JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 706-718 Copyright 0 1976 American Society for Microbiology

Vol. 127, No. 2 Printed in U.S.A.

Physiological and Genetic Regulation of the Aldohexuronate Transport System in Escherichia coli GEORGES NEMOZ, JANINE ROBERT-BAUDOUY,* AND FRANQOIS STOEBER Laboratoire de Microbiologie de l'Institut National des Sciences Appliquees de Lyon, 69621 Villeurbanne, France Received for publication 6 October 1975

In Escherichia coli K-12, the specificity of the aldohexuronate transport system (THU) is restricted to glucuronate and galacturonate. There is a relatively high basal-level activity in uninduced wild-type or isomeraseless strains. Supplementary activity is obtained with the inducers mannonic amide (fivefold), galacturonate (fourfold), fructuronate (fivefold), and tagaturonate (sevenfold). Specific THU- mutants were selected as strains unable to grow on either aldohexuronate but able to grow on fructuronate or tagaturonate. The remaining transport activity in uninduced and induced THU- strains represents less than 20o of that found in the wild type. Conjugation and transduction experiments indicate that all of the THU- mutations are located in a unique locus, exuT, half-way between the tolC (59 min) and argG (61 min) markers. exuT is closely linked to the uxaC-uxaA operon (60 min) and to the regulatory gene exuR (60 min), which controls the above-mentioned operon and the uxaB operon (45 min). Growth on either aldohexuronate and transport activity are fully recovered when exuT mutants are allowed to revert to exuT+ on galacturonate or glucuronate. Reversion on glucuronate alone may lead to the mutational derepression of the 2-keto-3-deoxygluconate transport system, which is uninducible in the wild type, which also takes up glucuronate, and whose structural gene belongs to the kdg regulon. Such strains, which remain unable to grow on galacturonate, are exuT and kdgR (constitutive allele of the regulatory gene kdgR of the kdg regulon). THU activity is superrepressed in an exuR mutant in which the uxaC-uxaA operon and the uxaB operon are superrepressed; exuR +/ exuR merodiploids are also superrepressed. In a thermosensitive exuR mutant in which the above-mentioned operons are constitutive at 420C, the THU activity is fully derepressed at this temperature. On the basis of these and other results, it is concluded that THU is coded for by the structural gene exuT, which is negatively controlled by the exuR gene product and which probably belongs to an operon distinct from the uxaA-uxaC operon.

The aldohexuronates glucuronate and galacturonate can serve as carbon sources for growth of Escherichia coli K-12. These sugars are degraded according to the pathway first discovered by Ashwell (3) and depicted in Fig. 1. Regulation of the overall system catabolyzing 13-hexuronides and hexuronates is under study in our laboratory (29). To facilitate understanding of the present paper, it seems appropriate to recall certain previous results. (i) When added to the wild-type strain, glucuronate is able to induce synthesis of the five central enzymes (II, IlI, III', IV, and IV' of Fig. 1), whereas galacturonate induces only the synthesis of the enzymes of the lowest branch (II, III', and IV'). By using strains blocked in different steps of the chain, fructuronate and tagaturonate were shown, in fact, to be the true inducers (26, 29).

(ii) The structural genes of all five central enzymes were identified and located on the chromosome in three presumed, distinct operons belonging to two regulatory units (26, 29): the exu regulon and the uxu operon. (The symbol exu concems functions involved in both glucuronate and galacturonate metabolism; the symbol uxu concerns functions involved only in glucuronate metabolism; and the symbol uxa concems functions involved only or mainly in galacturonate metabolism.) The exu regulon includes two operons: the uxaC-uxaA operon (structural genes of enzymes II and IV') (60 min) (17, 18) and the uxaB operon (structural gene of enzyme III') (45 min) (17). The exu regulon is subjected to the negative control of the product of the regulatory gene exuR (60 min) (29; R. Portalier, D.Sc. thesis, Universite 706

VOL. .127, 1976

ALDOHEXURONATE TRANSPORT IN E. COIl

Claude Bernard, Lyon, 1972; R. Portalier, J. Robert-Baudouy, and F. Stoeber, unpublished data). The uxu operon (87 min) (29) includes the structural genes uxuB (entyme III) and uxaA (enzyme IV); it is negatively controlled simultaneously by the products of two regulatory genes exuR and uxuR (previously named mga; 87 min) (15, 25, 29; J. Robert-Baudouy, R. Portalier, and F. Stoeber, unpublished data). The occurrence of an active transport system responsible for both glucuronate and galacturonate uptake was demonstrated by JimenoAbendano (6; personal communication). In this paper, we present results concerning the specificity and inducibility of this aldohexuronate transport system (THU). Deficient mutants were selected, and their genetic characterization was undertaken. The influence of several mutations affecting the regulation of the overall hexuronate system on THU was also studied. MATERIALS AND METHODS Bacterial strains. All strains used were Escherichia coli K-12 derivatives (auxotrophic for thiamine). Their relevant genetic markers are listed in Table 1. The exuRAU(ts2) allele is responsible for the constitutive synthesis of enzymes II, II, and IV' at high temperature. Hfr strain BJt2 [exuRAU(ts2)] was crossed with the F- strain EWlb (str his argG toiC) and, among the Arg+ (str) recombinants, a strain carrying his, toiC, and exuRAU(ts2) was selected. The presence of the exuRAU(ts2) allele was assessed by a colorimetric method, specific for altronate oxidoreductase (III') (19), on solid glycerol medium at 30 and 42°C. The mutation uxaC2 (uronate isomerase [II] negative) was further transduced into the preceding strain by using a P1 lysate made on strain MH2 (uxaC2). Among the tolC+ transductants, strain RXC [str his uxaC2 exuRAU(ts2)] was selected. To construct the diploid strain F122FUTH, Fstrain FU9 (his exuT9 argG str) was made thyA by using trimethoprim (12) and then mated with Hfr strain KL166 (recA). His+ (str) recombinants were selected and analyzed for recA inheritance by using ultraviolet light sensitivity. Strain KL11O carrying the F122 episome (covering the 54- to 62-min region on the Taylor-Trotter map [30]) was crossed, on solid medium, with strain FUTH9 (recA thyA exuT9 argG str), and strain F122FUTH was obtained as an independent Thy+ Arg+ type. For all diploid strains studied, the presence of the episome was checked by (i) curing with acridine orange (12) for the reappearance of the recessive characters of the covered region and (ii) for transmission of episomal markers at high frequency when crossed with other F- recA strains carrying markers recessive to those of the wild-type episome. We also examined whether the episome of the latter diploid carries the wild-type allele of the locus under consideration by selecting

