Vol. 163, No. 1JOURNAL OF BACTERIOLOGY, JUlY 1985, p. 37-450021-9193/85/070037-09$02.00/0Copyright X) 1985, American Society for Microbiology

Characterization of the Salmonella typhimurium mgl Operon and ItsGene Products

NORBERT MULLER, HANS-GEORG HEINE, AND WINFRIED BOOS*Department ofBiology, University of Konstanz, D-7750 Konstanz, Federal Republic of Germany

Received 22 January 1985/Accepted 9 April 1985

In Salmonella typhimurium and Escherichia coli the high-affinity galactose transport system, which containsa periplasmic galactose-binding protein as an essential component, is encoded by the mgl genes. The entire mglregion of S. typhimurium is contained on a 6.3-kilobase EcoRI restriction fragment, which has been cloned intoplasmid vectors. We determined the extent of the mgl region on this fragment by TnS mutagenesis, examinationof lacZ fusions to mgl genes, and subcloning smaller restriction fragments. Polyacrylamide gel electrophoresisof protein preparations derived from strains carrying different plasmids was used to identify the mgl geneproducts. We conclude that the mgl operon consists of four genes that form a single transcription unit: mglB,mglA, mglE, and mglC. The mglB gene codes for galactose-binding protein (33,000 daltons), mglA codes for amembrane-bound protein of 51,000 daltons, and mglC codes for a 29,000-dalton membrane protein. The mglEproduct was less well characterized. Its existence was inferred from a mglE-lacZ protein fusion located betweenmglA and mglC. In addition, the coupled transcription-translation in vitro system indicated that mglE codes fora 21,000-dalton protein.

The mgl genes of Salmonella typhimurium and Esch-erichia coli code for a binding-protein-dependent transportsystem with a high affinity for galactose (5, 6, 33). The bestcharacterized component is the periplasmic galactose-bind-ing protein (GBP), which represents the recognition site ofthe system (1, 28). Besides its function in galactose trans-port, GBP acts as the galactose chemoreceptor (17, 43).

Transport systems that depend on periplasmic-bindingproteins are characterized by several properties: (i) they aremulticomponent systems; (ii) they are primary pumps andare not driven by proton or other cation gradients; (iii) theyhave substrate affinities in the micromolar range and canestablish concentration gradients exceeding 1:104; and (iv)their primary substrate recognition site consists of "soluble"binding proteins positioned in the periplasmic space ofenteric gram-negative bacteria. These properties distinguishthem from other active transport systems, like the lactosesystem of E. coli, which consists of only one protein andwhose energy coupling is linked to the electrochemicalpotential of protons (18). The mechanism by which bindingprotein-mediated systems translocate substrate through thecytoplasmic membrane is not understood. In particular, it isintriguing how the binding protein catalyzes the transfer ofsubstrate through the cytoplasmic membrane with the helpof membrane proteins that by themselves do not appear toexhibit substrate binding. From well-analyzed systems, itappears that these systems consist, in addition to the bindingprotein, of three proteins that are associated with the innermembrane. The gene for the periplasmic binding protein is inmost cases promoter proximal (19, 20, 31, 39). The compo-nents of the transport system are not synthesized in equalamounts. Whereas the binding protein is synthesized in morethan 30,000 copies per cell (14) and establishes a 1 mMsolution in the periplasm, the membrane-bound componentsmay amount to only 500 copies or less per cell (37).The mgl-dependent transport system for galactose in E.

* Corresponding author.

coli maps at 45 min on the linkage map (26). Three genes,mglA, mglB, and mglC, have been defined by complementa-tion analysis (30), and the region has been cloned (15, 34). Inaddition to GBP (the mglB gene product) a membraneprotein with an apparent molecular mass of 51,000 daltonshas been identified as the mglA product by both studies,whereas the mglC gene product is claimed by Harayama etal. (15) to be a membrane protein with a molecular mass of38,000 daltons. We have cloned the corresponding regionfrom S. typhimurium (29). In our present paper we analyzedthis region by Tn5 mutagenesis, construction of lacZ fusionsto mgl genes, and subcloning of restriction fragments. Wefound the mglA and mglC products to be membrane-boundproteins of 51,000 and 29,000 daltons, respectively. In addi-tion, we identified a lacZ protein fusion within a fourth gene(mglE) that is located between mglA and mglC. From thelocalization of the hybrid protein in the membrane, weconclude that the mglE product is also a membrane-associ-ated protein.

