Vol. 133, No. 2

JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 549-557

0021-9193/78/0133-0549$02.00/0

Printed in U.S.A.

Copyright 0 1978 American Society for Microbiology

Isolation of Specialized Transducing Bacteriophages Carrying the Structural Genes of the Hexuronate System in Escherichia coli K-12: exu Region MIREILTLE MATA,* MARIE DELSTANCHE, AND JANINE ROBERT-BAUDOUY Laboratoire de Microbiologie de l'Institut National des Sciences Appliquees de Lyon, Laboratoire Propre du Centre National de la Recherche Scientifique no. 05421, 69621 Villeurbanne, France Received for publication 1 September 1977

In Escherichia coli HfrH 58, isolated by Shimada et al., a heat-inducible phage has been integrated in a secondary attachment site. We have characterized the nature of the A integration. The exuR regulatory gene is inactivated by prophage integration. Genetic and biochemical analysis indicated a gene order: uxaA-uxaC-exuT-(exuR')-ANRAJ (exuR"). By induction of HfrH 58, one class of deletions extending into the exu region was obtained. Analysis of these deletions confirms the exu region topography and the regulatory mechanism of the hexuronate system previously described. It has been possible to regenerate a functional exuR gene by prophage exision. Various lambda transducing particles, plaqueforming and defective transducing phages carrying the left part or the right part of the exu region, have been derived from the secondary site lysogen HfrH 58. A phage carrying the entire exuR region was constructed by a cross between these two types of phage. The construction and characterization of these exu transducing phages are presented. The aldohexuronates glucuronate and galacturonate are degraded according to the Ashwell pathway (1) (Fig. 1). Fructuronate (D-lyxo-5hexulosonate) is able to induce the synthesis of the five enzymes II, m, m', IV, IV' (14) and that of the aldohexuronate transport system THU (8), whereas tagaturonate (D-arabino-5hexulosonate) induces only the synthesis of the enzymes of the galacturonate branch (enzymes

I,I ml, IV") (14) and that of the aldohexuronate

transport system. Fructuronate and tagaturonate are the true inducers. The structural genes of galacturonate catabolizing enzymes are located in two distinct regions on the chromosome: the exu region (8, 9, 10) (min 66), which includes the exuR regulatory gene and the three structural genes uxaC (enzyme II), uxaA (enzyme IV"), and exuT (aldohexuronate transport system); and the uxaB operon (enzyme M') (min 52) (Fig. 1). The exu regulon involves all the above-mentioned genes and is under the negative control of the exuR regulatory gene product (21); (R. Portalier, J. Robert-Baudouy, and F. Stoeber, unpublished data). To study transcription in vitro, we tried to obtain trnsducing phages carrying different portions of the exu region. Shimada et al. (18) isolated several lysogens of Escherichia coli to analyze secondary attachment sites of phage A. In one such lysogen, strain HfrH 58, the A pro-

phage site was located near metC (18), i.e., near the exu region. Upon heat induction of this lysogen, we isolated several transducing phages carrying either the whole or a part of the exu region. This paper describes the isolation and characterization of these phages as well as deletions in the exu region obtained by induction of strain HfrH 58. MATERIALS AND METHODS Bacterial and phage strains. The genotypes of the bacterial and phage strains used in this study are shown in Table 1. The bacteriophage Xvir, Ximm21ccL

Xinm21b2c, and Xsus mutants were from the laboratory of P. Thomas in Brussels. Culture media. Media for growth were identical to those described by Portalier et al. (10). Minimal medium was M 63, pH 7.2 (20). Solid media contained glucose (5 mg/ml), glycerol (5 mg/ml), tagaturonate (3 mg/ml), and galacturonate, glucuronate, or altronate (2.5 mg/ml). Tetrazolium medium (7) contained galacturonate at 5 mg/ml; aldohexuronate MacConkey media (7) contained these sugars at 15 mg/ml. Enzyme induction and extraction. The conditions for induction and enzyme extraction were outlined previously (14). The inducer concentration was 5 mM. Enzyme assays. Aldonic oxidoreductases and hydrolyases were assayed according to previously published methods (11, 12, 15); hexuronic isomerase was measured by a coupling method described in a pre-

549

vious paper (9).