707

wild-type recombinants when crossing such a diploid with a F- recA + mutant in that locus. Mutagenesis. The method for nitrosoguanidine mutagenesis was that of Adelberg et al. (1) and was detailed previously (18). The conditions given by Loveless and Howarth (10) for ethyl methane sulfonate mutagenesis were adopted (30 mg/ml, 30 min). All THU- mutants selected grew as white colonies on MacConkey agar plates containing both aldohexuronates at 15 mg/ml. Reversions. Spontaneous revertants were obtained on solid or liquid medium at 370C unless specified otherwise. Conjugation and transduction. The methods used for conjugation and transduction were those of Miller (12). Culture media. Media for growth were identical to those described by Portalier et al. (18). Minimal medium was M63, pH 7.2 (28). Solid media contained glucose (5 mg/ml), glycerol (5 mg/ml), tagaturonate (3 mg/ml), galacturonate, glucuronate and mannonate (2.5 mg/ml), fructuronate, 2-keto-3deoxygluconate (KDG), or methyl-glucuronide (MeGlcU) (1 mg/ml). Eosin methylene blue (EMB) medium (12) contained sugars at 10 mg/ml. Media containing oleic acid as the sole carbon source were supplemented with Brij 35 by the method of Overath et al. (16). Induction and enzyme extraction. The conditions for induction and enzyme extraction were outlined previously (26). Inducers were used at 5 mM. Enzyme assays. Aldonic oxidoreductases and hydrolyases were assayed by methods published previously (20, 21, 27; J. Jimeno-Abendano, thesis, Universit6 Claude Bernard, Lyon, 1968). Uronate isomerase was measured by a coupling method described previously (17); /8-glucuronidase was also measured as described previously (14). Assay for uptake experiments. The rapid filtration technique described by Kepes (7) was used with minor modifications. Standard incubation medium (30C) contained cells (200 Zg of dry weight per ml), [U-'4C]glucuronate (1 mM; 0.5 Ci/mol), and medium M63. Samples (0.2 ml) were removed at time intervals and filtered through Millipore HA (0.45-tm) membrane filters. After being washed and dried, the filters were counted in a Packard Tri-Carb spectrometer. For calculating intracellular concentrations, the equivalence 2.18 ,lA of intracellular water per mg of bacterial dry weight was adopted (32). Chemicals. Intermediate substrates of the hexuronate pathway were synthesized in our laboratory (17). [U-'4C]glucuronate, potassium salt, was purchased from Radiochemical Centre, Amersham, England.

RESULTS Kinetic aspects of aldohexuronate uptake. When grown on glycerol, strains P4X (wild type, uronate isomerase is not induced) and MH2 (isomerase negative) take up glucuronate at about the same rate, and their steady-state values are similar (Fig. 2). The exchange and uptake inhibition of glucuronate by several in-

708

NEMOZ, ROBERT-BAUDOUY, AND STOEBER

J. BACTERIOL.

EXTRACELLULAR GLUCURONATE

AlI [i

|IIkcl |tEXTRACELLULAR w2-KETO-3-DEOXYCN2H O cm

H-1

I

H-C-OH § 8 t-O §|@ HO-f-HGLUCORATE N-C-ON Coo HO-U

NMYLGLUCURDNIIDE

I

o.M H

--OH

H-C-OOW

N

I HO-t-H mu4 H-NOH 1Ok NH%-OH

\

ON

20

LOH C

D-GLUCURONATE

D-FRUCTURONATE

H-C-O

rH2OW

H- -OH OH

C-0

MOC*O

I

\

2

11 I HO--H

HO-C-H

CN3

COON

D-MANNONATE

IT

I,k

_

H1-tOH

-Kl kdg

I H~ -C-OH o HO-C-H H- -OH

2-KETO-3-DEOXY

I

COON

D-GALACTURONATE

D-TAGATURONATE

2-KETO-3-DEOXY

6-PHOSPHOD-GLUCONATE t

D-GLUCOHATE

-i-OH

COOH

IH °P

CH2OP CN2ON ~~~~~~~~~~~~~~2O ~ C,2O2

HO-I-a HO-C-H NH-C-OH ux C UX H-c-ommH-C-OH N

I-

CH20H

D-ALTRDNATE

GLUCONATE EXTRACELLULAR

GALACTURONATE

min45

min 60

exuR

exuT

uxaB

uxaC uxaA

regulatory gene exuToperon uxaC_uxaA operon

uxa B

operon

exu regulon min86

uxu R reogatory gene

uxu A uxu

uxu B operon

min 76

min69

kdgT

kdgK

PYRUATE

- JHI1HfO? [Ik H-C-OW N-(j-O

IV

sea

VI

f

min36

kdgR regulatory gene

kdg regulon

kdgA

TRIOSE-3-

PNDOSPTE

ALDOHEXURONATE TRANSPORT IN E. COLI

VOL. 127, 1976

709

TABLE 1. Bacterial strainsa Strains

Sex

Genotype

P4X KL166 EWlb K63 KL110 (F122)

