JOURNAL

OF

Vol. 172, No. 11

BACTERIOLOGY, Nov. 1990, P. 6403-6410

0021-9193/90/116403-08$02.00/0 Copyright C 1990, American Society for Microbiology

EntG Activity of Escherichia coli Enterobactin Synthetase JANET F. STAABt AND CHARLES F. EARHART*

Department

of Microbiology,

The University of Texas

at

Austin, Austin, Texas 78712-1095

Received 10 April 1990/Accepted 29 August 1990

The last steps in the biosynthesis of the Escherichia coli siderophore enterobactin (Ent) are carried out by Ent synthetase, a multienzyme complex believed to be composed of the entD, -E, -F, and -G products (EntD to -G). However, sequencing data showed that there is no separate eniG gene and, unlike EntD to -F, no distinct EntG polypeptide has been identified. In this study, genetic, biochemical, and immunological approaches were used to study the anomalies associated with EntG activity. Two plasmids, pJS43 and pJS100, were isolated that had mutations resulting in truncated EntB proteins; both had the phenotype EntB+ EntG-. PJS43 had a TnS inserted 198 bp from the entB termination codon, and pJSlOO had the last 25 codons of entB deleted. Plasmids isolated with TnS insertions in the 5' half of entB had the phenotype EntB- EntG+. These latter TnS mutations were EntB- EntG- when moved to the bacterial chromosome. Polyclonal antiserum was prepared and shown to react only with intact EntB in Western immunoblots. Addition of anti-EntB antiserum to Ent synthetase assays resulted in complete inhibition of enzyme activity, whereas preimmune serum had no effect. Lastly, AN462, the type strain for entG which was derived by Mu insertion and which has the phenotype EntB-G-A-, was characterized. Southern blot data showed a Mu insertion, presumably with polar effects, in the vicinity of the 5' end of entB. In summary, EntG activity was found to be encoded by the entB 3' terminus. The evidence, while not rigorously eliminating the possibility that a separate EntG polypeptide exists, strongly supports the idea that EntB is a bifunctional protein.

Iron is an essential element for all microorganisms except certain lactobacilli. In environments deficient in iron, many aerobic and facultatively anaerobic bacteria obtain the metal by synthesizing and excreting low-molecular-weight ironchelating molecules termed siderophores. Specific transport proteins subsequently bring the ferrisiderophore complexes into the cells (29). Escherichia coli has a high-affinity iron transport system that uses the siderophore enterobactin (Ent) (reviewed in reference 12). Ent system genes are clustered at min 13.5 on the chromosome and are negatively regulated by the Fur protein (reviewed in reference 3). Ent is a cyclic trimer of 2,3-dihydroxy-N-benzoylserine (DBS); its biosynthesis (Fig. 1) originates from chorismic acid and is thought to require the products of seven genes (entA to -G). The entC, -B, and -A products (EntC, EntB, and EntA) convert chorismate to dihydroxybenzoic acid (DHB), and the entD to -G polypeptides (EntD, EntE, EntF, and EntG) synthesize Ent from DHB and L-serine. The reactions catalyzed by EntC, EntB, and EntA can be bypassed by DHB supplementation, but no intermediate can be added to growth medium to alleviate mutations in entD, -E, -F, or -G. The EntD to -G polypeptides are thought to exist as a complex (15) termed Ent synthetase, and no diffusible intermediates of the Ent synthetase reaction have been detected. EntD, -E, and -F have been identified (1, 8, 32). EntE dimer with a (2,3-dihydroxybenzoate-AMP ligase) is an monomer Mr of 58,299 that activates DHB (15, 36); its cognate gene has been sequenced (42), and EntE has been purified and characterized (36). EntF activates L-serine and is the largest polypeptide of the putative Ent synthetase complex; estimates of its apparent Mr vary from 115,000 (8)

to 160,000 (31);

