Deletion analysis of sucrose metabolic genes from a Salmonella plasmid cloned in Escherichia coli K12

142-155 (1987)

PLASMID18,

Deletion Analysis of Sucrose Metabolic Genes from a Salmonella Plasmid Cloned in Escherichia co/i K12 CYNTHIA HARDESTY, GRACE COLON, CLAUDIA

FERRAN AND JOSEPHM. DIRIENZO’

Department of Microbiology, School of‘Dental Medicine, and Center for Oral Health Research. University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received June 12, 1987; revised August 17, 1987 The sucrose operon from pUR400, a 78-kbp conjugative Salmonella plasmid, was cloned in Escherichia coli K12. The operon was located in a 5.7-kbp SalI restriction fragment and was subcloned, in each of two possible orientations, using the expression vector pUC18. The insert DNA was restriction mapped and duplicate restriction sites in the insert and in the polyhnker of the vector were used to create various deletions promoter distal in the operon sequence. Additional deletions were made with the restriction exonuclease Ba13 1. Cells containing hybrid plasmids with specified deletions lacked the ability to transport sucrose or were constitutive for hydrolase and/or uptake activities. The scrA (enzyme II”“) and scrR (regulatory) genes resided within 2900-bp SmaI-SalI DNA fragment and were assigned the order scr3, scrA, scrR. An amplified sucrose-inducible gene product, A4, 68,000, was detected only in the membrane fraction from recombinant cells that contained plasmid with the intact operon sequence. This protein represented 11% of the total membrane protein and was resistant to extraction with 0.5 M sodium chloride, 2% Triton X-100, and 0.5% sodium deoxycholate. The protein did not appear to be the product of either the scrA, scrE, or scrR gene and may therefore represent a previously unidentified membrane-bound sucrose protein. A new gene, scrC, is proposed. In addition, the cloned 5.7-kbp SalI and 2.5-kbp SmaI-SalI DNA fragments failed to hybridize to chromosomal DNA from Bacillus subtilis. Streptococcus lactis, Streptococcus mutans, and Lactobacillus acidophilus as well as to DNA from a sucrose plasmid from Salmonella tennessee. However, the probes showed weak homology with a 20-kbp EcoRI restriction fragment from Klebsiella pneumoniae. o 1987 Academic press, hc

The metabolism of specific di- and trisaccharides such as lactose (Johnson et al., 1976), sucrose (Alaeddinoglu and Charles, 1979; Palchaudhuri et al., 1977; Smith and Parsell, 1975; Wohlhieter et al., 1975) and raffinose (Schmid and Schmitt, 1976; Schmitt et al., 1979; Smith and Parsell, 1975) can be mediated, in Escherichia coli, by plasmids that carry genes required for the transport and hydrolysis of these sugars. Several sucrose metabolic plasmids have been recovered from strains of E. cofi (Alaeddinoglu and Charles, 1979; Palchaudhuri et al., 1977) and Salmonella species (Bartlett and ’ To whom correspondence should be addressed at Department of Microbiology, University of Pennsylvania, School of Dental Medicine, 400 1 Spruce St., Philadelphia, PA 19 104. 0147-619X/87

$3.00

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

142

Trust, 1980; Johnson et al., 1976; Wohlhieter et al., 1975) but one plasmid, designated pUR400 (SCR-53), has recently been the subject of more detailed investigations to elucidate its role in carbohydrate metabolism by these bacteria. The 78-kbp plasmid, originally recovered from a clinical isolate of S. typhimurium, was transferred to E. coli by conjugation (Wohlhieter et al., 1975). Analysis of chemically induced mutations indicated that the plasmid contained a phosphoenolpyruvate (PEP)-dependent carbohydrate, phosphotransferase system (PTS) enzyme IIs”’ (scrA), a /3-D-fructofuranoside fructohydrolase (scrB), and a regulatory protein (scrR) that controlled the inducible expression of the scrA and scrB genes (Schmid et al., 1982). It was suggested that the sucrose genes comprised an operon under the con-

DELETION

ANALYSIS

trol of a single promoter (scrP) and that these plasmid-coded gene products complemented the soluble PTS proteins, enzyme I, HPr, and an enzyme IIIG’c*Sc’, produced by the host cells (Lengeler et al., 1982; Schmid ef al., 1982). Strains of E. coli that do not contain the plasmid lack the ability to metabolize sucrose as a sole carbon source. Based on similarities between the sucrose plasmid system and sucrose fermentation in Klebsiella pneumoniae. it was proposed that the sucrose plasmid may have evolved from the Klebsiefla genome (Lengeler et al., 1982; Schmid et al., 1982). Within the Enterobacteriaceae only the Klebsielleae demonstrate a stable sucrose-positive phenotype. The sucrose operon from pUR400 was located in a 5.7-kbp Sal1 DNA fragment cloned in pBR328 (DiRienzo d al., 1986). Single BarnHI, C/al, EcoRI, and Kpnl sites were found in the insert DNA as well as two sites for each of the restriction enzymes SmaI, PvuI, and &I. Restriction analysis of the cloned insert DNA confirmed that the 5.7-kbp fragment resided within the IO-kbp EcoRI DNA fragment cloned, in pBR325, by Garcia (1985) in a separate study. In this study, we have subcloned the sucrose operon, in both orientations, in the expression vector pUC 18 and have used these constructs to create various deletions, within the operon, to locate the scrA and scrR genes. In addition, we have used the sucrose operon DNA sequence to determine homologies, by DNA/DNA hybridization, between the sucrose plasmid genes and chromosomal DNA from various sucrose-fermenting bacteria. MATERIALS

AND

METHODS

Strains, plasmids, and growth conditions.

