163 , No. 1 Salmonella typhimurium Gene Products mgl Operon and Its Characterization of the NORBERT MULLER , HANS-GEORG HEINE , AND WINFRIED BOOS * Department of Biology , 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 contains a periplasmic galactose-binding protein as an essential component , is encoded by the mgl genes .
The entire mgl region of S. typhimurium is contained on a 6.3-kilobase EcoRI restriction fragment , which has been cloned into plasmid vectors .
We determined the extent of the mgl region on this fragment by TnS mutagenesis , examination of lacZ-fusions to mgl genes , and subcloning smaller restriction fragments .
Polyacrylamide gel electrophoresis of protein preparations derived from strains carrying different plasmids was used to identify the mgl gene products .
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 a membrane-bound protein of 51,000 daltons , and mglC codes for a 29,000-dalton membrane protein .
The mglE product was less well characterized .
Its existence was inferred from a mglE-lacZ protein fusion located between mglA and mglC .
In addition , the coupled transcription-translation in-vitro system indicated that mglE codes for a 21,000-dalton protein .
The mgl genes of Salmonella typhimurium and Esch-erichia coli code for a binding-protein-dependent transport system with a high affinity for galactose ( 5 , 6 , 33 ) .
The best characterized component is the periplasmic galactose-bind-ing protein ( GBP ) , which represents the recognition site of the system ( 1 , 28 ) .
Besides its function in galactose transport , GBP acts as the galactose chemoreceptor ( 17 , 43 ) .
Transport systems that depend on periplasmic-binding proteins are characterized by several properties : ( i ) they are multicomponent systems ; ( ii ) they are primary pumps and are not driven by proton or other cation gradients ; ( iii ) they have substrate affinities in the micromolar range and can establish concentration gradients exceeding 1:104 ; and ( iv ) their primary substrate recognition site consists of `` soluble '' binding proteins positioned in the periplasmic space of enteric gram-negative bacteria .
These properties distinguish them from other active transport systems , like the lactose system of E. coli , which consists of only one protein and whose energy coupling is linked to the electrochemical potential of protons ( 18 ) .
The mechanism by which binding protein-mediated systems translocate substrate through the cytoplasmic membrane is not understood .
In particular , it is intriguing how the binding protein catalyzes the transfer of substrate through the cytoplasmic membrane with the help of membrane proteins that by themselves do not appear to exhibit substrate binding .
From well-analyzed systems , it appears that these systems consist , in addition to the binding protein , of three proteins that are associated with the inner membrane .
The gene for the periplasmic binding protein is in most cases promoter proximal ( 19 , 20 , 31 , 39 ) .
The components of the transport system are not synthesized in equal amounts .
Whereas the binding protein is synthesized in more than 30,000 copies per cell ( 14 ) and establishes a 1 mM solution in the periplasm , the membrane-bound components may amount to only 500 copies or less per cell ( 37 ) .
The mgl-dependent transport system for galactose in E. coli maps at 45 min on the linkage map ( 26 ) .
Three genes , mglA , mglB , and mglC , have been defined by complementation analysis ( 30 ) , and the region has been cloned ( 15 , 34 ) .
In addition to GBP ( the mglB gene product ) a membrane protein with an apparent molecular mass of 51,000 daltons has been identified as the mglA product by both studies , whereas the mglC gene product is claimed by Harayama et al. ( 15 ) to be a membrane protein with a molecular mass of 38,000 daltons .
We have cloned the corresponding region from S. typhimurium ( 29 ) .
In our present paper we analyzed this region by Tn5 mutagenesis , construction of lacZ-fusions to mgl genes , and subcloning of restriction fragments .
We found the mglA and mglC products to be membrane-bound proteins of 51,000 and 29,000 daltons , respectively .
In addition , we identified a lacZ protein fusion within a fourth gene ( mglE ) that is located between mglA and mglC .
From the localization of the hybrid protein in the membrane , we conclude that the mglE product is also a membrane-associ-ated protein .
MATERIALS AND METHODS Bacterial strains and growth-conditions and genetic methods .
Strains , phages , and plasmids are listed in Table 1 .
Luria broth ( LB ) and minimal-medium A ( MMA ) were made according to the method of Miller ( 27 ) and supplemented with carbon sources and other requirements .
DNA manipulations were according to the method of Maniatis et al. ( 24 ) .
Transformation was by the method of Lederberg and Cohen ( 23 ) .
The presence of the chloramphenicol resistance gene `` CAT cartridge '' 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 of chloramphenicol per ml .
X phage lysates were prepared with strain LE392 according to the method of Davis et al. ( 13 ) .