MATERIALS AND METHODS

Bacterial strains and growth conditions and genetic meth-ods. Strains, phages, and plasmids are listed in Table 1. Luriabroth (LB) and minimal medium A (MMA) were made ac-cording to the method of Miller (27) and supplemented withcarbon sources and other requirements. DNA manipulationswere according to the method of Maniatis et al. (24). Trans-formation was by the method of Lederberg and Cohen (23).The presence of the chloramphenicol resistance gene "CATcartridge" of pCM1 and pCM7 (12) in mgl hybrid plasmids(see Fig. 6) was screened by testing ampicillin-resistant (Apr)transformants for growth on LB plates containing 10 ,ug ofchloramphenicol per ml. X phage lysates were prepared withstrain LE392 according to the method of Davis et al. (13).

Isolation of mgl-lacZ fusions in vivo. mgl-lacZ proteinfusions were isolated in strain MC4100 carrying the mglhybrid plasmid pHG4 by using phage XplacMu3 as described

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38 MULLER, HEINE, AND BOOS

TABLE 1. Bacterial strains, plasmids, and phagesStrain, Reference

plasmid, or Known genotype or Originphage

MC4100

HfrG6LE392

DS410T

LA5440LA5709

LA6221

NM303

74/3

pACYC184pBR322pMLB524

pMLB1034

pBR327pCM1

pCM7

XplacMu3

XpMu5O7.3

X::Tn5

F- araDJ39 A(argF-lac)U169rpsL150deoCI relAl thiAptsF25flbB5301

Hfr hisF- hsdR514 supE44 supF58

A(lacIZY)6 galK2 galT22metBI trpR55 A-

F- minA minB ara lacYmalA mtl xyl rpsL thi tonAazi gyrA A (glpT-glpA)593

F- mglB550 gatAF- mgl512 lacY galE ptsF

arg recAl srl (strain doesnot contain GBP)

F- mglB501 lacY galE argfpk zee-700::TnJO

F- mg503 lacZ lacY+ recAl(strain does not containGBP)

Hfr thi glpT

(10)

Cmr TcrApr TcrApr; carries the wild-type

lacZ EcoRI site and thefollowing C-terminalportion of the lacZ gene

Apr, carries cloning sites in alinker replacing the firsteight condons of lacZ

Apr TcrApr, carries CAT cartridge in

Sall site of Tcr region ofpBR327

Apr, carries CAT cartridge inHinklIl site of Tcr regionof pBR327

X-Mu hybrid phage, imm21(for construction of I-

galactosidase proteinfusions)

X-Mu hybrid phage imm21Sam7 MuA+B+

Xb221 c1857 rex::TnS

by Bremer et al. (9). To 0.1 ml of logarithmically grown cellsresuspended in 10 mM MgSO4, 50 p.l of XplacMu3 (4 x 109phages per ml) and 50 pul of the helper phage XpMu5O7.3 (4 x

109 phages per ml) were added. After incubation at 37°C for20 min, LB (0.4 ml) containing 10 mM MgSO4 was added,and the cells were incubated at 37°C for at least another 3 huntil lysis occurred. This lysate was used to infect strainNM303. After phenotypic expression at 37°C for 1 h in LB,the mixture was plated on McConkey lactose plates contain-ing 5 p.g of tetracycline per ml. Red colonies were screenedon LB plates containing 5-bromo-4-chloro-3-indolyl-3-D-galactoside in the presence and absence of 0.2% glucose.Colonies that were less blue in the presence of glucose weregrown in LB and tested for galactose transport and binding.This allowed us to distinguish lacZ fusions in mglB fromfusions in mgl genes distal to mgiB.For subcloning, DNA from the large XplacMu3 hybrid

plasmids was digested with EcoRI, ligated with EcoRI-

digested pMLB524 (38), transformed into strain NM303, and

selected on McConkey lactose plates in the presence of 30

jig of ampicillin per ml. Red colonies were isolated, their

plasmid DNA was subjected to restriction analysis, and

mgl-lacZ fusions were identified (see Fig. 1). One mgl-lacZfusion (pNM101) was constructed by subcloning the EcoRI-

BgiII fragment of pHG4 carrying mgiA, mglB, and part of

mglE into plasmid pMLB1034 digested with EcoRI and

BamHI (38). This connected the mglE gene, which is cut byBgiII, to the eighth codon of the lacZ gene, creating an

in-frame fusion of lacZ to mgiE.TnS mutagenesis of the mgl hybrid plasmid pHG4. Tn5

mutagenesis of the mgl hybrid plasmid pHG4 was done byusing phage X::TnS (2) as described by Tommassen et al.