The bacterial strains and plasmids employed or constructed in this study are shown in Table 1. E. coli strains were grown in either LB (tryptone, 10 g; yeast extract, 5 g; sodium chloride, 5 g; deionized water, 1 liter; pH 7.5) M9 (sodium phosphate, 6 g; potassium phosphate, 3 g; sodium chloride, 0.5 g; ammonium chloride, 1 g: 1 M magnesium sul-

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143

fate, 2 ml; 1 M calcium chloride, 0.1 ml; deionized water, 1 liter; pH 7.4) or K medium (M9 medium including casamino acids 10 g/liter). All media were supplemented with either glucose, glycerol, or sucrose (0.4% final concentration) and ampicillin, tetracycline, or chloramphenicol (50 pg/ml). The M9 and K media were also supplemented with 0.1 pg/ml thiamine and the appropriate required amino acids (40 pg/ml). Depending upon the experiment, agar plates containing the above media or MacConkey agar base supplemented with 1% sucrose and ampicillin (50 &ml) were used. All other strains were grown in brain-heart infusion broth. Cells were grown by shaking at 37°C or, in the case of Srreptococcus mulans, in an anaerobic chamber (Forma Scientific, Marietta, OH) in a mixed gas containing nitrogen (80%), hydrogen ( 10%) and carbon dioxide (10%). Growth of bacteria was followed with a spectrophotometer at either A600 or A4%. Preparation of DNA and use of modqjing enzymes. Covalently closed circular plasmid DNA was recovered from E. coli by a sodium dodecyl sulfate (SDS)-salt precipitation method (Godson and Vapnek, 1973). The plasmid DNA was then purified on cesium chloride-ethidium bromide density gradients (Maniatis et ul., 1982) and dialyzed against TE (0.01 M Tris-hydrochloride [pH 8.01-l mM EDTA) buffer. Ethidium bromide was removed by butanol extraction. The rapid boiling method of Holmes and Quigley (198 1) was used for the small-scale recovery of plasmid DNA. Genomic DNA was isolated according to the procedure of Nakamura and co-workers (1979). In the case of S. mutans DNA extraction, the cells were grown in a medium containing tryptone, 20 g; monobasic potassium phosphate, 4 g; dibasic potassium phosphate, 1 g; sodium chloride, 2 g; magnesium chloride, 0.25 g; sucrose, 20 g; deionized water, 1 liter; and 20 mM DL.threonine to make the cells more susceptible to lysis (Chassy, 1976). DNA was extracted according to the procedure of Lunsford and Macrina ( 1986) except that mutanolysin

HARDESTY

144

(Sigma Chemical Co., St. Louis, MO) was substituted for lysozyme. The cells were incubated with 500 units of mutanolysin for 2 h at 37°C. The DNA preparations were stored in TE buffer at -70°C. Restriction endonucleases, Bal3 1 exonuclease, T4 DNA ligase, and DNA polymerase I were purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Reactions

ET AL.

were carried out according to the manufacturer’s instructions except for blunt-end ligations, which were carried out in the presence of polyethylene glycol 6000 (5%. final concentration) at 20°C for I8 h. The bluntend ligation procedure was adapted from that described in l“ocz~s (Vol. 8. pp. l-3 ( 1986): Bethesda Research Laboratories. Gaithersburg. MD).

TABLE I BACTERIAI.

Strain or plasmid Escherichia

AND

PLASMIDS

Relevant genotype or phenotype

source

coli

DS402 DHI CSR603

Prototroph gyrA recA relA en& the hsdR .supE thr IeuB proA phr recA argE thi uvr.4 IucY KalK ara xyl mtl gyr,4 rpsL tsx supE ,I(luc-pro) strA aru thi 08OdlacZAM I5 hsdR

TBI Salmonella

STRAINS

arizonae

R. Schmitt (Schmid ef ul. 1982) B. Bachmann (CGSC 6040) B. Bachmann (CGSC 5830) BRLh T. J. Trust (Bartlett and Trust.

47x253

1980)

B. Rosan B. Rosan Author’s collection Author’s collection

Streptococcus mutans GSS S. lactis Bacillus subtilis PR5 Lactobacillus acidophilus

PRl3 Klebsiella

pneumoniae

pUR4OO pJD2OOO

Scr’ Tet’ Scr’ Amp’ Cml’

pUC18 pCHI83 pcH186 pCH186A104 pCH 186A470 pCHl83A13 pCH 183A52 pCH183A21 pCHl83A154 pCH183A210 pITIC

Ampr LacZ Scr’ Amp’ Same as pCH 183 ScFn Amp’ Scr- Amp’ Same as pCH186Al04 El]%‘- Amp’ Same as pCH 186A52 Same as pCH186A104 Same as pCH I86A IO4 Scr’

Author’s collection R. Schmitt (Schmid et al.. 1982) J. DiRienzo (DiRienzo et al., 1986) BRL This study This study This study This study This study This study This study This study This study T. J. Trust (Bartlett and Trust, 1980)

Note. Ampicillin (Amp’), tetracycline (Tet’), chloramphenicol (Cml’), fi-galactosidase (LacZ). sucrose fermentation (Scr’), enzyme IIs” (EII*), sucrose constitutive (Scrco”“). a E. coli Genetic Stock Center (Yale University, New Haven. CN), CGSC. ’ Bethesda Research Laboratories (Gaithenburg, MD), BRL. ‘This is our own designation for a 7.6kbp sucrose plasmid previously isolated from S. arizonae47:r:253 (Bartlett and Trust, 1980).

