JOURNAL OF BACTERIOLOGY, Mar. 1979, p. 1253-1262 0021-9193/79/03-1253/10$02.00/0

Vol. 137, No. 3

Salmonella typhimurium newD and Escherichia coli leuC Genes Code for a Functional Isopropylmalate Isomerase in Salmonella typhimurium-Escherichia coli Hybrids PATRICIA N. FULTZ, DEBORAH Y. KWOH,t AND JOST KEMPER* Institute ofMolecular Biology, University of Texas at Dallas, Richardson, Texas 75080 Received for publication 20 November 1978

The supQ newD gene substitution system in Salmonella typhimurium restores leucine prototrophy to leuD mutants by providing the newD gene product which is capable of replacing the missing leuD polypeptide in the isopropylmalate isomerase, a complex of the leuC and leuD gene products. Mutations in the supQ gene are required to make the newD protein available. An Escherichia coli F' factor was constructed which carried supQ- newD+ from S. typhimurium on a P22-specialized transducing genome. This F' pro lac (P22dsupQ394newD) episome was transferred into S. typhimurium strains containing the leuD798-ara deletion; the resulting merodiploid strains had a Leu+ phenotype, indicating that supQ- newD+ is dominant over supQ+ newD+, and eliminating the possibility that the supQ gene codes for a repressor of the newD gene. Furthermore, transfer of the F' pro lac (P22dsupQ394newD) into E. coli leuD deletion strains restored leucine prototrophy, showing that the S. typhimurium newD gene can complement the E. coli leuC gene. Growth rates of the S. typhimurium-E. coli hybrid strains indicated that the mutant isopropylmalate isomerase in these strains does not induce a leucine limitation, as it does in S. typhimurium leuD supQ mutants. In vitro activity of the mutant isopropylmalate isomerase was demonstrated; the Km values for a-isopropylmalate of both the S. typhimurium leuC-newD isomerase and the S. typhimurium-E. coli hybrid isomerase were as much as 100 times higher than the Km value for a-isopropylmalate of the wild-type enzyme, which was 3 x 10-4 M. Mutagenesis of E. coli leuD deletion strains failed to restore leucine prototrophy, indicating that E. coli does not have genes analogous to the S. typhimurium supQ newD genes, or that, if present, activation of a newD gene is a rare event or is lethal to the cell.

The leucine operons in Salmonella typhimurium and Escherichia coli consist of four structural genes, leuABCD (16, 26, 27). Isopropylmalate isomerase, the second enzyme specific for the biosynthesis of leucine, is a multimeric enzyme, which interconverts a-isopropylmalate and f8-isopropylmalate, and is coded for by the leuC and leuD genes (6). In vivo complementation of these two genes has been demonstrated in both species by abortive transduction analysis (16, 27) as well as in S. typhimurium-E. coli hybrids (26). E. coli F' factors carrying leuC+Dor leuC-D+ were transferred into S. typhimurium strains which were leuC-D+ or leuC+D-, respectively. The resulting Leu+ phenotypes showed that the leuC and leuD genes in these species produce polypeptides which are not only genetically equivalent but also capable of forming a functional isopropylmalate isomerase (26), t Present address: Cold Spring Harbor Laboratory of Quantitative Biology, Cold Spring Harbor, NY 11724.

even though there is only 4% DNA homology between the two leucine operons (17). The supQ newD gene substitution system in S. typhimurium provides a polypeptide which is also functionally equivalent to the leuD gene product. leuD mutants, including those with deletions of the entire leuD gene, can be restored to leucine prototrophy if there is an additional mutation in the supQ gene which lies adjacent to newD and between proAB and proC. It was previously suggested (9, 10), in a model for the mechanism of suppression of leuD by supQ mutations, that the supQ and newD gene products form a complex which prevents the availability of the newD gene product (Fig. 1). An alternate possibility, that supQ coded for a repressor of the newD gene, was also consistent with the original data. The function(s) of the proteins produced by the supQ-newD genes is unknown. If the supQ gene is deleted or mutated in leuD mutants, then the newD gene product is made

1253

1254

J. BACTERIOL.

BULTZ, KWOH, AND KEMPER

leuC

louD

supO

louD

leuC

active IPM isomerase (leuC+ lOuD)

I.

leuC

leuD

newD

SUCQ

no leuC subunits available

leuC

I

active enzyme complex (?) (supQ+newD) no newD subunits available

supQv

I

pointmutation

new D

1

newD

I

pointmutation

or deletion I*****..l

or

deletion

U*******I

newD

active IPM isomerose

(leuC+newD)

,.

l

FIG. 1. Model for supQ suppression of leuD mutant strains.