(40). The mgl phenotype of TnS-carrying plasmids after

transformation into the mgl mutant LA5709 was screened bymeasuring galactose transport, as described below.

Galactose transport. Galactose transport was measured in

cells washed and resuspended to an optical density at 578 nm

of 0.5 in MMA without a carbon source as described byMuller et al. (29). Instead of 0.1 ,uM [14C]galactose, 10 ,uMwas used. This assured a clear distinction between active

transport, binding of galactose to GBP in the periplasmwithout transport, and lack of any mgl function (see Fig. 2).

Labeling of plasmid-encoded proteins. Labeling of plasmid-encoded proteins was done in minicells of strain DL410Tharboring different mgl hybrid plasmids. Minicells were

prepared by the method of Meagher et al. (25) modified

according to Reeve (32). Minicells (0.5 ml) with an opticaldensity at 578 nm of 0.5 were incubated at 37°C with 5 ,uCi of[35S]methionine (1,100 mCi/mmol) for 30 min in MMA con-

taining 0.2% glycerol. The minicells were then washed in

MMA, resuspended in 25 of sample buffer, heated to

100°C for 5 min before slab gel electrophoresis (21), and

autoradiography.Plasmid-directed in vitro protein synthesis was done ac-

cording to the methods of Zubay (44) and Schumacher and

Bussmann (36). Routinely, 1 to 5 ,ug of DNA per 200 of

incubation mixture was used. The S-30 extract used was

isolated from strain 74/3 (36).For the gel electrophoretic analysis of unlabeled proteins,

whole cells were dissolved in sample buffer (see Fig. 4), or

the cells were first fractionated. Two methods were used for

fractionation. The first method separates soluble proteinsfrom membrane-bound proteins by NaOH treatment (35);the second separates periplasmic, cytoplasmic, and mem-

brane-bound proteins (3). The second method was modified

in that spheroplasts were broken only by repeated freezing in

liquid nitrogen and thawing in 0°C water; sonification was

omitted. Before electrophoresis in sodium dodecyl sulfate

(SDS)-containing slab gels according to the method of Laem-

mli (21), the samples were routinely heated to 100°C in

sample buffer for 10 min. This method precluded the identi-

fication of the mglC gene product, since this protein appar-ently is not solubilized from membranes by SDS at 100°C. To

solubilize MglC, membranes isolated by the NaOH method

(35) were incubated in sample buffer for 1 h at 37°C before

heating to 65°C for 10 min followed by electrophoresis. Gels

were stained with either Coomassie brilliant blue or silver

stain according to the method of Wray et al. (42).f-Galactosidase assay. The ,B-galactosidase assay in

toluenized whole cells was done as described by Miller (27).For determining the P-galactosidase activity of membrane-

bound hybrid proteins, 10-ml cultures were grown overnightat 32°C in LB containing 30 p.g of ampicillin per ml. Mem-

branes were isolated according to the method of Boeke and

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THE S. TYPHIMURIUM mgl OPERON

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101

pMLB S24

Iac Z pMLB 1034

P/Om gl Genesand their FVducts l l

rngl8 mglA rngl rnglC

33kd SI kd 21 kd 29 kdGOP)

FIG. 1. Genetic analysis of the mgl region. Depicted is the subcloning of DNA containing parts of the mgl region, with the location of TnSinsertions in mgl genes and mgl-lacZ fusions shown. The mgl hybrid plasmids pHG30, pHG14, pHG16, and pHG15 were constructed withpBR322 as vector. Plasmid pHG4 is a derivative of pACYC184. pHG30 and pHG4 contain the complete mgl operon, pHG14 and pHG16contain mglE and mglC, and pHG15 contains mglB and mgIA. TnS insertions in pHG4 are indicated by triangles: insertions 11 and 5 are inmglB, insertions 32, 7, and 22 are in mglA, and insertion 14 is in mglE or mglC. lacZ fusions are indicated by shaded areas. Fusions 506 and515 are to mglB, fusions 512, 518, and 502 are to mglA, fusion 101 is to mglE, and fusion 514 is to mg1C.