DELETION

ANALYSIS

Construction of plasmid deletions and recombinant screening. Restriction enzyme deletions were made by completely digesting either pCH 183 or pCH 186 plasmid DNA with the appropriate restriction enzyme (PstI, KpnI, SmaI, or EcoRl), 2 units of enzyme/pg of plasmid DNA at 37°C for 60 min. The plasmid DNA was then rejoined with T4 DNA ligase. Bai3 1 exonuclease deletions of pCH 183 were constructed by completely digesting the plasmid DNA at the unique XbaI site. The linear DNA was then partially digested with Bai3I (0.1 units of enzyme/pg of plasmid DNA) at 30°C. Aliquots of the digestion mixture were removed at I-min intervals and the reaction was stopped by the addition of 5 M EDTA and buffer-saturated phenol. The DNA fragments were then extracted with phenol and chloroform and ethanol precipitated. The DNA fragments were rejoined with T4 DNA ligase. In each case, the desired constructs were selected by transforming E. coli TBl, made competent by the calcium chloride procedure (Morrison, 1979), and plating the transformants on MacConkey base agar sup plemented with sucrose and ampicillin. The appropriate deletions were verified by restriction mapping the plasmid DNA obtained from individual clones. Plasmid-directed gene products were identified according to the maxicell labeling method of Sancar et al. (1979). Preparation of bacterial cell fractions. Whole cell lysates of E. coli were prepared by resuspending washed cells ( 1.5 ml of culture) in 200 ~1 of a solution containing 2% SDS-80 mM Tris-hydrochloride (pH 6.8) and heating the suspension at 50°C for 20 min. Cell envelope and soluble protein fractions were isolated as reported previously (DiRienzo and Inouye, 1979). Briefly, cells harvested by centrifugation were washed, resuspended in 10 mM Tris-hydrochloride (pH 8.0), and disrupted by sonication. Unbroken cells were removed by centrifugation at 12,lOOg for 10 min and the envelope fraction was sedimented by centrifugation at 100,OOOg for 60 min. The protein that re-

OF

SUCROSE

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145

mained in suspension following the highspeed centrifugation was designated the soluble protein fraction and contained both cytoplasmic and periplasmic protein. In some experiments the cell envelope fraction (0.7 pg of protein/ml) was extracted with 0.5 M sodium chloride, 2% Triton X- 100, or 0.5% sodium deoxycholate in a buffer containing 20 mM Tris-hydrochloride (pH 8.0)-l mM dithiothreitol. The extractions were carried out at 20°C for 30 min. The various cell fractions were analyzed by SDS-polyacrylamide gel electrophoresis on 17.5% slab gels (DiRienzo and Inouye, 1979). Samples were solubilized, prior to electrophoresis, in a solution containing 2% SDS-80 mM Tris-hydrochloride (pH 6.8)-10% glycerol (with or without 1% pmercaptoethanol). The samples were heated at either 100°C for 10 min or 50°C for 20 min. The gels were stained with Coomassie brilliant blue and scanned with an LKB laser densitometer (LKB Instruments, Paramus, NJ). Total protein was determined according to the method of Lowry et al. ( 195 1) employing bovine serum albumin as a standard. Transport assay. For the sucrose uptake assay (Schmid et al., 1982) cells were grown overnight in K medium supplemented with 0.4% glycerol or sucrose. The cells ( I .5 ml of culture) were harvested by centrifugation for 30 set in an Eppindorf microcentrifuge and were washed three times with 100 mM sodium phosphate buffer, pH 6.6. All cell suspensions were adjusted to AdVO = 1.2 (0.5 mg/ml of cell protein). [‘4C]Sucrose, 12 &i (552 mCi/mmol, Amersham Corp., Arlington Heights, IL), was added to 1 ml of each cell suspension. Aliquots (250 ~1) were then collected on 0.22~pm GS filters (Millipore Corp., Bedford. MA) following 5,20,40, and 60 s of incubation with the radiolabeled sugar. The filters were washed three times with I ml of 100 mM sodium phosphate buffer (pH 6.6) containing 0.5 M unlabeled sucrose, placed in scintillation vials, dried, and counted with 5 ml of Scinti-Verse I1 (Fisher Scientific, Pittsburgh, PA). Uptake