Hfr Hfr FFF'

RP1 MH2 TH1 to TH11 TH9rf FU9 FUTH9 F122FUTH

Hfr Hfr Hfr Hfr FFF'

FUT6

F-

metB recA rif his-1 toiC argG6 str oldD88 his-1 str leu lac his recA mal xyl metB thyA argG str (F thyA + argG+) uxaAl metB uxaC2 metB exuT1 to exuTll, metB exuT9rf metB his exuT9 argG str recA thyA exuT9 argG str recA thyA exuT9 argG str (F thyA+ exuT+ argG+) his thyA exuT6 argG str

Source/reference

E. Wollman M. Hofnung E. Whitney P. Overath B. Low (11) 18 17 Mutants of P4X (this paper) TH9 spontaneous revertant (this paper) TH9 x EWlb (this paper) Derived from strain FU9 (this paper) KL110 x FUTH9 (this paper)

thyA derivative (12) of a TH6 x EWlb recombinant (tolC transductant) HJ1 Hfr exuRAl metB 26, 29 Hfr RC1 exuRAUl metB 26, 29 JMTH1 Fhis recA exuRAUl argG str recA derivative (12) of an RC1 x EWlb recombinant (toiC transductant) (Robert-Baudouy, Portalier, and Stoeber) F' his recA exuRAUl argG str F122JMTH KL110 x JMTH1 (Robert-Baudouy, Por(F exuR + argG+) talier, and Stoeber) BJt2 Hfr exuRAU(ts2) metB RC1 spontaneous revertant (Robert-Baudouy, Portalier, and Stoeber) RXC Fhis uxaC2 exuRAU(ts2) str This Paper PR1 FthyA str argH uxuRi uxuAl Derived from strain PM1 (25) a Besides the genetic symbols employed by Taylor and Trotter (30), we introduced the following: uxaA, structural gene of altronate hydrolyase (IV') (16); uxaC, structural gene of uronate isomerase (II) (20); exuT, structural gene of the hexuronate transport system (A) (this paper); uxuB, structural gene of mannonate hydrolyase (IV) (29); exuR and UxuR (previously named mga), regulatory genes of the hexuronate pathway (15, 25, 29; Portalier, D.Sc. thesis; Robert-Baudouy, et al., unpublished data).

termediates of the hexuronate pathway, used were also tested. The results (Fig. 3A) show that intracellular radioactivity is almost totally removed (90%) in strain MH2 when an excess of non-radioactive glucuronate is added to the reaction medium. Similar values are obtained in uninduced strain P4X (data not shown). All of these results suggest that glucuronate is incorporated only weakly or not at all in a strain carrying the isomerase, provided that the latter is not induced, or in an isomerase-negative strain. Galacturonate was shown previously to be a competitive inhibitor of glucuronate uptake (6). Me-GlcU and tagaturonate showed small but reproducible inhibiat 10 mM,

tion (20%). Fructuronate was shown to contain glucuronate as an impurity, estimated at 5 to 10%o by paper chromatography. However, this contamination could hardly account for the high inhibitory effect (70%/) caused by fructuronate. Further experiments are needed to de. termine whether this compound is a substrate of THU. The THU system cannot take up KDG at all (8). The apparent Km for glucuronate uptake (0.1 mM) is the same in strain P4X whether initial rates of uptake or steady-state levels are measured (Fig. 4). The Vma, for uptake is about 7 nmol/min per mg (dry weight), the capacity (maximal internal level at steady state) is 23

FIG. 1. Degradative pathway of hexuronides and hexuronates in E. coli K-12. The different steps are catalyzed by the following enzymes: I, (&glucuronidase (EC 3.21.31); H, uronate isomerase (EC 5.31 12); III, mannonate oxidoreductase (EC 11 1.57); IV, mannonate hydrolyase (EC 4.21.8); III', altronate oxidoreductase (EC 111.58); IV', altronate hydrolyase (EC 4.21.7); V, 2-keto-3-deoxygluconate kinase (EC 2.71.45); VI, 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 42114); A, aldohexuronate transport system (THU); B, 2-keto-3-deoxygluconate (KDG) transport system. The symbols under each roman numeral (or A and B) are the structural genes of the corresponding enzymes. At the bottom of the figure, the distribution of these genes in different regulons and/or operons is given together with the corresponding regulatory genes and the chromosomal location. Glucuronate and galacturonate are "aldohexuronates," and fructuronate and tagaturonate are "ketohexuronates."