Model (3) and resuspended in 0.5 ml of 20 mM Tris-hydro-chloride (pH 8.0) containing 4% sucrose. Samples werediluted 10-fold in Z buffer and assayed for ,-galactosidaseactivity in the standard test (27).

RESULTS AND DISCUSSIONAnalysis of the mgl region. We previously cloned the mgl

region as a 6.3-kilobase (kb) EcoRI fragment in pACYC184to generate plasmid pHG4 starting from a library of EcoRIfragments in Xgt7 (29). Figure 1 shows the restriction endo-nuclease analysis of this EcoRI fragment after recloning intopBR322 to generate plasmid pHG30. Subcloning yieldedthe plasmids pHG14, pHG15, and pHG16 (Fig. 1). Strain

LA5709, an mgl mutant carrying the last three plasmids, wasunable to transport galactose, but cells containing pHG15retained galactose by a process that was independent of timeand not inhibited by energy uncouplers. This rapid ac-cumulation represented binding to the large amount ofperiplasmic GBP (Fig. 2). Strains LA5440 (mglBSSO) andLA6221 (mglBSOI), which carry missense mutations in mglB(7, 8), were complemented to mgl+ by plasmid pHG15 byusing the criterion of restoration of wild-type transportactivity (data not shown). This result indicated that GBPfrom S. typhimurium could replace E. coli GBP and func-tionally interacts with membrane components of the E. colimgl transport system.

of pHG30

i ====i I I.

we

VOL. 163, 1985 39

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40 MULLER, HEINE, AND BOOS

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co

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LA0

0 10 30 60 120Time (sec)

FIG. 2. Uptake of galactose in mgl-hybrid plasmid-carryingstrains. The recipient strain LA5709 does not synthesize GBP. Weanalyzed strain LA5709 carrying the following plasmids: pHG30(mgl+) (0), pHG15 (containing only mglB+ and mglA+) (A), andpHG16 (containing only mglE+ and mglC+) (O). Uptake wasmeasured at a galactose concentration of 10 ,uM. The differencebetween pHG15 and pHG16, both transport negative, representsbinding of galactose to the large amount of GBP in the periplasm.

To further localize the extent of the mgl genes, we isolatedinsertions of the kanamycin resistance transposon TnS intopHG4. Of 30 independently isolated plasmids carrying Tn5,only five remained mgl+. These insertions mapped in thepACYC184 vector DNA. All other plasmids had become mgland had insertions located within a 2.6-kb DNA segment(Fig. 1).As another approach to identification of mgl genes, we

constructed six mgl-lacZ fusions integrating the XplacMu3phage (9) into pHG4 and subcloning the fusion carryingEcoRI fragments into the fusion cloning vector pMLB524(38). In addition, fusion 101 was constructed in vitro byligating the larger EcoRI-BgIII fragment of pHG4 withEcoRI-and BamHI-digested plasmid pMLB1034 (38). Thisplasmid contains the structural gene of lacZ, starting withcodon 8 of P-galactosidase in front of a linker DNA contain-ing EcoRI and BamHI. The resulting plasmid (pNM101)contained lacZ fused in frame to an mgl gene. All fusionswere Lac' and produced mgl-lacZ hybrid proteins. Theyhad lost the mgl-dependent galactose transport activity, andtheir fusion joints were within a 3.3-kb region on the left onthe 6.3-kb EcoRI fragment (Fig. 1). Accordingly, the begin-ning of the mgl operon was located within 0.7 kb between theleft EcoRI site and the fusion joint of the earliest mglB-lacZfusion (fusion 506). The end of the mgl operon was less welldefined. The most promoter distal mutation established bythe mglC-lacZ fusion (fusion 514) was located some 2.4 kbapart from the right EcoRI site of the 6.3-kb EcoRI fragmentpresent in pHG4 or pHG30.

