Utilization in Salmonella Ethanolamine can serve as the sole source of carbon and nitrogen for Salmonella typhimurium if vitamin-B ,2 is present to serve as a cofactor .
The pathway for ethanolamine utilization has been investigated in order to understand its regulation and determine whether the pathway is important to the selective forces that have maintained the ability to synthesize B12 in S. typhimurium .
We isolated mutants that are defective in ethanolamine utilization ( eut mutants ) .
These mutants defined a cluster of genes located between purC and cysA at 50 min on the Salmonella chromosome .
A genetic map of the eut region was constructed .
Included in the map are mutations which affect ethanolamine ammonia lyase , the first degradative enzyme , and mutations which affect the second enzyme in the pathway , acetaldehyde dehydrogenase .
Transcriptional regulation of the eat genes was studied by using eut-lac operon fusions created by insertion of Mu d lac .
Transcription is induced by the simultaneous presence of ethanolamine and B12 in the growth medium .
The eut genes constitute a single unit of transcription .
One class of mutations located at the promoter-distal end of the eut operon prevent induction of transcription .
Salmonella typhimurium can use ethanolamine as the sole source of carbon and nitrogen ( 13 ) .
Breakdown of ethanol-amine requires the enzyme ethanolamine ammonia-lyase ( EC 4.3.1.7 ) .
If provided with coenzyme B12 , this enzyme splits ethanolamine to acetaldehyde and ammonia ( 9 , 37 ) .
Acetaldehyde generated by this reaction can be converted to acetylcoenzyme A by acetaldehyde dehydrogenase ( EC 1.2.1.10 ) ( 26 ) .
Due to the B12 dependence of ethanolamine ammonia-lyase , S. typhimurium can use ethanolamine aerobically only if vitamin-B12 is provided .
Recently it was shown that S. typhimurium can synthesize B12 but only under anaerobic-growth-conditions ( 18 , 24 , 25 ) .
The endog-enous B12 synthesis which occurs anaerobically is sufficient to permit use of ethanolamine as the sole nitrogen source without added B12 .
Ethanolamine ammonia-lyase is one of only two B12-dependent enzymes known for Escherichia coli and S. typhimurium .
The other B12-dependent enzyme is a methyltransferase involved in the synthesis of methionine .
The B12-dependent transferase ( metH ) is not essential since , in the absence of B12 , an alternative transferase ( metE ) is capable of synthesizing methionine without B12 ( 19 ) .
Thus , neither of the B12-dependent functions is essential under most growth-conditions .
Ethanolamine ammonia-lyase is only required if cells are growing on ethanolamine ; the metH function is only required in mutants lacking the metE enzyme .
However , some growth advantage may be provided by metH because the alternative ( metE ) enzyme is inefficient and represents several percent of total protein in cells growing without B12 ( 42 ) .
If no other B12-dependent en-discovered , both of these `` nonessential '' zymes are one or contribute the selective forces that have enzymes must to maintained the extensive array of genes required for synthesis and transport of vitamin-B12 .
Perhaps one or both of these B12-dependent enzymes is of greater importance in nature .
than is immediately obvious .
Assimilation of ethanolamine may be an important part of the natural lifestyle of S. typhimurium .
Phosphatidylethanol-amine constitutes a large fraction of bacterial and animal phospholipids ( 34 , 41 ) .
The enzymatic activities necessary to convert phosphatidylethanolamine to component fatty-acids , glycerol , and ethanolamine have been identified in E. coli ( 28 , 33 ) .
Therefore , the host diet , the bacteria present in the environment of S. typhimurium , and the epithelial cells of a host animal 's intestinal tract could provide an abundant source of ethanolamine in nature .
The benefits of ethanol-amine assimilation may be a significant selective pressure for the maintenance of B12 biosynthesis .
Ethanolamine ammonia-lyase has been purified from Clos-tridium spp. , and its mechanism of catalysis has been studied extensively ( 1 ) .
Ethanolamine ammonia-lyase from E. coli has been purified and characterized by Blackwell and Turner ( 3 ) .
The E. coli enzyme is very large ( molecular weight , about 540,000 ) and is composed of subunits with molecular weights of 56,900 and 35,200 ( 3 , 26 ) .
In E. coli , synthesis of ethanolamine ammonia-lyase is induced by the simultaneous presence of ethanolamine and B12 , a phenomenon termed concerted induction ( 4 , 5 ) .
A coenzyme A-dependent acetal-dehyde dehydrogenase activity is coordinately induced with ethanolamine ammonia-lyase activity ( 26 ) .
Mutants of E. coli have been described that are unable to use ethanolamine as a nitrogen source ; these mutants lack ethanolamine ammo-nia-lyase activity in-vitro .
Mutants have also been described which synthesize both activities constitutively ( 4 , 26 , 27 ) .
Thus far , no detailed genetic study has been made of the system of ethanolamine utilization and the mechanism of its regulation .
The long-term goal of this study is to determine whether ethanolamine utilization is of sufficient physiological importance to account for the selective maintenance of the B12 biosynthetic capability .
We also hope to understand the mechanism by which B12 and ethanolamine regulate ethanol-amine ammonia-lyase synthesis .
In this paper we describe the isolation and characterization of mutants defective in ethanolamine utilization .