more recently a value of 140,000 was obtained (J. F. Staab, unpublished results). The entD gene has been sequenced (1, 9), and its product has been identified as an approximately 24,000-Da cytoplasmic membrane component (1). There is evidence that EntD associates with EntG and EntF (15, 45), and EntD has been proposed to mediate a loose association of Ent synthetase with the cytoplasmic membrane (15). The EntG activity is the least understood of those associated with Ent synthetase, and the existence of a separate entG gene and corresponding polypeptide is in doubt. The entG gene was postulated upon characterization of Entstrain AN462, which has functional entD to -F genes but fails to produce Ent even in the presence of DHB (44). AN462, the only EntG- strain known, arose from a Mu insertion and is also EntA- and EntB-. (The AN462 genotype was reported to result from Mu-induced polarity, and it is now clear that the rightmost operon of the Ent gene cluster consists of the genes entCEB[G]A and is transcribed from left to right [14, 20, 27, 30], but more recently Laird and Young [19] suggested that a Mu-induced deletion of entB [G]A was responsible for the AN462 characteristics. Some recent workers [27, 28] have adopted the latter suggestion.) Weak genetic complementation of the AN462 EntG defect occurs with plasmids containing an approximately 700-bp fragment that includes portions of entB and entA (28). However, recent sequencing data show that no separate and distinct open reading frame that could correspond to entG exists between entB and entA (20, 27). Thus, EntG activity is necessary for an uncharacterized terminal step in Ent synthesis, but no unique polypeptide corresponding to EntG has been detected and there is no separate entG gene. In this report, we demonstrate that EntG activity is encoded by the 3' terminus of the entB gene. (Portions of this work were presented at the 90th Annual Meeting of the American Society for Microbiology, Anaheim, Calif., 13-17 May 1990.)

x2

* Corresponding author. t Present address: Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75235-8895.

6403

J. BACTERIOL.

STAAB AND EARHART

6404

COOH CCH2

COOH P H

EntC

o C. COOH

OH CHORISMIC ACID

Isochorsmate synthetase

mg+2

° C' COOH ISOCHORISMIC ACID

Isochorismate-

pyruvate-hydrolase

EntB COOH

pyruvate

PJ2OH

OOH

EntA

ENTEROBACTIN

EntDEFG

L-sene FJ

COOH OH I

2,3-DIHYDRO-2,3DIHYDROXYBENZOIC ACID

2,3-dihydro-2,3dihydroxybenzoate dehydrogenase NAD+

o

enterobactin synthetase ATP

OH

2,3-DIHYDROXYBENZOIC ACID FIG. 1. Ent biosynthesis.

MATERIALS AND METHODS Bacteria, plasmids, and bacteriophages. The bacterial strains, all E. coli K-12 derivatives, and plasmids used are listed in Table 1. Bacteriophage X467 has the genotype b221 rex: :TnS c1187 Oam29 Pam8O (10) and was obtained from C. Prody. Bacteriophage P1 vir was from J. Walker. Materials. L broth (26), succinate-dipyridyl (low-iron) plates (34), and T medium (40) have been described before. Siderophore production was assayed on chrome azurol S plates (39). Antibiotics (Sigma Chemical Co., St. Louis, Mo.) were added to solid media in the following concentrations: kanamycin (neomycin), 25 jig/ml; ampicillin, 200 jig/ml; and tetracycline, 25 ,ug/ml. Supplements to minimal media were added as described elsewhere (33). L-[14C]serine was purchased from Amersham Corp. (Arlington Heights,

Ill.); [cx-32P]dCTP and [cx-35S]ATP were purchased from New England Nuclear Corp. (Boston, Mass.), respectively. The Sequenase DNA sequencing kit was purchased from United States Biochemical (Cleveland, Ohio). Restriction enzymes and T4 ligase were obtained from Bethesda Research Laboratories (Gaithersburg, Md.) and Promega Biotec (Madison, Wis.). DNA-purifying columns were obtained from 5 Prime-3 Prime, Inc. (West Chester, Pa.). Transposon TnS mutagenesis. The method described by de Bruijn and Lupski (10) was followed. Strain MC4100 harboring plasmid pCP410 was grown overnight at 30°C in L broth plus tetracycline and 0.2% maltose. The culture was then diluted 100-fold into fresh L broth containing tetracycline and maltose and grown at 30°C to early stationary phase (109 cells per ml). A 1-ml sample of cells was mixed with A::TnS

TABLE 1. E. coli K-12 strains and plasmids Strain

or

Genotype

plasmid

or Source reference

E. coli K-12 strains AB1515

AB1515.24 AB1515.43 AN93 AN192 AN462 JC7623

MC4100 P678-54 Plasmids

pACYC184 pGEMBlue pGEM4 pCP410 pJS5 pJS22, -23, -24 pJS43 pJS3.35' pJS100

pJS1200 pJSG750 a

purE42 proC14 leu-6 trpE38 thi-J JhuA23 lacYl mtl-l xyl-5 rpsL109 azi-6 tsx-67 As AB1515; entB::TnS entG As AB1515; entB::Tn5 entG As AB1515; purE+ entE405 As AB1515; purE+ entB402 pabA his4 arg-3 ilv-7 aroE purE str spc entA,B,G415 (Mu') thr-J leuB6 thi-J lacYl galK2 ara-14 xyl-5 mtl-1 proA2 his4 arg rpsL31 tsx-33 recB21 recC22