550

J. BATRIUOL.

MATA, DELSTANCHE, AND ROBERT-BAUDOUY A THU

atracLLular

sMuT

Dglucuronate

_

III

11

usaC

D-glucurn

=

uxuB

Dfructuronate

mannonate

=

IV

uA

2-I.tol3deoxy

Digluconate

A

/IV' agalacturonste

-

D-pgaLuronh

=

D4igaturont.

uxaC

extracellutar THU T

=

Datronate

uxa a

A

FIG. 1. Degradative pathway of hexuronides and hexuronates in E. coli K-12. Symbols: II, uronate isomerase (EC 5.3.1.12); III, mannonate oxidoreductase (EC 1.1.1.57); IV, mannonate hydrolyase (EC 4.2.1.8); IIr, altronate oxidoreductase (EC 1.1.1.58); IV', altronate hydrolyase (EC 4.2.1.7); A, aldohexuronates transport system (THU). TABLE 1. Strains Strains

E. coli HfrH 58

C600 (P2) EWlb RP1 MH2 TH6 TH9 HfrH HJ1 RC1 1573 1574 1576

1693 1694 1576 (P2)

Bacteriophages Apexul Xpexu2 Xpeux3 Apexu4 Apexu5 Adexul Adexu2

Source/reference

Relevant genotype

A C1857 integrated at exuR; M. Gottesman Plaques Spi- phage; Thomas E. Witney

A(gal-bio) F- his-i toiC argG6 str Hfr uxaAl metB Hfr uxaC202 metB Hfr exuT6 metB Hfr exut9 metB

(10) (9) (8) (8) Institut Pasteur

(14,21) (14, 21)

Hfr exuRAl metB Hfr exuRAUl metB

A(gal-bio) uxaA) A(gal-bio) uxaA) A(gal-bio) uxaA) A(gal-bio)

A(exuR'-exuT-uxaC-

Heat survivor of HfrH 58; this study

A(exuR'-exuT-uxaC-

Heat survivor of HfrH 58; this study

A(exuR'-exuT-uxaC-

Heat survivor of HfrH 58; this study

Heat survivor of HfrH 58; this study Heat survivor of HfrH 58; this study This study

A(gal-bio)

cI857 (exuR") cI857 (exuR')-exuT-uxaC-uxaA c1857 exuR-exuT-uxaC-uxaA

cI857 (exuR')-exuT c1857 (exuR'-exuT-uxaC cI857 (exuR'-exuT-uxaC-uxaA cI857 Sam7 (part of exuR)-exuTuxaC-uxaA

Chemicals. Intermediate substrates of the hexuronate pathway (tagaturonate, fructuronate, and al-

tronate) were synthesized in our laboratory (9). Lysogenization and transduction frequencies. Lysogenization frequency was determined as described by Shimada et al. (18). Transduction frequency was determined by the same procedure, except that infected cells were plated on minimal agar plates. Preparation of phage lysates. Low-frequency and high-frequency lysates (LFT and HFT) of lambda lysogens were prepared as described by Miller (7).

Derived from HfrH 58; this study Derived from HfrH 58; this study Derived from a cross between Xpexul and Apexu2; this study Derived from HfrH 58; this study Derived from HfrH 58; this study Derived from HfrH 58; this study Isolated by Schrenk and Weisberg method

Isolation of plaque-forming transducing phages. LFT lysates were plated on a lawn of galacturonate- recipients, using galacturonate tetrazolium plates. Plaques formed by nondefective exu transducing phages were easily identified by their halos and

colored centers after 36 h of incubation at 320C. Isolation of ASpi-. ASpi- (6) were isolated using fresh LFT lysates derived from HfrH 58, and 0.1 ml of the lysate, containing 10' plaque-forming units, was mixed with 0.1 ml of an exponential-phase culture of a P2 lysogen. After 15 min of adsorption, the phage-

VOL. 133, 1978

infected cells were poured onto tryptone plates. After overnight incubation at 370C, plaques of XSpi- appeared and were isolated as described by Boulter and Lee (3). Prophage curing procedure. The method for curing A lysogens by a thermal treatment (18) was modified as follows. The A lysogen cultures were spread at 420C and 32°C either on tryptone plates or galacturonate MacConkey medium or on minimal media containing galacturonate, altronate, or tagaturonate as the sole carbon source. On galacturonate MacConkey, mutants deleted in the exu region are easily screened. Wild-type survivors are red, while those having lost one or several genes, required for the galacturonate dissimilation, produce white colonies. On galacturonate inimal medium, survivors that had retained all the genes needed for galacturonate metabolism were selected. Altronate and tagaturonate media should favor the production of survivors displaying a functional uxaA gene but having lost exuT and uxaC genes.