146

IIARDESTY

was expressed as picomoles per minute per milligram of cell protein. PEP-dependent sugar phosphorylation. Membrane fractions, prepared from sucrosegrown cells as described above, were treated with 1% toluene at 20°C for 10 min. Assay mixtures (100 (~1total volume) contained 25 mM Tris-hydrochloride (pH 8.0) 5 mM magnesium chloride, 10 mM potassium flueride, 1 mM dithiothreitol, 10 mM PEP, 0.1 mM (‘4C]sucrose (540 mCi/mmol), saturating amounts of soluble PTS proteins (see below), and 2 IO pg of membrane protein (Jacobson et al., 1979). Mixtures were incubated for 30 min at 37°C and the reaction was stopped by adding 1 ml of 0.5 M nonradioactive sucrose. The samples were then collected on disks of DEAE-filter paper (DE81, Whatman. Clifton, NJ). The filters were dried, washed with 50 ml of water, redried, and counted. Phosphorylated sugar was expressed as picomoles per minute per milligram of membrane protein. The soluble PTS protein fraction (Jacobson et al.. 1979) was obtained by breaking washed E. coli TB I cells with glass beads and unbroken cells were removed by centrifugation. The resulting supernatant fluid was centrifuged at 100,OOOg for 90 min. Magnesium chloride was added (IO mM final concentration) and the sample was concentrated 20-fold in an Amicon ultrafiltration cell using a membrane with a :M, 5000 cutoff. The soluble fraction was dialyzed against 20 mM Tris-hydrochloride (pH 7.5)-l mM dithiothreitol10 mM magnesium chloride, standardized on the basis of activity in the in vitro phosphorylation assay, aliquoted, and stored at -20°C. Enzyme assays. A sucrase assay, as a measure of sucrose-6-phosphate hydrolase activity, was performed using cells grown overnight in 5 ml of K medium supplemented with either 0.4% glycerol or sucrose. The cells ( 1.5 ml of culture) were collected by centrifugation for 30 s in a microcentrifuge and were washed once with 100 mM sodium phosphate buffer, pH 6.6. All cell suspensions were adjusted to A4% = 2.0 (1.45 mg/ml of

ET AL.

cell protein) in the phosphate buffer. Toluene (20 ~1) was added to I ml of adjusted cell suspension and the preparation was incubated for IO min at room temperature. Each decryptified cell suspension then received 100 ~1 of 500 mM sucrose (50 mM final concentration) and was incubated for an additional 30 min at room temperature. The cell suspensions were heated in a boiling water bath for I min to stop the reaction. The samples were then centrifuged for 2 min and the supernatant fluid was collected for determining glucose and fructose concentrations with either a commercially available test kit obtained from Boehringer-Mannheim Biochemicals or reagents prepared as described (Schachter, 1975). The assay tubes contained 0.5 ml of cell supernatant fluid, 1 ml of ATP/NADP reagent (25 mM ATP, 5 mM NADP, 100 mM magnesium chloride, and 500 mM Tris-hydrochloride, pH 7.5) in a total volume of 3.0 ml. After 3 min at room temperature the A340 of the assay tubes and appropriate blank was measured to provide value A 1. Then, 8.6 ~1 of hexokinase (6 units) and 2.2 ccl of glucose-6-phosphate dehydrogenase (3 units) were added to each tube. The assay tubes were incubated for 15 min at room temperature and the A340 was determined (value n2). Following this reading, 4 ~1 of phosphoglucose isomerase (14 units) was added, the tubes were incubated for 15 min at room temperature and the A340 was determined (value n3). The concentration of glucose and fructose was then obtained by applying the formula Cone (g/liter) VX MW X AA(glc or fru), = txdxvx 1000 where V is the final volume (3.02 ml), v is the sample volume (0.5 ml), MW is the molecular weight of glucose or fructose ( 180.16 gm/ mol), d is the light path (1 cm), f is the absorption coefficient of NADPH at 340 nm (6.3 1 X mmol-’ X cm-‘), A&&) is A2 - A I, and hA(fru) is A3 - A2. Sucrase activity is

DELETION

ANALYSIS

expressed as nanomoles per minute per milligram of cell protein. Southern blotting and DNA/DNA hybridization. Genomic DNA preparations were restricted with either EcoRI or Hind111 (2 units of enzyme/pg of DNA) for 60 min at 37°C and the DNA fragments -were separated on a 0.7% agarose gel in TEB (89 mM Tris-89 mM borate-3 mM EDTA (pH 8.0)) buffer. The gel was stained with ethidium bromide and photographed and the DNA fragments were transferred to nitrocellulose (Millipore) by the procedure of Southern ( 1975). Probe DNA, labeled with 32P by nick translation (Rigby et al., 1977), was hybridized to the filters for 18 h at 42°C in 50% formamide as described (Silhavy et al., 1984). The probe DNA consisted of the 5.7kbp SalI DNA fragment isolated from pCH 183 and l-2 X lo6 cpm/filter were used. The hybridized filters were washed with 2X SSC (20X SSC contains 3 M sodium chloride-O.3 M sodium citrate, pH 7.0)- 1X Denhardt’s solution (100X Denhardt’s solution contains Ficoll, 2 g; polyvinyl pyrrolidone, 2 g; bovine serum albumin, 2 g; 3X SSC to 100 ml) for 30 min at room temperature followed by 2X SSC-0. 1% SDS for 90 min at the same temperature. The final wash consisted of 0.1 X SSC-0. 1% SDS for 90 min at 65°C.

OF SUCROSE GENES

147

key plates, were picked and the plasmid DNA was purified by cesium chloride-ethidium bromide density gradient centrifugation. The plasmids were then restriction mapped to verify the orientation of each insert. Two clones, TBl (pCH183) and TBl (pCH186), were selected for further study. Both clones contained 8A-kbp plasmids consisting of the

V BamHl

Bgl II

RESULTS

Deletion Analysis of the 5.7-kb SaN DNA Fragment Containing the Sucrose Operon A 5.7-kbp SalI DNA fragment from a hybrid sucrose plasmid was subcloned in the San site of the expression vector pUC 18 and transformed in competent cells of E. coli TB 1. The cloning strategy is shown in Fig. 1, This experiment was performed to employ the restriction sites in the polylinker of the vector to construct deletions by restriction enzyme digestion and to attempt to increase the amount of the cloned sucrose gene products. Transformants were screened, as before, on MacConkey agar plates containing sucrose and ampicillin. Several clones, which produced dark red colonies on the MacCon-