We show that the genes for etha-nolamine utilization ( eut ) are located in a single operon mapping between cysA and purC at 50 min and that transcription of the eut operon is induced by the simultaneous presence of ethanolamine and B12 .
metE205 ara-9 purC7 metE864 : : TnlO zeb-609 : : TnlO supD501 zeb-609 : : TnlO recAl/F ' 128 zzf-1066 : : Mu dA TT10288 hisD9953 : : Mu dJ Lab collection hisA9944 : : Mu dl TT10508 cysA1585 : : Mu dA Lab collection TT10509 cysA1586 : : Mu dA Lab collection TT11567 zfa-3644 : : TnlOdTet This study TT11568 zfa-3645 : : TnJOdTet This study TT13438 zfa-3646 : : TnlO This study TT13440 zfa-3648 : : TnlO This study TT13744 eut-162 : : Mu dJ This study TT13749 btu-l : : Mu dJ This study TT13767 eut-189 : : Mu dJ This study TT13775 btu-2 : : Mu dJ This study TT13894 btu-3 : : TnlO This study a Nomenclature for Mu d and TnlOdTet strains is described in Materials and Methods .
MATERIALS AND METHODS Bacterial strains and transposons .
All stains used are derivatives of S. typhimurium LT2 .
Strains used in this study are described in Table 1 .
Some eut mutants are listed only in Fig. 3 by allele number and phenotype .
We used two derivatives of the specialized transducing phage Mu dI ( Ampr lac cts ) of Casadaban and Cohen ( 10 ) .
The first derivative , Mu dl-8 , carries two mutations which make it conditionally defective for transposition ( 21 ) .
In this paper Mu dl-8 will be referred to as Mu dA .
The second derivative , Mu d11734 , specifies Kanr and lacks the Mu A and B genes necessary for transposition ( 11 ) .
In this paper Mu dI1734 will be referred to as Mu dJ .
The complex medium was nutrient broth ( 0.8 % ; Difco Laboratories ) with NaCI ( 0.5 % ) .
The minimal-medium was E medium of Vogel and Bonner ( 39 ) with glucose ( 0.2 % ) as the carbon source .
The carbonfree minimal-medium was NCE ( 17 ) , and the carbon-and nitrogen-free minimal-medium was NCN ( 35 ) .
Ethanolamine hydrochloride ( 0.2 % ; Aldrich ) was used as the carbon source in NCE medium , as the nitrogen source in NCN medium with glycerol ( 0.2 % ) , or as both the carbon and nitrogen source in NCN medium .
Glycerol ( 0.2 % ) or acetate ( 0.2 % ) was used as the carbon source in NCE medium .
Cyanoco-balamin ( Sigma Chemical Co. ) was used as the exogenous B12 source ( 100 , ug/liter ) .
Amino acids and purines were added to minimal media as required at the concentrations recommended previously ( 17 ) .
Cystine ( 0.058 mM ) was added to nutrient broth used for growth of Cys auxotrophs .
Antibiotics were used at the following concentrations in minimal and complex media , respectively : ampicillin , 15 or 30 , ug/ml ; kanamycin , 125 or 50 , ug/ml ; tetracycline , 10 or 20 , ug/ml .
Solid medium contained agar ( 1.5 % ; Difco ) or , when a nitrogen source other than ammonia was used , Noble agar ( 1.5 % ; Difco ) .
Cells were grown aerobically at 37 °C unless indicated otherwise .
NCE medium containing glycerol and 25 , ug of 5-bromo-4-chloro-3-indolyl - , - D-galactoside ( Xgal ; dissolved in N,N-dimethylformamide before addition ) per ml was used in indicator plates for , B-galactosidase activity .
The tetrazolium indicator medium of Bochner and Sava-geau ( 7 ) was modified and used with ethanolamine to identify mutants ( Eut - ) unable to use ethanolamine .
Ethanolamine indicator medium contained KH2PO4 ( 0.56 % ) , K2HPO4 ( 0.24 % ) , Bactopeptone tetra - ( 0.2 % ; Difco ) , MgSO4 ( 1 mM ) , zolium chloride ( 0.0025 % ) , cyanocobalamin ( 0.1 , ug/ml ) , ethanolamine hydrochloride ( 1 % ) , and agar ( 1.5 % ) .
On eth-anolamine indicator plates containing many colonies , Eutstrains form red colonies , while Eut + strains form white colonies .
All transductional crosses were performed with the high-frequency generalized transducing phage mutant P22 HT105/1 int-201 ( 38 ) .
Most transductions were carried out by mixing 2 x 108 cells with 108 to 109 phage directly on the selective medium .
When Kanr transductants were selected , the cell culture and phage were incubated together nonselectively in liquid for 45 min before plating on the solid selective medium .
Transductants were purified and made phage-free by streaking for single colonies on nonselective green indicator plates ( 12 ) .
Transductants were tested for phage sensitivity by cross-streaking against the clear-plaque mutant P22 H5 .
In construction of the deletion map , many transductional crosses were done on a single plate with a nail block to apply 25 recipient strains to nutrient broth medium seeded with the donor P22 lysate .
After 6 to 10 h of incubation , the plate was replica printed to NCN medium containing ethanolamine and B12 to select recombinants .
The map was refined with higher-resolution crosses by using a whole plate for a single donor-recipient pair .