available to complex with the leuC gene product and isopropylmalate isomerase activity is restored (9-11). Because of the complementation between the S. typhimurium leuD and E. coli leuC gene products, we wanted to see (i) whether the S. typhimurium newD gene product could also complement the E. coli leuC polypeptide and (ii) whether E. coli carries genes analogous to supQ and newD. To test whether the S. typhimurium newD can complement the E. coli leuC gene, we took advantage of the fact that E. coli carries an attachment site for the S. typhimurium phage P22 (8) and also that supQ newD specialized transducing particles generated by P22 had been previously identified (12). However, since E. coli does not adsorb P22, an F' factor carrying the proAB-attP22-lac region of the E. coli chromosome and residing in S. typhimurium was utilized. Such F' factors were used previously to demonstrate pro-specialized transduction by P22 (8). The isolation of an F' factor carrying the supQ- newD+ genes also allowed us to construct S. typhimurium strains which are merodiploid for supQ newD. Using these strains, we demonstrated that supQ- newD+ is dominant over

supQ+ newD+, thus eliminating the possibility that the supQ gene product is a repressor of the newD gene and supporting the model of a supQnewD gene-product complex. The presence of an active isopropylmalate isomerase in leuDsupQ- mutant strains, previously proven by genetic analysis (9, 10), was verified by demonstrating in vitro isopropylmalate isomerase activity. It should be pointed out that the E. coli gene supQ, characterized in 1971 (22), is unrelated in map position and function to the supQnewD system of S. typhimurium discussed in this report. MATERIALS AND METHODS Bacterial and phage strains. The bacterial strains used are listed in Table 1. All S. typhimurium strains are derivatives of LT2, and all E. coli strains are derivatives of B/r. Phages used were P22 wild type and mutant strains c2-5, inm, and c2ts29, c2ts29 obtained from M. Levine. Media. Bacteria to be used in transductions and for preparation of phage lysates were grown in Luria broth (13). For growth rate determination and preparation of cell-free extracts, bacteria were grown in minimal medium (MM), which contained, per liter of water: K2HP04, 10.5 g; KH2PO4, 4.3 g; Na3-citrate * 3H20, 0.47 g; (NH4)2S04, 1.0 g; Mg2SO4.7H20, 0.1 g; and 0.2%

VOL. 137, 1979

leuC-newD ISOPROPYLMALATE ISOMERASE

1255

TABLE 1. Bacterial strains Strain

Source

Genotype

S. typhimurium ara9 (wild type) ara9 leuB 698 leuB698 A(leuD-ara)798 JK63 A (leuD-ara) 798 fol_Olla JK66 A(leuD-ara) 798 fol-101 proAB47 JK74 proAB47purE66/F'proAB+ lac+ JK194 proAB47purE66/F' lac+ proC+ JK195 A(leuD-ara)798fol-lOlproAB47/F'proAB+ lac+ JK254 A(leuD-ara) 798 fol-101 proAB47/F' proAB+ JK352 (P22dsupQ394newD+) (P22c2ts29) A(leuD-ara)798 supQ394 (P22c2ts29) JK367 A(leuD-ara) 798 fol-101 proAB47 (P22) JK415 A(leuD-ara) 798 supQ394 JK1394 ilvA- purC- purI proA rpsLb JK393

lac+

JK414

A(leuD-ara) 798 fol-101 ilvA- purC- purI- proA- rpsL-

JK417

A(leuD-ara)798 fol-101 ilvA- purC- purI- proA- rpsL(P22) A(leuD-ara)798 fol-101 ilvA- purC- purlI proA- rpsL(P22)/F' proAB+ lac+ (P22dsupQ394newD+) (P22c2ts29) proAB47purE66 A(leuD-ara) 798 fol-101 proAB47purE66

JK476 JK404 JK412 JK416 JK481 JK134 JK468 E. coli B/r JK518 JK519 JK520 JK522

A(leuD-ara) 798 fol-101 proAB47purE66 (P22) A(leuD-ara) 798 fol-101 proAB47 purE66 (P22)/F' proAB+ lac+ (P22dsupQ394newD+) (P22c2ts29) A(leuD-ara) 798 fol-101 proB25 supQ394 A(leuD-ara) 798 fol-101 proB25 supQ394 (P22)

K. Sanderson P. Margolin J. Kemper J. Kemper J. Kemper J. S. Gots, E66F' pro lac J. S. Gots, E66F'13 JK74 x JK194, select Lac' JK254 x P22 induced from JK367, select Leu+ Lysogen of JK1394 Lysogen of JK74 J. Kemper J. S. Gots, SL751 cured of F'pro lac JK393 x P22 on JK66, select TMP' Lysogen of JK414

JK417 x JK352, select Pro' Strr JK194 cured of F' pro lac JK404 x P22 on JK66, select TMP' Lysogen of JK412 JK416 x JK476, select

Pro' JK79 x P22 on JK1394, select Leu+ Lysogen of JK134

Wild type H. Bremer leuD1198 = A(leuD-ara) thr-1 araD139 rpsL-dau-5 E. Englesberg, SB1598 leuD1188 = A(leuD-ara) thr-1 araD139 rpsL- dau-5 E. Englesberg, SB1588 leuCD1195 = A(leuCD-ara) thr-1 araD139 rpsL- dau-5 E. Englesberg, SB1595 leuD1198 thr-1 araD139 rpsL- dau-5/F' proAB+ lac+ JK518 x JK476, select (P22dsupQ394newD+) (P22c2ts29) Leu+ JK523 leuD1188 thr-1 araD139 rpsL- dau-5/F' proAB+ lac+ JK519 x JK476, select (P22dsupQ394newD+) (P22c2ts29) Leu+ afol-101 is co-transducible with A(leuD-ara) 798 and renders the cell resistant to trimethoprim (i.e., TMPr). brpsL- renders the cell resistant to streptomycin (i.e., Strr).