FlG. I. Cloning of the sucrose plasmid genes from pUR400. A 17.1-kbp Bg/II DNA fragment was inserted into the BQ~HI site of pBR328. A 6.8-kbp Hind111 fragment was deleted from one of the sucrose-positive, ampicillin-resistant transformants (pJD143) to create pJD1086. A 5.7-kbp Sun DNA fragment from the insert DNA in this plasmid was then subcloned in pBR328 to construct pJD2000. Cells containing pJD2000 were inducible for sucrose fermentation and were ampicillin resistant. For subsequent deletion analysis the insert DNA from pJD2000 was subcloned in each orientation in pUC 18 to construct pCH 183 and pCH 186. pUR400 DNA is represented by the open areas in the plasmid maps. Amp and Cm1 represent the ampicillin and chloramphenicol resistance genes, respectively.

148

HARDESTY

2.7-kbp vector sequence and the 5.7-kbp DNA insert (Fig. 1). The DNA insert in pCH 183 is in the orientation opposite that in pCH 186. Analysis of scrA and .wB gene expression in these clones showed that [‘“Clsucrose uptake and sucrose-6-phosphate hydrolase activities were similar to those obtained from DHl (pJD2000) (Table 2). Based on comparable levels of hydrolase and uptake activities in both TB 1 (pCH 183) and TB 1 (pCH 186) it appeared that transcription of the scrA and scrB genes was under control of the operon promoter (scrP) rather than under the control of the luc promoter in the vector. In addition, both the hydrolase and uptake activities in cells containing these plasmids were induced by growth on sucrose. It appeared that the 5.7-kbp DNA fragment contained a functional scrR gene as reported by Schmid et al. (1982) in the Salmondla plasmid. The 5.7-kbp of DNA thus was the minimum-size fragment containing a functional sucrose operon.

ET Al.

Subcloning of the 5.7-kbp Safl restriction fragment in both orientations in pUC I8 permitted the mapping of the .scrA and scrB gene loci by restriction deletion analysis due to the availability of duplicate restriction sites, one in the insert DNA and a second site in the pUC 18 polylinker. Various size deletions were made in pCH 183 and pCH 186 (Fig. 2). The removal of a 1.6-kbp PstI fragment from pCH I86 yielded transformants, represented by A104. that still demonstrated a sucrose-positive phenotype on MacConkey plates. Therefore, the deleted region appeared to lack essential genes for sucrose fermentation. However, the sucrose uptake activity was significantly reduced in these cells (Table 2). A 1.4-kbp KpnI deletion. in pCH 183, resulted in transformants which also expressed low uptake activity as shown for A I3 in Table 2. A 2.9-kbp S’maI deletion, in the same hybrid plasmid, produced pink colonies on MacConkey sucrose plates and provided transformants (A52) that expressed high hydrolase activity (approximately six

TABLE 2 PHENOTYPESOF HYBRID SUCROSEPLASMIDS Sucrose-6-phosphate hydrolase activity“ (nmol/ min per mg of protein)

Size (kg) Strain/plasmid designation DS402(pUR400) DH l(pJD2000) TBI(pUC18) TBl(pCH183) TBl(pCH186) TBl(pCH183A13) TBI(pCH186AlO4) TBl(pCH186A470) TBI(pCH183A52) TBI(pCH183621) TBI(pCH183Al54) TBI(pCHl83A210)

[‘4C]Sucrose uptake” (pmol/min per mg of protein)

Plasmid

Insert

No inducer

Inducer

No inducer

Inducer

78.0 10.6 2.7 8.4 8.4 7.0 6.8 5.8 5.5 5.3 8.2 6.2

5.7 5.7 5.7 4.3 4.1 3.1 2.8 2.6 5.5 4.6

18 ND
I92 34
102 ND 17 I40 23 59 113 25 53 67 652 120

917 851 31 I296 903 143 86 26 56 52 509 123

IVofe. Values represent the average of three experiments. ’ Cells were grown in K medium supplemented with 0.4% glycerol (no inducer) or sucrose (inducer). ’ Uptake was measured at 20 PM [‘4C]sucrose (552 mCi/mmol). ’ ND, not determined. Clone DH l(pJD2000) did not grow in glycerol or galactose-supplemented (DiRienzo er al., 1986).

medium

DELETION

ANALYSIS

149

OF SUCROSE GENES

pCH 186

Al04 A470

pCH183

,-------

L ___-

-----

&, ____

--------

-____

-____

I ,-----

I1

14

0

12

Ifi

1 I

I1

I

3

4

5

I

I1

I

6

7

I

I 6

1

Al3

1

A52

1

A21

I

Al54

1

A210

I

11

I

9

IO

kbp

scale

FIG. 2. Restriction and deletion maps of the hybrid sucrose plasmids pCH 183 and pCH186. The deletion plasmids were constructed by complete digestion with restriction enzymes, followed by ligation and transformation of the plasmids. The enzymes used for the digestions are presented in the text. In the case of A 154 and A2 10 the plasmid was digested with XbuI followed by Ba13 1 as described in the text. Each deletion was confirmed by restriction mapping. For simplicity, only the insert DNA is depicted for each deletion plasmid. Deleted regions are indicated by the dashed lines. 0, pUC 18 DNA; DNA; -, insert DNA.