710

NEMOZ, ROBERT-BAUDOUY, AND STOEBER

J. BACTERIOL.

-cII 0

0

rE

40

0 720

30

time ( min ) FIG. 2. Glucuronate uptake in strains P4X (O) and MH2 (a) grown on glycerol medium. Transport activity was assayed as described in the text. [14C]glucuronate was added at I mM.

mM, and the factor of concentration (inside/ outside) may reach 360 at low external substrate concentrations. Inducibility of the THU system. The differential rate of THU synthesis (13) was measured in strain MH2 (isomerase negative) growing on glycerol plus different intermediates of the hexuronide-hexuronate pathway. Neither glucuronate nor Me-GlcU is able to induce the system (Fig. 5). On the contrary, galacturonate increases the basal level about fourfold without detectable delay. Since the strain used is isomeraseless, this is taken to mean that it is a true inducer. Mannonic amide is not metabolized by E. coli, and it has been demonstrated to behave as a gratuitous inducer of the central enzymes of hexuronate metabolism (26). It leads to a fivefold induction of the transport system. Fructuronate and tagaturonate or their degradation products are responsible for a fivefold and sevenfold induction, respectively. Despite a relatively high basal level, THU appears to be weakly inducible, and the effectors of the induction are identical to those identified previously for the three central enzymes of the pathway: enzymes II, III', and IV' (26). In addition, the induced synthesis of THU is very sensitive to catabolite repression exerted by glucose (80 to 90% decrease), and the effect is totally reversed by adenosine 3',5'-monophosphate (data not

Eleven independent mutants (TH1 to TH11) selected for the inability to grow on either glucuronate or galacturonate were found to be defective in the THU activity (Table 2). The characteristics of such mutants are as follows. (i) They grew well on glucose plates, and the fermentation of galactose, lactose, or maltose (EMB medium) was not affected at all. However, the 11 mutants did not grow on glucuronate, galacturonate, or Me-GlcU (after 48 h) in contrast to the wild-type P4X. Growth on fructuronate was the same as for the wild-type strain but, contrary to the wild-type growth on tagaturonate, was somewhat reduced for certain mutants (Table 2). (ii) Residual transport activity found in glycerol-grown mutant strains was 5 to 20% that of the wild-type strain under the same conditions. When grown in the presence of glucuronate, galacturonate, fructuronate, or tagaturonate, the transport activity was also less than 20% of the induced level found in strain MH2. (iii) To be sure that the leaky tagaturonate growth phenotype did not result from a defect in one of the enzymes of the pathway, activities were assayed in mutants TH6 and TH9 (Table 3). Central enzymes are either weakly induced or not inducible in the presence of aldohexuronates (glucuronate or galacturonate) but are normally induced in the presence of ketohexuronates (fructuronate or shown). tagaturonate). Interesting conclusions may be Characterization of transport mutants. drawn from these results: in TH6 and TH9, the

