Identification and Initial Characterization of the eutF Locus of Salmonella typhimurium investigate growth on ethanolamine as the sole carbon source .
No-carbon , no-nitrogen medium ( NCN ) ( 17 ) supplemented with glycerol and ethanolamine was used to investigate growth on ethanolamine as the sole nitrogen source .
When present in the culture medium , and unless otherwise stated , the final concentrations of the following compounds were as follows : ethanolamine hydrochloride ( Gold Label , .99 % pure ; Aldrich Chemical Co. , Milwaukee , Wis. ) , 27 mM ; MgSO4 , 1 mM ; glycerol , 22 mM ; methionine , 0.5 mM ; tryptophan , 0.1 mM ; cyanocobalamin ( CN-B12 ) or Ado-B12 ( Sigma Chemical Co. , St. Louis , Mo. ) , 15 nM ; and 5-bromo-4-chloro-3-indolyl-p-D-galactoside ( X-Gal ) ( Bachem , Inc. , Torrance , Calif. ) , 40 mg/liter .
The final concentrations of antibiotics ( micrograms per milliliter ) in the rich-medium were as follows : tetracycline , 20 ; ampicillin , 30 .
Those in the minimal-medium were 10 and 15 , respectively .
For solid medium , Bacto-Agar ( Difco ) was added to a final concentra-of tion 1.5 % ( wt/vol ) .
Cultures were incubated at 37 °C and shaken vigorously ( 400 rpm ) , and growth was monitored with a Spectronic 20D spectrophotometer at 650 nm .
All experiments were done under aerobic conditions .
( i ) Phage P22-mediated transduction .
All transductions were performed with lysates of phage P22 ( 7 ) carrying the HTJ05/1 and int-201 mutations ( 22 , 23 ) .
A multiplicity of infection of approximately 1 was used in all crosses .
( ii ) Isolation of a deletion-in the eutF locus .
A deletion in the eutF was in locus generated vivo by the method of Bochner et al. ( 4 ) as modified by Maloy and Nunn ( 13 ) .
Fusaric acid was dissolved in N,N-dimethylformamide prior to its addition to the medium .
The culture medium was prepared immediately before use , and the plate cultures were incubated at 42 °C for 24 h .
A TnJODEL16DEL17Tcr ( TnJOdc [ 26 ] ) element inserted between the trp and tonB loci ( JE1121 [ 24a ] ) was used to generate deletion DEL903 ( JE1418 ) ( Table 1 ) .
( iii ) Isolation of hydroxylamine-generated mutations .
Hy-droxylamine-generated mutations in eutF were isolated by localized mutagenesis ( 9 ) as previously described ( 7 ) .
A lysate of phage P22 grown on a strain carrying the selectable marker trp-3484 : : TnJOdTc ( JE1291 ) was chemically mutagenized with hydroxylamine and used as a donor to transduce strain JE1684 ( eutE18 : : Mu dl-8 ) to tetracycline resistance .
EutF MATERIALS AND METHODS Bacteria , media , and growth-conditions .
The bacterial strains used in this study are derivatives of S. typhimurium LT2 , and their genotypes are listed in Table 1 .
Nutrient broth ( Difco , Detroit , Mich. ) ( 0.8 % [ wt/vol ] ) supplemented with 85 mM NaCl was used as a rich-medium .
Minimal medium E of Vogel and Bonner ( 25 ) was supplemented-with-glucose ( 11 mM ) as the carbon source .