Recombination between a point mutation and a deletion was scored as negative if two whole-plate crosses produced no eut + recombinants .
Two such plates yield at least 2,000 eut + recombinants when the donor lysate is grown on a wild-type strain .
Hfrs were constructed as described by Chumley et al. ( 14 ) except that integration of the temperature-sensitive plasmid F'ts 114 lac zzf-20 : : TnJO occurred by recombination between the plasmid lac sequences and the lac sequences present in eut-18 : : Mu dA .
These recombinants were obtained by selecting for Tetr at 42 °C .
Phage P22 lysates for localized mutagenesis experiments were concentrated and mutagen-ized with hydroxylamine by the method of Hong and Ames ( 20 ) .
Two donor strains were used , one containing a TnIO insertion adjacent to the promoter-proximal end of the eut region ( TT13438 ) and the other containing a TnJO insertion adjacent to the promoter-distal end of the eut region ( TT13440 ) .
These mutagenized lysates were used to transduce LT2 to Tetr on ethanolamine indicator plates , and eut mutants were identified among the recombinants .
Diethyl sulfate ( DES ) mutagenesis of cell cultures was performed by incubating approximately 2 x 108 cells in 5 ml of E medium with 20 Rll of DES for 20 to 60 min at 37 °C .
The cells were diluted 1,000-fold into E medium containing glucose , allowed to grow overnight , diluted again , and spread on complex medium to obtain single colonies .
Eutmutants were identified as those which did not grow on NCE medium containing ethanolamine and B12 but did grow on NCE medium containing acetate .
Tn1O and TnlOdTet mutagenesis .
TnJO insertions were obtained by transducing pools of random TnJO insertions into the eut-6 : : Mu dA or eut-18 : : Mu dA fusion strain on Xgal indicator plates which contained tetracycline , ampicillin , ethanolamine , and B12 or on Xgal indicator plates whic contained only tetracycline and ampicillin .
TnWO insertion mutants which formed lighter or darker blue colonies than the parent eitt : : Mu dA fusion strain were saved as potential eit mutants .
TnIO Dell6 Dell7 Tet ' r ( Tn/OdVet ) is a small derivative of TnWO from which the transposase genes have been deleted ( 40 ) .
TnlOdTet will transpose when introduced into strains containing the TnWO transposase gene on a multicopy plasmid .
TnIOdTet insertions were isolated from a pool of random Tn/OdTet insertions obtained from Tom Elliott .
Insertions of TnOO or TnIOdrTet adjacent to each end of the eiat region were obtained by transducing the promoterproximal euit-18 : : Mu dA mutant and the promoter-distal eut-34 : : Mu dA mutant to Tetr with a P22 lysate grown on a pool of random TnWO or TnlOdTet insertions .
The Tet ' transductants were screened for those which had become Amp ' and Eut ' .
Deletion mutations in eiat were isolated as tetracyclinesensitive derivatives of the TnJOdTet strains TT11567 and TT11568 ; these were selected on complex medium by a modification ( 29 ) of the medium described by Bochner et `` al. ( 6 ) .
The selection and screening of tetracycline-sensitive derivatives was done at 40 °C .
Tetracycline-sensitive colo-nies which had become Eut-were identified by replica printing the colonies to minimal-medium selective for tetracycline sensitivity prepared as described previously ( 6 ) .
except that ethanolamine and B12 were substituted for glucose .
Random insertions of Mu dA were obtained by transducing the amber suppressor strain TT7610 ( supDSO ) with phage P22 grown on the Mu dA insertion strain TT8388 on rich-medium containing ampicillin .
The Mu dA prophage in TT8388 can not be inherited in the Salmo-niella chromosome by homologous recombination because the donor strain has Mu dA inserted in an E. coli F ' which contains no sequence homology with Salmonella .
The Amp '' transductants were replica printed to NCE glycerol medium containing ampicillin and Xgal and to NCE glycerol medium containing ampicillin , Xgal , ethanolamine , and B12 .
E'ach pair of replica plates was screened for colonies which were blue on one plate and white on the other .
Mu dA insertion strains containing an amber suppressor mutation were grown at 30 °C to prevent inactivation of the temperature-sensitive Mu repressor .
To prevent further transposition of Mu dA , each insertion of interest was transduced into a suppressorfree background .
Mu dJ is defective for transposition .
To isolate new Mu dJ insertion mutants , Mu dJ was transduced into a recipient on a transduced fragment that also included the Mu A and B genes of a Mu dl present near Mu dJ in the donor strain ( 23 ) .
Random insertions of Mu dJ were obtained by infecting LT2 with a P22 lysate grown on the Mu dl Mu dJ double lysogen TT10288 and selecting for Kan ' transductants .
Strain TT10288 contains an insertion of Mu dJ in the hisD gene and an insertion of Mu dl in the hisA gene .
P22 transducing particles which contain Mu dJ frequently contain the transposase genes of Mu dl as well , permitting Mu dJ to transpose ( 23 ) .
Insertions of Mu dl are not inherited along with Mu dJ because a complete Mu dl is too large to be packaged in a single P22 particle which also contains Mu dJ ( 22 ) .
Mutants unable to use ethanolamine were identified by replica printing the Kan ' transductants to NCE medium containing acetate and to NCE medium containing ethanolamine and B12 .