glucose. Amino acids, when required, were added to give a final concentration of 40 jtg/ml. Solid MM contained 1.5% agar and, when enriched for transductions and mutagenesis, 1.25% (vol/vol) reconstituted Difco nutrient broth. Transductions. Recipient cells grown overnight in Luria broth were incubated for 6 min at 37°C with an equal volume of diluted phage lysate to yield a multiplicity of 15 PFU/bacterium. The transduction mixture was plated on selective plates and incubated at 37°C. To obtain phage-sensitive (i.e., nonlysogenic) recombinant strains, P22int4, an integration-defective mutant was used. Phage lysates were prepared either by lytic infection with wild-type or int4 phage or by thermal induction of lysogens carrying prophages with a temperature-sensitive mutation in the repressor gene C2.

F' transfer. The recipient and donor strains used

for F' transfer were grown in Luria broth. A 2-ml amount of a log-phase culture (absorbancy at 460 nm 0.7) of the donor strain and 1 ml of a stationaryphase culture of the recipient strain were added to 2 ml of Luria broth and incubated at 37°C for 1 h with no aeration. After an additional 1 h with aeration, the mixture was plated on selective media and incubated at 37°C. Curing of F' episomes by acridine orange was done by the method of Miller (18). Preparation of extracts. Cells were grown in 500 ml of MM to an absorbancy at 520 nm of 0.8, centrifuged, washed, and resuspended in 5 ml of cold 0.1 M potassium phosphate, pH 7. The resuspended cells were disrupted by sonic oscillation with an MSE sonicator at 21 kc per s for 30 s and were centrifuged at 30,000 x g for 20 min to remove cell debris. Enzyme extracts were prepared at 4°C, kept on ice, and assayed immediately after centrifugation. Protein concentra-

1256

BULTZ, KWOH, AND KEMPER

J. BACTERIOL. crassa (4) and S. typhimurium (P. Fultz et al., in preparation); some a- and,t8-isopropylmalate were generously provided by J. Calvo. Mutagenesis. Mutagenesis by 2-aminopurine and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was done as previously described (9). Mutagenesis by nitrous acid followed the procedure of Miller (18).

tion was determined by the method of Lowry et al. (15), with bovine serum albumin as the standard. Enzyme assays. Isopropylmalate isomerase was assayed with a- or,B-isopropylmalate as substrate and by following the appearance of the intermediate dimethylcitraconate at 235 nm in a Gilford recording spectrophotometer. The procedure of Gross et al. (6) was modified such that the reaction mixture contained 5 ,Lmol of neutralized a-isopropylmalate, 20 ,umol of potassium phosphate, pH 7, and 0.1 ml of diluted extract (approximately 0.1 mg of protein) in a total volume of 1 ml. The reaction temperature was 320C. For in vitro assays of mutant isopropylmalate isomerase, concentrations of a-isopropylmalate as high as 150 ,umol per assay were used. /B-Isopropylmalate dehydrogenase activity was determined by measuring the production of a-ketoisocaproate colorimetrically as described by Parsons and Burns (19). The substrate a-isopropylmalate was purified from Neurospora

S.typhimurium

E. coili

r:pcpD

pepD

gpt

gpt

proBA

proBA

'

-l

7.3

into an S. typhimurium strain carrying the leuD798-ara deletion and the proAB47 mutation which deletes proAB, the P22 attachment site, ataA, and the supQ newD genes. This strain, JK254, was infected with phage from a

FI8pro loc

F

5,

i

>. to a

RESULTS Construction of an FP factor carrying the newD gene. F'pro lac, which carries the E. coli wild-type genes proAB and lac as well as the P22 attachment site (see Fig. 2), was transferred

atoA

0 nowD

attP22

6

argF cxm trmB

7.

locAYZO

U otbB 8J hemB

loci hemB brnR

8

brnQ

brnQphoA proC

* proC * hsdL

phoB R tsx

U

9x * * thiC opeB

argF

13

F

IEoc

I

'lac proC

tsx

Ion

*U omk aptU U U

Il

lc-e

I:Iq

i1

:. I

gpt proBA

attP22 supQ U newD

0

genes

attP22 pro-

phoge

attP22 argF

dnaZ popA 11.

F28 pro Jac (P22d supQ newD) (P22)

iF

minA acrA

10

11-3

I

ottP22

srnA

0 U

U

codBA

gpt proBA

pisA

*opt Al

F

* suf F, 12-

U U

purE

i2. I

purE

purE

!U FIG. 2. Comparison of the S. typhimurium and E. coli linkage maps. Gene sequence and symbols are based on the 100-min map for E. coli K-12 (1) and on the 100-min map for S. typhimurium (23). The continuity of the S. typhimurium chromosome is interrupted for better alignment with the E. coli chromosome. The exact end points of the E. coli F factors are not known (14). The arrow indicates the point of F-factor integration into the parental chromosome, as well as the direction of transfer. The size of a phage P22 genome or of a transducing genome corresponds to approximately 1 min (1%) of the bacterial chromosome.