times that of DS402 (pUR400) but lacked uptake activity. Finally, a 3. I-kbp EcoRI deletion (A21) displayed activities similar to those expressed by A52 except that the sucrose-6-phosphate hydrolase activity in pCH183 A21 was reduced, approximately one-half, relative to the activity expressed by pCH183A52. The sucrose uptake and hydrolase activities in all of the deletion mutants just described were constitutively expressed. The results obtained from the analysis of deletion mutants in previous studies provided tentative evidence that both the scrA and scrB genes were under the control of a single promoter and that the promoter was located in the region of the ClaI and BamHI restriction sites (DiRienzo et al,, 1986; Garcia, 1985). Analysis of the restriction deletion mutants demonstrated that the scrB gene re-

sided immediately downstream from the C/a1 site. A 2.6-kbp SmaI deletion (A470), containing the proposed promoter region and SUB gene, abolished sucrose hydrolase and uptake activities. To clarify the locations of the scrA and scrR genes, smaller deletions were made, promoter distal, using a strategy based on digestion of the plasmid with Ba13 1. Several of these deletions are illustrated in Fig. 2. A single XbaI site in pCH 183 was used to make the plasmid linear. The plasmid DNA was then digested for increasing periods of time with B&31. The restricted fragments were then each rejoined and transformed into competent cells. The transformants were plated on MacConkey medium supplemented with ampicillin and sucrose and representative colonies were picked and the plasmid DNA was purified. Deletions ranging from approximately 100

150

HARDESTY

to 2000 bp were obtained and the cells were assayed for hydrolase and sucrose uptake activities. The results showed that deletions that extended downstream from the promoter distal PstI site resulted in the constitutive expression of both the scrA and scrB genes. Deletion A 154, in which only approximately 50 bp were removed from the 5.7-kbp MI fragment, constitutively expressed sucrose uptake and hydrolase activities. When approximately 1000 bp were removed (A21O) promoter distal in the insert DNA, sucrose uptake activity was low but within detectable limits.

ET AL. A

BC

Kd 97* 66,

24~

Arnplijcation of Gene Products In previous studies attempts were made to use a maxicell system to label and identify the sucrose plasmid gene products (Garcia, 1985; Postma and Lengeler, 1985). However, the data produced in these studies were conflicting and inconclusive. We attempted to identify the sucrose gene products by cloning the sucrose genes in an expression vector and analyzing the subsequently constructed deletion mutants for functional activity correlated with the visual detection of the gene products by SDS-polyacrylamide gel electrophoresis. The sucrose-6-phosphate hydrolase and sucrose uptake activities of the various deletion mutants have been presented in Table 2 and, as noted, cells carrying pCH 183 and pCH186 appeared to contain all of the genes necessary for the inducible expression of sucrose transport and catabolism. Therefore, whole cell lysates of E. coli TBl (pUC18), TBl (pCH183), and TBl (pCH 186) were prepared from cells grown in K medium supplemented with either glycerol or sucrose. Total cell protein in these samples was then examined by SDS-polyacrylamide electrophoresis. A new protein (M, 68,000) was observed only in cells that contained either pCH183 or pCH186 and that were grown in sucrose supplemented medium. Based upon a previously reported molecular weight estimation of 55,000 for the scrB gene product (Schmid et al., 1982)

FIG. 3. Detection of amplified sucrose gene products by SDS-polyacrylamide gel electrophoresis. Total membrane fractions were prepared from cells containing hybrid sucrose plasmids and were examined on an SDS-gel. The samples were solubilized at 50°C and the gel was stained with Coomassie blue. Lane A, TBl (pUCl8); lane B, TBI (pCH183A52); lane C, TBl (pCH 183). The large arrow shows the position of a new polypeptide, A4,68,000. The position of the major outer membrane protein OmpA is also indicated.

this newly expressed protein appeared to represent either the scrA (enzyme 11%) or scrR gene product. Since the enzyme II proteins are the only PTS components that are integral membrane proteins (Lee and Saier, 1983; Saier, 1985) the membrane fractions from E. coli TBl (pUCl8), TBl (pCHl83), and TBl (pCH183852) were prepared and the proteins were separated by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 3, the A4,68,000 protein was recovered only in the membrane fraction from pCH 183. This protein represented 11% of the total membrane protein (in sucrose-induced cells) and was not detected in the soluble protein fraction from the same cells. Since sucrose-6-phosphate hydrolase activity was recovered only in the soluble protein fractions from pCH183 and pCH183A52, the M, 68,000 membrane protein was thought to be the scrA gene product. How-

DELETION

ANALYSIS

TABLE 3 PEP-DEPENDENT MEMBRANE

SUGAR PHOSPHORYLATION FRACTIONS FROM HYBRID

BY

SUCROSEPLASMIDS

Strain TBl(pCH186) TBl(pCHl83A154) TBl(pCHl83A210) TBl(pCH186A104) TBl(pCH183A13) TBI(pUC18)

pm01 of sucrose phosphate/mm per mg of membrane protein’ 70.6 85.1 67.6 115.5 15.7 7.9

LICells were grown in K medium supplemented with 0.4% sucrose and the membrane fractions were obtained as described under Materials and Methods. The assay mixtures contained 2 10 pg of membrane protein. Phosphorylation in all positive membrane samples was dependent upon the addition of PEP and soluble PTS proteins.