CGSCa This study This study I. G. Young I. G. Young 44 J. R. Walker

sbcB15 sbcC201 araD A(argF-lac)205 flbB ptsF relA rpsL deoC thr leu thi supE lacYfhuA gal mal xyl ara mtl minA minB

CSGC J. R. Walker

Cmr Tcr Apr; lac a peptide and multiple cloning site arrangement of pUC19 Apr; multiple cloning site arrangement of pUC18 entEB(G)A+ EcoRI 6.6-kb insert in pACYC184 TnS insertion in pCP410; ent+ TnS insertions in pPC410; entB Tn5 insertion in pPC410; entG pJS211 exonuclease III deletion derivative entEB+ 2.7-kb insert in pGEMBlue entB+ HindIII-HpaI 1.2-kb insert from pJS3.35' in pGEM4 0.75-kb PvuII-EcoRI insert from pJS1200 in pGEM4

7 Promega Corp. Promega Corp. 32 This study This study This study 42 42 This study This study

CGSC, Coli Genetic Stock Center, Yale University, New Haven, Conn.

VOL. 172, 1990

phage at a multiplicity of infection of 10 and placed at 30°C for 2 h. Infected cells were then plated onto L-broth plates containing kanamycin and tetracycline. The plates were incubated at 30°C for 48 h. Kmr Tcr colonies were washed off the plates with a sterile glass spreading rod after addition of 3 ml of L broth to each plate. The cell suspensions were transferred to a test tube and dispensed into microfuge tubes. Plasmid DNA was then isolated (5) and used to transform strains AN462 and AN192. Kmr Tcr transformants were screened for the ability to grow on iron-poor plates. Succinate-dipyridyl plates for AN462 growth contained 10 ,uM DHB to circumvent the need for EntA and EntB; this allowed detection of EntG activity. Polypeptide analysis. Strain P678-54 was transformed with plasmids of interest, and minicells were then isolated and labeled with L-[35S]methionine by the method of Meagher et al. (25). The plasmid-encoded polypeptides were examined on 15% sodium dodecyl sulfate (SDS)-polyacrylamide gels as described previously (42). The molecular size standards lysozyme, ot-lactoglobulin, P-chymotrypsinogen, ovalbumin, phosphorylase b, and myosin (H chain) were obtained from Bethesda Research Laboratories. DNA cloning and sequencing. Plasmid DNA was digested as recommended by the enzyme suppliers. DNA ligations were done as described by King and Blakesley (17). A pJS211 (42) deletion derivative, pJS3.35', was used as source of ent DNA in the construction of an entB subclone, pJS1200. A 1.2-kb HindIII-HpaI fragment of pJS3.35' was cloned into the HindIII-SmaI sites of pGEM4. A 0.75-kb PvuII-EcoRI fragment from pJS1200 was cloned into the Smal-EcoRI sites of pGEM4 (pJSG750). A 1.3-kb EcoRVHindIII fragment of pJS43 carrying the junction between TnS and entB sequences was cloned into M13mpl9 (46) for DNA sequencing. Nucleotide sequences were determined by the dideoxy-chain termination method (38). [ax-35S]dATP was used as label, and parallel reactions were run with dITP in place of dGTP. Southern blots. Chromosomal DNA was purified by the procedure of Marmur (23). Restricted DNA was electrophoresed in 1% agarose (Sigma) gels, and the fragments were transferred (41) to Magnagraph nylon membranes (Micron Separations, Inc., Westborough, Mass.). The membranes were hybridized with [32P]dCTP-labeled probes made as outlined in the Bethesda Research Laboratories nick translation protocol. Unincorporated nucleotides were removed by chromatography through a Sephadex G-50 (Pharmacia LKB Biotechnology Ltd., Piscataway, N.J.) column as described by Maniatis et al. (22). Hybridizations were carried out at 68°C overnight (22). Washes were of 500 ml and were performed as follows: 2x SSC (lx SSC is 0.15 M NaCl plus 0.15 M trisodium citrate, pH 7)-0.5% SDS for S min at room temperature; 2x SSC-0.1% SDS for 15 min at room temperature with occasional agitation; 0.1 x SSC-0.5% SDS at 68°C for 2 h with gentle agitation; and fresh 0.1 x SSC0.5% SDS at 68°C for an additional 30 min. The dry membranes were exposed to X-ray film (Kodak XR-P) overnight and developed as recommended by Kodak. Marker exchange experiments. The TnS insertion mutations of pJS43 and pJS24 were transferred to the chromosome by homologous recombination. Strain JC7623, which is unable to digest linear DNA, was transformed with EcoRIdigested plasmid DNA; Kmr transformants were screened for the Tcs Kmr phenotype by picking isolated colonies onto L broth-tetracycline and L broth-kanamycin plates. Tcs Kmr colonies were tested on chrome azurol S plates for loss of Ent production. The ent mutations in JC7623 strains were