No-carbon medium E ( NCE ) ( 7 ) supplemented with ethanolamine was used to TR6583 LT2 metE205 ara-9 J. R. Roth JE1121 zdd-3703 : : Tn10DEL16DEL17Tcr Laboratory collection JE1418 DEL903 JE1291 trp-3484 : : TnJODEL16DELJ7Tcr Laboratory collection JE1293 cobA366 : : TnJODEL16DEL17TcF Laboratory collection pyrF696 : : TnJO JE1372 Laboratory collection JE1445 DEL902 Laboratory collection JE1684 eutE18 : : Mu dl-8 Roof and Roth ( 19 ) JE1685 eutEI8 : : Mu dl-8 derivative of JE1418 JE1687 eutEI8 : : Mu dl-8 eutF1115 derivative of JE1291 JE1690 eutF1115 derivative of JE1291 JE1692 eutE18 : : Mu dl-8 eutF1113 derivative of JE1291 derivative of JE1291 JE1693 eutF1113 JE1917 trp43 P. Margolin via J. Roth a All strains used were derivatives of S. typhimurium LT2 .
Unless otherwise stated , strains were constructed during the course of this work .
mutants were identified by their X-Gal phenotype ( white colonies ) on NCE containing glycerol , MgSO4 , ethanol-amine , CN-B12 , tryptophan , and X-Gal .
All spectrophotometric measurements were performed with a Perkin-Elmer Lambda 6 UV-VIS spectrophotometer .
The Perkin-Elmer UV Data Manager program was used to acquire and analyze data with an Epson Equity II + computer interfaced with the spectrophotometer .
( i ) ,3-Galactosidase activity assays .
P-Galactosidase activity assays were performed by the method of Miller ( 14 ) as described elsewhere ( 7 ) .
Units of activity was defined in nanomoles of o-nitrophenol produced per minute .
Units of,-galactosidase were normalized to cell density measured at 650 nm .
( ii ) EAL activity assay .
The level of ethanolamine ammo-nia-lyase ( EAL ) activity in cells was assayed by a modification of the method described by Sawicki et al. for the determination of aliphatic-aldehydes ( 21 ) .
The assay is based on the reaction of aliphatic-aldehydes with 3-methyl-2-benzothiazolinone hydrazone ( MBTH ) , which results in a stable adduct with a strong A305 ( e = 14.7 x 103 mol-1 cm-1 ) .
Cultures to be assayed were grown to the mid-log phase in NCE supplemented with glycerol , MgSO4 , ethanol-amine , tryptophan , and CN-B12 ( 150 nM ) .
Cells were harvested by centrifugation ( 8,000 x g for 10 min ) , resuspended in cold potassium phosphate buffer ( 0.35 M , pH 8.0 ) , and broken in a French pressure cell ( SLM/Aminco , Urbana , Ill. ) at 18,000 lb/in2 .
The cell extracts were extensively dialyzed ( dilution factor 10-5 ) against potassium phosphate buffer ( 0.35 M , pH 8.0 ) at 4 °C .
The reaction mixture contained ethanolamine at 200 , umol , potassium phosphate buffer ( pH 8.0 ) at 70 ixmol , KCI at 1 mmol , Ado-B12 at 30 nmol , and 0.03 to 0.05 mg of protein from the cell extract in a final volume of 333 RI .
The reaction was started by the addition of Ado-B12 and protected from light to minimize photolysis of Ado-B12 .
The reaction mixture was incubated at 37 °C for 10 min , the reaction was terminated by the addition of potassium citrate ( 200 , umol , pH 3.6 ) and MBTH ( 5 , umol ) , and the reaction mixture was incubated at 37 °C for an additional 10 min .
The reaction mixture was brought to a final volume of 1.2 ml with water , and the A305 was measured .
One unit of EAL activity was defined as the amount of enzyme needed to produce 1 nmol of acetaldehyde per min , and activity was normalized to milligrams of protein .
( iii ) Ethanolamine uptake assay .
Uptake of radiolabeled ethanolamine was determined by the method of Myers and Malloy ( 15 ) with the modifications as described below .
Strains to be tested were grown to the mid-log phase in NCE supplemented with MgSO4 , CN-B12 , glycerol , tryptophan , and ethanolamine .