VOL. 137, 1979

leuC-newD ISOPROPYLMALATE ISOMERASE

lysate made by thermal induction of the lysogenic strain JK367, which carries a P22c2ts29 prophage. JK367 also contains the leuD798-ara deletion and the supQ394 deletion which brings the newD gene into close proximity to ataA. Upon induction, specialized transducing genomes composed of phage DNA, a hybrid attachment site, the supQ394 mutation, and the newD gene are generated and can be used to transduce leuD mutants to Leu+. (For strain constructions see also Table 1). Since there is no region of homology between the transducing genome and the chromosome of the recipient, JK254, Leu+ colonies can be obtained only if the transducing genome integrates into the bacterial chromosome at a secondary attachment site or into the F' at attP22. Leu+ transductants were selected on enriched MM (glucose) plates and then replica-plated onto a lactose medium to see if they had retained the F' factor (Lac' phenotype). A Leu+ Lac' Pro' clone (JK352) which was identified as a stable lysogen (i.e., carrying a complete P22 prophage) by use of special indicator plates (25) was characterized as follows. (i) After acridine orange curing, the strain segregated LeuLac- Pro- colonies, indicating that the P22dsupQ394newD transducing genome had integrated into the F' at attP22 and not at a secondary attachment site on the bacterial chromosome. These segregants were P22 sensitive, which suggests that the strain did not contain a prophage in the bacterial chromosome and that the complete prophage was carried on the F' factor. (ii) JK352 was shown to transfer the F' to a Pro- Leu- S. typhimurium strain which carried a stable P22 prophage to prevent zygotic induction. The Leu+ colonies obtained were simultaneously Lac+ Pro'. (iii) Growth of JK352 under nonselective conditions segregated LeuPro' Lac+ derivatives, indicating loss of the P22dsupQ394newD specialized transducing genome from the F' pro lac. Construction of JK476, an S. typhimurium strain diploid for supQ newD. The F' pro lac (P22dsupQ394newD) was transferred into a Pro- S. typhimurium strain, JK417, containing the leuD798-ara deletion. Pro+ colonies were selected on enriched leucine plates, and the Leu phenotype of the colonies was determined by replica plating. All of the Pro' colonies obtained were also Leu+, indicating that the supQnewD+ genotype is dominant over the supQ+ newD+. This also suggested that, if E. coli did possess genes equivalent to supQ and newD, then selection for Leu+ colonies upon transfer of the F' pro lac (P22dsupQ394newD) into leuD mutants might be possible.

1257

Transfer ofFpro lac(P22dsupQ394newD) into E. coli Using JK476 as a donor, F' pro lac (P22dsupQ394newD) was transferred into two different E. coli leuD deletion strains (JK518 and JK519) by selecting for Leu+ colonies, the only selection possible with these strains. Leu+ colonies were obtained with both recipient strains, but the number of Leu+ colonies was low. The low efficiency could have been due to zygotic induction and/or to differences in the restriction and modification systems of S. typhimurium and E. coli. One Leu+ colony from each mating was selected, and the presence of the F' was verified by (i) the ability to transfer the F' back to JK415, which simultaneously acquired a Leu+ Pro' Lac' phenotype, and (ii) segregating Leu- colonies after acridine orange curing. Each of the parent leuD deletion strains was also tested for spontaneous reversion to Leu+. No spontaneous Leu+ colonies were found, which supported the conclusion that the strains were Leu+ because of the F' pro lac (P22dsupQ394newD). Also, since the S. typhimurium newD gene product can substitute for the missing E. coli leuD polypeptide, we conclude that E. coli has no function, e.g., a repressor, which interferes with newD gene expression. As an additional control, JK476 was mated with an E. coli strain which carried a deletion of both the leuC and leuD genes. No Leu+ colonies were found, which implies that, as in S. typhimurium, both the leuC and newD (or leuD) genes must be present for a functional isopropylmalate isomerase. In vivo expression of newD in S. typhimurium and E. coli It was previously shown that S. typhimurium leuD supQ mutants have a longer doubling time in minimal than in leucinesupplemented medium (9). Table 2 indicates that this is true whether the newD gene is carried on the bacterial chromosome or on an F' factor (JK1394, JK481). The ratio of the doubling time in leucine-supplemented medium to that in MM, L/M (Table 2), reflects the efficiency with which the leuC-newD isopropylmalate isomerase can provide ,B-isopropylmalate for the biosynthesis of leucine. [The doubling times in leucine-supplemented medium of strains carrying the F'pro lac (P22dsupQ394newD) are longer than the doubling time of JK1394, the strain without an F', because of their lysogenic state (compare with JK468, Table 2).] Because the two E. coli leuD deletion mutants containing the F' pro lac (P22dsupQ394newD) (Table 2, JK522 and JK523) have the same doubling times in minimal and leucine-supplemented medium, we conclude that the newD gene product is not limiting the growth of these

1258

BULTZ, KWOH, AND KEMPER

J. BACTERIOL.

TABLE 2. Growtha of leuD supQ mutants Doubling timeb

supQ newD allele Strains

S. typhimurium ara9 JK1394 JK468C JK476

F' factor/chromosome

-/supQ+ newD+ -/supQ394 newD+ -/supQ394 newD+ supQ394 newD+/ supQ+ newD+ supQ394 newD+/-