RESULTS Location of phage A in the eu region of strain HfrH 58. Strain HfrH 58, provided by M. Gottesman, was induced at 420C, and several independent LFT lysates were obtained. These phages transduced various galacturonate- point and deletion mutants to galacturonate+ (see below), but none of them was able to transduce the exuR gene. These results suggest that, in strain HfrH 58, phage A has inserted near or into the exu region. HfrH 58 grew on aldohexuronates, thus indicating that the presence of the prophage does not inactivate any of the exuT, uxaA, and uxaC structural genes. The level of the different enzymes, II, III', and IV', involved in the exu regulon was measured in the presence or absence of the inducer at 320C in strain HfrH 58 (Table 2). Synthesis of the three inducible enzymes, which are regulated by the exuR regulatory gene and belong to different operons, is constitutive in that strain. This character shows that exuR expression is altered by A insertion (Fig. 2). Isolation of plaque-forming and defective exu transducing phages. Plaque-forming exu phages. LFT lysates made by heat induction of HfrH 58 were used to transduce cells of TH6 (exuT) at 320C on galacturonate tetrazolium plates. Twenty-four galacturonate+ transductants picked up from halos of colored plaques were purified on homologous media and used to prepare HFT lysates. Each HFT lysate was spotted on strain 1576 (class B deletion, see Fig. 6). Of the 24 HFT lysates, 6 transduced exuT+ (like Xpexu4), 16 transduced exuT-uxaC (like Apexu5), and 2 transduced exuT-uxaC-uxaA (like Apexu2) (Fig. 3 and Table 3). All these

phages were plaque-forming.

551

SPECIALIZED TRANSDUCING PHAGES IN E. COLI

TABLE 2. Activity of the exu regulon enzymes in E. coli K-12 in differential rate of synthesis Strain

Inducer

Relative differential rate of synthesis (%) in enzymea:

IV' m' THU

II

Wild type

None Fructuronate

HfrH 58

None Fructuronate

A class A

None Fructuronate

A classB

None Fructuronate

A class C

None

TH9

None

3

6

4

100

100

100

74 38

49

235

4 102

5 128

87

0 0

0 0

383

75

74

410

100

70 422 7 398

18

18

of maximal value induced in the wild type. The activities of strain HfrH grown with fructuronate are taken as 100 baais for activities of enzymes II, M', and IV'. The wild-type strain for enzyme THU is MH2 (uxaC202) (8). THU activity, which is determined by the amount of intracellularly accumulated glucuronate, cannot be measured in HfrH and in class A and C deletions, because in these strains enzyme II synthesis is derepressed, and glucuronate cannot accumua Percentage

late.

Defective exu transducing phages. These phages were obtained by two different procedures. First, an LFT lysate of HfrH 58 was used to transduce E. coli RP1 (uxaAl) on galacturonate MacConkey plates at 320C. One such defective transducing phage, Adexul (Table 3), carried the bacterial genes exuT, uxaC, and uxaA. By marker rescue, we have shown that phage markers from K to J were deleted (Fig. 3). This result shows that the prophage in HfrH 58 is oriented clockwise and suggests the following gene sequence: uxaA-uxaC-exuT (exuR') XNRAJ (exuR") (Fig. 2). This is substantiated by the results obtained with prophage deletions (Fig. 6). The other defective exu transducing phage, Xdexu2 (Table 3), was obtained by induction of a mixed culture of abnormal lysogens as described by Schrenk and Weisberg (16). The mixed lysate was used to transduce strain 1576. The phage Xdexu2 that we obtained transduced exuT, uxaC, and uxaA. ASpi- carrying the right part, (exuR"), of exuR gene. XSpi- obtained from HfrH 58 by abnormal excision (see above) has lost phage genes from the left end of the prophage map. The lost phage genes may be replaced by bacterial DNA adjacent to the right prophage end (6), i.e., the (exuR") part of the exuR gene. The presence of (exuR") in the XSpi- was demonstrated by an indirect method (see below).