ALDOHEXURONATE TRANSPORT IN E. COLI

VOL. 127, 1976 -

A -o 4 .E0)40 0 E c

//W0

.E o 20,

0 c

01

,

~ 10

20

~~~ 30

U.

40

time (min)

_b°~

-C .6.

-0 0)

E 0

0-.4 a

30 time (min) FIG. 3. Exchange and uptake inhii ['4C]glucuronate with different intermedic

btieonfoH2tof -aen

bition Of

hexuronate pathway. Glycerol-grown str was used. (A) ['4C]glucuronate (1 mM) wais allowed to accumulate for at least 25 min before the 12C inhibitors (10 mM) were added at the times indicated by the arrows. (B) [14C]glucuronate (1,mM) and various 12C compounds (10 mM) were addhid at time zero. Symbols: 0, no inhibitor added; 0, Me-GlcU; A, tagaturonate; A, fructuronate; *, gala(cturonate; glucuronate. U,

uptake of aldohexuronates, but not sulbsequent steps, is affected; fructuronate and tUagaturonate are very likely able to penetrate the cells through channels independent froim THU. Since tagaturonate does not affect gluicuronate uptake strongly (Fig. 3), the THU systtem probably does not, or at least not actively, transport

711

tagaturonate. Accordingly, delay of growth of THU- mutants on this sugar is probably due to a deficiency of its specific transport system. Since growth on fructuronate remains unchanged, it is suggested that the tagaturonate and fructuronate transport systems are distinct from each other. Mapping of the exuT mutations. The linkage between several THU- mutations (written exuTl to exuTll), the uxaC2 mutation (17), the exuRAUl mutation (26, 29; Portalier, D.Sc. thesis; Robert-Baudouy et al., unpublished data), and one THU- mutation, exuT6 (origin: strain TH6), taken as a reference mutation, was estimated by mating the Hfr strains THU-, MH2, RC1, and P4X with the F- strain FUT6 (str argG exuT6). Arg+ (str) and Arg+ glucuronate+ (str) recombinants were selected. In each experiment, the "normalized" recombination frequencies [number of Arg+ glucuronate+ (str)/number of Arg+ (str)] were analyzed. In crosses between the F- strain FUT6 (exuT6) and the Hfr strain RC1 (exuRAUl) (phenotype: glucuronate- galacturonate-), the normalized frequency of recombination was 180-fold lower than in similar crosses with the wild-type Hfr P4X. When Fstrain FUT6 was crossed with Hfr MH2 (uxaC2; phenotype: glucuronate- galacturonate-), the normalized recombination frequency was 90fold lower than in similar crosses with Hfr P4X. Finally, in crosses between the F- strain FUT6 and different THU- Hfr strains, the normalized recombination frequency fluctuated between 0 and V/i8o that obtained with Hfr P4X. These results indicate that all the exuT mutations are closely linked and are close to exuRAUl and uxaC2 (near min 60 on the Taylor-Trotter map [30]). They define a unique locus, exuT, with mutated alleles leading to a deficiency in transport activity and an inability to use either glucuronate or galacturonate for growth. By mating Hfr TH9 (exuT9) with F- EWlb (str argG his tolC exuT+), the exuT locus has been located between the markers argG (61 min) and tolC (59 min) (Table 4). The glucuronate- galacturonate- phenotype is strongly cotransmitted with argG and toiC, since directly selected Arg+ Tol+ recombinants are 89% glucuronate- galacturonate-. Among the Tol+ recombinants, the class represented the least is argG+ exuT+ (10% quadruple crossing-over). The suggested order is then tolC-exuT-argG. Percentages of cotransduction between exuT6 or exuT9 mutations and different markers of the 59- to 61-min region are given in Table 5. exuT cotransduces very tightly with exuR (26, 29; Portalier, D.Sc. thesis; Robert-Baudouy,

712

NEMOZ, ROBERT-BAUDOUY, AND STOEBER

J. BACTERIOL.

/

E -

0

0

o150

1/Extemal Glucuronote (mM -1) FIG. 4. Lineweaver-Burk plot of initial rates (Vin) of uptake of ['4C]glucuronate and steady-state levels

("internal glucuronate") of accumulated [14C]glucuronate. Symbols: (a) Vin, wild-type strain (P4X); (0) steady-state level (P4X) (external ['4C]glucuronate concentrations ranged in both cases from 0.005 to 1 mM); (A) Vin, TH9rf mutant (external ['4C]glucuronate concentrations ranged from 0.01 to 1 mM).

Portalier, and Stoeber, unpublished data) and with uxaA (18). exuT is probably located between these two loci, near min 60, as shown in Fig. 6. The nature of the exuT9 mutation has been determined by constructing merodiploid strains carrying the exuT9 mutation on the chromosome and the wild-type allele on the episome. F' strain F122FUTH normally grows on both aldohexuronates, and the transport activity is about 60 to 70% of that found in the F- exuT+ strain EWlb and threefold that found in the FexuT9 strain FUTH9. Since exuT9 is recessive to the wild-type allele, it is not a superrepressed allele (31) of a regulatory gene, and exuT is likely a structural gene coding for a THU component. On the other hand, since a simultaneous decrease of the constitutive and inducible transport activities was observed in exuT mutants, it seems most probable that both activities depend upon a single structural gene.

Reversion of the THU- mutants. exuT mutants could revert by two different modes. (i) Among 40 revertants of TH9 isolated at high frequency (10-6)

on

solid media with glucuro-

nate or galacturonate, all are glucuronate- and

galacturonate+. Such a result suggests that exuT9 is a point mutation. In no case was the THU activity of revertants greater than that of