Each Eut-insertion mutation was transduced into strain LT2 before characterization .
Directed deletion formation with Mu d prophages .
Deletions generated by recombination between eitt and cvsA Mu dA insertions were isolated by three different crosses .
The first cross , previously described by Hughes and Roth ( 22 ) , is shown in Fig. 1A .
The transduction is performed with a mixture of phage lysates made on two different Mu dA insertion strains .
Recombination between the two Mu dA insertions can create a deletion or duplication of the material between the insertion sites ( Fig. 1A ) .
P22 lysates made on the Lac ' ( eysA1585 : : Mu dA insertion strain TT10508 or the Lac cysA1586 : : Mu dA insertion strain TT10509 were mixed with P22 lysates made on eut : : Mu dA insertion strains and used to transduce LT2 to Amp ' .
The transductants were replica printed to minimal-medium and NCE medium containing ethanolamine , B12 , and cystine to identify deletion ( elt ( vs ) and duplication ( eltt ( ys 1/4 ) strains .
Deletions ending at eut : : Mu dJ insertions were also constructed in the cross shown in Fig. 1B .
The eut : : Mu dJ recipient strains were transduced to Ampr with P22 made on the cysA1585 : : Mu dA strain or the cysAI586 : : Mu dA strain .
The transductants were replica printed to minimal-medium and NCE medium containing ethanolamine , B12 , and cystine to identify the deletion ( Kan ' eut ) strains .
Deletions were formed between the two Mu d insertions in 5 to 10 % of the transductants when the eut : : Mu d and the cysA : : Mu dA insertions were in the same orientation .
Additional deletions ending at eut : : Mu d insertions wer constructed as shown in Fig. 1C .
A strain containing a large Mu dA deletion ( generated by either of the above methods ) was transduced to Tetr with P22 made on an eut : : TnJO eut : : Mu d double mutant .
The eut : : TnJO insertion could only be inherited by replacing the large deletion .
Frequently this occurred by recombination between the two Mu d insertions , generating a shorter deletion with an endpoint at the donor Mu d insertion site .
This technique has the advantage that all of the Tetr transductants contain the expected new deletion .
P22 lysates grown on the Tetr colonies were used to transduce LT2 to Ampr to obtain the new Mu dA deletion in a background without the eut : : TnJO insertion .
Strains to be assayed for P-galactosidase activity were grown at 37 °C to mid-log phase in NCE glycerol medium containing other additions as indicated in Table 3 .
, - Galactosidase activity was assayed in permeabilized cells as described previously ( 31 ) .
Strains to be assayed for ethanolamine ammonia-lyase activity and acetaldehyde dehydrogenase activity were grown at 37 °C with vigorous gyratory shaking to an A650 of 0.6 in NCE glycerol medium containing other additions as indicated in Table 2 .
Cells were harvested by centrifugation , washed in 20 mM Tris hydro-chloride ( pH-7.6 ) , suspended in 50 mM HEPPS ( N-2-hydrox-yethylpiperazine-N ' -3-propanesulfonic acid , pH 8 ) -200 mM KCl-1 mM dithiothreitol , and lysed by passage through a French pressure cell at 18,000 lb/in2 .
The extract was first centrifuged at 24,000 x g for 45 min , and the supernatant was then centrifuged at 150,000 x g for 60 min .
Portions of the second supernatant were frozen in dry ice-ethanol and stored at -70 °C for up to 3 months before use .
Ethanolamine ammonia-lyase was assayed by using a procedure similar to an assay described previously ( 8 ) .
Reaction mixtures ( 0.1 ml ) contained 400 nmol of [ 2-14C ] ethanolamine hydrochloride ( 0.113 uCi4 , Lmol ; Amersham ) , 2 nmol of adenosylcobalamin ( Sigma ) , 5 , imol of Tris hydrochloride ( pH 7.5 ) , 200 nmol of NADH , 10 U of yeast alcohol dehydrogenase ( Sigma ) , and extract .
The reactions were started by the addition of the adenosylcobalamin in dim red light and were incubated for 6 to 18 min at 37 °C .
The reactions were terminated by the addition of about 200 mg of Dowex 50W-X8 ( hydrogen form ) cation-exchange resin in 0.5 ml of 0.01 N HCl .
The cation-exchange resin formed a complex with the unreacted etha-nolamine , which was then removed by centrifugation .
To determine the quantity of labeled acetaldehyde and ethanol formed , 0.2 ml of the supernatant was counted in a scintillation counter .
Acetaldehyde dehydrogenase was assayed spectrophotometrically by following production of NADH at 340 nm as described previously ( 16 ) .
Cell protein was determined with the Bio-Rad protein assay with bovine serum albumin as the standard .
RESULTS Mutants defective in ethanolamine utilization .
Mutants unable to use ethanolamine as the sole source of nitrogen or carbon for growth fall into two phenotypic classes .
Mutants unable to use ethanolamine as either the sole carbon or nitrogen source are designated Eut - ( N-C - ) .
Mutants able to use ethanolamine as the sole nitrogen source but not as the sole carbon source are designated Eut - ( N+C - ) .
We identified insertions of Mu dA which caused lacZ to be induced by the presence of ethanolamine and B12 in the growth medium .
Among 25,000 mutants with random Mu dA insertions screened , 41 formed blue colonies on Xgal indicator plates only when ethanolamine and B12 were present ( mutations eut-3 through eut43 ) .