EntG ACTIVITY OF ENTEROBACTIN SYNTHETASE

6405

moved to the chromosome of AB1515 by P1 transduction as described previously (34). Production of antisera and immunoprecipitation procedures. New Zealand White rabbits were given subcutaneous primary injections of 250 ,ug of purified EntE and EntB (provided by F. Rusnak and C. Walsh) emulsified in Freund complete adjuvant (Sigma). Four weeks after the primary inoculations, subcutaneous booster injections of 100 ,ug of protein emulsified in Freund incomplete adjuvant (Sigma) were given, and this was continued every 2 to 3 weeks. The rabbits were bled 7 to 10 days after booster inoculations, and the serum was collected and tested for reactivity in Western immunoblots (6) of AN102 and AB1515 cell extracts. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Bio-Rad Laboratories, Richmond, Calif.) served as the secondary antibody to assay for the binding of the antiserum to EntE or EntB via an enzyme-linked immunosorbent assay. The protocol outlined by the Bio-Rad immunoblotting kit was followed for color development of the goat anti-rabbit antibody conjugate. Ent synthetase assays. Ent synthetase activity was measured as described previously (15). Cell extracts were prepared by growing 1-liter cultures of E. coli strains to stationary phase overnight in T medium without added iron (Ent' strains) or with 3 ,uM FeCl3 (Ent- strains). The pellets from 1-liter cultures were suspended to 5 ml with cold 100 mM Tris hydrochloride (pH 8)-5 mM dithiothreitol, and the cells were broken with a French pressure cell (18,000 lb/in2). Cell debris was pelleted at 12,000 x g for 10 min at 4°C, and the supernatant was saved and used as the cell extract. The incorporation of L-['4C]serine into DBS-containing compounds was determined per milligram of protein. Inhibition of Ent synthetase activity was done by adding 0.5 mg of polyclonal antisera to reaction mixtures containing 0.5 mg of cell extract. The mixtures were preincubated for 10 min at room temperature before initiation of the assay (370C, 15 min).

RESULTS Construction of mutant plasmids. Plasmid pCP410 (entE+B+[G+]A+) (Fig. 2) was mutagenized with transposon TnS to produce insertional inactivations of ent genes. One isolate, pJS43, did not complement strain AN462 (EntB[G-]A-) when growth was on low-iron plates supplemented with DHB, indicating that the mutation abolished EntG activity. This plasmid complemented strain AN192 (entB) despite the fact that restriction mapping localized the insertion site to the 3' end of entB. The exact location of the TnS insertion was determined by DNA sequencing, since both the entB (20, 27) and TnS inverted repeat (IR) (2) sequences are known. A TnS IR started 201 bp upstream of the last base pair of the entB termination codon (data not shown), in good agreement with the restriction mapping results. The entB gene was fused in frame to an IR sequence capable of coding for nine amino acids, making the predicted size of the EntB fusion protein encoded by pJS43 27,755 Da. Three additional plasmids with TnS insertions were isolated that failed to complement AN192 but did complement AN462. These EntB-G+ plasmids, pJS22, pJS23, and pJS24, had insertions in the 5' half of entB (Fig. 2). A subclone of pCP410 containing an entB gene lacking the 74 nucleotides of the 3' terminus was constructed by ligating the 2.7-kb EcoRI-EcoRV fragment to pGEMBlue that had been digested with EcoRI and SmaI. The recombinant plasmid, pJS100 (Fig. 2), has the same insert as pITS33 (28)

6406

~ ~ ~I

STAAB AND EARHART

A.

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entE

a I

pJS1 00

entB _

a I

= >

I I I

I entE

1 kb

TIpJSl1200

entB

I

J. BACTERIOL.

I entB

IT

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I T

>-

pCP41 0

I entA IP15 I

5 RI

Hcll NI

HpII~~~~~~~~~~~~~~~

I

entC I

I

entE

Pll

RV

entB

I

I I

I

24 2322

I

Hol

Al

AI

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entA

43

B. Z

I

I

I

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I

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< Im

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] pCP41 0

I entB I entA IPI51 1KM\\m entB probe M7277170MMM17//Al entEBA probe

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YQ-I ac

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entE

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:

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1III I I I

][][

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Tn5 probe

1 kb FIG. 2. Partial restriction maps of transposon TnS and relevant E. coli DNA. (A) Bacterial DNA present in pCP410, pJS100, and pJS1200 and positions of Tn5 insertions in pCP410. The 3'-terminal HindlIl site of pJS1200 was derived from the multiple cloning site of plasmid vector pGEM4 after exonuclease III digestion of ent DNA. Sites of the Tn5 insertions of pJS22, pJS23, pJS24, pJS43, and pJS5 are shown ( T ). A pertinent HinclI site is shown in the enlarged region. (B) DNA fragments of pCP410 and Tn5 used as probes. Tn5, the complete sequence of which is known (1, 4, 24), is shown in the orientation in which it is inserted in pJS43. Restriction sites: Al, AccI; HcII, HincII; HpI, HpaI; KI, KpnI; NI, NruI; Pll, PvuII; RI, EcoRI; RV, EcoRV; and SI, SmaI.

and, like pITS33, was entE+B+G in complementation tests. A subclone (pJS1200) carrying the entire entB gene was constructed from pJS3.35'; a 1.2-kb HindIII-HpaI fragment was cloned into HindlIl-Smal-digested pGEM4. Both entB and entG mutations were complemented by pJS1200 (Fig. 2). The results indicated that either insertion (pJS43) or deletion (pJS100) mutations in the 3' end of entB eliminated EntG but not EntB activity. The absence of polar effects on EntG activity in plasmids pJS22, pJS23, and pJS24 was surprising but later attributed to copy number considerations (see below).

Analysis of plasmid-encoded polypeptides. Plasmid pCP410 derivatives were introduced into the minicell-producing strain P678-54 by transformation, and the polypeptides synthesized in minicells were examined (Fig. 3). Four Ent cluster polypeptides are encoded by pCP410: EntE (59,299 Da), EntB (32,554 Da), EntA (26,249 Da), and P15 (14,970 Da). Plasmids carrying TnS insertions produced five transposon-encoded polypeptides of 58,000, 54,000, 53,000, 49,000, and 27,500 Da (4, 35). Plasmid pJS43 produced a 25,700-Da truncated EntB polypeptide (Fig. 3, lane 2); this apparent size is in reasonable agreement with that predicted

EntG ACTIVITY OF ENTEROBACTIN SYNTHETASE

VOL. 172, 1990 1

2

3

4

5

6

TABLE 2. Genetic complementation assays

7

Plasmid

qg~

-8-u ~~~~

4S .A -

Complementation AN462

AN192

AB1515.24

(entB)

(entABG)

AB1515.43

+

-

-

-

pCP410 +DHB -DHB pJS100

NTa +

+

+

+

+

+

+

+DHB -DHB

NT +

-

-

+DHB NT + -DHB a NT, Not tested.

+ -

+ -

4_ None +DHB -DHB ~~~~~~~~~~~~~~~~~~~~~.

6407

pJS1200 NMpj

FIG. 3. Autoradiogram of L-[5S]methionine-labeled polypeptides synthesized in minicells and examined on a 15% SDS-polyacrylamide gel. Lanes: 1, pCP410; 2, pJS43; 3, pJS24; 4, pJS23; 5, pJS22; 6, pCP410; 7, pJS5. Ent gene products are identified.

from sequencing data. A truncated EntB polypeptide was also observed with pJS100 (data not shown). Detection of these shortened polypeptides supports the earlier complementation data and confirms the suggestion (28) that truncated EntB retains some EntB activity. The protein profiles of pJS22, pJS23, and pJS24 all lacked the 32,554-Da EntB polypeptide, and no EntB fragments were detected (lanes 3 to 5). The four plasmids with TnS insertions in entB all showed an apparent lack of EntA and P15, which presumably resulted from polarity. Construction and characterization of bacterial mutants. The transposon mutations of pJS24 (EntB-G+) and pJS43 (EntB+G-) were first transformed into JC7623 and then moved onto the AB1515 chromosome by P1 transduction to yield strains AB1515.24 and AB1515.43, respectively. Even in the presence of DHB, neither strain grew on low-iron medium or produced Ent on chrome azurol plates, indicating that both were deficient in EntG activity. The entB insertion of the high-copy-number plasmid pJS24 apparently exerted obvious polar effects to abolish EntG activity when moved to the chromosome. AB1515.24, AB1515.43, and AN462 were transformed with pCP410, pJS100, and pJS1200, and complementation tests were then performed (Table 2). The results indicated that AB1515.24 and AB1515.43 were EntG-A-; AB1515.24 presumably has a genotype identical to that of AN462 (entBGA), but EntB activity of this strain was not determined. In contrast to the effect of insertion mutations in entB, the N-methyl-N'-nitro-N-nitrosoguanidine-induced entB mutation of AN192 (47) is nonpolar on entA and was complemented by pJS100 and pJS1200. Blot hybridizations. The site of TnS insertion in AB1515.43 was confirmed by blot hybridization (see Fig. 2 for predicted fragment sizes). AB1515 and AB1515.43 DNA was cut with NruI and EcoRV, electrophoresed, transferred to nylon membranes, and hybridized with TnS and entB probes. The TnS probe contained approximately 340 nucleotides of IR1 (2) plus the entire neomycin phosphotransferase gene (4), and the entB probe contained 600 nucleotides of entB (Fig. 2B). Plasmids pCP410 and pJS43 were used as controls. The