Cells were harvested ( 8,000 x g for 10 min ) , washed with cold NCE , resuspended in cold NCE supplemented with glycerol ( 1 mM ) and chloramphenicol ( 50 , ug/ml ) , and stored on ice .
Cells were starved for ethanol-amine immediately before the assay by being shaken at room temperature for 15 min .
The reaction was started by the addition of 0.35 ml of cell suspension ( A650 , 1.0 to 1.2 ) to an equal volume of a 2x reaction mixture ( glycerol , 2 mM ; chloramphenicol , 100 , ug/ml ; [ 1,2-14C ] ethanolamine , 6.84 , uCi [ Amersham Corp. , Arlington Heights , Ill. ; specific radioactivity , 100 mCi/mmol ] ; and ethanolamine , 20 , uM ; final volume , 12 ml in NCE [ pH 7.0 ] ) , and the reaction mixture was mixed rapidly and incubated at room temperature .
Samples ( 100 , ul ) were removed from the reaction mixture at designated intervals and added to 4 ml of stop buffer ( morpholineethanesulfonic acid , 5 mM ; Tris , 5 mM ; KCl , 300 mM ; HgCl2 , 2 mM [ pH 7.0 ] ) .
Cells were filtered quickly through 0.45-p .
m-pore-size nitrocellulose filters ( Hoefer Scientific Instruments , San Francisco , Calif. ) prewetted with stop buffer by use of a Hoefer filter manifold ( model FH 224V ) , and the filters were rinsed with an additional 4 ml of stop buffer .
Filters were dried under a 150-W lamp .
Radio-activity retained by the filters was determined by counting for 1 min in a scintillation counter ( model 4530 ) from Packard Instruments , Downers Grove , Ill. .
RESULTS Isolation of EutF mutants .
A deletion in the eutF region ( DEL903 ; JE1418 ) was generated in-vivo by the method of Bochner et al. ( 4 ) with a TnJOdTc element located between the trp and tonB loci at 34 min on the S. typhimu-rium linkage map .
One of the strains isolated ( JE1418 ; Table 1 ) was a tryptophan auxotroph and had a functional cobA locus ( 8 ) but could not use ethanolamine as a sole carbon source when either CN-B12 or Ado-B12 was provided exog-enously .
These results suggested that JE1418 ( DEL903 ) was unable to utilize ethanolamine because of a deficiency in cobalamin transport caused by the deletion of the nearby tonB locus .
This possibility was unlikely for two reasons .
First , JE1418 could use CN-B12 or Ado-B12 for the synthesis of methionine at a concentration of 15 nM .
A tonB mutant strain requires micromolar concentrations of cobalamin to overcome the transport defect ( 1 ) .
Second , JE1418 was sensitive to infection by bacteriophage ES18 , which requires a functional tonB locus for absorption and subsequent infection of the cell ( 24 ) .
These results suggested that DEL903 did not delete the tonB locus .
Genetic analysis confirmed that the eutF locus was missing in deletion DEL903 .
A pyrF mutant strain , JE1372 , was transduced to pyr + with strain JE1418 ( DEL903 ) as the donor .
Pyr + transductants were screened for tryptophan auxotrophs ; tryptophan auxotrophy indicated the inheritance of DEL903 .
All the transductants that became tryptophan auxotrophs could no longer utilize ethanolamine as a carbon source ( 28 of 28 ) , indicating that DEL903 spanned the eutF locus .
Hydroxylaminegenerated mutations in eutF were isolated by localize JE1418 DEL903 NG 0.10 ( 0.26 ) JE1693 eutF113 NG 0.25 ( 0.79 ) JE1690 eutFII15 NG 0.13 ( 0.32 ) TR6583 eutF + 0.16 0.33 ( 0.85 ) a NCE was supplemented with ethanolamine ( 27 mM ) , MgSO4 , CN-B12 , and tryptophan .
b NCN was supplemented with glycerol , ethanolamine ( 27 mM ) , MgSO4 , CN-B12 , and tryptophan .