No mutants were identified which formed blue colonies only when ethanolamine and B12 were absent .
All but two of the Mu dA insertions conferred the Eut - ( N-C - ) phenotype .
The two exceptional insertion strains ( eut-38 and eut4J ) displayed no defect in growth on ethanolamine as a carbon or a nitrogen source .
All of the eut : : Mu dA insertions were linked by P22 transduction to the eut46 : : TnJO insertion ( described below ) and therefore mapped at the same chromosomal locus .
Mu dJ insertions in eut were isolated by screening pools of random insertion mutants for mutants unable to grow on ethanolamine as the sole carbon source .
This strategy was used to obtain Eut-Mu dJ insertions in both chromosomal orientations , in genes whose expression was not necessarily regulated by ethanolamine plus B12 and in genes whose expression was abolished by the insertion .
Among 52 Mu dJ mutants that are unable to use ethanolamine as the carbon source , 48 showed linkage to eut46 : : TnJO and thus carried mutations in the eut region .
All 48 Mu dJ insertions linked to the eut locus conferred the Eut - ( N-C - ) phenotype ; 14 of these Mu dJ mutants showed induction of lacZ by ethanol-amine plus B12 .
Most of the unexpressed fusions were in the wrong orientation , but one , discussed later , appeared to affect a regulatory function .
The four mutations unlinked to the eut operon will be discussed later .
TnWO insertions in eut were isolated as Lac-derivatives of eut : : Mu dA fusion strains .
This was done by looking for white colonies on Xgal indicator plates which contained ethanolamine and B12 .
Several of the mutants were Lac-because of the polarity of their TnWO insertion on expression of the lacZ gene of a promoter-distal Mu dA insertion .
Two strains became Lac-because the TnJO insertions disrupted genes whose activity is required for expression of the eut operon ( discussed below ) .
Several eut : : TnJO insertions were isolated which apparently caused lacZ to be transcribed from a promoter located within the TnWO element ( 15 ) .
These insertions were identified as blue colonies on Xgal indicator plates lacking ethanolamine and B12 .
All but one of the TnWO insertions isolated were linked by P22 transduction to the eut-18 : : Mu dA insertion ; the unlinked TnWO insertion will be described later .
The phenotype of each TnWO insertion in the absence of the eut : : Mu dA insertion is indicated in Fig. 3 ; both Eut - ( N-C - ) and Eut - ( N+C - ) insertion strains were isolated .
A large number of hydroxylamine-induced point mutations in the eut region were isolated by localized mutagenesis as described in Materials and Methods .
Several Eut-point mutants induced by DES were also isolated .
These point mutations , which included both Eut - ( N-C - ) and Eut - ( N+C - ) types , are included in Fig. 3 .
Deletions extending into the eut region were isolated for use in the construction of a deletion map .
The deletions were obtained by applying positive selection for tetracycline sensitivity to strains containing TnJOdTet inserted next to the eut region ( on the purC side of eut ; see below ) .
Some mutants survived the selection because the TnJOdTet element had been deleted .
TnJOdTet was used instead of TnWO to avoid the creation of deletions with the nonrandom endpoints characteristic of deletions induced by the TnWO transposase ( 32 ) .
We identified fifteen independent Eutdeletion strains .
All of the deletions conferred the Eut - ( N-C - ) phenotype because these deletions all remove the promoter end of the operon ( see below ) .
A few Eut-mutants are defective in B12 transport .
The mutant searches were done aerobically ; under these conditions , S. typhimurium requires B12 for ethanolamine utiliza tion .
Therefore , mutants defective in B12 transport would be expected among Eut-mutants .
Mutants which are unable to transport B12 can not use the B12-dependent methyltransferase enzyme ( metH ) , one of the two enzymes which can methylate homocysteine to give methionine ( 2 ) .
We checked the Eut-mutants for B12 transport function by eliminating the alternative B12-independent methyltransferase enzyme ( metE ) and testing for growth-on-minimal-medium supplemented only with B12 .
All of the metE strains containing eut : : Mu d and eut : : TnJO insertions located in the eut region were able to grow on minimal-medium supplemented with B12 .
This indicates that mutations at the eut locus do not impair B12 transport .
Three Eut-mutations unlinked to the eut region did seem to prevent B12 uptake .
The metE strains containing one of the unlinked Eut-Mu dJ insertions ( btu-J : : Mu dJ ) or the unlinked Eut-TnJO insertion ( btu-3 : : TnlO , isolated as a Lac-derivative of eut-18 : : Mu dA ) were unable to grow on minimal-medium containing the usual concentration of B12 ( 0.1 , g/ml ) .
These strains exhibited full growth-on-minimal-medium containing a high concentration of B12 ( 10 , ug/ml ) , a phenotype characteristic of many B12 uptake mutants ( 2 ) .
These two insertions showed close linkage to each other ( 94 % cotransduction by P22 ) .
The metE strain containing a third Eut-mutation ( btu-2 : : Mu dJ , unlinked to the eut region ) was able to grow on minimal-medium containing the usual concentration of B12 but not on minimal-medium containing a lower than usual B12 concentration ( 20 pg/ml ) ( C. Grabau , personal communication ) , suggesting that btu-2 : : Mu dJ is also defective in B12 uptake .