(min) + L/M Leu- Minicine mal

(L)

(M)

52 52 63 60

52 58 70 67

1.00 0.90 0.90 0.90

JK481 58 66 0.88 E. coli JK522 supQ394 newD+/?d 68 68 1.00 JK523 supQ394 newD+/?d 70 70 1.00 a Doubling times were determined by growing the strains in minimal and leucine-supplemented media at 37°C in a New Brunswick water-bath shaker and following the absorbancy at 460 nm in a Zeiss M4 QIII spectrophotometer; all media contained any additional supplements required by the individual strains. bValues given are the average of at least three determinations. independent C P22 lysogen. d It is not known whether E. coli contains supQ newD alleles; see text.

strains. This could be due to a higher level of expression of the newD gene in E. coli than in S. typhimurium, a greater affinity of the E. coli leuC than the S. typhimurium leuC polypeptide for the newD gene product, or a greater affinity of the E. coli leuC-newD complex for the enzyme

substrate, a-isopropylmalate. In vitro activity of the leuC-newD isopropylmalate isomerase. From the growth rate difference of S. typhimurium leuD supQ mutant strains in miniimal versus leucine-supplemented medium, we theorized that the leucine operon would be maximally derepressed. This was verified by in vitro assays of the ,8-isopropylmalate dehydrogenase (coded for by the leuB gene) (compare lines 1, 3, and 4, Table 3). The in vitro specific activity of the mutant isopropylmalate isomerase, however, was expected to be significantly lower than the specific activity of the wild-type isopropylmalate isomerase. Also, the mutant isopropylmalate isomerase, a complex of the leuC protein and the newly recruited newD protein, might have a lower affinity than the wild-type enzyme for the substrate. Both assumptions were shown to be true, for only very small amounts of isomerase activity were detected when the mutant enzyme was assayed under the same conditions used for the wild-type enzyme (line 4, Table 3). However, when in-

creased levels of a-isopropylmalate were used in assaying crude extracts of leuD supQ mutants, isopropylmalate isomerase activity was easily demonstrated. Assays using crude extracts of strains with a wild-type isopropylmalate isomerase contain a-isopropylmalate at a concentration of 5 x 1iO3 M, sufficient for substrate-excess conditions since the Km for a-isopropylmalate of the wild-type enzyme is 3 x 1O' M (unpublished data). Preliminary data indicate that the S. typhimurium leuC-newD mutant isomerase has an approximately 100-fold higher Km (-3 x 102 M); the assays using extracts of these mutant strains are run with an a-isopropylmalate concentration of 1.5 x 10-1 M, which should represent a condition of substrate excess. The enzyme

activity observed when using the high concentrations of a-isopropylmalate was shown to be specific for the isopropylmalate isomerase because no activity was detected when a crude extract of a leuD mutant strain was assayed with a-isopropylmalate at high concentrations (line 3, Table 3). A more detailed determination of Km values and characterization of the mutant isopropylmalate isomerase activity is difficult because of the apparent high instability of this enzyme, which appears to be significantly more unstable than the wild-type enzyme. In addition, Table 3 shows comparative values of the specific activity of isopropylmalate isomerase in S. typhimurium as well as in E. coli and S. typhimurium-E. coli hybrid strains. Strain leuB698, grown under conditions which result in derepression of the leucine operon, shows approximately six-fold higher levels of wild-type isopropylmalate isomerase activity (compare lines 1 and 2). The specific activity of the leuCnewD mutant isopropylmalate isomerase is lower than the isopropylmalate isomerase activity for strain ara9, even though the mutant strains have a fully derepressed leucine operon. This correlates well with the fact that the mutant strains are limited in their growth by expression of the leucine operon. Isopropylmalate isomerase assays of crude extracts ofJK1394 and JK476, a heterozygous merodiploid strain containing supQ+ newD+ on the chromosome and supQ394 newD+ on an F' factor, show approximately the same in vitro specific activity. However, JK481, containing supQ- newD+ alleles on an F' factor and no supQ newD genes on the chromosome, seems to have in vitro a somewhat higher specific activity, although in vivo this strain does not show an improved L/M value. The higher specific activity is probably the result of less loss of enzyme activity than occurred with JK1394 and JK476 after sonic treatment and before the assays were conducted;

leuC-newD ISOPROPYLMALATE ISOMERASE

VOL. 137, 1979

TABLE 3. Specific activitiesa of isopropylmalate (IPM) isomerase and f-IPM dehydrogenase in wildtype and leuD-supQ mutant strains

Strain'

IPM isomerae

IPM isomerase

ersp

sp act

a-IPM used

,B-IPM dehy-

per asy

gena-se

dro-

(pamol) sp act S. typhimurium 5.12 5 3.2 1. ara9 WT 0 5 2. leuB698d WT 20.4 25.3 150 3. JK63 d None 0 33.8 4. JK1394 0.1 5 Mutant 5. JK1394 150 Mutant 1.5 NTe 150 6. JK476 Mutant 2.0 NT 150 Mutant 3.4 7. JK481 E. coli WT 5.7 5 8. B/r 4.3 8.5 35 Mutantf 1.2 9. JK522 a Specific activity is expressed as micromoles of product formed per hour per milligram of protein. b Cell-free extracts were prepared from cultures grown in MM with appropriate supplements and in the absence of leucine, except leuB698 and JK63 (see

d). eIPM isomerase: WT is a complex of the leuC and leuD gene products; mutant is a complex of the leuC and newD gene products. d Strains leuB698 and JK63 were grown under leucine limitation (10 ,ug of leucine/ml and 50 ,ug of isoleucine/ml added to MM) to insure derepression of the leucine operon. ' NT, Not tested. f Hybrid IPM isomerase: E. coli leuC and S. typhimurium newD gene products; see text.