552

J. BACTER1OL.

MATA, DELSTANCHE, AND ROBERT-BAUDOUY

:1

uaA uxaC

toL C

exuT

exu R

argG

-simEmo-

T(9'

R

uxa A uxa C exu

a

(exu R)

I int FIG. 2. The integration of ACI857 into the exuR gene and the formation of Aexu transducing phages. The transducing phages are of two types: Apexul (bio-type), carrying the right part of the exu region, and Apexu2 (gal-type), carrying the left part of the exu region. The left end and right end of the exuR gene are called, respectively, (exuR') and (exuR").

uxa A uxa C

exu T

.

(exu R') int N P Q S R A C D E F K(exuR") Hfr H 58 Xpexu 1

...

iz::i

iX::-?~p exu 2 Apxu 4 p exu S

..._

x d exu 1 FIG. 3. Different types of exu transducing phages obtained by induction ofHfrH 58. Broken lines indicate that the exact end point of the phage has not been determined.

Construction of X recombinant carrying the entire exuR gene (Xpexu3). A cross between a phage carrying the left part of the exu region and a second phage which transduces the right portion of the exu region should yield a A recombinant tranaducing the entire exuR gene. A method of constructing this phage is illustrated in Fig. 4. Since it was not possible to check whether the phages XSpi- carry (exuR"), ten independent XSpi- were crossed with Xpexu2 carrying (exuR')-exuT-uxaC-uxaA. Only A re-

combinants (Apexu3-type) produced red plaques on a lawn of the P2 lysogen 1576 growing on galacturonate tetrazolium plates. Several red plaques were obtained with 3 out of the 10 A crosses and were purified. The corresponding lysates were used to transduce strain 1576 (deleted in the exu region) to galacturonatel. In these transductants, the constitutive synthesis of enzyme Il' was abolished and no longer detectable on colonies by a colorimetric method (13). Since the synthesis of enzyme m' is con-

SPECIALIZED TRANSDUCING PHAGES IN E. COLI

VOL. 133, 1978

This result proves that the three ASpiwhich are able to recombine carry the right part, (exuR"), of the exuR gene (Apexul-type, see above). Lysogenization of strain 1576 by phages Apexu2 and Adexu2. Strain 1576 lysogenized by Apexu2 presents two genotypes as described in Fig. 5. Two different events lead to lysogenization of strain 1576. On the one hand, integration at specific sites restores a whole and functional exuR gene (93% of lysogens). On the other hand, integration by homology between sequences on the phage and the chromosome leaves exuR nonfunctional (57% of lysogens), and enzyme III' is synthetized constitutively. In the second alternative, 100% of the transductants obtained by transduction of 1576 with Adexu2, isolated by the method of Schrenk and Weisberg (16), retain constitutivity for the synthesis of enzyme III'. At this point, we cannot conclude that Adexu2 carries a part of exuR, but, if it does, this part is probably different from (exuR') because the entire exuR gene is never restored by integration of Xdexu2. exu transducing phage and the exuR superrepressible mutations. Two types of superrepressible mutation have been isolated for the exu regulon, type RC1 (genotype exuRAUl) and type HJ1 (genotype exuRAl) (14, 21; Portalier et al., unpublished data). These pleiotropic mutations have been located in the exuR gene. Phage Apexu2 (gal-type) and Apexul (bio-type) carry, respectively, the left part and the right part of the exuR gene. To determine the position of these exuR transdominant mutations in one or the other part of exuR, strains RC1 and HJ1 were infected by the two phage types, and recombinants able to grow on galacturonate minimal medium were selected. Reversion frequency of these exuR mutations was 6 x 10'. Revertants were distinguished from wild-type recombinants by estimation of enzyme III' con-

stitutive in strain 1576 (see class B deletion, below), the presence of a functional exuR gene should render this strain inducible. Table 4 shows the level of enzymes II, III', and IV' in strain 1576 transduced by Apexu3, measured in the presence and absence of galacturonate as inducer. The synthesis of these enzymes is no longer constitutive, but they present a wild-type induction pattern, thus proving that a functional exuR gene has been restored. A recombinants (Apexu3-like phages) carry the entire exuR gene and the exuT-uxaC-uxaA

genes.