The metE strains containing the two other Mu dJ insertions unlinked to the eut region ( eut-162 and eut-189 ) exhibited methionine-independent growth-on-minimal-medium containing 2 pg of B12 per ml and were presumed to be normal for B12 transport .
We have not investigated the basis for their Eutphenotype .
Enzymatic defects in eut mutants .
Extracts of several eut mutants were assayed for the two enzymes required for ethanolamine utilization , ethanolamine ammonia-lyase and acetaldehyde dehydrogenase ( Table 2 ) .
As in E. coli ( 5 ) , the enzyme activities were detected only in extracts of cells grown in the presence of ethanolamine and B12 .
The genes for both ethanolamine ammonia-lyase and acetaldehyde dehydrogenase appeared to be located in the eut region .
A strain containing a deletion of the entire eut region ( eut-237 ) lacked both enzyme activities .
Table 2 contains examples of eut point mutants which lacked one or both of the enzyme activities .
Location of the eut region .
We located the eut region on the S. typhimurium map by using Hfr mapping crosses ( 14 ) .
The Hfr was formed by integrating F'tsll 4 lac into the eut-18 : : Mu dA insertion by recombination between the plasmid and Mu dA lac sequences as described previously ( 30 ) .
This Hfr transferred the cysA locus at a high frequency and transferred markers located further counterclockwise from cysA with gradually decreasing frequency ( data not shown ) .
These results indicate that the insertion is located between cysA at 50 map units and guaA at 52 map units on the chromosome ( 36 ) .
The eut region was more precisely located with P22-mediated cotransduction crosses .
The eut region was found to be located midway between cysA and purC at approximately 50.5 map units .
A map of cotransduction frequencies between these markers is presented in Fig. 2 .
Using the formula described by Wu ( 43 ) , we estimate from the cotransduction data that the eut region is between 12 and 15 kilobases long .
While this is a rough estimate , it suggests that the operon is rather large .
Direction of transcription of eut .
The eut operon was found to be transcribed in a counterclockwise orientation .
The orientation of the eut-18 : : Mu d insertion was inferred from the Hfr mapping crosses described above .
The gradient of chromosome transfer in these Hfr crosses indicated that the lac genes of eut-18 : : Mu dA are transcribed in the counterclockwise direction ( 30 ) .
The lac genes of eut-18 : : Mu dA are expressed from the eut promoter , indicating that transcription of the eut operon is also counterclockwise .
The eut promoter is indicated in Fig. 2 on the purC side of the eut region .
Orientation of Mu d insertions and generation of deletion by transductional crosses .
Deletions can be generated by homologous recombination between Mu d insertions in eut and cysA only when the two Mu d insertions are in the same chromosomal orientation , as described in Materials and Methods and shown in Fig. 1 .
In these deletion strains , the sequences between the two parental Mu d insertions were deleted , leaving a single Mu d insertion at the deletion join point .
Mu d deletions were constructed with 29 eut : : Mu d insertions which showed induction of the lac genes by ethanolamine plus B12 and with eight eut : : Mu d insertions which did not show induction .
The inducible eut : : Mu d insertions only formed deletions with the Lac ' cysA1585 : : Mu dA insertion , while seven of the uninducible eut : : Mu d insertions only formed deletions with the Lac-cysA1586 : : Mu dA insertion .
This indicates that all of the inducible eut : : Mu d insertions are in the same chromosomal orientation and that all but one of the uninducible eut : : Mu d insertions are in the opposite orientation .
One unusual uninducible eut : : Mu d insertion ( eut-156 : : Mu dJ ) formed a deletion to the cysA1585 : : Mu dA insertion and is therefore in the same orientation as the inducible eut : : Mu d insertions .
Evidence presented below suggests that this a in the eut exceptional insertion disrupts gene region required for induction of the eut promoter .
Genetic map of the eut region .
The linear arrangements of 130 point and insertion mutation sites in eut and 50 deletions extending into eut are presented in Fig. 3 .
The data were obtained by transducing a series of deletion mutants with P22 lysates made on pciint or insertion mutants and scoring the frequencies of eut + recombinants produced .
In addition , several intervals in the map were defined by crosses between two deletions .
Four different regions , defined by mutant phenotypes , are indicated on the genetic map ( Fig. 3 ) .
The promoter-proximal third of the operon encodes activities necessary only for use of ethanolamine as the sole carbon source .
All of the point mutations which conferred the Eut - ( N+C - ) phenotype mapped in the promoter-proximal third of the map , region I ( Fig. 3 ) .
The two TnWO insertions which conferred the Eut - ( N+C - ) phenotype , eut44 : : TnJO and eut49 : : TnlO , also mapped in this region .
These strains could grow on ethanolamine as a nitrogen source and complete the first step in ethanolamine utilization , catalyzed by ethanolamine ammonia-lyase .
The enzyme which catalyzes the second step in ethanolamine utilization , acetaldehyde dehydrogenase , seems to be encoded in region I. Three of the Eut - ( N+C - ) point mutants , eut-55 , eut-56 , and eut-91 , lacked in-vitro acetaldehyde dehydrogenase activity but retained in-vitro ethanolamine ammonia-lyase activity ( Table 2 ) .