Isolation of mgl-lacZ-fusions in-vivo .
mgl-lacZ protein fusions were isolated in strain MC4100 carrying the mgl hybrid plasmid pHG4 by using phage XplacMu3 as described by Bremer et al. ( 9 ) .
To 0.1 ml of logarithmically grown cells resuspended in 10 mM MgSO4 , 50 p.l of XplacMu3 ( 4 x 109 phages per ml ) and 50 pul of the helper phage XpMu5O7 .3 ( 4 x 109 phages per ml ) were added .
After incubation at 37 °C for 20 min , LB ( 0.4 ml ) containing 10 mM MgSO4 was added , and the cells were incubated at 37 °C for at least another 3 h until lysis occurred .
This lysate was used to infect strain NM303 .
After phenotypic expression at 37 °C for 1 h in LB , the mixture was plated on McConkey lactose plates containing 5 p.g of tetracycline per ml .
Red colonies were screened on 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 were grown in LB and tested for galactose transport and binding .
This allowed us to distinguish lacZ-fusions in mglB from fusions 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-lacZ fusion ( 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 by BgiII , 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 was done mutagenesis of the mgl hybrid plasmid pHG4 by using 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 by measuring galactose transport , as described below .
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 by Muller et al. ( 29 ) .
Instead of 0.1 , uM [ 14C ] galactose , 10 , uM was used .
This assured a clear distinction between active transport , binding of galactose to GBP in the periplasm without 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 DL410T harboring 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 optical density at 578 nm of 0.5 were incubated at with 5 37 °C , uCi of [ 35S ] methionine ( 1,100 mCi/mmol ) for 30 min in MMA containing 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 according 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 proteins from 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 identification of the mglC gene product , since this protein apparently 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 ) .
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 overnight at 32 °C in LB containing 30 p.g of ampicillin per ml .
Membranes were isolated according to the method of Boeke an 3 J. BACTERIOL .
Bacterial strains , plasmids , and phages Reference or Origin Known genotype plasmid , or phage MC4100 ( 10 ) F-araDJ39 A ( argF-lac ) U169 rpsL150 deoCI relAl thiA ptsF25flbB5301 Hfr his F-hsdR514 supE44 supF58 A ( lacIZY ) 6 galK2 galT22 metBI trpR55 A-F-minA minB ara lacY malA mtl xyl rpsL thi tonA azi gyrA A ( glpT-glpA ) 593 F-mglB550 gatA F-mgl512 lac Y galE ptsF arg recAl srl ( strain does not contain GBP ) F-mglB501 lacY galE arg fpk zee-700 : : TnJO F-mg503 lacZ lacY + recAl ( strain does not contain GBP ) Hfr thi glpT HfrG6 LE392 DS410T LA5440 LA5709 LA6221 NM303 74/3 Cmr Tcr Apr Tcr pACYC184 pBR322 pMLB524 Apr ; carries the wild-type lacZ EcoRI site and the following C-terminal portion of the lacZ gene Apr , carries cloning sites in a linker replacing the first eight condons of lacZ Apr Tcr Apr , carries CAT cartridge in Sall site of Tcr region of pBR327 Apr , carries CAT cartridge in HinklIl site of Tcr region of pBR327 X-Mu hybrid phage , imm21 ( for construction of Igalactosidase protein fusions ) X-Mu hybrid phage imm21 Sam7 MuA + B + Xb221 c1857 rex : : TnS pMLB1034 pBR327 pCM1 pCM7 XplacMu3 XpMu5O7 .3 X : : Tn5 I .
A U ¬ I fit CO , tX-t = ¬ UB i a w t x O I X121 Tet Restr , ctionmap of pHG30 1 Amp .
Amp i A IF Tot ' pHG 14 ' a = Xa .
C cm lii-s - `` Iamo o th d ==== i I Amp I. pHG 16 Tot , a , a pHG 15 .
Tet Amp e = 8 W 00 3 A A pACYC 18L U ,1 ¬ x CD Tn 5 Insertions in pHG A1 A A 221 35 11 5 32 7 22 14 Er 0 w mgl-lac Z Proteinfusions 515 w pMLB S24 ; - ac 518 Z 502 ; 514 Z pMLB 1034 1 % w0e1 Iac P/O m gl Genes and their FVducts l l rngl8 mglA rngl rnglC 33kd SI kd 21 kd 29 kd GOP ) FIG. 1 .
Genetic analysis of the mgl region .
Depicted is the subcloning of DNA containing parts of the mgl region , with the location of TnS insertions in mgl genes and mgl-lacZ-fusions shown .
The mgl hybrid plasmids pHG30 , pHG14 , pHG16 , and pHG15 were constructed with pBR322 as vector .