ever, when the membrane and soluble protein fractions from TB 1 (pCH 183A 154) were examined, the new protein was not detected (data not shown) and both the hydrolase and uptake activities, as directed by this deletion plasmid, were constitutively expressed (Table 2). Activity indicative of a functional enzyme II’“’ was also obtained when the membrane fractions from these cells were assayed for PEP-dependent sucrose phosphorylation (Table 3). In fact, cells carrying the deleted plasmids A2 10 and A 104 also exhibited PEP-dependent sucrose phosphorylation. Since the deletion in pCH183A154 was only approximately 50 bp it could not have extended beyond the scrR gene. This was supported by the observation that a truncated M,. 68,000 protein gene product was not found in either the membrane or soluble protein fractions from these cells. In maxicell experiments the M, 68,000 protein was labeled only when cells contained plasmid having the intact 5.7-kbp S&I DNA fragment or the deletion plasmid A 154 (Fig. 4). The new protein could not be extracted with either Triton X-100 or deoxycholate, detergents that usually solubilize cytoplas-

151

OF SUCROSE GENES

mic membrane proteins. According to these results the M, 68,000 polypeptide appeared to be the product of a previously unidentified sucrose gene. Sucrose Gene Homologies The 5.7-kbp MI fragment, containing the sucrose operon from pCH186, was isolated by excision from an agarose gel. The DNA fragment was labeled with 32P by nick translation and hybridized to chromosomal DNA fragments on Southern blots. Chromosomal DNA was prepared from a variety of bacteria that were known to ferment sucrose and restriction fragments were generated by complete digestion with either EcoRI and HindII1. The sucrose gene probe showed weak homology with an EcoRI fragment (approximately 20 kbp) from K. pneumoniae but failed to hybridize to the DNA from S. mutans, S. lactis, Bacillus subtilis, and Lactobacillus acidophilus (Fig. 5). Probe prepa-

Kd 97,

ABC

DE

68,

-Amp 24~

FIG. 4. Detection of the M, 68,000 sucrose gene product in maxicells. Escherichia coli CSR603 was transformed with various hybrid sucrose plasmids and deletion plasmids. The plasmid-coded gene products were then labeled with [35S]methionine as described (Sancar et al., 1979) separated by SDS-polyacrylamide gel electrophoresis, and detected by autoradiography. Approximately 1.4 X 10’ cpm were added to each well. Lane A, CSR603; lane B, CSR603 (pUC18); lane C, CSR603 (pCH186); lane D, CSR603 (pCH183A154) lane E, CSR603 (pCH 186A 104). The large arrow shows the position of the kt, 68,000 polypeptide.

152

HARDESTY

rations routinely contained trace amounts of vector DNA which were exceedingly difficult to remove (Fig. 5B, lane B). However, pUCl8 DNA did not hybridize to K. pneuthe moniae DNA. In other experiments, probe also failed to hybridize to chromosomal DNA obtained from the gram-negative bacteria E. coli DHl, Fusobacterium nucleatum, Bacteroides intermedius, Capnocytophaga gingivalis, and a 7.6-kbp sucrose plasmid (pTT 1) from S. arizonae. DISCUSSION

In this study, a sucrose operon

from a

Salmonella plasmid was subcloned in the ex-

pression vector pUC 18 and examined by restriction enzyme deletion analysis to locate the scrA (enzyme 11%‘) and scrR (regulatory protein) genes and to identify the gene products. The results are consistent with the gene order scrB, scrA, scrR. Based on previous studies employing deletion mutants (DiRienzo et al., 1986; Garcia, 1985), the sucrose operon promoter (serf’) locus appeared to map within the region of the BamHI and

A ABCDEFG

ET AL.

ClaI restriction

sites in the 5.7-kbp Sal1 DNA fragment (see Fig. 2). Cells carrying a hybrid plasmid containing the subcloned 4.7-kbp ClaI-Sal1 DNA fragment from pJD2000 failed to express either the scrA or scrB genes, as determined from an analysis of enzyme activities (DiRienzo et al., 1986). In addition, neither of the structural genes were expressed when a 1.2-kbp BamHI DNA fragment was deleted from pJD2000. Garcia (1985) found that the scrB gene was located just downstream from the BamHI restriction site by subcloning a 2.6-kbp SafI-EcoRI DNA fragment. However, amplification of the scrB gene was not obtained in the high copy number plasmids pBR325 and pCD5. In the present study, we have confirmed that the scrB gene resides within the same DNA fragment on the basis of results obtained with the plasmid deletions pCH 183A52 and pCH183A21. Since the scrB gene product has been reported as M, 55,000 (Schmid et al., 1982) and 60,000 (Garcia, 1985) the 2.6kbp DNA fragment can readily contain this gene and the promoter.

B ABCDEFG

kb 23.1) g-$c 414 w 2.3% 2.0

FIG. 5. Homology between sucrose plasmid genes and K. pneumoniae DNA. The 5.7-kbp SaA insert fragment, containing the sucrose genes, was isolated from pCH 186, labeled with “P, and hybridized to chromosomal DNA fragments on a Southern blot. Chromosomal DNA was prepared from a variety of bacteria that were known to ferment sucrose. (A) Ethidium bromide-stained 0.7% agarose gel. Lane A, XDNA digested with HindIII; lane B, pCH 186 digested with SalI; lane C, K. pneumoniae DNA; lane D, S. Zactis DNA; lane E, S. mutans DNA; lane F, L. acidophilus DNA; lane G, B. subtih DNA. DNA samples in lanes C-G were digested with EcoRI. (B) Autoradiogram of the Southern blot of the gel in (A) following hybridization.