this is probably due to an overall higher protein concentration in the crude extract, a factor which has been shown to stabilize the isomerase (unpublished data). It is also important to remember that the intracellular concentration of a-isopropylmalate is not comparable to that used to assay for the mutant enzyme; therefore, the fact that JK481 has the same in vitro specific activity as strain ara9 has no relationship to the growth of these two strains or to isopropylmalate isomerase activity in vivo. Table 3 also shows that the mutant isopropylnalate isomerase was detectable in an E. coli strain (JK522) carrying the S. typhimurium newD gene on an F' factor. This E. coli-S. typhimurium hybrid strain grows with approximately the same growth rate in minimal as in leucine-supplemented media, suggesting that the growth is not limited by the leuC-newD mutant isopropylmalate isomerase. This was reflected in the in vitro assays of the hybrid isopropylmalate isomerase, which indicated a Km value for a-isopropylmalate that is intermediate between the Km values of the wild-type and the

1259

S. typhimurium leuC-newD enzyme. The specific activities of the E. coli and S. typhimurium wild-type isopropylmalate isomerases (lines 1 and 8, Table 3) are comparable, as are those of both mutant isopropylmalate isomerases (lines 5 and 9, Table 3). This may indicate that, in the E. coli strains with the F' factor, the L/M value of 1 reflects a greater affinity of the hybrid isopropylmalate isomerase for a-isopropylmalate and is not due to a difference in transcription and/or translation of the newD gene in E. coli as compared to its expression in S. typhimurium. Specific activity values obtained when fl-isopropylmalate was used as a substrate in the in vitro assays paralleled those found with a-isopropylmalate. However, crude extracts of all strains were not assayed with f-isopropylmalate because only limited amounts were available. Mutagenesis of an E. coli leuD mutant strain. To determine whether E. coli has the potential to provide a polypeptide analogous to the S. typhimurium newD gene product, JK518 was mutagenized with 2-aminopurine, NTG, and nitrous acid. To determine the efficiency of the mutagenesis procedures, S. typhimurium strain JK63 was treated in an identical way. Selection, at 24 and 370C, was for Leu+ on enriched prolinethreonine plates. Proline was added since many supQ mutations in S. typhimurium are deletions which extend into the proAB genes. No E. coli Leu+ colonies were found either spontaneously or after treatment with the mutagens; however, Leu+ colonies did arise after treatment of the S. typhimurium strain JK63 with all three mutagens. For example, 424 Leu+ revertants of JK63 were found after nitrous acid mutagenesis, which represents a mutation frequency of 2 x 10'8. No E. coli Leu+ revertants were found after an identical treatment of 6 x 10"1 cells. Therefore, either E. coli does not have genes similar to supQ and newD or, if the potential for leuD suppression does exist, its activation is a very rare event. It might require either multiple mutations or the inactivation of a gene essential for growth. If multiple mutations are required in genes that are contiguous, then mutagenesis with nitrous acid or NTG could have resulted in Leu+ colonies, since nitrous acid can cause deletions (24) and NTG is known to cause multiple, neighboring mutations (7). If the generation of an E. coli newD gene product inactivates an essential gene, then selection for a mutation analogous to supQ might be possible in an S. typhimurium background. Because of considerable homology between S. typhimurium and E. coli with respect to the map position of genes (see Fig. 2), we utilized two F' factors containing the portion of the E. coli

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J. BACTERIOL.