Plasmid pHG4 is a derivative of pACYC184 .
pHG30 and pHG4 contain the complete mgl operon , pHG14 and pHG16 contain mglE and mglC , and pHG15 contains mglB and mgIA .
TnS insertions in pHG4 are indicated by triangles : insertions 11 and 5 are in mglB , 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 and 515 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 were diluted 10-fold in Z buffer and assayed for,-galactosidase activity in the standard test ( 27 ) .
RESULTS AND DISCUSSION Analysis of the mgl region .
We previously cloned the mgl region as a 6.3-kilobase ( kb ) EcoRI fragment in pACYC184 to generate plasmid pHG4 starting from a library of EcoRI fragments in Xgt7 ( 29 ) .
Figure 1 shows the restriction endo-nuclease analysis of this EcoRI fragment after recloning into pBR322 to generate plasmid pHG30 .
Subcloning yielded the plasmids pHG14 , pHG15 , and pHG16 ( Fig. 1 ) .
Strain LA5709 , an mgl mutant carrying the last three plasmids , was unable to transport galactose , but cells containing pHG15 retained galactose by a process that was independent of time and not inhibited by energy uncouplers .
This rapid accumulation represented binding to the large amount of periplasmic GBP ( Fig. 2 ) .
Strains LA5440 ( mglBSSO ) and LA6221 ( mglBSOI ) , which carry missense mutations in mglB ( 7 , 8 ) , were complemented to mgl + by plasmid pHG15 by using the criterion of restoration of wild-type transport activity ( data not shown ) .
This result indicated that GBP from S. typhimurium could replace E. coli GBP and functionally interacts with membrane components of the E. coli mgl transport system 518 Z 502 ; 514 Z pMLB 1034 1 % w0e1 Iac P/O m gl Genes and their FVducts l l rngl8 mglA rngl rnglC 33kd SI kd 21 kd 29 kd GOP ) FIG. 1 .
Genetic analysis of the mgl region .
Depicted is the subcloning of DNA containing parts of the mgl region , with the location of TnS insertions in mgl genes and mgl-lacZ-fusions shown .
The mgl hybrid plasmids pHG30 , pHG14 , pHG16 , and pHG15 were constructed with pBR322 as vector .
Plasmid pHG4 is a derivative of pACYC184 .
pHG30 and pHG4 contain the complete mgl operon , pHG14 and pHG16 contain mglE and mglC , and pHG15 contains mglB and mgIA .
TnS insertions in pHG4 are indicated by triangles : insertions 11 and 5 are in mglB , 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 and 515 are to mglB , fusions 512 , 518 , and 502 are to mglA , fusion 101 is to mglE , and fusion 514 is to mg1C .
To further localize the extent of the mgl genes , we isolated insertions of the kanamycin resistance transposon TnS into pHG4 .
Of 30 independently isolated plasmids carrying Tn5 , only five remained mgl + .
These insertions mapped in the pACYC184 vector DNA .
All other plasmids had become mgl and 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 XplacMu3 phage ( 9 ) into pHG4 and subcloning the fusion carrying EcoRI fragments into the fusion cloning vector pMLB524 ( 38 ) .
In addition , fusion 101 was constructed in-vitro by ligating the larger EcoRI-BgIII fragment of pHG4 with EcoRI-and BamHI-digested plasmid pMLB1034 ( 38 ) .
This plasmid contains the structural gene of lacZ , starting with codon 8 of P-galactosidase in front of a linker DNA containing EcoRI and BamHI .
The resulting plasmid ( pNM101 ) contained lacZ fused in frame to an mgl gene .
All fusions were Lac ' and produced mgl-lacZ hybrid proteins .
They had lost the mgl-dependent galactose transport activity , and their fusion joints were within a 3.3-kb region on the left on the 6.3-kb EcoRI fragment ( Fig. 1 ) .
Accordingly , the beginning of the mgl operon was located within 0.7 kb between the left EcoRI site and the fusion joint of the earliest mglB-lacZ fusion ( fusion 506 ) .
The end of the mgl operon was less well defined .
The most promoter distal mutation established by the mglC-lacZ fusion ( fusion 514 ) was located some 2.4 kb apart from the right EcoRI site of the 6.3-kb EcoRI fragment present in pHG4 or pHG30 .
Expression and localization of mgl gene products .
Strain LA5709 carrying the plasmids pHG30 , pHG15 , pHG16 , or pBR322 was fractionated into cytoplasm , periplasm , and membranes .
Protein content was analyzed by SDS-polyacrylamide gel electrophoresis .
Only cells carrying pHG15 and pHG30 synthesized GBP ( mglB gene product ) as f c protein ( Fig. 3 ) .