In addition to acetaldehyde dehydrogenase , a second function must also be encoded in region I because the Eut - ( N+C - ) point mutants eut-64 , eut-101 , and eut-124 retained both enzyme activities in-vitro .
We have not yet determined the nature of this function .
About one-quarter of the hydroxylamine-induced point mutations , three TnWO insertion mutations , and all of the Mu d insertion mutations mapping in region I conferred a Eut - ( N-C - ) phenotype .
These mutations are probably polar on downstream genes whose function is needed for growth on ethanolamine as a nitrogen source .
The promoter-distal half of the operon encodes activities required for use of ethanolamine as either a carbon or a nitrogen source .
All mutations in the promoter-distal half of the map ( regions II and III ) conferred only the Eut - ( N-C - ) phenotype .
Two Eut - ( N-C - ) mutants carrying point mutations in region II , eut-107 and eut-llO , lacked ethanolamine ammonia-lyase activity in-vitro but retained much of the acetaldehyde dehydrogenase activity ( Table 2 ) .
This suggests that the genes for ethanolamine ammonia-lyase are located in region II .
Several other strains ( eut-91 , eut-66 , and eut-106 ) had greatly reduced or eliminated ethanolamine ammonia-lyase activity in-vitro as well .
It is possible that the mutations in these strains exert a polar effect on transcription of a downstream gene ( s ) for ethanolamine ammonia-lyase .
Complementation tests will be required to resolve these ambiguities .
As in region I , an additional , undetermined function must also be encoded in region II because the Eut - ( N-C - ) point mutants eut-Jll and eut-114 retained both enzyme activities in-vitro .
The Eut - ( N-C - ) strain containing the eut-100 point mutation located at the far promoter-distal end of the operon ( region III ) lacked both enzyme activities in-vitro .
This strain did not seem to express the eut operon .
Some mutations in the promoter-distal part of the operon prevent transcription of eut .
Several lines of evidence indicate that an activity encoded in the promoter-distal third of the operon ( region III ) is required for expression of eut .
The eut-205 : : TnlO insertion ( in region III ) was isolated as a Lac-derivative of the eut-18 : : Mu dA fusion strain .
Since this TnWO element mapped on the promoter-distal side of the affected Mu d insertion , we infer that the TnWO insertion can not be exerting a polar effect and must be preventing initiation of transcription from the eut promoter .
The unusual eut-156 : : Mu dJ insertion , which was properly orientated but did not show induction of lac in the presence of ethanolamine and B12 , also mapped in region III .
In addition , expression of the eut operon was prevented by deletions which removed the right ( promoter-distal ) end of the operon .
The strains carrying eut-cys deletions made with inducible eut : : Mu d insertions retained the structural integrity of the eut promoter and the lac operon fusion but lost material distal to the fusion ( Fig. 1 ) .
Induction of lacZ was examined by using Xgal indicator plates .
In all but two of these deletion strains , lacZ was no longer inducible by ethanol-amine plus B12 ( the two exceptions are discussed below ) .
Therefore , deletion of material between the eut : : Mu d and the cysA : : Mu d insertions appears to remove a function required for induction of the eut promoter .
To further define the extent of region III , we examined the effect of a series of point mutations on transcription of the eut operon .
The promoter-proximal eut-18 : : Mu dA insertion was introduced into a series of point mutants by P22 transduction on Xgal indicator plates containing ampicillin , etha-nolamine , and B12 .
Point mutation eut-100 and all point mutations located to the right of eut-100 ( Fig. 3 ) prevented expression of the eut operon , as judged by the failure of the double mutants to express the promoter-proximal lac fusion .
Point mutations located between the lac fusion and eut-100 ( left of eut-1OO ) had no effect on expression or regulation of lac expression .
Therefore , the eut-100 point mutation defines the border between regions II and III .
Since mutations in region III seemed to abolish a function required for expression of the eut genes , we hypothesize that this region is essential for activity of the operon 's main promoter .
If a protein is encoded in region III , its gene must be transcribed independently from the upstream genes in the operon , because Mu d insertions in the upstream genes did not appear to abolish the function of this region .
Further evidence for an independent promoter is the fact that mutants carrying eut-lac fusions in region III retained a low level of expression in strains with a polar TnWO insertion in region I ( data not shown ) .
The two exceptional eut-cys deletions that retained induc-ible expression of transcription were constructed by using the phenotypically Eut + Mu d insertions ( eut-38 : : Mu dA and eut41 : : Mu dA ) as their left endpoint .
The deletion strains remained Eut + , and the lac fusions were still inducible by ethanolamine and B12 .
This indicates that the Eut + insertions are located on the cysA side of all the genes required for ethanolamine utilization ; deletions extending from the Eut + Mu d insertion sites to the right did not remove the region needed for induction of the eut promoter .
The two Eut + Mu d insertions define region IV ( Fig. 3 ) .
All eut : : lac fusions are in the same transcription unit .
To determine whether all eut : : Mu d fusions which showed induction of lacZ by ethanolamine plus B12 are located in the same transcription unit , we introduced a promoter-proximal TnJO insertion ( eut46 : : TnlO ) into 41 eut : : Mu dA insertion strains ( eut-3 through eut43 ) .
Each of the double mutants was tested for transcriptional polarity of the TnWO insertion on the expression of lacZ by using Xgal indicator plates .
