i c p h o s p h a t e s t r o n g l y interf e r i n g in t h e c h r o m a t o g r a p h y a n d c o l o r a t i o n o f o r g a n i c p h o s p h a t e s , a l o w p h o s p h a t e m e d i u m L P M P I P E S ( R u c h et al., 1974) and buffered by PIPES containing 1 mM KzHPO4, was used w h e n e v e r l a b e l l e d d e r i v a t i v e s w e r e to be e x t r a c t e d f r o m ceils. A 100 ml c u l t u r e w a s p r e g r o w n in L P M P I P E S g a l a c t i t o l to 1 x 109 b a c t e r i a / m l , c e n t r i f u g e d a n d r e s u s p e n d e d in L P M P I P E S

Thin Layer and Paper Chromatography. P o l y o l - a n d h e x o s e - p h o s phates were develloped by one run on Whatman Nr. 1 or by two to t h r e e c o n s e c u t i v e r u n s o n t h i n l a y e r cellulose p l a t e s a n d p h o s ~l)ate s p o t s r e v e a l e d b y m o l y b d a t e - p e r c h l o r i c a c i d s p r a y as des c r i b e d ( L e n g e l e r , 1975b). R a d i o a c t i v e b a n d s w e r e l o c a t e d in a p a p e r strip s c a n n e r B e r t h o l d L B 280 or in a t h i n l a y e r p l a t e s c a n n e r

T a b l e 1. O r i g i n , g e n o t y p e a n d p h e n o t y p e o f b a c t e r i a l s t r a i n s Strain

Origin or reference

Genotype or phenotype

mtl

236 L141 L146 L150 L153 L193 L251 KL-16 L253 L192 AM-1 AM-l-R20 N P 315 KLF3-1 KL96 K12

S o l o m o n + L i n (1972) L e n g e l e r (1975 a) Lengeler (1975a) this s t u d y

L150 L153 R.T. Vinopal L e n g e l e r (1975 a) this s t u d y

this study V i n o p a l + F r a e n k e l (1975) V i n o p a l + F r a e n k e l (1975) R u f f l e r + B 6 c k (1973) Lengeler (1975a) Lengeler (1975a) J. L e d e r b e r g

gut

gat

C

A

D

C

A

D

C

A

D

+ + + + + + + + + + + + + + +

A A A A A A + + + + + + + + + +

+ + + + + + + + + + + + + + + +

+ +

A + A50 A A A + + + + + + + + + +

+ + + + + + D50 + + + + + + + + +

--

+ + A50 + + + + A + + A A A + + +

+ + + + + D51 + + + + + + + + + +

+ + + + + + + + + + + +

pfkA

pfkB

fda

+ + + + + + + + + A1 A1 A1 + + + +

+ + + + + + + + + + + B1 + + + +

+ + + + + + + + + + + + ts + + +

kba

ts ts ts ts tr tr ts ts tr ts ts ts ts ts ts ts

G e n e t i c m a r k e r s a r e a c c o r d i n g to B a c h m a n n et al. (1976) a n d L e n g e l e r ( 1 9 7 5 a ) (see a l s o Fig. 1). F o r fda a n d kba the t e m p e r a t u r e sensitive o r r e s i s t a n t p h e n o t y p e is i n d i c a t e d b y ts o r tr. T h e sign " - " i n d i c a t e s c o n s t i t u t i v e o p e r o n e x p r e s s i o n

T a b l e 2. G r o w t h o f g a l a c t i t o l m u t a n t s o n d i f f e r e n t c a r b o h y d r a t e s Strain

236 L237 L241 L242 L14l L146 L146 gat + L150 L153 L193 L194 KL-16 KL-16 gat + L253 The symbol "-"

Origin

AB313 236 L237 L241 L141 L146 L150 L153 L193 Kl-16 KL-16 gat +

Mutation or Phenotype

gat + K b a t~ gatA K b a ts gat + K b a ts gat + K b a tr gat + K b a ts gatA K b a t~ gat* K b a t~ gat + K b a ts gat + K b a tr gatD K b a tr gatA,D gatA K b a t~ gat + K b a ts gat + K b a ~r

denotes no growth, and '"+"

Growth on MM Gut 42 °

Gly 42 °

Gly/Gat 42 °

Gat 28 °

Gat 42 °

CAA/Gat 42 °

+ + + + + + + +

+ + + + + + + + + +

+ + + + .

+ + + + -+ + +

+ +

+ + + + + + + + +

--

+

+

--

+

+ + +

+ + +

+ +

+

+ + +

.