TABLE 3. Mapping data for the Xexu transducing phagese Phage

Xpexul Xpexu2

Apexu3 Xpexu4 Xpexu5 Xdexul Xdexu2

Galacturonate, TH6,

Galacturonate, MH2,

Galacturonate, RP1,

exuT 0

uxaC 0

+ + + + + +

+ + 0 + + +

uxaA 0 + + 0 0 + +

Galacturon-

ate, 1576 A

Glucuronate

1576a,

B

clam classB clasB 0

+ + 0

0 + +

0 + + +

a Xpexul, -2, 4, and -5 and Adexul were obtained by induction of HfrH 58 (see Fig. 3), and Xdexu2 was isolated as described by Schrenk and Weisberg (16). Apexu3 is a recombinant from Xpexu2 and Xpexul. A fresh lysate containing 1iO to 1010 plaque-forming units per ml was prepared for each ransducing phage and was spotted on galacturonate or glucuronate minimal plate seeded with E. coli K-12 galacturonate- mutants. The plates were examined for galacturonate+ or glucuronate+ transductants after 48 h of growth at 32°C. Symbols: 0, no transductants obtained; +, tranaductants obtained. The transduction frequency of the point mutants Aatt+ (TH6-MH2-RP1) with Xpexu2, -4, and -5 was about 1%. The transduction frequency of mutant 1576 with the same phages was less than 0.1%.

J uxa A uxa C

A

exu T

J

b2

opexul A

exu R ) int N Cl

R

P

X pexu 2 A

553

J uxaA uxaC

exuT

PI& (exR exuR

N Cl

R

NCI

R

XpLxu 3 FIG. 4. Diagrammatic representation of the phage cross Apexul x Apexu2 yielding Apexu3 carrying exuRexuT-uxaC-uxaA.

554

MATA, DELSTANCHE, AND ROBERT-BAUDOUY

stitutivity. For both exuR mutations, recombinants were obtained with Xpexu2 but not with Xpexul. This result suggests that the two exuR mutations were located in the left part, (exuR'), of the exuR gene. The same experiment was carried out with Xdexu2. Recombinants were obtained with RC1 infected by Xdexu2. HJ1 gave no recombinants. This result is consistent with the view that Xdexu2 carries a part of the exuR gene but that this portion is smaller than the (exuR') part. These two exuR mutations are not located at the same point of the exuR gene, which corroborates unpublished results (Portalier et al. and Robert-Baudouy et al., unpublished data) and places the exuRAUl mutation closer to exuT than exuRAl. TABLE 4. Activity of the exu regulon enzymes in differential rates of synthesis (percent of maximal value induced in the wild type) in strain 1576 before and after transduction by Apexu3 (k recombinant phage carrying exuR-exuT-uxaA-uxaC) Relative differential Strain

Inducer

rate of synthesis (%) in enzyme:

II

IV' 6

None Galacturonate

3 100

100

IHl 4 100

1576

None Galacturonate

0 0

0 0

395 320

1576, (Apexu3)

None Galacturonate

0 113

4 110

112

HfrH

2

J. BACTERIOL.

Deletion mutants formed on induction of HfrH 58. When HfrH 58 is incubated at 420C, most cells die because phage functions lethal to the host are induced. In the rare heat-resistant survivors, phage genes specifying or controlling these lethal functions have been deleted (17). Among the survivors, one would expect to find deletions extending into adjacent bacterial genes, i.e., in the exu region. For such an experiment, a Xvir-resistant derivative of strain HfrH 58 was examined. Survivors were selected on tryptone broth or galacturonate MacConkey medium or on minimal media supplemented with galacturonate, tagaturonate, or altronate as the sole carbon source. The growth of deletion mutants on these minimal media requires the presence of genes involved in hexuronate dissimilation. Deletions to various lengths in the exu region were expected by this selection (see above), but it was not possible to obtain deletions extending into exuT or exuT-uxaC while keeping uxaA undamaged. To explain this behavior, hypotheses are given in the discussion section.