+

600-nucleotide entB fragment was present in AB1515 and pCP410, but the entB probe recognized two fragments (2.9 and 2.6 kb) in both AB1515.43 and pJS43 DNA, thereby demonstrating that TnS was present in the same site in both replicons. Hybridization was stronger to the 2.9-kb fragment because it carried approximately four times more entB sequence than did the 2.6-kb fragment. Neither AB1515 nor pCP410 sequences were recognized by the Tn5 probe. In AB1515.43 and pJS43, the TnS probe recognized the same two fragments that hybridized to the entB probe. In this case, the 2.6-kb fragment hybridized more strongly, since the 2.9-kb fragment contained only the 340 nucleotides of IRr that could hybridize to the TnS probe. The pattern of hybridization intensities demonstrated that TnS is inserted as shown in Fig. 2B. Blot hybridizations were also performed to characterize the mutation in entG type strain AN462. AB1515 and AN462 DNA was restricted with EcoRl or HindIII and hybridized to a 1.9-kb probe (Fig. 2B) that extended from mid-entE to mid-entA. When AB1515 was cut with EcoRI (Fig. 4), a single 6.6-kb fragment, identical to that cloned in pCP410, was detected. In contrast, two AN462 fragments, both larger A

B 2

2

7.1 61.6:_

3..2

FIG. 4. Southern blots of AB1515 (lanes 1) and AN462 (lanes 2) probed with entEBA sequences. (A) Chromosomal DNA digested with EcoRI; (B) chromosomal DNA digested with HindIII. Molecular sizes are in kilobases.

6408

STAAB AND EARHART

J. BACTERIOL.

TABLE 3. Ent synthetase assays Sample

A AB1515 ............. No ATP ............. No DHB ............. AB1515.43 ............. No ATP ............. No DHB ............. AN93 ............. AB1515 (dialyzed) ............. No ATP ............. No DHB ............. B AB1515 Preimmune EntE ............. Immune EntE ............. Preimmune EntB ............. Immune EntB ............. No serum .............

Sp acta

9.4 2.7 1.9 2.1 1.2 1.0 1.3 4.2 0.3 0.3 3.2 0.3 3.0 0.4 3.3

a Expressed as nanomoles of L-[14C]serine converted to DBS-containing compounds in 15 min at 37°C per milligram of cell extract protein. Values are means

of two reactions.

than 6.6 kb (7.1 and approximately 19 kb) were recognized, demonstrating that an insertion was present. A single 11-kb AB1515 HindIII fragment (the fragment present in pMS112 [18]) and two HindIII fragments (3.2 and approximately 20 kb) of AN462 were detected. Phage Mu (37 kb) has EcoRI sites at 5.1 and 23.8 kb and HindIll sites at 1.0 and 26.7 kb, reading left to right from the C gene (16). These data therefore showed that AN462 has a Mu insertion within entEBA; specifically, the insertion is in the vicinity of the 5' entB terminus, in the same general region as the TnS insertion of pJS24. Ent synthetase assays. Ent synthetase assays, which measure the combined activities of EntD, -E, -F, and -G, were performed on extracts of AB1515, AN93, and AB1515.43 (Table 3). AB1515 extracts incorporated L-[14C]serine into DBS-containing compounds with a specific activity greater than 9. AB1515.43 extracts were inactive; the residual activity was similar to that of AN93 (entE) extracts or AB1515 extracts with no added ATP or DHB. Dialysis of extracts to remove small molecules such as ATP and DHB resulted in a reduction in residual activity; since Ent synthetase activity is labile, more than half of the activity was lost during this 24-h dialysis step. The data provided biochemical evidence that AB1515.43 lacks EntG activity. Assays were also carried out in the presence of antisera raised against purified EntE and EntB proteins. The amount of extract used in these experiments was half that used in reactions reported in Table 3 (sample A). This concentration is below the linear range of the Ent synthetase assays, which accounts for the comparatively low specific activity in the control samples containing no serum or preimmune serum. In addition, at this lower extract concentration, the specific activity of assays to which no ATP or DHB was added could not be further reduced by dialysis. Preincubation of extracts with either anti-EntB or anti-EntE antiserum inhibited the reaction and to approximately the same extent, whereas preimmune serum had little effect. Attempts to detect an EntG polypeptide. The genetic analyses described above are consistent with the possibility that the 3' portion of entB encodes a polypeptide with EntG activity. Additional genetic evidence for this possibility can be inferred from the report that a pBR328-based plasmid