. + +

.

i n d i c a t e s g r o w t h a t the t e m p e r a t u r e s a n d in the m e d i a i n d i c a t e d

J. Lengeler : Mutations of Galactitol Dissimilation

85

Berthold LB 2723, eluted with water and rechromatographed. Commercial D-mannitol-l-P, D-glucitol-6-P, D-fructose-6-P, Dfructose-l,6-bisphosphate and galactitol-l-P prepared from D-galactose-6-P as described were used as references. Bacterial Strains. The origin, phenotypes and genotypes of the strains used in this paper are listed in Tables 1 and 2 (see also Fig. 1). strain L150 is a galactitol positive recombinant from the cross L146 x Hfi" 236. Strains HfrH gat +, Kl-16 gat +, KL98-3 gat+ and L146 gat+ are galactitol positive transductants obtained from a transduction with P1.L153, while L205, AM-1/F' gat +, and AMl-R20 gat+ have been obtained after F-duction ofphenocopies of NP 315, AM-1 or AM-I-R20 with strain KLF3-1 (B6ck and Neidhardt, 1966; Lengeler, 1975a; Vinopal and Fraenkel, 1975). Strain L192 met + pfkA was constructed by conjugating Hfr AM-I metB + pjkA 1 and L173 metB pfk + (Lengeler, 1975a). RT-70 is a gutD mutant of HfrC, isolated by R.T. Vinopal. Culture Media and Growth Conditions. These are as described before (Lengeler, 1975a; Ruch et al., 1974). When sensitivity toward hexitols was to be tested substrates highly purified by a biological scavenging method (Lengeler, 1975b) were utilized. Genetical Techniques. Mutagenesis, Penicillin selection, conjugations and Pl-transductions were performed as described previously (Lengeler, 1975 a). Uptake and Enzyme Assays. Standard uptake assays as well as the preparation of cell extracts and the enzyme II-complex activity tests have been described (Lengeler, 1975a, b). To test the galactitoM-phosphate dehydrogenase the assay mixture contained in a total volume of 0.5 ml: Water, 0.25 ml; Na2CO3, 1 M, pH 10, 0.05 ml; nicotinamide adenine dinucleotide, 20 mM, 0.05 ml; galactitol-1-phosphate, 10 mM, 0.100 ml; and cell extract. When the reduction of ketose-6-phosphates was to be measured the mixture contained in 0.5 ml: potassium phosphate buffer pH 6.5, 0.1 M, 0.25 ml; ketose-6-P, 10 raM, 0.1 ml; NADH, 50 mM, 0.05 ml; and cell extract. Reactions were measured at 340 nm and 25 ° C. Phosphofructokinase activities were tested according to Kotlarz et al. (1975) in 1 ml mixture of: tris (hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.2, 0.2 M, 0.4 ml; MgC12, 0.1 M, 0.1 ml; NADH, 2 mM, 0.1 ml; ATP, 10mM, 0.1 ml; 0.1 ml auxiliary enzyme solution [containing 0.5 ml fructose-l,6-bisphosphate aldolase (E.C.4.1.2.13) (10mg/ml), 0.5 ml triosephosphate isomerase (E.C.5.3.1.1), sn-glycerol-3-phosphate dehydrogenase (E.C.I.I.I.8) (2 mg/ml) and 4 ml Bovine albumin (2.5 mg/ml) in potassium phosphate buffer, 0.05 M, pH 7.5]; cell extract. To test the enzyme phosphofructokinase A (E.C. 2.7.1.11) 10 mM fructose-6-P (0.1 ml) were added; to test the pfkB regulated activity 2 mM fructose-6-P, 0.1 ml with and without 20 mM PEP, 0.1 ml were added and the reaction followed at 340 nm and 25 ° C. The coupled aldolase test was according to Stribling and Perham (1973). Uptake and enzymeII-complex, phosphofructokinase and aldolase activities are expressed as nmoles per min. per mg of protein, other enzymes as ~tmoles per min. per mg protein. Protein determinations were as described (Lengeler, 1975a).

Results Properties of Galactitol Positive Strains

A t 37 ° C wild t y p e strains o f E. coli K12 a l m o s t invaria b l y are u n a b l e to g r o w o n g a l a c t i t o l as sole c a r b o n source. H o w e v e r , as was d i s c o v e r e d by accident, m o s t