Expression and localization of mgl gene products. StrainLA5709 carrying the plasmids pHG30, pHG15, pHG16, orpBR322 was fractionated into cytoplasm, periplasm, andmembranes. Protein content was analyzed by SDS-polyacrylamide gel electrophoresis. Only cells carryingpHG15 and pHG30 synthesized GBP (mglB gene product) as

the major periplasmic protein (Fig. 3). The membrane frac-tion of pHG15 and pHG30 contained two additional proteinsof 51,000 and 38,000 daltons. Both proteins also could befound, albeit in smaller amounts, in the cytoplasmic fraction.They correspond to the mglA and mglC gene products asidentified by Rotman and Guzman (34) and Harayama et al.(15) in E. coli.By labeling plasmid-encoded proteins in minicells, we

found that GBP and the 51,000-dalton MglA protein, but notthe 38,000-dalton protein, were synthesized in pHG30,pHG4, and pHG15. No mgl-specific proteins were observedwith pHG14 and pHG16 (Fig. 4). Figure 4 also shows theminicell analysis of plasmids carrying TnS in mgl genes.Plasmids containing the insertions 32, 7, and 22 could directsynthesis of GBP, but not the MglA protein, whereas plas-mids containing insertions 5 and 11 synthesized neither. Aplasmid with insertion 14 synthesized both proteins.

Plasmids carrying mgl-lacZ fusions (Fig. 1) were alsoanalyzed for their capacity to synthesize GBP and the MgIAprotein. The proteins of whole cells carrying these fusionplasmids were analyzed by SDS-polyacrylamide gel electro-phoresis. Plasmids with fusions 506 and 515 synthesizedneither GBP nor the MglA protein, indicating that the fusionoccurred in mglB. Plasmids with fusions 512, 518, and 502still synthesized GBP but not the MglA protein. Theyrepresent fusions to mgIA. Fusions 101 and 514 producedboth proteins and must be in genes distal to mglA (data notshown).

peripla srn membrane cytoplasm

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(-LID

co CoL Ca CiL

a bc

'D C'N C14 Lfl _o C,4 fL 0D C:lo r) m , .-

m--

roD WU -1 LO U aCU aLWa) -,

LC-LCo CrLCL1- Co C1d e f g h i j k I m n

kd

51 k -

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333~ ~ 4. . se-rf

_ _ _ _ 628

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FIG. 3. Cellular localization of mglB and mglA gene products inmgl-hybrid plasmid-carrying strains. The recipient strain LA5709carrying the plasmids pHG30 (mgl+), pHG15 (mglB+ and mglA+),and pHG16 (mglE+ and mglC+) or the vector pBR322 was separatedinto periplasmic-membrane and cytoplasmic fractions by themethod of Boeke and Model (3). The gel was stained with silvernitrate (42). As indicated by the arrowheads, GBP was found only inthe periplasm of strains carrying pHG30 and pHG15. The mglAproducts, 51,000- and 38,000-dalton proteins, were present in strainscarrying pHG30 and pHG15. Both proteins were partially membranebound and partially cytoplasmic.

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THE S. TYPHIMURIUM mgl OPERON

A C 3a E a b c d e f g h i j k I r

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-43s In

51kI

B

kd

a b C d e f g h j k I.

68

43 - " 4s40 --

25.7 - m m

;Tn5

_pGBP-- --- -- GBP

--- -Tn 5G BP

225.7-Uar_ --...- wm* * ..... ...AM -

14.3 a-.

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~~pp -_In_.__lit- 1 4.3

-_ ... .F i. *... _

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FIG. 4. Identification of mgl gene products in strains carrying mgl-hybrid plasmids. The minicell strain DS410T contained the followingplasmids: pHG30 (mgl+) (lane a); pHG14 (mglE+ and mglC+) (lane b); pHG15 (mglB+ and mglA+) (lane c); pHG16 (mglE+ and mglC+) (laned); and pHG4 (mgl+) (lane e). TnS insertions in pHG4 are the following: insertion 5 (in mgIB) (lane f); insertion 32 (in mgIA) (lane g); insertion11 (in mglB) (lane h); insertion 7 (in mglA) (lane i); insertion 14 (in mglE or mglC) (lane j); insertion 22 (in mgIA) (lane k); and insertion 35(in pACYC184) (lane 1). The minicells were labeled with [35S]methionine and then solubilized in SDS at 60°C and subjected to polyacrylamidegel electrophoresis (15% acrylamide). (A) Coomassie blue-stained gel; (B) autoradiogram of the dried gel. GBP, its precursor (pGBP), the51,000-dalton mglA gene product, and TnS-encoded proteins are indicated.