The membrane fraction of pHG15 and pHG30 contained two additional proteins of 51,000 and 38,000 daltons .
Both proteins also could be found , albeit in smaller amounts , in the cytoplasmic fraction .
They correspond to the mglA and mglC gene products as identified 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 not the 38,000-dalton protein , were synthesized in pHG30 , pHG4 , and pHG15 .
No mgl-specific proteins were observed with pHG14 and pHG16 ( Fig. 4 ) .
Figure 4 also shows the minicell analysis of plasmids carrying TnS in mgl genes .
Plasmids containing the insertions-32 , 7 , and 22 could direct synthesis of GBP , but not the MglA protein , whereas plasmids containing insertions 5 and 11 synthesized neither .
A plasmid with insertion 14 synthesized both proteins .
Plasmids carrying mgl-lacZ-fusions ( Fig. 1 ) were also analyzed for their capacity to synthesize GBP and the MgIA protein .
The proteins of whole-cells carrying these fusion plasmids were analyzed by SDS-polyacrylamide gel electro-phoresis .
Plasmids with fusions 506 and 515 synthesized neither GBP nor the MglA protein , indicating that the fusion occurred in mglB .
Plasmids with fusions 512 , 518 , and 502 still synthesized GBP but not the MglA protein .
They represent fusions to mgIA .
Fusions 101 and 514 produced both proteins and must be in genes distal to mglA ( data not shown ) .
the major periplasm e-ri tn4 co 0 7a - Co 0 .
LA 0 0 10 30 60 120 Time ( sec ) FIG. 2 .
Uptake of galactose in mgl-hybrid plasmid-carrying strains .
The recipient strain LA5709 does not synthesize GBP .
We analyzed strain LA5709 carrying the following plasmids : pHG30 ( mgl + ) ( 0 ) , pHG15 ( containing only mglB + and mglA + ) ( A ) , and pHG16 ( containing only mglE + and mglC + ) ( O ) .
Uptake was measured at a galactose concentration of 10 , uM .
The difference between pHG15 and pHG16 , both transport negative , represents binding of galactose to the large amount of GBP in the periplasm .
peripla srn membrane cytoplasm-1 - ] r F C4 'D C'Nr C14 Lfl o C ,4 fL 0D rCo : LID lo ) m , m. - -- ( - U U a a co Co D W -1 LO CU LW a ) L Ca CiL LC-L Co CrLCL1-Co C1 a bc d e f g h i j k I m - , n kd 51 k-38 kc-GBP 4 333 ~ ~ .
Cellular localization of mglB and mglA gene products in mgl-hybrid plasmid-carrying strains .
The recipient strain LA5709 carrying the plasmids pHG30 ( mgl + ) , pHG15 ( mglB + and mglA + ) , and pHG16 ( mglE + and mglC + ) or the vector pBR322 was separated into periplasmic-membrane and cytoplasmic fractions by the method of Boeke and Model ( 3 ) .
The gel was stained with silver nitrate As indicated by the arrowheads , GBP was found only in ( 42 ) .
the periplasm of strains carrying pHG30 and pHG15 .
The mglA products , 51,000-and 38,000-dalton proteins , were present in strains carrying pHG30 and pHG15 .
Both proteins were partially membrane bound and partially cytoplasmic A C 3 B * w1 .
, I b c d e f g h i j k I f h a b C d k e g j a E a r .
; Tn5 68 ~ 68 4s 43 - '' 40 51k pGBP -- GBP -- Tn 5 ¬ - ¬ -- -43 s In I 25.7-m m G BP 25.7 mar. ... .
AM - ¬ ¬ - U w - * * ¬ 14.3 a ¬ ... - * .
ll - ~ ~ pp it mmm - .
w w-w * W4A Am * -- l - '' I '' ow , ' .4 , ll. , = ¬ im-1 4.3 FIG. 4 .
Identification of mgl gene products in strains carrying mgl-hybrid plasmids .
The minicell strain DS410T contained the following plasmids : pHG30 ( mgl + ) ( lane a ) ; pHG14 ( mglE + and mglC + ) ( lane b ) ; pHG15 ( mglB + and mglA + ) ( lane c ) ; pHG16 ( mglE + and mglC + ) ( lane d ) ; and pHG4 ( mgl + ) ( lane e ) .
TnS insertions in pHG4 are the following : insertion 5 ( in mgIB ) ( lane f ) ; insertion 32 ( in mgIA ) ( lane g ) ; insertion 11 ( 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 polyacrylamide gel electrophoresis ( 15 % acrylamide ) .
( A ) Coomassie blue-stained gel ; ( B ) autoradiogram of the dried gel .