The double mutants containing Mu d insertions located on the promoter-distal side of eut46 : : TnJO displayed no induction of lacZ by ethanolamine plus B12 .
A double mutant containing the eut46 : : TnJO insertion and the single promoterproximal Mu d insertion ( eut-18 : : Mu dA ) showed normal expression and regulation of the lacZ gene .
Transcriptional regulation of eut .
We used the eut : : lac operon fusions created by Mu d insertion to quantitatively examine transcriptional regulation in the eut region .
In the fusion strains , changes in the level of P-galactosidase activity reflect changes in the level of eut transcription .
The effect of ethanolamine and B12 on the level of , B-galactosidas activity in five lac fusion strains is presented in Table 3 .
Induction of P-galactosidase activity requires the combination of ethanolamine plus B12 in all but one of the strains tested .
The exceptional strain showed no induction of lacZ because it contained the eut-156 : : Mu dJ insertion in the promoter-distal region ( region III ) inferred to be essential for transcription induction .
DISCUSSION We isolated a large collection of S. typhimurium mutants unable to use ethanolamine as a carbon source ( eut mutants ) .
Nearly all of the eut mutations mapped between cysA and purC at 50 min on the chromosome ( Fig. 2 ) .
The eut mutations in this region fell into two phenotypic classes .
Some mutants , designated Eut - ( N-C - ) , are unable to use ethanolamine as the sole carbon source or the sole nitrogen source .
Other mutants , designated Eut - ( N+C - ) , are able to use ethanolamine as the sole nitrogen source but not as the sole carbon source .
The genetic map of the operon is divided into four regions , distinguished by the phenotype of mutations mapping in each region .
All of the mutations which conferred the Eut - ( N+C - ) phenotype are located in the promoter-prox-imal third of the eut operon , region I ( Fig. 3 ) .
This region contains the gene for the second enzyme in ethanolamine utilization , acetaldehyde dehydrogenase .
This gene was defined by several Eut - ( N+C - ) mutants which lacked in-vitro acetaldehyde dehydrogenase activity but retained in-vitro ethanolamine ammonia-lyase activity ( Table 2 ) .
Several Eut - ( NC - ) mutants carrying mutations in region I retained high levels of each activity in-vitro , indicating that an undetermined function , required for use of ethanolamine as a carbon source , is also encoded in region I. Region II , the central ` third of the operon , contains only mutations which conferred the Eut - ( N-C - ) phenotype .
The genes for ethanolamine ammonia-lyase are located ' in this region , defined by several mutants ` which lacked ethanol-amine ammonia-lyase activity in-vitro but retained in-vitro acetaldehyde dehydrogenase activity .
Several other region II mutants retained both in-vitro activities , indicating that another undetermined function , required for use of ethanol-amine as either a carbon or a nitrogen source , is encoded in region II .
Two eut : : Mu d insertions mapping at the far distal end of the eut operon ( region ` IV ) had lac genes that were regulated in response to ethanolamine and B12 but caused no detect-able defect in ethanolamine utilization or in eut transcription .
These insertions may disrupt a gene whose function is related to ethanolamine utilization but which is not required for use of ethanolamine under the conditions tested , or they may be located between the last gene of the operon and the transcription terminator .
The genetic linkages suggest that the length of the operon is 12 to 15 kilobases .
This implies that the operon could contain 10 to 15 genes , but characterization of mutants ( and complementation data to be presented elsewhere ) gives evidence for only six genes necessary for ethanolamine utilization .
The discrepancy suggests that additional genes are present in the operon whose loss does not lead to the Eut-phenotype .
An alternative and likely explanation is that the transductional crosses overestimated distances .
For example , if a favored P22 packaging site exists within the operon , fewer transductants would inherit markers at both ends of the operon .
Transcription of the eut genes required the simultaneous presence of ethanolamine and B12 .
While it is unusual that induction of an operon requires a cofactor of one of the enzymes in that operon , B12 is an unusual cofactor .
The cell only makes B12 anaerobically ; under aerobic conditions the availability of B ,2 depends on its presence in the medium .
Thus , B12 is a cofactor whose presence depends on growth-conditions ; it seems appropriate that its presence might be a for prerequisite gene expression .
Strains containing mutations in region HII displayed the Eut - ( N-C - ) phenotype and lacked both in-vitro activities .
These mutations prevented induction of transcription of the eut genes by ethanolamine and B12 .
Region III could encode genes required to transport the ethanolamine necessary for induction , or it could encode a positive regulator of eut transcription .
Work currently in progress ` indicates that region III is not necessary for use of ethanolamine as a nitrogen source when the eut genes are transcribed from a plasmid promoter ( D. Roof and J. Roth , unpublished results ) .
This result suggests that region III is not directly required for ethanolamine utilization and is more likely to encode a positive regulatory element .
It appears that region III is transcribed from a weak promoter which is independent of the promoter for the upstream genes , because polar insertion mutations in the upstream genes did not completely abolish the function of region III .
Mu d lac operon fusions downstream of region III ( in region IV ) appeared to be transcribed from the primary promoter as well as the weak promoter for region III .
This suggests that region III is transcribed from both the primary promoter and a weak internal promoter This work was supported by Public Health Service grant GM-34804 to J.R.R. and training grant GM-07464 to D.M.R. from the National Institutes of Health .
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