The above analysis of these various plasmids containingparts of the mgl region, with TnS insertions as well as lacZfusions to mgl genes, allowed the following conclusions (Fig.1). mglB is the first gene in an operon that contains mglA asthe second gene. The product of mglA is a 51,000-dalton

kd a b c d

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66.2

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membrane protein. A 38,000-dalton membrane protein (Fig.3) also appears to be an mgl gene product. Since it issynthesized by pHG15, which contains mglB and mglA asthe only intact genes, and since the mglB gene product isonly 33,000 daltons, the 38,000-dalton protein can only be aproduct of mgIA. From the observations that the 38,000-dalton protein was only sometimes present and that it wasnot observed in minicells, we feel that it is likely to be arelatively stable proteolytic fragment of MglA. PlasmidpHG15 is too small to code for the 33,000-dalton GBP, the51,000-dalton mglA product, and an additional 38,000-daltonprotein, which itself would need more than 1 kb of DNA.TnS insertions 5 and 32 define a region of less than 0.5 kb ofDNA between mglB and mglA. Therefore, the 38,000-daltonprotein cannot be encoded by this region. The region be-tween the fusion joint of fusion 502, the last fusion in mglA,and the HindIII site of pHG15 is also less than 0.5 kb. Thus,the 38,000-dalton protein cannot be encoded by a gene distalto mgIA.

Identification of the mglC gene product as a membrane'bound protein of 29,000 daltons. Since a TnS insertion(insertion 14 in Fig. 1) and two lacZ fusions (fusions 514 and

*t .. 4

31.0 *w1 .. ,21.5

FIG. 5. Identification of the mgIC gene product. Membranes ofstrain LA5709 carrying different mgl-hybrid plasmids and mglC-lacZ fusions were isolated by the method of Russel and Model (35).They were solubilized in SDS at 370C for 1 h, followed by heating to65°C and gel electrophoresis. The acrylanlide concentration of thegel was 15%. The following plasmids were analyzed: pNM514(carrying the mglC-lacZ fusion) (lane b); pHG30 (mgl+) (lane c);pBR322 (lane d); pHG15 (containing mglB and inglA) (lane f);pHG16 (containing mglE and mg1C) (lane g); and pBR322 (lane h).Lanes a and e contain marker proteins. Arrowheads indicate the51,000-dalton MglA protein in lane b and the 29,000-dalton MglCprotein in lane c.

.w w-w *W4A Am*- --l-" I" ow,'.4, ll.,= -mmm

im

VOL. 163, 1985 41

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a...4 i.. ..j, k, .,.4 -.11,11. 6-. . - ;".'o 16.1 -* O-W

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42 MULLER, HEINE, AND BOOS

kd

925

66.2 -

-MGL A45.0

31.0

21.5-

14.4-

FIG. 6. Identification of the mglE gene product by in vitrosynthesis with mgl containing DNA as template. Proteins were

labeled with [355]methionine, and samples were treated as describedin the legend to Fig. 5. After electrophoresis, the gel (15%acrylamide) was dried and autoradiographed. The following DNApreparations were used as template: pNM101 (mglE-lacZ) (lane a);pNM514 (mglC-lacZ) (lane b); pNM514-1 (mglC-lacZ, deletion inmglE) (lane c); pBR322 (lane d); pHG30 (mgl+) (lane e); and controlsynthesis without DNA template (lane f). The arrow indicates theposition of the mgl-lacZ fusion proteins in lanes a, b, and c.

Arrowheads indicate the 21,000-dalton MglE protein in lane b andthe 16,000-dalton MglE protein carrying the internal deletion causedby the removal of the Hindlll and BglII fragment.

101 in Fig. 1) distal to mglA destroyed mgl-dependenttransport activity, one or more genes distal to mglA mustbelong to the mgl system. Analyzing the SDS-polyacrylamide gel electrophoresis protein patterns of plas-mids carrying the entire mgl region, we detected largeamounts of GBP and Mg1A, but no other mgl product.Several changes in the experimental protocol allowed us todetect a third component, the mglC gene product. StrainLA5709 carrying the various plasmids grown in LB had to becultivated to an optical density at 578 nm exceeding 1.2. Ata lower density the mgl operon is not strongly expressed, atleast when grown in LB. In addition, the plasmid copy

number increases when the culture enters the stationary

phase. The isolated membranes of such cells were firstsolubilized in SDS for 1 h at 37°C before incubation for 10min at 65°C. Apparently, boiling in SDS results in theformation of aggregates of some highly hydrophobic pro-teins, preventing their entry into the acrylamide gel, and thussuch proteins escape detection.The analysis of membrane proteins solubilized without