the wild type (revertants of a superrepressed allele of a regulatory gene often have a greater activity than the wild type [31]). (ii) Revertants of TH6 obtained on galacturonate are all glucuronate+ and galacturonate+, but only 1/4 Of the revertants obtained on glucuronate (frequency 10-9) exhibit such a phenotype, the remaining strains being glucuronate+ galacturonate-. The latter "pseudo" revertants acquired the ability to grow on KDG and on mannonate. Pouyssegur and Stoeber (22, 24) demonstrated that the Kdg+ phenotype is associated with the mutational derepression of a transport system responsible for KDG uptake, which is uninducible in the wild type; its structural gene belongs to the kdg regulon, which is negatively controlled by the kdgR regulatory gene (24). This transport system exhibits a low affinity for glucuronate (but not for galacturonate) (8). Since the Kdg+ and mannonate+ phenotypes are often linked (J. Pouyssegur and G. Novel, unpublished data), it became clear that the abovementioned revertants were not THU+ revertants but rather kdgR" mutants (with a constitutive allele of kdgR). A P1 lysate made on one TH6 "pseudo" revertant (glucuronate+ galacturonate- Kdg+ mannonate+) served to transduce the Kdg+ character into the -recipient strain K63 (oldD Kdg- mannonate-). Among 200 Kdg+ transductants, 100% are able to grow on

ALDOHEXURONATE TRANSPORT IN E. COLI

VOL. 127, 1976

C,

Nt

I

0

100

200

AB ( pgdrywerghtilmcuiture) FIG. 5. Differential rate of synthesis of the aldohexuronate transport system with different intermediates ofthe hexuronate pathway and mannonic amide. Separate cultures (strain MH2) were grown on glycerol (4 mM). At the beginning of the experiment, inducers (5 mM) were added: 0, none; glucuronate; A, Me-GlcU; +, galacturonate; A, fructurontagaturonate. Abbreviaate; 0, mannonic amide; tions: MF, increase ofglucuronate transport activity after induction; AB, increase of bacterial dry weight after induction. The structural formula of mannonic amide is: OH OH H H

713

and that this gene codes for a modified THU in TH9rf. This is in agreement with exuT being a structural gene. TH9 revertants exhibiting thermosensitive growth on aldohexuronates were sought by culture in liquid medium plus galacturonate at 30°C, followed by counterselection with penicillin (4) at 42°C. Among 1,000 clones analyzed, four mutants were found to possess the expected phenotype: they are able to grow on glucuronate or galacturonate at 30°C but not at 42°C. They were lactose' galactose' maltose+ at both temperatures on EMB media. However, growth on tagaturonate and fructuronate is also thermosensitive at 420 C (although normal at 300C). The four temperature-sensitive revertants took up glucuronate at about the same rate whether grown at 30 or 420C. Consequently, it was suspected that the original mutation reverted and that, in addition, thermosensitivity emerged from a second mutation affecting tagaturonate and fructuronate utilization. Indeed, growth on gluconate was found to be normal at 30°C but absent at 42°C after 48 h. The degradative pathway for gluconate joins the hexuronate pathway at the level of 2-keto-3deoxy-6-phosphogluconate (KDPG), which is converted by KDPG-aldolase to pyruvate plus triose phosphate (enzyme VI in Fig. 1). Because of the relatively high propensity for this aldolase becoming thermolabile (23), it was inferred

U,

0,

CH20H-C (b)

C OH

C-C H

H

NH2

mannonate, and 70% are able to grow on oleic acid (oldD+). This is in agreement with the fact

that the regulatory gene kdgR is cotransducible (55 to 78%) (24) with the oldD marker (16). In a TH9 partial revertant, TH9rf (glucuronate+ galacturonate+), 50% of the THU activity was recovered (reduced V,",,), and the affinity of THU for glucuronate was decreased (K,,, = 0.2 mM instead of 0.1 mM for the wild type) (Fig. 4). A P1 lysate made on TH9rf (argG+) served to transduce the glucuronate+ galacturonate+ character into the recipient F- FU9 (exuT9 argG). Among THU recombinants, 4% are argG+. Since argG and exuT9 were shown to be 2% cotransducible (Table 5), it is likely that the reversion occurred in the exuT gene

TABLE 2. Characteristics of aldohexuronate transport-negative mutants Growthc on tagaturonate THU activityb Strainr 16h

P4X TH1 TH2 TH3

48h

+ + 100 + + 6 ± + 8 + + 7 + + TH4 5 + + 5 TH5 + 10 TH6 + 7 TH7 + + 7 TH8 + 18 +TH9 + + 10 TH10 + + 16 TH11 methwere induced ethyl by aTH1 through TH3 ane sulfonate; TH4 through TH8, TH10, and TH11 were induced by N-methyl-N'-nitro-N-nitrosoguanidine; and TH9 was a spontaneous mutant. bResidual transport activity was measured with glycerol-grown cultures (percentage of the basal level of reference strain P4X). c Growth was observed on solid media after 16 and 48 h.

714

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NEMOZ, ROBERT-BAUDOUY, AND STOEBER

TABLz 3. Enzyme activities in TH6 and TH9 mutants in the presence of various inducers Differential rate of synthesisa Uronate

Altronate

Altronate oxidoreductase

Mannonate hydrolyase hyrlae

Mannonate oxidore-

None Galacturonate Glucuronate Tagaturonate Fructuronate

8 268 254 228 186

3 140 195 123 174

99 3,300 5,090 3,517 3,740

3 _b 49 37

340 _ 5,690 4,497

Galacturonate Glucuronate Tagaturonate Fructuronate

72 76 244 190

21 41 125 162

165 255 2,880 4,420

0 -

417 4,708

Strain

Inducer

P4X

TH6

iomerase isomrashyrolase hyd(rIV)ase

54

ductase

273 33 35 Galacturonate 464 0.6 420 35 36 Glucuronate 3,641 128 265 Tagaturonate 5,050 37 3,678 175 154 Fructuronate Differential rates of synthesis were measured under the same conditions as given in the legend of Fig. 5 and are given in milliunits (nanomoles of product per minute) per milligram of dry weight. b Not determined.

TH9

The Me-GlcU- phenotype cannot be genetically dissociated from the galacturonate- glucuronate- phenotype. When strain EWlb is Inheritance of unselected markers crossed with strain TH9 (Table 4), all glucuroSelected markersa Percent Class nate+ galacturonate+ recombinants are also argG+exuT+ 101 54 (argG+) Me-GlcU+ and, conversely, all glucuronatetolC+ str argG+ exuT- 44j) 7 eu galacturonate- recombinants are Me-GlcU-, agrG exuT- 3478(exuT) suggesting that a unique mutation was responargG- exuT+ 13 sible for the dual phenotype. However, among 45 tolC+ argG+ str 40 glucuronate+ galacturonate+ revertants of exuT- 60 exuT- 89 tolC+ argG+ str TH9 selected on glucuronate or galacturonate, exuT+ 11 50% were Me-GlcU+ and 50% were Me-GlcU-. All 29 TH9 revertants selected on Me-GlcU (frea Donor, TH9 (exuT9); recipient, EWlb (argG toiC str); 156 recombinants of each selected class were analyzed. quency about 10-7) were glucuronate+ galactuTABLE 4. Conjugation location of exuT9 between argG and toiC