GBP , its precursor ( pGBP ) , the 51,000-dalton mglA gene product , and TnS-encoded proteins are indicated .
The above analysis of these various plasmids containing parts of the mgl region , with TnS insertions as well as lacZ-fusions to mgl genes , allowed the following conclusions ( Fig. 1 ) .
mglB is the first gene in an operon that contains mglA as the second gene .
The product of mglA is a 51,000-dalton membrane protein .
A 38,000-dalton membrane protein ( Fig. 3 ) also appears to be an mgl gene product .
Since it is synthesized by pHG15 , which contains mglB and mglA as the only intact genes , and since the mglB gene product is only 33,000 daltons , the 38,000-dalton protein can only be a product of mgIA .
From the observations that the 38,000-dalton protein was only sometimes present and that it was not observed in minicells , we feel that it is likely to be a relatively stable proteolytic fragment of MglA .
Plasmid pHG15 is too small to code for the 33,000-dalton GBP , the 51,000-dalton mglA product , and an additional 38,000-dalton protein , which itself would need more than 1 kb of DNA .
TnS insertions 5 and 32 define a region of less than 0.5 kb of DNA between mglB and mglA .
Therefore , the 38,000-dalton protein can not be encoded by this region .
The region between 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 can not be encoded by a gene distal to 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 .
w w-w * W4A Am * -- l - '' I '' ow , ' .4 , ll. , = ¬ im-1 4.3 FIG. 4 .
Identification of mgl gene products in strains carrying mgl-hybrid plasmids .
The minicell strain DS410T contained the following plasmids : pHG30 ( mgl + ) ( lane a ) ; pHG14 ( mglE + and mglC + ) ( lane b ) ; pHG15 ( mglB + and mglA + ) ( lane c ) ; pHG16 ( mglE + and mglC + ) ( lane d ) ; and pHG4 ( mgl + ) ( lane e ) .
TnS insertions in pHG4 are the following : insertion 5 ( in mgIB ) ( lane f ) ; insertion 32 ( in mgIA ) ( lane g ) ; insertion 11 ( 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 polyacrylamide gel electrophoresis ( 15 % acrylamide ) .
( A ) Coomassie blue-stained gel ; ( B ) autoradiogram of the dried gel .
GBP , its precursor ( pGBP ) , the 51,000-dalton mglA gene product , and TnS-encoded proteins are indicated .
kd b a c d 925 0 On .
Identification of the mgIC gene product .
Membranes of strain 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 to 65 °C and gel electrophoresis .
The acrylanlide concentration of the gel 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 the 51,000-dalton MglA protein in lane b and the 29,000-dalton MglC protein in lane c 31.0 21.5 101 in Fig. 1 ) distal to mglA destroyed mgl-dependent transport activity , one or more genes distal to mglA must belong to the mgl system .
Analyzing the SDS-polyacrylamide gel electrophoresis protein patterns of plasmids carrying the entire mgl region , we detected large amounts of GBP and Mg1A , but no other mgl product .
Several changes in the experimental protocol allowed us to detect a third component , the mglC gene product .
Strain LA5709 carrying the various plasmids grown in LB had to be cultivated to an optical density at 578 nm exceeding 1.2 .
At a lower density the mgl operon is not strongly expressed , at least when in LB .
In addition , the plasmid grown copy number increases when the culture enters the stationary-phase .
The isolated membranes of such cells were first solubilized in SDS for 1 h at 37 °C before incubation for 10 min at 65 °C .
Apparently , boiling in SDS results in the formation of aggregates of some highly hydrophobic proteins , preventing their entry into the acrylamide gel , and thus such proteins escape detection .
The analysis of membrane proteins solubilized without boiling is shown in Fig. 5 .
Strain LA5709 carrying a plasmid with lacZ fusion 514 did not synthesize a protein that appeared as a diffuse band with an apparent molecular mass of ca. 29,000 daltons in strains carrying the entire mgl region ( pHG30 ) .
Strains carrying the plasmid pHG15 , which contains only mglB and mglA , and pHG16 , which lacks the mgl promoter , did not synthesize this protein ( Fig , 5 ) .
In addition , LA5709 carrying pHG4 with TnS insertion 14 ( mgIEIC : : TnS ) also did not synthesize this protein .
The observation that the expression of the 29,000-dalton protein was repressed in strains carrying pHG30 by growth in the presence of glucose ( data not shown ) further supports its identification as an mgl gene product .
We conclude this protein is the mglC product .
Fusion 514 , therefore , is in mglC .
Fourth gene ( mglE ) located between mglA and mglC .
Removal of a small HindIII-BglII fragment from pNM514 resulting in pNM514-1 ( Fig. 7 ) did not eliminate the synthesis of the mglC-lacZ fusion protein ( Table 2 ) .