(pITS441), which contains a PvuII-HpaI fragment now known to be 728 bp (Fig. 2) that includes the 3' portion of entB and the 5' end of entA, weakly complements the entG mutation of AN462 (28). The presumed EntG polypeptide therefore must have a translational start site between the PvuII site and Tn5 insertion in pJS43. In fact, in this region there are five Met codons and four GUG valine codons in the same reading frame as that used for EntB synthesis. One Met codon, located 296 nucleotides from the end of entB, has an appropriately positioned ribosome-binding site and could direct the synthesis of an 11,083-Da polypeptide composed of the carboxy-terminal 98 amino acids of EntB. We confirmed the experiment demonstrating that the PvuII-HpaI fragment weakly complements the entG defect (28). The fragment was ligated into pGEM4, and the resulting plasmid (pJSG750) weakly complemented the entG lesion in AB1515.43. Weak complementation in this case was faint growth after 4 days, compared with the good growth seen after 36 h in normal complementation. In contrast, plasmid pJS13, which consists of the 728-bp fragment plus an additional downstream 2.6 kb inserted into the lower-copynumber vector pACYC184 (J. F. Staab, Ph.D. thesis, The University of Texas at Austin, Austin, Tex., 1990), did not complement AB1515.43. No EntG polypeptide has been detected. A polypeptide of the expected size was not observed among the pCP410encoded proteins (Fig. 3), which is consistent with the data of others (27, 32). Additional efforts using gel systems better able to resolve low-Mr polypeptides or nondenaturing gels were similarly unsuccessful (data not shown). Also, antiEntB antiserum failed to detect a peptide other than intact EntB in Western blots even though this antiserum was able to detect breakdown products of the purified protein (Staab, Ph.D. thesis, 1990). DISCUSSION Current and previous genetic and biochemical data show that an EntG activity exists, that it functions in the terminal stages of Ent biosynthesis, and that an intact 3' end of entB is a necessary but not always sufficient condition for its expression. In addition to the relationship between entB and EntG, analyses of entB mutations are also complicated by the facts (discussed below) that some truncated EntB polypeptides have isochorismatase (EntB) activity and that polar effects of entB insertion mutations are suppressed when the mutation is in a high-copy-number plasmid. The results obtained in complementation tests with a given mutation therefore depend on its location within entB, whether it exerts polar effects, and whether it is located in a plasmid or on the chromosome. With these considerations in mind, three of the four predicted phenotypes arising from entB chromosomal mutations have now been identified: (i) EntB-G+A+ (AN192), (ii) EntB-G-A- (AB1515.24) (the EntB- condition is assumed knowing the properties of pJS24), and (iii) EntB+G-A- (AB1515.43). The precise nature and location of the mutation in entB type strain AN192 are unknown, but it is most likely a missense (nonpolar) mutation in the 5' half of the gene. A fourth phenotype, EntB+G-A+, which would arise from a nonpolar mutation in the entBl 3' end, can be anticipated but has not yet been reported. In contrast, when mutations are in entB genes present on high-copy-number plasmids, only two phenotypes would be predicted (EntB-G+A+ and EntB+G-A+), and both have been found (pJS24 and pJS43, respectively).