boiling is shown in Fig. 5. Strain LA5709 carrying a plasmidwith lacZ fusion 514 did not synthesize a protein thatappeared as a diffuse band with an apparent molecular massof ca. 29,000 daltons in strains carrying the entire mgl region(pHG30). Strains carrying the plasmid pHG15, which con-tains only mglB and mglA, and pHG16, which lacks the mglpromoter, did not synthesize this protein (Fig, 5). In addi-tion, LA5709 carrying pHG4 with TnS insertion 14(mgIEIC: :TnS) also did not synthesize this protein. Theobservation that the expression of the 29,000-dalton proteinwas repressed in strains carrying pHG30 by growth in thepresence of glucose (data not shown) further supports itsidentification as an mgl gene product. We conclude thisprotein is the mglC product. Fusion 514, therefore, is inmglC.

Fourth gene (mglE) located between mglA and mglC. Re-moval of a small HindIII-BglII fragment from pNM514resulting in pNM514-1 (Fig. 7) did not eliminate the synthesisof the mglC-lacZ fusion protein (Table 2). The plasmid alsostill produced an intact MgIA protein; thus, fusion 514cannot be in mgIA. The deletion could be located in theamino-terminal portion of the mglC-lacZ fusion, causing anin-frame shortening of the hybrid protein. In this case theribosomal binding site of mglC has to be located to the left ofthe HindIll site. Another explanation is more likely: theHindIII-BglII fragment belongs to an mgl gene different frommglA and mg1C, and removal of the fragment in pNM514leaves mg1C-1acZ fusion 514 intact. On SDS-polyacrylamidegel electrophoresis the size of this hybrid protein is notaltered in comparison with the fusion produced by plasmidpNM514 (Fig. 6). The gel shown in Fig. 6 contained 15%acrylamide, which was not optimal for the separation of thefusion proteins. A similar gel with 10% acrylamide showedno difference between the fusions in lanes b and c, but aslightly smaller size in lane a (data not shown). The existenceof mglE is further supported by the following. Using acoupled transcription-translation in vitro system accordingto the method of Zubay (44) and plasmids containing dif-

TABLE 2. Localization of ,B-galactosidase activity of mgl-lacZfusion proteins in cytoplasmic and membrane fractionsa

P-Galactosidase activity in:Plasmid Fusion in: Membrane Cytoplasmic

fraction fraction

pNM506 mglB 4.43 2.41pNM512 mglA 3.14 0.64pNM502 mglA 2.62 0.61pNM514 mglC 0.12 <0.01pNM101 mglE 3.97 1.01pNM514-1 mglC 0.61 0.12Control; HfrG6 grown 6.18 57.43

in LB containing2 - 10-4 M IPTGa Strain NM303 carrying the various mgl-lacZ fusion plasmids was grown at

32°C overnight in LB containing 50 ,ug of ampicillin per ml. Membrane andcytoplasmic fractions were prepared as described in the text. f3-Galactosidaseactivity is given as (micromoles per minute per 109 cells) x 10-2. IPTG,Isopropyl-13-D-thiogalactopyranoside.

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THE S. TYPHIMURIUM mgl OPERON 43

'U4t-)

0

(ac Z M pMLB 524

0

w

514-3LL.-

iac Z

0

I pMLB 524

FIG. 7. The mgl region is one operon. Replacement of the Sall-Sall fragment of pNM514 still allowed the synthesis of the mglC-lacZprotein. Removal of the EcoRI-EcoRI fragment of pNM514-2 eliminated the mgl promnoter and no longer permitted the synthesis of themglC-lacZ fusion protein. The analogous experiments with pNM101 (not shown here) proved that the mglE-lacZ fusion also is under thecontrol of the mgl promoter.