The frequency of thermal curing of HfrH 58 was about 10- for survivors selected on rich media and about 10' for survivors selected on minimal media. Survivors were purified and analyzed for residual phage and hexuronate genes (Fig. 6). Their growth phenotypes were examined, and activities of enzymes II, m', and IV' were measured in the presence or absence of inducer (Table 2). Three classes of survivors, A, B, and C, were obtained. Class B mutants were isolated

deletion ex

1576 exuT

F

AexuR"

uxaC

Apexu2

R,

uxaC I uxa A

bexuT

exu R

F

J uxaA uxaC

exuT

(exuR) int Cl RA J (exuW)

FIG. 5. Diagrammatic representation of the events leading to lysogenization of strain 1576 by phage Apexu2. Integration occurs (a) by reciprocal recombination between AP on the phage and PA', nucleotide sequence of strain 1576; or (b) by a reciprocal recombinational event involving homologous sequences on the phage and chromosome (F to J).

SPECIALIZED TRANSDUCING PHAGES IN E. COLI

VOL. 133, 1978 HfrH

uxaA uxaC

exuT

(exuR')N P Q S R A C D E F

1576

_ _ _ _ R

1573

1574 XE-

_

_

_

_4 _+

T

+ +4

K(exuR")

E + -.E1+

+ + + +

+ + +

555

4

+

class B

4-

B

4 +

B

+ r-N+ + C + + + 1693, 4 1694 ' FIG. 6. Prophage deletion mapping of HfrH 58. Derivatives of strain HfrH 58 able to grow at 42°C were scored for phage and bacterial markers as follows. Detection of residual phage markers is as described by Shimada et aL (18). The bacterial DNA deletions were deternined by the growth phenotype on hexuronates and by measure of enzymes II, IIr, IV' activity (Table 2). Class B deletion was studied by cross-streaking the deleted mutants with mutants containing various point mutations in the structural genes of the exu region. *, Part of this gene is present.

only on galacturonate MacConkey medium, and classes A and C could be obtained on each medium mentioned above. Class A (95% of survivors). These mutants have wild-type behavior and catabolize galacturonate and glucuronate. The product of the exuR gene is functional; enzymes II, 111, and IV' are inducible by fructuronate, and their differential rates of synthesis are identical to those obtained with a wild-type strain (Table 2). The deletion has eliminated the entire prophage. Class B (0.6% of survivors). This class grows on fructuronate but not on aldohexuronates and tagaturonate. Enzymes II and IV' are not inducible by fructuronate, and the synthesis of enzyme IH' is constitutive (Table 2). The aldohexuronate transport activity is much lower than that of the wild-type strain (18%). This basal level is identical to that of a strain bearing a mutation in the structural gene of the permease (8).

For genetic analysis, 15 independent mutants were examined. The mutations were transferred in a F- strain, EWlb. Presence of bacterial genes uxaA, uxaC, and exuT was tested by crossstreaking the F- class B strains with Hfr mutants bearing various independent point mutations in the structural genes of the exu region; 10 uxaA, 4 uxaC, and 11 exuT Hfr were used (8, 9, 10). Recombinants were selected on galacturonate and glucuronate minimal media. F- class B mutants were unable to recombine with uxaC and exuT Hfr. The class B deletion extends at least into exuT and uxaC genes. The same F- deletion mutants crossed with uxaA- Hfr gave no recombinants able to grow on galacturonate, but recombinants able to grow on glucuronate were

obtained. Class B strains transduced by a Apexu carrying exuT and uxaC but not uxaA genes (Xpexu5, Table 1) have the same behavior, transductants catabolize glucuronate but not galacturonate. All these data indicate that the class B deletion encompasses exuT, uxaC, and uxaA (Fig. 6). Presence of phage markers was analyzed for three class B strains (Fig. 6). Some phage genes were rescued in tests with Xsus lines; N, P, and Q A genes at least were deleted. Consequently, the left end, (exuR'), of the exuR gene, which is located between exuT and X insertion, is encompassed by the deletion. Class C (4% of survivors). This class can grow on aldohexuronates, thus indicating that none of the exuT, uxaA, and uxaC structural genes is deleted. The synthesis of enzymes II, I', and IV' is constitutive and identical to that of HfrH 58. Some phage markers are deleted (Fig. 3), and the exuR gene is inactivated as in HfrH 58 by the residual X genes.