The plasmid also still produced an intact MgIA protein ; thus , fusion 514 can not be in mgIA .
The deletion could be located in the amino-terminal portion of the mglC-lacZ fusion , causing an in-frame shortening of the hybrid protein .
In this case the ribosomal binding site of mglC has to be located to the left of the HindIll site .
Another explanation is more likely : the HindIII-BglII fragment belongs to an mgl gene different from mglA and mg1C , and removal of the fragment in pNM514 leaves mg1C-1acZ fusion 514 intact .
On SDS-polyacrylamide gel electrophoresis the size of this hybrid protein is not altered in comparison with the fusion produced by plasmid pNM514 ( Fig. 6 ) .
The gel shown in Fig. 6 contained 15 % acrylamide , which was not optimal for the separation of the fusion proteins .
A similar gel with 10 % acrylamide showed no difference between the fusions in lanes b and c , but a slightly smaller size in lane a ( data not shown ) .
The existence of mglE is further supported by the following .
Using a coupled transcription-translation in-vitro system according dif-to the method of Zubay ( 44 ) and plasmids containing pNM502 pNM514 pNM101 pNM514-1 Control ; HfrG6 grown in LB containing-2 10-4 M IPTG a 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 and cytoplasmic fractions were prepared as described in the text .
f3-Galactosidase activity is given as ( micromoles per minute per 109 cells ) x 10-2 .
IPTG , Isopropyl-13-D-thiogalactopyranoside ferent regions of mgl as DNA templates , we could identify a protein that fits the expected properties of the mglE products ( Fig. 6 ) .
The synthesis of a protein of 21,000 daltons was directed by pNM514 ( mglE + ) but not by pNM101 , which contains a mglE-lacZ fusion .
The MglE protein was reduced by some 5,000 daltons after introducing a 0.15-kb in-frame deletion between the HindIII and BglII sites in pNM514 .
The plasmid directed in-vitro synthesis of MglA , and the precursor of MglB ( GBP ) can easily be seen in Fig. 6 .
MglC can not be clearly identified .
Also , it is puzzling that MglE is only visible as a band in preparations that do not contain the MglC protein .
It appears that MgIE and MglC form complexes that either do not enter the gel or are covered by unrelated protein bands .
On the DNA level mglE begins to the left of the HindIII site and extends to the right of the BglII 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 pNM514 was replaced by a Sall-SalI fragment containing the chlor-amphenicol transacetylase gene without the original CAT promoter ( pNM514-2 ) .
This introduced a new EcoRI site .
Subsequent partial digestion with EcoRI , followed by religation , yielded pNM514-3 , in which the mgl promoter , mglB , and part of mglA were removed .
This resulted in the loss of expression of the mg1C-1acZ fusion protein .
The analogous experiment ( not shown in Fig. 6 ) with pNM101 demonstrated that the mglE-1acZ fusion 101 protein also is not 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 prompted us to study the regulation , as well as the localization , of these fusion proteins .
Table 3 shows that the specific Igalactosidase activity as measured in toluenized cells showed 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 mgl system , was weak but consistent .
More prominent was the repression 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 of the E. coli mgl operon ( 15 , 34 ) , no similarities could be observed .
Nevertheless , at least two gene products are very similar .
The GBPs produced by both species are of nearly identical size , and both cross-react with antibodies against the E. coli protein ( 29 ) .
Furthermore , S. typhimurium GBP was fully active in transport and chemotaxis , when the remaining mgl gene products and the chemotactic signal transducer Trg ( taxis to ribose and galactose ) came fromn E. coli .
Similarly , the mglA gene product , a 51,000-dalton membrane protein , appears to be the same in both organisms .
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 discussed we feel that the 38,000-dalton protein , which we also above , observed , must be encoded by the mglA gene .
Either it is translated from only part of mglA or it is a degradation product of the normal 51,000-dalton mglA gene product .
Finally , applying the gene fusion technique and an in-vitro protein-synthesizing system , we could identify mglE as a fourth gene , not yet reported in E. coli , that is located between mglA and mglC and codes for a 21,000-dalton protein kd 925 66.2 ¬ - MGL A 45.0 31.0 21.5 ¬ 14.4 ¬ FIG. 6 .
Identification of the mglE gene product by in-vitro synthesis with mgl containing DNA as template .
Proteins were labeled with [ 355 ] methionine , and samples were treated as described in the legend to Fig. 5 .
After electrophoresis , the gel ( 15 % acrylamide ) was dried and autoradiographed .