substrate, suggesting a lower K,m,, and (ii) the finding that shorter doubling times in minimal medium are obtained for leuD- supQ- mutant strains carrying additional mutations which result in overproduction of a-isopropylmalate (manuscript in preparation). It might be interesting to see whether secondary mutations can be selected which lower the Km of the leuCnewD isopropylmalate isomerase and whether such mutations are alterations of the leuC or the newD gene. Such experiments are currently in DISCUSSION progress. Failure to obtain Leu+ colonies after extensive The construction of an F' factor carrying a supQ394 newD specialized transducing genome mutagenesis of E. coli leuD mutants indicates: has made it possible to characterize the suppres- (i) that E. coli has no genes analogous to supQ sion of leuD mutants in S. typhimurium and E. and newD; or (ii) that, if present, the wild-type coli. The finding that supQ- newD+ is dominant supQ gene in E. coli has a function essential for over supQ+ newD+ eliminates the idea proposed growth, in contrast to the supQ gene in S. typhiearlier that the supQ protein could be a repres- murium; or (iii) that multiple mutations are sor of the newD gene, and strengthens the pro- required to make the newD gene product availposed model (10) that the supQ and newD gene able. The latter is possible since in S. typhimurium supQ mutations showing high efficiency of products form a complex (Fig. 1). The slower growth rate of leuD- supQ- mu- suppression of leuD mutations were found more tant strains in MM than in leucine-supple- frequently after mutagenesis with nitrous acid mented medium indicates that the activity of than with 2-aminopurine, and many of these the mutant isopropylmalate isomerase is were found to be deletions (9). Furthermore, growth-rate limiting. This is apparently due to temperature-sensitive supQ mutations were isoa decreased affinity of the mutant enzyme for its lated that allow growth only at 240C but which substrate (higher K,,,) and/or due to the presence spontaneously acquire secondary mutations that of limited amounts of enzyme. The latter is allow growth at 370C (unpublished data). Since, supported by the finding that, when two copies in general, a high degree of homology in gene of the newD gene are present in S. typhimurium, sequence exists between E. coli and S. typhithe doubling times in minimal and leucine-sup- murium (17), one would expect that putative plemented medium are identical (to be reported supQ newD genes in E. coli would map in the elsewhere). This growth limitation is not seen in region betweenproAB and proC. This particular E. coli-S. typhimurium hybrid strains, although region, however, exhibits major differences bethe specific activity of the isopropylmalate isom- tween E. coli and S. typhimurium (Fig. 2), so erase in vitro was approximately the same as for that it is quite possible that genes analogous to the S. typhimurium strain. It is not known supQ and newD are absent in E. coli. This is whether the better growth rate is due to differ- supported by the failure to obtain supQ mutaences in the enzyme activity or to the amount of tions in E. coli and also in S. typhimurium enzyme present. It should be pointed out that strains containing F' factors, each of which carry the differences in the specific activity of mutant part of the E. coli region between proAB and isopropylmalate isomerase detected by in vitro proC. (It should be noted [1] that the leuD assays (Table 3) might not necessarily reflect deletion strains are E. coli B/r derivatives, the actual differences in specific activity of these whereas the F' pro lac is derived from E. coli Kenzymes in vivo. The mutant enzyme might be 12, and [ii] that B/r strains lack the argF gene more labile than the wild-type enzyme so that, (21) which lies adjacent to attP22 between the in independent preparations of crude extracts, proAB and proC genes on the K-12 chromodifferent fractions of isopropylmalate isomerase some. No other differences between E. coli B/r and K-12 are known to exist in this region.) That activity might be lost prior to assay. One further explanation for the growth rate E. coli has no function which interferes with the limitation could be that these cells cannot pro- expression of a potential newD gene was shown vide high enough levels of a-isopropylmalate for by the complementation of the S. typhimurium the mutant enzyme. This possibility is supported newD and E. coli leuC gene products to produce by (i) the fact that the in vitro assays of the a functional isopropylmalate isomerase. In vivo complementation of leuC and leuD isopropylmalate isomerase of the faster growing E. coli-S. typhimurium hybrids required less mutants in S. typhimurium, E. coli, and S. tygenome between proAB and proC, the region where supQ and newD map in S. typhimurium. F' proAB lac and F' lac proC were transferred into an S. typhimurium strain carrying the leuD798-ara and proAB47 deletions and both of the resulting hybrids were mutagenized with NTG. No Leu+ colonies arose, indicating that E. coli does not have the potential for supQ mutations in these two regions of the genome, or both regions are required simultaneously.

VOL. 137, 1979

leuC-newD ISOPROPYLMALATE ISOMERASE

phimurium-E. coli hybrids, as well as the supQ newD gene substitution system, suggests that the isopropylmalate isomerase is a complex enzyme. Whether the leuC and leuD gene products form a complex to produce the functional isopropylmalate isomerase has recently been questioned (3), since the purification of this enzyme from yeast (2) and N. crassa (19) has shown that in both of these species the isopropylmalate isomerase is composed of only one polypeptide with a molecular weight of 90,000. Also, the previous failure to demonstrate in vitro complementation between leuC and leuD mutants of S. typhimurium was taken to suggest that only one of the genes, leuC or leuD, codes for the isopropylmalate isomerase and the other one has some other function (e.g., regulatory), or that the leuC and leuD region of the chromosome is only one gene. The latter possibility would imply that all observed in vivo complementation was intragenic, which seems unlikely, especially since it included chain-terminating mutations and deletions. Furthermore, the complexity of the suppression pattern of different leuD mutations (missense, chain-terminating, and deletions) by the same supQ mutations (11) can only be satisfactorily explained by assuming that leuC and leuD are two separate genes whose protein products form a complex. We have recently shown in vitro complementation of the leuC and leuD polypeptides (to be published elsewhere), demonstrating that the products of these genes are fairly stable and are able to form an active isopropylmalate isomerase complex. Further support for a multimeric enzyme comes from our tentative identification of the polypeptide products of the S. typhimurium leuC and leuD genes by sodium dodecyl sulfatepolyacrylamide gel electrophoresis of fractions obtained after partial purification of the isopropylmalate isomerase. Crude extracts of leuDand leuC-D- deletion strains were purified in the same manner as crude extracts of a wildtype strain and after each purification step were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides, present in wild-type extracts but absent in extracts of leuD- and leuC-D- deletion strains, co-purified in sequential steps and were tentatively identified as the polypeptides of the leuD and leuC genes, having molecular weights of 25,000 and 51,000, respectively. The co-purification suggests that the isopropylmalate isomerase in S. typhimurium is a complex enzyme. The fact that in yeast and N. crassa the isopropylmalate isomerase is composed of only one polypeptide may be due to gene fusion during evolutionary development. Similar gene fusion has