DELETION

ANALYSIS

We have placed the scrR gene promoter distal in the operon sequence on the basis of results obtained with deletion pCH 183A 154. In this deletion plasmid a 50-bp fragment was removed resulting in the constitutive expression of both hydrolase and sucrose uptake activities, PEP-dependent sucrose phosphorylation, and a significant reduction in the amount of M, 68,000 protein produced. When maxicells of CSR603 (pCH 183A154) were labeled the M, 68,000 protein was observed. The scrR gene product has not been clearly identified in earlier studies. Maxicell labeling experiments have tentatively indicated a gene product having an M, of 37,000 (Postma and Lengeler, 1985) or 41,000 (Garcia, 1985). Therefore, this deletion was too small to extend beyond a single gene and most likely resides in the scrR gene. These results indicate that the .scrA and M, 68,000 protein (designated .scrC) genes reside between the scrB and scrR genes. The data suggest that the scrA gene may reside immediately downstream from the scrB gene. Cells containing plasmids with deletions extending downstream from the scrB locus (pCH183A13, pCH186A104, and pCH183A2 10) lack the M, 68,000 protein, exhibit the low constitutive expression of sucrose hydrolase and uptake activities, and contain membrane-associated sucrose phosphorylation activity. However, it is not clear at the present time why TB 1 (pCH 183A 13) lacks sucrose phosphorylation activity. It is possible but not likely that the M, 68,000 protein is the product of the scrR gene based on its identification in maxicells containing the deletion plasmid pCH 183A154, the reported tentative size of the regulatory protein (Garcia, 1985; Postma and Lengeler, 1985) and our finding that the M, 68,000 protein is tightly associated with the membrane. Even though large molecular weight regulatory proteins have been reported, such as in the case of the IV, 94,000 mulT gene product that functions as a positive activator of the maltose regulon (Debarbrouille and Schwartz, 1979), these proteins are not located in the cell membrane. A simi-

OF

SUCROSE

GENES

153

lar argument can be made in the case of the scrA gene product. Since the enzyme 11% has not been purified nor has the scrA gene been sequenced, the true size of the gene product is not known. Maxicell labeling experiments, from independent studies, have yielded conflicting results with the size of the gene product assigned as M, 25,000 (Garcia, 1985) or 60,000 (Postma and Lengeler, 1985). However, it was not clearly delineated in these studies which maxicell-labeled polypeptides represented sucrose or sucrose/vector fusion gene products. The M, 68,000 protein identified in our study is not likely to be the product of fused genes since it was produced by cells that had plasmids containing sucrose gene inserts in each of two orientations relative to the vector DNA. Enzymes II that require an enzyme III for substrate phosphorylation have an apparent molecular weight in the range of 25,000-45,000 (Saier, 1985; Schachter, 1975). In addition, overexpression of the recently cloned bg!C gene, coding for the enzyme IIB@, was observed as a very diffuse and broad band in SDS-gels (Bramley and Kornberg, 1987). This anomalous mobility appears to be a characteristic of hydrophobic sugar transport proteins such as the enzyme II Bg’ (Bramley and Kornberg, 1987) and the lactose permease (Ehring et al., 1980). We have noted a broad, diffuse band, apparent M, 39,000, in SDS-gels of the membrane fraction from TBI (pCH 183) and TB 1 (pCH 186) (see Fig. 3, lane C). This diffuse band may represent the overexpressed scrA gene product. The estimated size of this protein would be appropriate for the enzyme II*’ since the enzyme IIIG’c~S”’ is it4,20,000 (Meadow and Roseman, 1982). It has been proposed that for those enzymes II that require an enzyme III the sum of the molecular weights of these proteins equals about 68,000 (Saier, 1977, 1985). For example, the molecular weights for the glucose pair are 45,000 and 20,000 (Erni et al., 1982; Meadow and Roseman, 1982). It is interesting, however, to speculate as to the function of the M, 68,000 protein. It does not appear to be an enzyme 111~’ based on the known

IIARDES l-Y ET AL

154

molecular weights for other sugar-specific enzymes III (Saier, 1977, 1985). Other possibilities under investigation include its role as a sucrose-binding protein. Due to the similarities between the sucrose metabolic system of the Sulmonellu plasmid and that of K. pneumoniae it has been suggested that the sucrose plasmid originated from the Kfehsiella chromosome (Lengeler d al., 1982; Schmid et al.. 1982). We showed that the sucrose operon DNA sequence from pUR400 hybridized to a K. pneumoniuc~ chromosomal DNA fragment, thus providing strong support for the proposed evolutionary hypothesis. Several DNA probes containing the entire sucrose operon sequence (5.7-kbp MI DNA fragment) and a 2.5kbp SmaI DNA fragment, containing the scrA gene, hybridized to the DNA from Kiebsielfu but not to DNA from other grampositive sucrose-fermenting bacteria. The use of the cloned sucrose gene sequences as hybridization probes should facilitate the cloning of analogous sucrose metabolic genes of K. pneumoniae. ACKNOWLEDGMENTS We thank B. Bachmann, B. Rosan, R. Schmitt, and T. J. Trust for supplying bacterial strains and plasmids. This work was supported by Public Health Service Grants DE02623 and DE07 I I8 from the National Institute of Dental Research. During the course of this work G.C. was supported by Biomedical Research Support Grant RR05337.

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ANALYSIS

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Dunny