that in the double mutants a thermosensitive mutation in the kdgA locus, structural gene for aldolase (23), was responsible for the resultant phenotype. This conclusion is borne out by the fact that no cotransduction between the glucuronate- galacturonate- character at 420C and any marker of the region exuT uxaA exuR argG tolC was obtained. On the contrary, the temperature-sensitive phenotype was located by noninterrupted mating in the his region, i.e., close to kdgA (23). THU- mutation and Me-GIcU phenotype. It was surprising that all THU- mutants lost the ability to grow on Me-GlcU (see above). Since THU was shown to exhibit no significant affinity for Me-GlcU and since it is suspected that Me-GlcU has its own transport system, we attempted to elucidate this puzzling point.

ronate+. Two interpretations could account for these results. (i) 8-Glucuronidase (enzyme I, Fig. 1), which converts ,8-glucuronides to glucuronate, is mutated or superrepressed in THU- mutants. Such an hypothesis does not hold since the enzyme in TH6 and TH9 was shown to be induced by 3 mM mannonic amide (weak inducer; M.A. Berthelot, unpublished data) at a level comparable to that found in the wild-type strain. Specific activities were 100, 93, and 74 U/mg (dry weight) in strains P4X, TH6, and TH9, respectively. In contrast, Me-GlcU, which is a strong inducer in the wild type (14), has lost this property in strains TH6 and TH9. Such a result can be explained by hypothesis (ii): MeGlcU cannot penetrate the cell because its own transport system is either absent or inactive. Although several models may be put forward,

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VOL. 127, 1976

715

TABLE 5. Transduction study of exuT6 and exuT9 mutations Inheritance of unselected Donor (P1)

Recipient

TH6 (exuT6)a

EWlb (argG tojC)b

RP1 (uxaAl ) HJ1 (exuRA1)' TH9 (exuT9)a

TH6 (exuT6)a TH6 (exuT6 )a EWlb (argG tolC)b

&lected marker tolC+ argG+ exuT+" exuT+" tolC+ argG+ uxaA +9

RP1 (uxaAl )' TH9 (exuT9)f Phenotype: glucuronate- galacturonate-. Phenotype: glucuronate+ galacturonate+. Phenotype: glucuronate+ galacturonate-. "Selected phenotype: glucuronate+. Phenotype: glucuronate- galacturonate- tagaturonate+. ' Phenotype: glucuronate+ galacturonate- tagaturonate-. D Selected phenotype: tagaturonate+.

markers

No. analyzed

96 1t6 259 142 156 104 56

Class

Percent

exuTexuTuxaAl' exuRAl" exuTexuTexuT-

5 0.6 95 93 2 2 84

a b

min 59

men 61

FIG. 6. Genetic map of the tolC-argC segment of the E. coli K-12 chromosome. Distances are expressed as cotransduction frequencies with phage Pl (data taken from Table 5). Complementary values (in parentheses) were taken from references 17 and 18 and from the D.Sc. thesis of R. Portalier, Universite Claude Bernard, Lyon, 1972.

experimental evidence cannot be given at pres- is not inducible. Ruling out the possibility that ent, i.e., until Me-GlcU transport activity is inducers have lost the ability to penetrate the cell (there is a basal level for THU, and RC1 measured. Genetic regulation of THU. Two features can grow on fructuronate; Robert-Baudouy et suggested that THU and the three enzymes al., unpublished data), it is likely that THU is determined by the exu regulon (enzymes II, also superrepressed. (ii) In merodiploid F' III', and IV') are under the control of a common exuR +lexuRA UJ strains, the superrepressed regulatory gene: inducers are identical and the allele is dominant for enzyme II, III', and IV' exuT gene is very close to the presumed operon activities (29; Robert-Baudouy et al., unpubuxaC-uxaA (enzymes II and IV') (17, 18, 26). lished data) and also for THU (Table 6). (iii) In Additional, stronger evidence is given in Table the thermosensitive revertant BJt2, the allele 6, where THU activity is measured in several exuRAU(ts2) synthesizes a thermolabile remutants affected in the regulation of the hexu- pressor. During growth at 300C, enzyme III' is ronate pathway. (i) In strains RC1 and JMTH1 superrepressed and enzymes II and IV' are parcarrying the exuRAUl allele, controlling the tially constitutive, but at 42°C all three enexu regulon, and where enzymes II, III', and zymes are constitutive (29; Portalier, D.Sc. theIV' are superrepressed (26, 29; Portalier, D.Sc. sis; Robert-Baudouy et al., unpublished data). thesis; Robert-Baudouy et al., unpublished Similarly, in the F- strain RXC LexuRAU(ts2) data), the basal level of THU is similar to that uxaC2I, THU is largely derepressed at 300C found in the exuR+ strains MH2 and EWlb but (6-fold the level found in the F- strain EWlb

716

J. BACTERIOL.

NEMOZ, ROBERT-BAUDOUY, AND STOEBER

TABLE 6. Aldohexuronate transport activity in mutants affected in regulation of the hexuronate pathway Glucuronate uptake (nmollmin per mg of dry wt) Growth Genotype Strain Strain (°C)temp

None'

Galactu-

ronate'

Fructu-

ronate'

TagaturonateW

42 37 6 25 30 5.2 7.9 6.9 6.3 37 3 30 3 37 2.2 42 18 30 usaC2 exuRAU(ts2) RXC (F-) 42 23 2.6 5.3 5.3 37 exuRAUl JMTH1 (F-) 3.7 37 2.2 3.3 F exuR+lexuRAUl F122JMTH (F') 2 37 uxuRl PR1 (F-) a Inducer (5 mM) was added to glycerol-medium at the beginning of the culture, and cells were harvested at the end of the exponential phase. MH2 (Hfr) RC1 (Hfr) EWlb (F-)

uxaC2 exuR+ uxuR+ exuRAUl exuR+ uxuR+

exuR+) and fully derepressed at 42°C (10-fold). (iv) The second regulatory gene, uxuR, of the hexuronate system, which primarily controls the uxu operon and also the structural gene of enzyme I but not the exu regulon, does not seem to govern THU biosynthesis: in the mutant strain PR1 (uxuR-), which exhibits a constitutive synthesis only of enzymes I, III, and IV (15, 25, 29; Robert-Baudouy et al., unpublished data), THU is not derepressed in the absence of any inducer.