DISCUSSION We have demonstrated that, in strain HfrH 58, phage A was inserted into the exuR gene and rendered it nonfunctional. Two classes of transducing phage were isolated by abnormal excision, Agal-type, transducing the left part of the exu region, and Xbio-type, transducing the right part. We isolated a X recombinant carrying the entire exu region (Xpexu3) by a cross between these two types of phage. The Xpexu3 isolation is sinilar to that described by Shimada et al. (19) for XptrpEDCBA phage. The formation of this phage occurs by an int-xis promoted recombination between secondary attachment

556

J. BACTERIOL.

MATA, DEISTANCHE, AND ROBERT-BAUDOUY

sites of the bacterium and phage chromosome, AP' and PA'. The length of bacterial DNA carried by each type of phage is being investigated using restriction enzymes and heteroduplex mapping. Our results indicate that the ?gal-type transducing phages derived from HfrH 58 and Xdexu2 isolated by Schrenk's method both carry a different portion of the exuR gene. It appears that phage X can integrate in the exuR gene at two different locations as in the araB gene (3). Isolation of X exu transducing particles and deletions is proving to be extremely useful in studies on the expression of the exu regulon. We have shown that strain 1576 (class B deletion) transduced by Xpexu5 (XpexuT-uxaC) is unable to dissimilate galacturonate because it lacks the uxaA gene, but grows on glucuronate. Since metabolism of glucuronate requires the expression of isomerase (enzyme II) coded for by the uxaC gene, the growth of 1576 (Xpexu5) on glucuronate suggests that the promoter of the uxaC-uxaA operon is located between exuT and uxaC, and that the transcription direction might be from uxaC to uxaA. Heat survivors selected from HfrH 58 were able to regenerate a functional exuR gene (consistent with a mechanism of int-promoted recombination [4]) or provided large deletions (class B) in the exu region involving (exuR')exuT-uxaC and uxaA with variable end points into the prophage. Another set of deletions extending only in (exuR')-exuT-uxaC was expected by selection on tagaturonate or altronate. Growth on these sugars requires only the expression of the uxaA gene. We assume that this selection was inadequate, since our results suggest that the promotor of the uxaC-uxaA operon is located between exuT and uxaC. It should be possible to obtain every kind of deletion on galacturonate MacConkey medium. We did not succeed in selecting them because the number of deletions analyzed was too small (genetic analysis was carried out on for 15 independent

deletions). Class B deletion was unable to catabolize galacturonate, tagaturonate, and glucuronate, but grew on fructuronate. This result confirms that the fructuronate transport system, which has not yet been located, is not affected by the deletion. In this same class of deletion, the basal level of enzyme THU is identical to that of a strain bearing a mutation in the structural gene of the transport system. Among negative exuT point mutations, none was found to completely abolish hexuronate transport activity (8). Since all the deletions also exhibit a residual THU activity, the result would suggest that another

transport system is responsible for slow aldohexuronate uptake in the cells. In the deletion strains as well as in strain HfrH 58, the activity of enzyme Il' is strongly derepressed, corresponding to a three- to fourfold higher level than the fully induced wildtype strain (Table 2). This suggests that the whole regulatory system involved in the synthesis of that enzyme is not functional in these strains. Consequently, the transcription of the structural gene of enzyme III' is probably subjected to the action of the exuR product. ACKNOWLEDGMaENS This work was supported by gants from the Centre National de la Recherche Scientifique (laboratoire propre du C.N.R.S. no. 05421) and from the Fondation pour la Recherche Medicale Fran9aise. We thank F. Stoeber for the encouragement he has given to this study. We are very grateful to M. E. Gottesman for the strain HfrH 58 and for helpful disctussions. We also thank C. Marchal and M. Hofnung of Institut Pasteur for fruitful advice, D. Atlan and S. Ottomani for skillful technical assistance, and Le Thy Bich Thuy.

LITERATURE CTE 1. Ashwell, G. 1962. Enzymes of glucuronic and galacturonic acid metabolism in bacteria. Methods Enzymol.