The following DNA preparations were used as template : pNM101 ( mglE-lacZ ) ( lane a ) ; pNM514 ( mglC-lacZ ) ( lane b ) ; pNM514-1 ( mglC-lacZ , deletion in mglE ) ( lane c ) ; pBR322 ( lane d ) ; pHG30 ( mgl + ) ( lane e ) ; and control synthesis without DNA template ( lane f ) .
The arrow indicates the position of the mgl-lacZ fusion proteins in lanes a , b , and c. Arrowheads indicate the 21,000-dalton MglE protein in lane b and the 16,000-dalton MglE protein carrying the internal deletion caused by the removal of the Hindlll and BglII fragment .
Localization of , B-galactosidase activity of mgl-lacZ fusion proteins in cytoplasmic and membrane fractionsa P-Galactosidase activity in : Membrane Cytoplasmic fraction fraction 4.43 2.41 3.14 0.64 2.62 0.61 0.12 < 0.01 3.97 1.01 0.61 0.12 6.18 57.43 Plasmid Fusion in : pNM506 pNM512 mglB mglA mglA mglC mglE mglC 0 UJ ` U4 0 M t - ) ( ac Z pMLB 524 514 * -2 t 0 0 w iac Z I pMLB 524 514-3LL .
The mgl region is one operon .
Replacement of the Sall-Sall fragment of pNM514 still allowed the synthesis of the mglC-lacZ protein .
Removal of the EcoRI-EcoRI fragment of pNM514-2 eliminated the mgl promnoter and no longer permitted the synthesis of the mglC-lacZ fusion protein .
The analogous experiments with pNM101 ( not shown here ) proved that the mglE-lacZ fusion also is under the control of the mgl promoter .
P-Galactosidase activity of mgl-lacZ fusion proteins in toluene-treated cellsa p-Galactosidase activity Fusion in : Induced by Repressed by Uninduced ~ 103 M fucose 0.2 % glucose pNM506 mglB 5.80 7.51 1.73 pNM515 mglB 5.71 7.80 2.11 pNM512 mglB 4.23 6.01 1.14 pNM518 mglA 6.62 8.53 1.30 pNM502 mglA 3.32 4.81 0.61 0.92 0.47 pNM514 mglC 0.92 pMLB524 Vector < 0.01 < 0.01 < 0.01 pNM101 mglE 8.14 17.00 2.28 pMLB1034 Vector < 0.01 < 0.01 < 0.01 a Strain NM303 harboring the various hybrid plasmids was grown at 32 °C overnight in MMA plus 0.2 % glycerol , 0.2 % Casamino Acids , and 50 , ug of ampicillin per ml .
The activity is given as ( micromoles per minute per 109 cells ) x 10 - .
Plasmid We are grateful to E. Bremer for his help in the lacZ fusion technique .
Financial support was obtained by the Deutsche Forschungsgemeinschaft ( SFB 156 ) and the Fond der Chemischen Industrie .
Transport of sugars and amino-acids in bacteria .
Detection of transposable antibiotic resistance determinants with phage lambda , p. 555-558 .
In A. I. Bukhari , J. A. Shapiro , and S. L. Adhya ( ed .
) , DNA insertion elements , plasmids and episomes .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 3 .
Boeke , J. D. , and P. Model .
A procaryotic membrane anchor sequence : carboxyl terminus of bacteriophage fl gene III protein retains it in the membrane .
Bolivar , F. , R. L. Rodriguez , P. J. Greene , M. C. Betlach , H. L. Heyneker , and H. W. Boyer .
Construction and characterization of new cloning vehicles .
A multipurpose cloning system .
The galactose-binding protein and its relationship to the 3-methylgalactoside pernlease from Escherichia coli .
Boos , W. , and M. 0 .
Close linkage between a galactose-binding protein and the 3-methylgalactoside permease in Escherichia coli .
Boos , W. , I. Steinacher , and D. Engelhardt-Altendorf .
Mapping of mgiB , the structural gene of the galactose-binding protein of Escherichia coli .
Brass , J. M. , U. Ehmann , and B. Bukau .
Reconstitution of maltose transport in Escherichia coli : conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells .
Bremer , E. , T. J. Silbavy , J. M. Weiseman , and G. M. Weinstock .
X placMu : a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step .
Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu .
Chang , A. C. Y. , and S. N. Cohen .
Construction and characterization of amplifiable DNA cloning vehicles derived from the P1SA cryptic miniplasmid .
Close , T. J. , and R. L. Rodriguez .
Construction and characterization of the chloramphenicol-resistence gene cartridge : a new approach to the transcriptional mapping of extrachromosomal elements .
Davis , R. W. , D. Botstein , and J. R. Roth .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 14 .
Dietzel , I. , V. Kolb , and W. Boos .