Numbers in parentheses are the final A650 values of the cultures measured after 42 h of growth ( all cultures had reached the stationary-phase ) .
To facilitate the isolation of eutF mutants , we took advantage of the lack of expression of a lacZ-transcriptional-fusion to the eutDEABCR operon in genetic backgrounds in which the eutF gene was defective ( see below ) .
Strain JE1684 carries an operon fusion of the lacZ + gene to the eutDEABCR promoter and displays an X-Gal + phenotype on indicator plates containing X-Gal , ethanolamine , and cobalamin .
A hydroxylamine-mutagenized lysate of bacteriophage P22 grown on strain JE1291 ( trp-3484 : : Tn1OdTc ) was used as the donor in a transductional cross with strain JE1684 ( eutEJ8 : : Mu dl-8 ) as the recipient ( hereafter referred to as eutE-lacZ ) .
We isolated tetracycline-resistant transductants and screened them for mutations which resulted in a lower expression of the eutE-lacZ fusion .
This protocol effectively eliminated mutations in the eutDEABCR operon which would result in the same X-Gal phenotype .
Ten independent mutations , 19 % cotransducible with the tetracycline resistance marker in trp , were isolated .
Two of the mutants , JE1693 ( eutF113 ) and JE1690 ( eutFI115 ) , were selected for further analysis .
Table 2 shows the growth phenotypes of eutF mutants .
These strains did not use ethanolamine as a sole carbon source and retained only a limited ability to utilize ethanolamine as a sole nitrogen source .
Growth studies showed that eutF + strains can use ethanolamine as a nitrogen source at concentrations as low as 0.27 mM ; all eutF mutants tested failed to grow at this concentration of ethanolamine ( data not shown ) .
When the concentration of ethanolamine was raised to 27 mM , all mutants grew , and the specific growth-rate ( doublings per hour [ , u ] ) for cultures grown on ethanolamine as the sole nitrogen source ranged from 0.33 in the wild type to 0.10 in the deletion mutant ( JE1418 ) .
Cultures of eutF mutants did not reach cell densities as high as did the wild type ( Table 2 ) .
One of the mutants ( JE1693 [ eutF1113 ] ) was unable to use ethanolamine as a sole carbon source but used ethanolamine as a sole nitrogen source at a rate ( , u = 0.25 ) that was only slightly slower than that of the wild-type strain .
The behavior of this mutant may have been the result of a mutation which only partially inhibits eutF function .
We also investigated the ability of eutF mutant strains to grow on acetate as the sole carbon and energy source , since acetate is a final product of ethanolamine catabolism .
Growth studies demonstrated that eutF mutant strains can utilize acetate as the sole source of carbon and energy at growth-rates identical to that of the wild type .
These results suggested that eutF mutations do not affect the utilization of acetate .
Deletion mapping of the eutF locus .
Three different classes of deletions were used to identify the position of the eutF locus relative to nearby ordered markers at 34 min , cobA , trp , and tonB ( 20 ) .
A mutant ( JE1445 ) with a deletion spanning the trp and cobA loci did not display a EutFphenotype , suggesting that eutF does not lie between these two markers .
A mutant with deletion trp43 , a partial deletion of the trp operon extending out of trp and toward tonB ( 3 ) , did not display a EutF-phenotype .
Finally , JE1418 ( DEL903 ) displayed Trp-and EutF-phenotypes , but the locus .
mutation did not affect either the cobA or the tonB Taken together , these results located the eutF gene between trp and tonB .
A three-factor cross experiment was performed to confirm the location of the eutF locus .
Donor strain JE1293 ( cobA366 : : TnlOdCm trp + eutF + ) was crossed with recipient strain JE1687 ( cobA + trp-3484 : : TnlOdrc eutFII15 eutEJ8 : : Mu dl-8 ) , and Cmr transductants were selected .