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been suggested to explain the differences in the tryptophan operons of bacterial and eucaryotic species (5). Supportive evidence is given by the fact that the N. crassa isopropylmalate isomerase is preferentially cleaved into two major fragments of approximately 56,000 and 37,000 daltons (20). ACKNOWLEDGMENTS This investigation was supported by Biomedical Research Support grant RR-07133 from the General Research Support Branch, Division of Research Resources, National Institutes of Health, and by the University of Texas at Dallas Research Fund. We thank Joyce Lee for excellent secretarial assistance.

LITERATURE CITED 1. Bachman, B. J., K. B. Low, and A. L. Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 2. Bigelis, R., and H. E. Umbarger. 1975. Purification of yeast a-isopropylmalate isomerase. High ionic strength hydrophobic chromatography. J. Biol. Chem. 250: 4315-4321. 3. Bigelis, R., and H. E. Umbarger. 1976. Yeast a-isopropylmalate isomerase. Factors affecting stability and enzyme activity. J. Biol. Chem. 251:3545-3552. 4. Calvo, J. M., and S. R. Gross. 1970. Isolation and chemical estimation of a-isopropylmalate and ,f-isopropylmalate. Methods Enzymol. 17A:791-793. 5. Crawford, I. P. 1975. Gene rearrangements in the evolution of the tryptophan synthetic pathway. Bacteriol. Rev. 39:87-120. 6. Gross, S. R., R. 0. Burns, and H. E. Umbarger. 1963. The biosynthesis of leucine. II. The enzymic isomerization of fl-carboxy-/?-hydroxyisocaproate and a-hydroxy-fi-carboxyisocaproate. Biochemistry 2:10461052. 7. Guerola, N., J. L. Ingraham, and E. Cerda-Olmeda. 1971. Induction of closely linked multiple mutations by nitrosoguanidine. Nature (London) New Biol. 230:122125. 8. Hoppe, I., and J. Roth. 1974. Specialized transducing phages derived from Salmonella phage P22. Genetics 76:633-654. 9. Kemper, J. 1974. Evolution of a new gene substituting for the leuD gene of Salmonella typhimurium: characterization of supQ mutations. J. Bacteriol. 119:937-951. 10. Kemper, J. 1974. Evolution of a new gene substituting for the leuD gene of Salmonella typhimurium: origin and nature of supQ and newD mutations. J. Bacteriol. 120:1176-1185. 11. Kemper, J., and P. Margolin. 1969. Suppression by gene substitution for the leuD gene of Salmonella typhimurium. Genetics 63:263-279. 12. Kwoh, D. Y., and J. Kemper. 1978. Bacteriophage P22mediated specialized transduction in Salmonella typhimurium: identification of different types of specialized transducing particles. J. Virol. 27:535-550. 13. Levine, M. 1957. Mutations in the temperate phage P22 and lysogeny in Salmonella. Virology 3:22-41. 14. Low, K. B. 1972. Escherichia coli K-12 F-prime factors, old and new. Bacteriol. Rev. 36:587-607. 15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 16. Margolin, P. 1963. Genetic fine structure of the leucine operon in Salmonella. Genetics 48:441-457.

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17. Middleton, R. B. 1971. The genetic homology of Sabnonella typhimurium and Escherichia coli. Genetics 69: 303-315. 18. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor,

N.Y. 19. Parsons, S. J., and R. 0. Burns. 1970. ,B-Isopropylmalate dehydrogenase (Salnonella typhimurium). Methods Enzymol. 17A:793-799. 20. Reichenbecher, V. E., and S. R. Gross. 1978. Structural features of normal and complemented forns of the Neurospora isopropylmalate isomerase. J. Bacteriol. 133:802-810. 21. Riley, M., and A. Anilionis. 1978. Evolution of the bacterial genome. Annu. Rev. Microbiol. 32:519-560. 22. Russell, R. R. B., and A. J. Pittard. 1971. New suppres-

J. BACTERIOL. sor in Escherichia coli. J. Bacteriol. 107:736-740. 23. Sanderson, K. E., and P. Hartman. 1978. Linkage map

of Salmonella typhimurium, edition V. Microbiol. Rev. 42:471-519. 24. Schwartz, D. O., and J. R. Beckwith. 1969. Mutagens which cause deletions in Escherichia coli. Genetics 61: 371-376. 25. Smith, H. O., and M. Levine. 1967. A phage P22 gene controlling integration of prophage. Virology 31:207216. 26. Somers, J. M., A. Amiallag, and R. B. Middleton. 1973. Genetic fine structure of the leucine operon of Escherichia coli K-12. J. Bacteriol. 113:1268-1272. 27. Yang, H.-L, and D. P. Kessler. 1974. Genetic analysis of the leucine region in Escherichia coli B/r: geneenzyme assignments. J. Bacteriol. 117:63-72.