DISCUSSION The role played by specialized transport systems in the overall metabolic flow of nutrients in bacteria does not need to be exemplified. It is clear that the correct functioning of a particular enzymatic sequence is governed initially by the permeability of the membrane for the external substrate. It was the aim of the present work to extend our knowledge of the complex genetic regulation of the f8-glucuronide-hexuronate pathway in E. coli K-12 (29) by investigating the hexuronate uptake step essentially from a genetic and physiological standpoint. Since analysis of kinetics and energy coupling is given elsewhere (6), the first part of our study was restricted to defining the proper conditions for assaying glucuronate uptake (use of isomeraseless mutants) and to delineating the transport parameters. The aldohexuronate transport system exhibits a substrate specificity limited primarily to glucuronate and galacturonate. It is uncertain whether fructuronate can also be taken up by the system. The ability of THU- mutants to grow on fructuronate and tagaturonate indicates that these intermediates do enter the cell through separate transport systems. In the second and main part of this work, we

studied the mechanisms of the physiological and genetic regulation of the aldohexuronate transport system. Several lines of evidence support the contention that THU belongs to the previously defined regulatory unit, the exu regulon controlled by the exuR regulatory gene, which governs the synthesis of the uronate isomerase (II), altronate-oxidoreductase (III'), and altronate-hydrolyase (IV') (29). (i) Inducers are identical for the four activities: galacturonate, fructuronate, tagaturonate, and mannonic amide. (ii) Mutations leading to a defect in transport activity are all located in a unique locus, exuT, which is closely linked to the structural genes uxaC (isomerase) and uxaA (hydrolyase) and to the regulatory gene exuR between the tolC (59 min) and argG (61 min) markers. (iii) In the RC1 mutant (exuRAUl), the four activities are superrepressed; i.e., no induction is obtained with any inducer. Superrepression remains in merodiploids F' exuRAUllexuR+. (iv) In the thermosensitive BJt2 mutant [exuRAU(ts2)] synthesizing a thermolabile repressor, the four activities are fully derepressed at 420C but not at 300C. (v) No derepression for any activity appears in a uxuR- mutant in which only enzymes I, III, and IV are constitutive. Therefore, THU and enzymes II, III', and IV' are under the same negative control exerted by the exuR regulatory gene, and the exuT gene also belongs to the exu regulon. However, the biosynthesis of isomerase, hydrolyase, and THU activities is not coordinated: the basal level for THU is somewhat high and its inducibility is weak (7-fold) compared to a 40-fold induction for isomerase and hydrolyase (26). Therefore, the structural gene exuT does not seem to belong to the uxaC-uxaA operon, although it is linked very closely to it. We expect that the selection for operator-constitu-

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VOL. 127, 1976

tive mutants ofboth classes will help clarify the situation. The mono- or polycistronic nature of the exuT gene remains to be assessed because substrate uptake is a necessary multistep process requiring the interaction of several molecules. At least the participation of a periplasmic binding protein (5) in the hexuronate uptake may be ruled out since no such activity was detected in a concentrated shock fluid (A. Lagarde, unpublished data); no chemotaxis towards glucuronate or galacturonate was observed (2); the D(- )-lactate-driven glucuronate accumulation in isolated membrane vesicles occurred (9). ACKNOWLEDGMENTS We thank A. Lagarde for fruitful discussion and criticism, and M. Mata, M. Bergon, and S. Ottomani for skillful technical assistance. This work was supported by the Delegation Generale a la Recherche Scientifique et Technique (Action Compl& mentaire Coordonne "Interactions Moleculaires en Biologie"), by the Centre National de la Recherche Scientifique (E.R.A. no. 177), and by the Fondation pour la Recherche Medicale Francaise. LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. Chein Ching Chen. 1965. Optimal conditions for mutagenesis by Nmethyl-N'-nitro-N-nitrosoguanidine in Escherichia coli K12. Biochem. Biophys. Res. Commun. 18:788795. 2. Adler, J., G. L. Hazelbauer, and M. M. Dahl. 1973. Chemotaxis towards sugars in Escherichia coli. J. Bacteriol. 111:824-847. 3. Ashwell, G. 1962. Enzymes of glucuronic and galacturonic acid metabolism in bacteria, p. 190-208. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 5. Academic Press Inc., New York. 4. Gorini, L., and H. Kaufman. 1960. Selecting bacterial mutants by the penicillin method. Science 131:604605. 5. Heppel, L. A., B. P. Rosen, I. Fnedberg, E. A. Berger, and J. H. Weiner. 1972. Studies on binding proteins, periplasmic enzymes and active transport in Escherichia coli, p. 133-156. In J. F. Woesner and F. Huiing (ed.), The molecular basis of biological transport. Academic Press Inc., New York. 6. Jimeno-Abendano, J., and A. Kepes. 1973. Sensitization of D-glucuronic acid transport system of Escherichia coli to protein group reagents in presence of substrate or absence of energy source. Biochem. Biophys. Res. Commun. 54:1342-1346. 7. Kepes, A. 1971. The ,B-galactoside permease of Escherichia coli. J. Membrane Biol. 4:87-112. 8. Lagarde, A., J. Pouyss6gur, and F. Stoeber. 1973. A transport system for 2-keto-3-deoxy-D-gluconate uptake in Escherichia coli K 12. Eur. J. Biochem. 36:328-341. 9. Lagarde, A., and F. Stoeber. 1974. Transport of 2-keto3-deoxy-D-gluconate in isolated membrane vesicles of Escherichia coli K 12. Eur. J. Biochem. 43:197-208. 10. Loveless, A., and S. Howarth. 1959. Mutation of bacteria at high levels of survival by ethyl methane sulfonate. Nature (London) 184:1780-1782. 11. Low, B. 1972. Escherichia coli K-12 F-prime factors, old and new. Bacteriol. Rev. 36:587-607.

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