The Tc and X-Gal phenotypes of 960 Cmr recombinants were determined .
Four classes of recombinants were found , as follows ( followed by the total number of recombinants and their relative frequency ) : Tcs X-Gal - ( 721 , 75.1 % ) ; Tcr X-Gal - ( 166 , 17.2 % ) ; Tcs X-Gal + ( 62 , 6.5 % , the donor class ) ; and Tcr X-Gal + ( 11 , 1.2 % ) .
If the gene order inferred from deletion mapping experiments is correct , the rare class of Cmr recombinant would be one displaying Tcr and X-Gal + phenotypes ( this class requires four recombination events to be generated ) .
Such a class of transductant was obtained at a frequency of 1.2 % , a frequency lower than those obtained for other classes in the experiment .
These data were consistent with the gene order cobA trp eutF tonB .
Effect of the eutF locus on the transcription of the eut operon .
In their studies of the transcriptional regulation of the eutDEABCR operon , Roof and Roth documented that ethanolamine and cobalamin were necessary for induction ( 19 ) .
To assess any effect that a mutation in eutF would have on the expression of the eutDEABCR operon , we measured the expression in early-log-phase cultures ( A650 , 0.2 to 0.3 ) of the eutE-lacZ operon fusion in genetic backgrounds with either a wild-type or a defective eutF locus under conditions in which ethanolamine was not required for growth .
A strain carrying a deletion of eutF ( JE1685 ) showed an 18-fold reduction in the levels of 3-galactosidase activity measured ( 9 U/A650 unit ) when compared with a strain with a functional eutF + locus ( JE1684 ) ( 170 U/A650 unit ; < 2 unless both ethanolamine and CN-B12 were provided in the medium ) .
Hydroxylamine-generated mutations in eutF yielded similar results , although levels of P-galactosidase activity in these backgrounds ( eutF1113 [ 3 U/A650 unit ] and eutFIllS [ < 2 U/A650 unit ] ) were three-to five-fold lower than those in strains carrying DEL903 .
A second approach was used to assess the effect of a eutF mutation on the transcription of a eutE-lacZ operon fusion .
The expression of the eutE-lacZ operon fusion was determined as a function of the concentration of ethanolamine in strains with a functional or defective eutF locus .
Induction of the eutE-lacZ fusion in a eutF + background was tested for ethanolamine concentrations of up to 56 mM and found to be linear in the range of 0 to 100 , uM ethanolamine .
In a eutE-lacZ eutF + ( JE1684 ) strain , as little as 25 , uM ethanol-amine induced levels of , B-galactosidase fivefold higher than background levels , and no significant increase in induction was observed at concentrations higher than 1 mM ethanol-amine .
In contrast , only background levels of 3-galactosi-dase ( < 2 U A650 unit - ' ) were detected in a eutE-lacZ eutFll15 ( JE1687 ) strain grown in medium containing up t Strain acta Relevant genotype Sp activity TR6583 eutF + 199 100 JE1418 DEL903 23 12 JE1690 eutFI15 7 4 a Expressed in nanomoles of acetaldehyde produced per minute per milli-gram of protein .
Data shown are the averages of two separate experiments , and each determination was performed in duplicate .
The values shown are corrected for background .
56 mM ethanolamine , a concentration which exceeds the minimal concentration necessary for induction by > 2 orders of magnitude .
Taken together , these transcriptional studies indicated that the eutF mutation was responsible for the drastic reduction in the transcription of the eutDEABCR operon .
Effect of eutF on EAL activity .
EAL catalyzes the initial step in the catabolism of ethanolamine , an Ado-B12-depen-dent reaction that yields acetaldehyde and ammonia .
We compared the steady-state level of EAL activity present in eutF mutants to that present in the wild-type strain ( Table 3 ) .
The level of enzyme activity present in the mutants was low ( 10 % of the wild-type activity ) but was reproducibly detect-able .