,,._

..,

/

i

Fig. 1. Simplified genetic map of E. coli KI2. Unless indicated otherwise the genetic symbols are according to Bachmann et al. (1976) and Lengeler (1975a). Genes coding for enzyme II-complexes (En) are designated by the letter A; genes coding for hexitolphosphate dehydrogenases (DH) are designated by the letter D, both preceded by the name of their operon: mtl for D-mannitol utilization, gat for galactitol utilization and gut (formery sbl or srl from D-sorbitol) for D-glucitol utilization, D-glucitol being the correct systematic designation for this hexitol. Genes coding for phosphofructokinase A or B are designated pfkA or pfkB, the structural gene for the D-fructose-l,6-P 2 aldolase is designated by fda while the new symbol kba is given to the genetic locus controlling the D-ketose-l,6-P2 aldolase activity

oso

I

I

I

I

I

I

z

P,

002

TIME IN HOURS Fig. 2. Growth on galactitol at 28 ° C and 42 ° C. Cultures of L153 (m- m), KL-16 gat ÷ ( ~ i ) , and L150 (o--o) growing exponentially at 28°C on galactitol and of strain LI50 ( ( ~ o ) growing also on D-glucitol were shifted rapidly at the time indicated by the arrow to 42° C

F - s t r a i n s ( i n c l u d i n g L141 a n d its d e r i v a t i v e L 1 5 0 ) a n d f e w H f r o r F ' s t r a i n s (e.g. K L - 9 6 , A B 3 1 3 , K L F 3 1, D F F - 1 a n d t h e o r i g i n a l E. coli K 1 2 f r o m t h e collect i o n o f J. L e d e r b e r g ) will g r o w o n g a l a c t i t o l a t t e m p e r a t u r e s b e l o w 3 0 ° C ( T a b l e 2). B y c o n t r a s t a series o f H f r s t r a i n s (e.g. H f r H , H f r C , K L - 1 6 o r K1-98) is u n a b l e t o g r o w o n g a l a c t i t o l a t a n y t e m p e r a t u r e . At 30°C galactitol positive revertants or recombi-

86

J. Lengeler: Mutations of Galactitol Dissimilation

nants can usually be isolated (Table 2, Fig. 3). At 42 ° C galactitol positive strains not only are unable to grow on galactitol but growth on glycerol is rapidly inhibited by the addition of galactitol. This inhibition is neither seen in wild type cells during growth at 30° C nor at 42 ° C in mutants lacking the galactitol transport system (Table 2, Fig. 3). Thus an unspecific toxicity seems to be ruled out. From the different galactitol positive strains, mutants able to grow at 42 ° C on galactitol and/or resistant against the galactitol promoted inhibition can be isolated (Table 2, Fig. 2 and 3).

~- 2.0

I

i I I I II

//

1.0 -

,~
9 5 % ) linked to gatA (Table 6). Frequently reverting point mutations in gatA (e.g. LI46 or KL-16 in Table 3) exhibit a strong polar effect on the gatD coded dehydrogenase activity. This is not due to a lack of induction of the dehydrogenase in gatA mutants, since the gat operon is constitutive in all K12 strains including Lederbergs original K12 strain. The genes gatA and gatD thus apparently are clustered in a gat operon. Obviously the enzymeII-complexG a t and the galactitol-l-P dehydrogenase are necessary to generate Dtagatose-6-P from galactitol. Since both enzymes are synthezised at low and high temperatures the putative temperature sensitive enzyme must be one involved in the following steps of D-tagatose-6-P catabolism.

c) Mutations in the Genes pfleA and pfkB. In E. coli K12 three phosphofructokinases controlled by at least three different genes are known (Doelle, 1975; Vinopal and Fraenkel, 1975). The effect of the available mutations pfkA-1 and pfkB-1 on galactitol metabolism was tested. Since strain AM-1 pfkA-1 in contrast to AM-1R20 pfkA-1 pfkB-1 could not be made galactirol positive its pfkA-1 mutation was introduced by

87 Table 4. G row t h of different p f k A , pfkB, fda and kba mutants Genotype or Phenotype

Strain

L150 L153 L173 L192 A M - l - R 2 0 gat T L205 ~30 :Rc #5 R /j9 T

/431 R

D -ma nni t ol

gat + K ba ts gat + Kba tr gat + K ba u pflcA1 pfkAlpfleB1 Fdat~Kba t~ Fda 'r K ba ts F da ts K ba 'r F da 's K ba tr Fda ts K ba tr

Galactitol

28 °

42 °

28 °

42 °

n.g." n.g. 120 n.g. 246 176 126 210 177 141

n.g. n.g. 74 n.g. 84 n.g. 72 600 n.g. 1000

170 b 138 165 n.g. 390 225 177 300 279 180

n.g. 114 n.g. n.g. n.g. n.g. n.g. 345 510 250

a n.g. no growth; b generation times in min; ° R = revertant; T = transductant

I

I

I

I

I

I

I

I

f

]

J

_ 2.0 ~ 1.0 o'" Z