5:190-208. 2. Bachmann, B. J., K. B. Low, and A. L Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 3. Boulter, J., and N. Lee. 1975. Isolation of specialized transducing bacteriophage lambda carrying genes of the L-srabinose operon of Escherichia coli B/r. J. Bacteriol. 123:1043-1054. 4. Franklin, N., W. Dove, and C. Yanofsky. 1965. The linear insertion of a prophage into the chromosome of E. coli by deletion mapping. Biochem. Biophys. Res. Commun. 18:910-923. 5. Kayajanlian, G. 1968. Studies in the genetics of biotintransducing, defective variants of bacteriophage A. Vi-

rology 36:30-41. 6. Lindahl, G., G. Sironi, IL Bialy, and R. Calendar. 1970. Bacteriophage lambda; abortive infection of bacteria lysogenic for phage P2. Proc. Natl. Acad. Sci.

U.SAL 66:587-594.

7. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spnng Harbor Laboratory, Cold Spnng Harbor, N.Y. 8. Nemoz, G., J. Robert-Baudouy, and F. Stoeber. 1976. Physiological and genetic regulation of the aldohexuronate transport system in Escherichia coli. J. Bacteriol. 127:706-718. 9. Portalier, R., J. Robert-Baudouy, and G. Nemoz. 1974. Etude de mutations affectant les genes de structure de l'isom6rase uronique et de l'oxydor6ductase altronique chez Escherichia coli K 12. Mol. Gen. Genet.

128:301-319. 10. Portalier, R., J. Robert-Baudouy, and F. Stoeber. 1972. Localisation genetique et caract6risation biochimique de mutations affectant le gene de structure de l'hydrolyase altronique chez Escherichia coli K 12. Mol. Gen. Genet. 118:335-350. 11. Portalier, R., and F. Stoeber. 1974. La D-altronate: NAD-oxydoreductase d'Escherichia coli K 12: purification, proprietes et individualite. Eur. J. Biochem. 26:50-61.

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SPECIALIZED TRANSDUCING PHAGES IN E. COLI

12. Portalier, R., and F. Stoeber. 1972. La D-mannonate: NAD-oxydoreductase d'Escherichia coli K 12: purification, propri6tes et individualite. Eur. J. Biochem. 26:290-300. 13. Portalier, R., and F. Stoeber. 1972. Dosages colorimetriques des oxydor6ductases aldoniques d'Escherichia coli K 12: applications. Biochim. Biophys. Acta 289:19-27. 14. Robert-Baudouy, J., RE Portalier, and F. Stoeber. 1974. R6gulation du metabolisme des hexuronates chez Escherichia coli K 12: modalit6s de l'induction des enzymes du systeme hexuronate. Eur. J. Biochem. 43:1-15. 15. Robert-Baudouy, J., and F. Stoeber. 1973. Purification et proprietes de la D-mannonate hydrolyase d'Escherichia coli K 12. Biochim. Biophys. Acta 309:474-485. 16. Schrenk, W. J., and R. A. Weisberg. 1975. A simple method for making new transducing lines of coliphage A. Mol. Gen. Genet. 137:101-107. 17. Shapiro, J. A., and S. L Adhya. 1969. The galactose

operon of E.

coli K 12.

1.

557

A deletion analysis of operon

structure and polarity. Genetics 62:249-264.

18. Shimada, K., R. A. Weisberg, and KL E. Gottesman. 1972. Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J. Mol. Biol. 63:483-503.

19. Shimada K., R. A. Weisberg, and K. E. Gottesman. 1973. Prophage lambda at unusual locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K 12. J. Mol. Biol. 80:297-314. 20. Sistrom, W. R. 1958. On the physical state of the intracellularly accumulated substrates of ,B-galactoside permease in Escherichia coli. Biochim. Biophys. Acta 29:579-587. 21. Stoeber, F., A. Lagarde, G. Nemoz, G. Novel, M. Novel, R. Portalier, J. Pouysegur, and J. RobertBaudouy. 1974. Le metabolisme des hexuronides et des hexuronates chez Escherichia coli K 12: aspects physiologiques et genetique de sa regulation. Biochimie 56:199-213.

Isolation of specialized transducing bacteriophages carrying the structural genes of the hexuronate system in Escherichia coli K-12: exu region.

Vol. 133, No. 2 JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 549-557 0021-9193/78/0133-0549$02.00/0 Printed in U.S.A. Copyright 0 1978 American Society...
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