This low level of activity may account for the limited ability of the mutants to use ethanolamine as a nitrogen source ( Table 2 ) .
Effect otf eutF on the uptake of ethanolamine .
The possibility that the phenom o a ut typeufteaFnt was due to a defect in the trarnsport of ethanolamine was addressed by performing uptakie assays with radiolabeled ethanolamine ( Fig. 1 ) .
On the basis of these data , it was clear that the transport of ethanolamine by eutF mutants was drastically reduced .
The rates of uptake were 56 nmol of ethanolamine s-A650 unit-1 for the wild type and 1.6 and 0.3 nmol s-1 A650 unit-1 for deletion mutant JE1418 ( DEL903 ) and a hydroxylaminegenerated mutant ( JE1690 [ eutFII15 ] ) , respectively .
Strain JE1693 ( eutF113 ) , which showed significant growth on ethanolamine as the sole nitrogen source , had a rate of uptake of 4.6 nmol of ethanolamine s-1 A650 unit-1 , approximately 10-fold lower than that of the wild type .
The rate of ethanolamine uptake , as well as the transcription of a eutE-lacZ operon fusion ( see above ) and EAL activity ( Table 3 ) , were consistently higher in the strain thought to carry a deletion of eutF ( JE1418 [ DEL903 ] ) than in the strain carrying a hydroxylamine-generated mutation in this locus ( eutFI115 ) .
It is difficult to explain these results without a molecular analysis of the nature of these mutations .
It is important to note that Roof and Roth ( 19 ) reported that the rate of ethanolamine uptake decreased in strains with any lesion in the eutDEABCR operon .
On the basis of this result , these authors concluded that the transport of ethanolamine may be coupled to its degradation ( 19 ) .
We confirmed their observation by performing ethanolamine uptake assays with strain JE1684 ( eutEJ8 : : Mu dl-8 ) ; this strain had no detectable uptake of ethanolamine .
DISCUSSION We have isolated mutations in the region of 34 min on the S. typhimurium chromosome , between the trp and tonB loci , which define the new genetic locus , eutF .
A eutF mutant can not use ethanolamine as a sole source of carbon and/or energy and retains only a limited ability to utilize ethanol-amine as a sole nitrogen source .
Our data are consistent with two possible roles for the EutF protein : ( i ) as an ethanol-amine permease or ( ii ) as a transcription factor required for the of the the mental evidence available at this point is not sufficient expression eutDEABCR operon .
However , to distinguish between these two models .
Roof and Roth ( 19 ) showed that any lesion in the eut DEABCR operon impaired transport and concluded that degradation and transport of ethanolamine were coupled .
Coupling between degradation and transport of a substrate has previously been demonstrated for proline utilization ( 12 , 16 , 27 ) .
If the eutF gene product is a transcription factor required for the expression of the eutDEABCR operon , a mutation in this locus would result in decreased expression of the eutE-lacZ operon fusion and low EAL activity and consequently abolish the transport of ethanolamine into the cell .
However , the data presented are also consistent with the model that eutF codes for a permease .
That is , a defect in the ethanolamine permease would prevent the uptake of a required coinducer of the transcription of the eutDEABCR operon , explaining the 18-fold decrease in expression of the eutE-lacZ operon fusion and the low levels of EAL activity measured .
Formally , eutF could code for a factor required for transcription of the ethanolamine permease and not the actual permease .
This model would also be consistent with our results .
Molecular analysis of the eutF locus is needed to establish its role in the catabolism of ethanolamine .
This work was supported by Public Health Service grant GM40313 to J.C.E.-S .
from the National Institute of General Med ical Sciences .
G.A.O. is a predoctoral trainee supported by NIH biotechnology training grant 1 T32 GM08349 .
We thank D. M. Roof and J. R. Roth for providing us with strains and for valuable comments on this work .
We also thank B. M. Cali and J. Silver for critical reading of the manuscript .
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