VOL. 172, 1990

Strain AN462 (EntE+B-G-A-), the only strain previously described which lacks EntG activity, was shown to have a Mu insertion with polar effects, as originally postulated. Limitations of the Southern blot data, however, prevent a definitive statement that the Mu insertion in AN462, like the TnS insertion in AB1515.24, is in entB. The insertion in AN462 was localized to an approximately 350-bp region extending from the 3' end of entE into the entB 5' end. Although AN462 is entE+, truncated EntE proteins can be functional (Staab, Ph.D. thesis, 1990). The EntE polypeptide encoded by pJS4.1 (42) is active (Staab, unpublished results) although its entE gene is deleted by 61 bp at its 3' terminus. Carboxy-terminal deletions of EntE cannot extend as far as those in EntB without causing inactivation, however, since pJS3.2 (42), whose entE gene lacks the 3' 150 bp, is EntE-. Evidence that isochorismatase activity does not require the complete entB product has been reported (28); we confirmed this result with plasmid pJS100, which, according to recent sequencing data, encodes an EntB protein lacking the normal 24 carboxy-terminus amino acids. In addition, results with pJS43 and AB1515.43 showed that 66 amino acids can be lost from the carboxy terminus without eliminating all EntB activity. EntB activity in these experiments was measured only by complementation assay; however, F. Rusnak and C. T. Walsh (personal communication) have evidence that a truncated EntB protein retains full isochorismatase activity in vitro. Several lines of evidence indicate that entB 3' sequences are essential for EntG activity. Truncated EntB proteins lack EntG activity, EntG activity is lost when TnS is inserted in the 3' entB end, and weak complementation of cells with entG mutations occurs with high-copy-number plasmids (e.g., pJSG750) containing the 3' half of entB. This last observation is likely to be related to the fact that plasmids with TnS insertions in the 5' half of entB are EntG+. Previous reports (11, 21) have noted the absence of polar effects by TnS when genes present on high-copy-number plasmids are examined. Also, the ability of Ent cluster genes cloned on multicopy plasmids to be expressed when lacking their normal promoters has been discussed (9). Whether or not a separate entity with only EntG activity exists is still unclear. That EntG can be inhibited by antiEntB antibodies indicates that (i) it is not an RNA and (ii) it is a polypeptide encoded by entB and read in the same frame as EntB. Either EntB is a bifunctional protein, with both isochorismatase and EntG activities, or there is a distinct EntG polypeptide synthesized from an intragenic translational start site within entB. A precedent for the latter possibility is the CheA. protein (43). (A third possibility, complementation of cells making a truncated EntB polypeptide by a plasmid-encoded fragment of EntB, such as might occur in AB1515.43 harboring pJSG750, could occur by a mechanism akin to a complementation of ,B-galactosidase. Such a process would not occur normally and is unlikely to occur in AN462, however, as our characterization of AN462 indicates that it could synthesize either an amino-terminal EntB fragment of perhaps 23 amino acids or an intact EntB polypeptide in such low amounts as to be undetectable even by biological assay.) Data on the sizes of the two activities do not resolve the issue. The native Mr of EntB is about 159,000 (37), indicating that it exists as a pentamer, but no definitive evidence on EntG is available. (Partially purified EntG activity, which may have some EntD associated with it, elutes at a position indicative of an Mr greater than that of an EntB monomer [15].) That no EntG polypeptide has been detected either immu-

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nologically or as a product encoded by a multicopy plasmid argues that EntB is bifunctional. Failure to observe EntG is hardly conclusive, however, particularly when another component of the putative Ent synthetase complex, EntD, is difficult to detect (1, 9). Attempts to restore Ent synthetase activity by adding purified EntB (1 and 5 ,ug) to crude extracts of AB1515.43 were unsuccessful (Staab, unpublished results) despite the fact that enzymatic complementation occurs with crude extracts (13, 44) and partially purified Ent synthetase components (15). The result is again not conclusive because of the lack of information regarding the assembly of Ent synthetase and the reactions it carries out and the relative insensitivity of the Ent synthetase assay. For example, pure EntB pentamers may be unable to associate with the putative Ent synthetase complex or may not exhibit EntG activity. Evidence for a separate EntG polypeptide comes from complementation results with plasmids such as pJS24 and pJSG750. Because these are high-copy-number plasmids, we favor the idea that the complementation detected is an artifact. In summary, the data indicate that the entB gene encodes a bifunctional protein. How the cellular complement of intact EntB polypeptides normally carries out the isochorismatase step necessary early in Ent biosynthesis and also participates, presumably as part of a complex, in the latter stages of Ent synthesis is unknown. ACKNOWLEDGMENTS We thank F. Rusnak and C. Walsh for purified samples of EntB and EntE, E. Marcotte for constructing and characterizing E. coli AB1515.24, and N. Davis for training in catheterization and inoculation techniques. This work was supported by Public Health Service grant A122203 from the National Institutes of Health and by a biomedical research support grant from The University of Texas at Austin. LITERATURE CITED 1. Armstrong, S. K., G. S. Pettis, L. J. Forrester, and M. A. McIntosh. 1989. The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product. Mol. Microbiol. 3:757-766. 2. Auerswald, E. A., G. Ludwig, and H. Schaller. 1981. Structural analysis of TnS. Cold Spring Harbor Symp. Quant. Biol. 45:107113. 3. Bagg, A., and J. B. Neilands. 1987. Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiol. Rev. 51:509-518. 4. Beck, E., G. Ludwig, E. A. Auerswald, and H. Schaller. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon TnS. Gene 19:327336. 5. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 6. Burnette, W. N. 1981. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195-203. 7. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134: 1141-1156. 8. Coderre, P. E., and C. F. Earhart. 1984. Characterization of a plasmid carrying the Escherichia coli K-12 entD, fepA, fes, and entF genes. FEMS Microbiol. Lett. 25:111-116. 9. Coderre, P. E., and C. F. Earhart. 1989. The entD gene of the Escherichia coli K-12 enterobactin gene cluster. J. Gen. Micro-

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