ferent regions of mgl as DNA templates, we could identify aprotein that fits the expected properties of the mglE products(Fig. 6). The synthesis of a protein of 21,000 daltons wasdirected by pNM514 (mglE+) but not by pNM101, whichcontains a mglE-lacZ fusion. The MglE protein was reducedby some 5,000 daltons after introducing a 0.15-kb in-framedeletion between the HindIII and BglII sites in pNM514. Theplasmid directed in vitro synthesis of MglA, and the pre-cursor of MglB (GBP) can easily be seen in Fig. 6. MglCcannot be clearly identified. Also, it is puzzling that MglE isonly visible as a band in preparations that do not contain theMglC protein. It appears that MgIE and MglC form com-plexes that either do not enter the gel or are covered byunrelated protein bands. On the DNA level mglE begins tothe left of the HindIII site and extends to the right of theBglII site in Fig. 1.To demonstrate that mglE and mglC belong to the same

operon as mglB and mgiA, the experiments outlined in Fig.7 were done. The SalI-SalI restriction fragment of pNM514was replaced by a Sall-SalI fragment containing the chlor-amphenicol transacetylase gene without the original CATpromoter (pNM514-2). This introduced a new EcoRI site.Subsequent partial digestion with EcoRI, followed by religa-tion, yielded pNM514-3, in which the mgl promoter,

TABLE 3. P-Galactosidase activity of mgl-lacZ fusion proteins intoluene-treated cellsa

p-Galactosidase activityPlasmid Fusion in: Induced by Repressed by

Uninduced ~103 M fucose 0.2% glucose

pNM506 mglB 5.80 7.51 1.73pNM515 mglB 5.71 7.80 2.11pNM512 mglB 4.23 6.01 1.14pNM518 mglA 6.62 8.53 1.30pNM502 mglA 3.32 4.81 0.61pNM514 mglC 0.92 0.92 0.47pMLB524 Vector <0.01 <0.01 <0.01pNM101 mglE 8.14 17.00 2.28pMLB1034 Vector <0.01 <0.01 <0.01

a Strain NM303 harboring the various hybrid plasmids was grown at 32°Covernight in MMA plus 0.2% glycerol, 0.2% Casamino Acids, and 50 ,ug ofampicillin per ml. The activity is given as (micromoles per minute per 109cells) x 10-.

mglB,and part of mglA were removed. This resulted in theloss of expression of the mg1C-1acZ fusion protein. Theanalogous experiment (not shown in Fig. 6) with pNM101demonstrated that the mglE-1acZ fusion 101 protein also isnot expressed after removal of the mgl promoter. Therefore,it is clear that mglB, mglA, mglE, and mg1C, in that order,constitute an operon.

Regulation and localization of the mgl-lacZ fusion proteins.The availability of protein fusions to all mg! genes promptedus to study the regulation, as well as the localization, ofthese fusion proteins. Table 3 shows that the specific I-

galactosidase activity as measured in toluenized cellsshowed the expected variations. The highest activity was

found with the mglE-lacZ fusion, the lowest with the mglC-lacZ fusion. Induction by D-fucose, the inducer of the mglsystem, was weak but consistent. More prominent was therepression by glucose. Table 2 shows that all fusions were

partially membrane associated.Comparison of the mgl operon of S. typhimurium and E.

coli. In comparing our restriction analysis of the S.typhimurium mgl operon with the corresponding analysis ofthe E. coli mgl operon (15, 34), no similarities could beobserved. Nevertheless, at least two gene products are verysimilar. The GBPs produced by both species are of nearlyidentical size, and both cross-react with antibodies againstthe E. coli protein (29). Furthermore, S. typhimurium GBPwas fully active in transport and chemotaxis, when theremaining mgl gene products and the chemotactic signaltransducer Trg (taxis to ribose and galactose) came fromn E.coli. Similarly, the mglA gene product, a 51,000-daltonmembrane protein, appears to be the same in both organ-isms. The S. typhimurium mglC gene product that we

identified differs from that reported for E. coli. Whereas we

find a 29,000-dalton membrane protein, Harayama et al. (15)reported a 38,000-dalton membrane protein. As discussedabove, we feel that the 38,000-dalton protein, which we alsoobserved, must be encoded by the mglA gene. Either it istranslated from only part of mglA or it is a degradationproduct of the normal 51,000-dalton mglA gene product.Finally, applying the gene fusion technique and an in vitroprotein-synthesizing system, we could identify mglE as a

fourth gene, not yet reported in E. coli, that is locatedbetween mglA and mglC and codes for a 21,000-daltonprotein.

0

UJ514*-2 t

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44 MULLER, HEINE, AND BOOS

ACKNOWLEDGMENTSWe are grateful to E. Bremer for his help in the lacZ fusion

technique.Financial support was obtained by the Deutsche Forschungsge-

meinschaft (SFB 156) and the Fond der Chemischen Industrie.

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