Pole cap formation in Escherichia coli following induction of the maltose-binding protein .
Harayamal S. , J. Bollinger , T. Iino , and G. L. Hazelbauer .
Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning .
Harfield , D. , M. Hofnung , and M. Schwartz .
Genetic analysis of the maltose A region in Escherichia coli .
Hazelbauer , G. L. , and J. Adler .
Role of the galactosebinding protein in chemotaxis of E. coli toward galactose .
Nature ( London ) New Biol .
Hiengge , R. , and W. Boos .
Maltose and lactose transport in Escherichia coli : examples of two different types of concentrative transport systems .
Higgins , C. F. , P. D. Haag , K. Nikaido , F. Ardeshir , G. Garcia , and G. Ferro-Luzzi Ames .
Complete nucleotide sequence and identification of metnbrane components of the histidine transport operon of Salmonella typhimurium .
Higgins , C. F. , and M. M. Hardie .
Periplastnic protein associated with oligopeptide permeases of Salmonella typhimurium and Escherichia coli .
Cleavage of structural proteins during the assembly of the head of bacteriophage T4 .
Larson , T. J. , G. Schumacher , and W. Boos .
Identification of the gipT-encoded sn-glycerol-3-phosphate permease of Esch-erichia coli , an oligomeric integral membrane protein .
Lederberg , E. M. , and S. N. Cohen .
Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid .
Maniatis , T. , E. F. Fritsch , and J. Sambrock .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 25 .
Meagher , R. B. , R. C. Tait , M. Betlach , and H. W. Boyer .
Protein expression in E. coli minicells by recombinant plasmids .
Middendorf , A. , H. Schweizer , J. Vreemann , and W. Boos .
Mapping of markers in the gyrA-his region of Escherichia coli .
Experiments in molecular genetics .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 28 .
Mowbray , S. L. , and G. A. Petsko .
The X-ray structure of the periplasmic galactose-binding protein from Salmonella typhimurium at 3.0 A resolution .
Muller , N. , H. G. Heine , anid W. Boos .
Cloning of mglB , the structural gene for the galactose-binding protein of Salmo-nella typhimurium and Escherichia coli .
Ordal , G. W. , and J. Adler .
Isolation and complementation of mutants in galactose taxis and transport .
Oxender , D. L. , J. J. Anderson , C. J. Daniels , R. Landick , R. P. Gunsalus , G. Zurawski , and C. Yanofsky .
Structural and functional analysis of cloned DNA containing genes responsible for branched chain amino-acid transport in Escherichia coli .
Use of minicells for bacteriophage directed polypeptide synthesis .
Rotman , B. , A. K. Ganesan , and R. Guzman .
Transport systems for galactose and galactosides in Escherichia coli .
Substrate and inducer specifities .
Rotman , B. , and R , Guzman .
Identification of the mglA gene product in the 3-methylgaloctoside transport system of Escherichia coli using plasmid deletions generated in-vitro .
Russel , M. , and P. Model .
Filamentous phage pre-coat is an integral membrane protein : analysis by a new method of membrane preparation .
Schumacher , G. , and K. Bussmann .
Cell-free synthesis of proteins related to sn-glycerol-3-phosphate transport in Esch-erichia coli .
Shuman , H. A. , T. J. Sihavy , and J. R. Beckwith .
Labeling of proteins with P-galactosidase by gene fusion .
Identification of a cytoplasmic membrane component of the Esch-erichia coli maltose transport system .
Silhavy , T. J. , M. L. Berman , and L. W. Enquist .
Experiments with gene fusions .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 39 .
Silhavy , T. J. , E. Brickman , P. J. Bassford , M. J. Casadaban , H. A. Shuman , V. Schwartzf L. Guarente , M. Schwartz , and J. Beckwith .
Structure of the malB region in E. coli K12 .
Genetic map of the malE , F , G operon .
Tommassen , J. , P. van der Ley , A. van der Ende , 1 .
Bergmans , and B. Lugtenberg .
Cloning of ompF , the structural gene for an outer membrane pore protein of Escherichia coi K-12 : physical localization and homology with the phoE gene .
West , R. W. , and R. L. Rodriguez .
Construction and characterization of E. coli promoter probe plasmid vectors .
pBR322 derivates with deletions in the tetracycline resistence promoter region .
Wray , W. , T. Boulikas , V. P. Wray , and P. Hancock .
Silver staining of proteins in polyacrylamide gels .
Zukin , R. S. , P. G. Strange , L. R. Heavy , and D. E. Koshland , Jr. 1977 .
Properties of the galactose-binding protein of Salmo-nella typhimurium and Escherichia coli .
In vitro synthesis of protein in microbial systems .